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Sample records for clotting factor viii

  1. EFFECT OF ORAL CONTRACEPTIVES (LD AND CILEST ON CLOTTING FACTORS VIII AND IX

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    H.R. Sadeghipour Roudsari

    1997-10-01

    Full Text Available Based on epidemiologic data, women who take oral contraceptives seem to have an increased risk of developing thromboembollic disease. The thrombotic effects of oral contraceptive (OC are probably mediated, at least partly through their effects on the coagulation system. Plasma levels of several clotting factors have been shown to be elevated in OC users, and this increase is graduated according to the dose of estrogen. In this study, fifty healthy and non smoking women, aged 18-35 years, were randomly assigned to treatment with 2 different OCs: a monophasic pill containing 30 pg of ethinyl estradiol plus 150µg levonorgestrel (LD and a monophasic pill containing 35µg ethinylestradiol plus 250pg norgestimate (Cilest. Factor VIII plasma values were significantly decreased (P<0.05 only in women treated with the preparation LD, but the levels of factor VIII were not significantly different in the group treated with Cilest. Factor IX plasma values were significantly increased (P<0.05 only in women treated with the preparation Cilest, but the levels of factor Ix were not significantly different in the group treated with LD. In LD and cilest users factors VIII and IX were not significantly changed (P<0.05 in overweight and obese subjects in comparison to normal weight.

  2. Improvement of fibrin clot structure after factor VIII injection in haemophilia A patients treated on demand.

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    Antovic, Aleksandra; Mikovic, Danijela; Elezovic, Ivo; Zabczyk, Michael; Hutenby, Kjell; Antovic, Jovan P

    2014-04-01

    Patients with haemophilia A have seriously impaired thrombin generation due to an inherited deficiency of factor (F)VIII, making them form unstable fibrin clots that are unable to maintain haemostasis. Data on fibrin structure in haemophilia patients remain limited. Fibrin permeability, assessed by a flow measurement technique, was investigated in plasma from 20 patients with severe haemophilia A treated on demand, before and 30 minutes after FVIII injection. The results were correlated with concentrations of fibrinogen, FVIII and thrombin-activatable fibrinolysis inhibitor (TAFI), and global haemostatic markers: endogenous thrombin potential (ETP) and overall haemostatic potential (OHP). Fibrin structure was visualised using scanning electron microscopy (SEM). The permeability coefficient Ks decreased significantly after FVIII treatment. Ks correlated significantly with FVIII levels and dosage, and with ETP, OHP and levels of TAFI. SEM images revealed irregular, porous fibrin clots composed of thick and short fibers before FVIII treatment. The clots had recovered after FVIII replacement almost to levels in control samples, revealing compact fibrin with smaller intrinsic pores. To the best of our knowledge, this is the first description of fibrin porosity and structure before and after FVIII treatment of selected haemophilia patients. It seems that thrombin generation is the main determinant of fibrin structure in haemophilic plasma.

  3. Tranexamic acid combined with recombinant factor VIII increases clot resistance to accelerated fibrinolysis in severe hemophilia A

    DEFF Research Database (Denmark)

    Hvas, Anne-Mette; Sørensen, Hanne Thykjær; Norengaard, Lisbeth

    2007-01-01

    BACKGROUND: Most patients with severe hemophilia A suffer from a profoundly compromised hemostatic response. In addition to both the delayed and slow development of a clot, previous studies have documented that severe hemophilia A is also associated with reduced clot stability. OBJECTIVES: We...... examined whether the clot stability in hemophiliacs could be improved by treatment with tranexamic acid (TXA) in combination with recombinant factor VIII (rFVIII). PATIENTS/METHODS: Baseline blood samples were obtained from eight males with severe hemophilia A. Thereafter, a bolus injection of r...... the elasticity curve increased 5-fold after rFVIII and 24-fold after addition of TXA. CONCLUSIONS: The study demonstrates that simultaneous treatment with TXA and rFVIII significantly improves the clot stability in patients with hemophilia A. Udgivelsesdato: December...

  4. Comparison of Clot-based, Chromogenic, and Fluorescence Assays for Measurement of Factor VIII Inhibitors in the U.S. Hemophilia Inhibitor Research Study

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    Miller, Connie H.; Rice, Anne S.; Boylan, Brian; Shapiro, Amy D.; Lentz, Steven R.; Wicklund, Brian M.; Kelly, Fiona M.; Soucie, J. Michael

    2015-01-01

    Summary Background Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety, and assessment of population trends. Methods Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA), and a novel fluorescence immunoassay (FLI). Results NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU)<0.5 and positive on 42/42 specimens (100%) with NBU≥2.0 and 43/80 specimens (53.8%) with NBU 0.5–1.9. Among specimens with positive NBA and negative CBA, 58.1% were FLI-negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0–1.9 NBU specimens and 43.1% and 50.0% of 0.5–0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P=0.004). Among 21 new inhibitors detected by NBA, 5 (23.8%) with 0.7–1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5–1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%). Conclusions FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5–1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays. PMID:23601690

  5. Impact of quality control matrix effect: application to the calculation of uncertainty of measurement in one-stage clotting factor VIII assay.

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    Sobas, Frédéric; Benattar, Norbert; Bellisario, Audrey; Marin, Sylvie; Nougier, Christophe; Lienhart, Anne; Négrier, Claude

    2010-07-01

    In Europe, the ISO 15 189 standard requires uncertainty of measurement to be calculated for all measurands. We calculated the analytical imprecision and bias of our factor VIII coagulometric assay method between 5 and 80 U/dl, using plasmas expected to be at 5, 30 and 80 U/dl of factor VIII. We implemented Meijer et al.'s [Clin Chem 2002; 48:1011-1015] long-term coefficient of variance, bias and also uncertainty of measurement calculations. Assessments used reference plasma diluted in severe haemophilic plasma, in immunodepleted factor VIII-deficient plasma and in bovine serum albumin. With plasmas diluted in severe haemophilic and immunodepleted factor VIII-deficient plasma, calculated uncertainty of measurement was 10% compared with 15% (i.e., 50% greater) for plasma diluted in albumin buffer or as calculated from European Concerted Action on Thrombosis consensus values. It is thus important to approximate the patient sample matrix to obtain as precise an estimation as possible of assay method uncertainty of measurement.

  6. Presentation and management of a stage Ia lung cancer patient with a paraneoplastic factor VIII inhibitor.

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    Laselle, Brooks T; Boggio, Lisa N; Blum, Matthew G

    2006-01-01

    Acquired inhibitors to clotting factors most commonly involve factor VIII and are associated with autoimmune disease. Factor VIII inhibitors can cause severe spontaneous and iatrogenic bleeding that is difficult to manage. Factor VIII inhibitors are rarely associated with solid tumors and only three cases of adenocarcinoma of the lung have been reported. This report describes the multidisciplinary management of a factor VIII inhibitor-producing stage Ia lung adenocarcinoma that ultimately resulted in complete resectability.

  7. Advances in clotting factor treatment for congenital hemorrhagic disorders.

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    Tarantino, Michael D; Aledort, Louis M

    2004-06-01

    During the last 50 years, clotting factor replacement has evolved from the use of frozen plasma in the 1950s, through the serendipitous discovery of cryoprecipitate in the 1960s and the development of purified clotting factors in the 1970s and 1980s, to the era of recombinant clotting factors beginning in the 1990s. The dawn of the new millennium has seen the refinement of recombinant factor (rF) VIII with enhanced safety via the elimination of plasma-derived culture media or product stabilizers. During the last decade of the 20th century, a cure for hemophilia through gene therapy became a possibility. This was, in part, facilitated by availability of large (dogs) and small (mice) animal models for hemophilia A and B. Although this review will focus primarily on clotting factor replacement, the reader may refer to recent discourse on gene therapy for hemophilia.

  8. Coagulation factor VIII activity in diabetic patients

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    Nermina Babić

    2011-02-01

    Full Text Available Aim To examine coagulation factor VIII activity in plasma, as a risk factor for thrombosis, in the patients with diabetes mellitus (DM. Also, to assess its relationship with ibrinogen and fasting blood glucose concentrations and with body mass index. Methods The plasma coagulation factor VIII activity, plasma levels of ibrinogen and blood glucose concentrations were measured in 30 patients with DM type 1, 30 patients with DM type 2 and in 30 healthy subjects. Body weight and body height were also measured and BMI was calculated.Results The plasma factor VIII activity in patients with DM type 1 and patients with DM type 2 was signiicantly higher than the values measured in healthy subjects. There was no signiicant difference in the factor VIII activity between patients with DM type 1 and type 2. The concentrations of ibrinogen and blood glucose in both groups of patients were signiicantly higher than in the group of healthy subjects. Patients with DM type 2 had a signiicantly higher BMI compared to healthy subjects, as well as compared to patients with DM type 1. There was a signiicant positive correlation between plasma factor VIII activity and plasma level of ibrinogen and a signiicant negative correlation between factor VIII activity and BMI in patients with DM type 2. Conclusion Diabetic patients have the elevated plasma coagulation factor VIII activity and increased ibrinogen concentration thus an increased risk of thrombosis and vascular diseases.

  9. Blood coagulation factor VIII: An overview

    Indian Academy of Sciences (India)

    G M Bhopale; R K Nanda

    2003-12-01

    Factor VIII (FVIII) functions as a co-factor in the blood coagulation cascade for the proteolytic activation of factor X by factor IXa. Deficiency of FVIII causes hemophilia A, the most commonly inherited bleeding disorder. This review highlights current knowledge on selected aspects of FVIII in which both the scientist and the clinician should be interested.

  10. Acquired factor VIII inhibitor syndrome: A rare cause of hematuria

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    Muthuvel Seral Kannan

    2015-01-01

    Full Text Available A 50-year-old woman presented with gross hematuria for 1 month. Clinical examinations, laboratory investigations, ultrasound and contrast computed tomography were normal, except anemia. Cystoscopy revealed bloody efflux from the right side. Retrograde pyelogram showed filling defect in the renal pelvis and biopsy was inconclusive. Renal angiogram was normal. She developed ecchymosis on the right thigh and arm with elevated activated partial thromboplastin time. The partial thromboplastin time correction study and Bethesda study confirmed the presence of acquired factor VIII inhibitor (acquired hemophilia. With flexible ureterorenoscopy, the mass in the renal pelvis was removed and its histopathology revealed clotted blood. The patient was subsequently managed with steroids and Factor eight inhibitor bypass activity.

  11. Game, set, match for factor VIII mismatch?

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    Miller, Connie H

    2015-08-13

    In this issue of Blood, Gunasekera et al provide evidence that the high rate of factor VIII (FVIII) inhibitors seen in black hemophilia A (HA) patients is not due to a mismatch between the structure of treatment products and FVIII genotypes common in blacks.

  12. Venom Concentrations and Clotting Factor Levels in a Prospective Cohort of Russell's Viper Bites with Coagulopathy.

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    Geoffrey K Isbister

    Full Text Available Russell's viper envenoming is a major problem in South Asia and causes venom induced consumption coagulopathy. This study aimed to investigate the kinetics and dynamics of venom and clotting function in Russell's viper envenoming.In a prospective cohort of 146 patients with Russell's viper envenoming, we measured venom concentrations, international normalised ratio [INR], prothrombin time (PT, activated partial thromboplastin time (aPTT, coagulation factors I, II, V, VII, VIII, IX and X, and von Willebrand factor antigen. The median age was 39 y (16-82 y and 111 were male. The median peak INR was 6.8 (interquartile range [IQR]: 3.7 to >13, associated with low fibrinogen [median,3 at 6 h post-antivenom but had reduced to <2, by 24 h. The aPTT had also returned to close to normal (<50 sec at 24 h. Factor VII, VIII and IX levels were unusually high pre-antivenom, median peak concentrations of 393%, 307% and 468% respectively. Pre-antivenom venom concentrations and the INR (r = 0.20, p = 0.02 and aPTT (r = 0.19, p = 0.03 were correlated (non-parametric Spearman analysis.Russell's viper coagulopathy results in prolonged aPTT, INR, low fibrinogen, factors V, VIII and X which recover over 48 h. Severity of clotting abnormalities was associated with venom concentrations.

  13. Acquired Factor VIII Inhibitors: Three Cases

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    Tay Za Kyaw

    2013-03-01

    Full Text Available Acquired hemophilia A is a rare, but devastating bleeding disorder caused by spontaneous development of autoantibodies directed against coagulation factor VIII. In 40%-50% of patients it is associated with such conditions as the postpartum period, malignancy, use of medications, and autoimmune diseases; however, its cause is unknown in most cases. Acquired hemophilia A should be suspected in patients that present with a coagulation abnormality, and a negative personal and family history of bleeding. Herein we report 3 patients with acquired hemophilia A that had different underlying pathologies, clinical presentations, and therapeutic responses. Factor VIII inhibitor formation in case 1 occurred 6 months after giving birth; underlying disorders were not identified in cases 2 or 3. The bleeding phenotype in these patients’ ranged from no bleeding tendency with isolated prolongation of APTT (activated partial thromboplastin time to severe intramuscular hematoma and hemarthrosis necessitating recombinant activated factor VII infusion and blood components transfusion. Variable responses to immunosuppressive treatment were also observed.

  14. Analysis of mutations in the entire coding sequence of the factor VIII gene

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    Bidichadani, S.I.; Lanyon, W.G.; Connor, J.M. [Glascow Univ. (United Kingdom)] [and others

    1994-09-01

    Hemophilia A is a common X-linked recessive disorder of bleeding caused by deleterious mutations in the gene for clotting factor VIII. The large size of the factor VIII gene, the high frequency of de novo mutations and its tissue-specific expression complicate the detection of mutations. We have used a combination of RT-PCR of ectopic factor VIII transcripts and genomic DNA-PCRs to amplify the entire essential sequence of the factor VIII gene. This is followed by chemical mismatch cleavage analysis and direct sequencing in order to facilitate a comprehensive search for mutations. We describe the characterization of nine potentially pathogenic mutations, six of which are novel. In each case, a correlation of the genotype with the observed phenotype is presented. In order to evaluate the pathogenicity of the five missense mutations detected, we have analyzed them for evolutionary sequence conservation and for their involvement of sequence motifs catalogued in the PROSITE database of protein sites and patterns.

  15. Development and characterization of recombinant ovine coagulation factor VIII.

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    Zakas, Philip M; Gangadharan, Bagirath; Almeida-Porada, Graca; Porada, Christopher D; Spencer, H Trent; Doering, Christopher B

    2012-01-01

    Animal models of the bleeding disorder, hemophilia A, have been an integral component of the biopharmaceutical development process and have facilitated the development of recombinant coagulation factor VIII (fVIII) products capable of restoring median survival of persons with hemophilia A to that of the general population. However, there remain several limitations to recombinant fVIII as a biotherapeutic, including invasiveness of intravenous infusion, short half-life, immunogenicity, and lack of availability to the majority of the world's population. The recently described ovine model of hemophilia A is the largest and most accurate phenocopy. Affected sheep die prematurely due to bleeding-related pathogenesis and display robust adaptive humoral immunity to non-ovine fVIII. Herein, we describe the development and characterization of recombinant ovine fVIII (ofVIII) to support further the utility of the ovine hemophilia A model. Full-length and B-domain deleted (BDD) ofVIII cDNAs were generated and demonstrated to facilitate greater biosynthetic rates than their human fVIII counterparts while both BDD constructs showed greater expression rates than the same-species full-length versions. A top recombinant BDD ofVIII producing baby hamster kidney clone was identified and used to biosynthesize raw material for purification and biochemical characterization. Highly purified recombinant BDD ofVIII preparations possess a specific activity nearly 2-fold higher than recombinant BDD human fVIII and display a differential glycosylation pattern. However, binding to the carrier protein, von Willebrand factor, which is critical for stability of fVIII in circulation, is indistinguishable. Decay of thrombin-activated ofVIIIa is 2-fold slower than human fVIII indicating greater intrinsic stability. Furthermore, intravenous administration of ofVIII effectively reverses the bleeding phenotype in the murine model of hemophilia A. Recombinant ofVIII should facilitate the maintenance of

  16. Development and characterization of recombinant ovine coagulation factor VIII.

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    Philip M Zakas

    Full Text Available Animal models of the bleeding disorder, hemophilia A, have been an integral component of the biopharmaceutical development process and have facilitated the development of recombinant coagulation factor VIII (fVIII products capable of restoring median survival of persons with hemophilia A to that of the general population. However, there remain several limitations to recombinant fVIII as a biotherapeutic, including invasiveness of intravenous infusion, short half-life, immunogenicity, and lack of availability to the majority of the world's population. The recently described ovine model of hemophilia A is the largest and most accurate phenocopy. Affected sheep die prematurely due to bleeding-related pathogenesis and display robust adaptive humoral immunity to non-ovine fVIII. Herein, we describe the development and characterization of recombinant ovine fVIII (ofVIII to support further the utility of the ovine hemophilia A model. Full-length and B-domain deleted (BDD ofVIII cDNAs were generated and demonstrated to facilitate greater biosynthetic rates than their human fVIII counterparts while both BDD constructs showed greater expression rates than the same-species full-length versions. A top recombinant BDD ofVIII producing baby hamster kidney clone was identified and used to biosynthesize raw material for purification and biochemical characterization. Highly purified recombinant BDD ofVIII preparations possess a specific activity nearly 2-fold higher than recombinant BDD human fVIII and display a differential glycosylation pattern. However, binding to the carrier protein, von Willebrand factor, which is critical for stability of fVIII in circulation, is indistinguishable. Decay of thrombin-activated ofVIIIa is 2-fold slower than human fVIII indicating greater intrinsic stability. Furthermore, intravenous administration of ofVIII effectively reverses the bleeding phenotype in the murine model of hemophilia A. Recombinant ofVIII should facilitate

  17. Continuous infusion of factor VIII for surgery and major bleeding.

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    Hay, C R; Doughty, H I; Savidge, G F

    1996-03-01

    In a clinical trial, 24 patients with haemophilia A who needed surgery or had suffered severe bleeding were treated by continuous infusion of Monoclate P, a factor VIII concentrate that is immunopurified by monoclonal antibodies. Continuous infusion of Monoclate P began with a dose of 2 U/kg per h that was adjusted according to the results of factor VIII assays to achieve a factor VIII target level of 100 IU/dl for 2 days and then 80 IU/dl for 5 days. The safety, efficacy, and economics of this approach were assessed. No haemorrhagic episodes were observed. The continuous infusion was convenient and had the advantage of producing steady-state levels of factor VIII. With a single-compartment model, we found median factor VIII clearance values of 3.11 (range 1.79-7.78) x 10(3) litres/kg per h, elimination rates of 5.0-19.4 x 10(-2)/h and a median half-life of 9.9 h (range 4.8-20.0 h). Clearance and the elimination rate appeared to decline over the infusion period, as judged by the decreasing infusion rate required to maintain the target concentration of factor VIII. An economic comparison with bolus therapy, using theoretically derived bolus dosages, indicated that the potential saving was related inversely to the factor VIII half-life. Potential savings of 75% were predicted on the first postoperative day, averaging 35% over the full course of therapy.

  18. Blood Clots

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    ... or prevent blood clots from dissolving properly. Risk factors for excessive blood clotting include Certain genetic disorders Atherosclerosis Diabetes Atrial fibrillation Overweight, obesity, and metabolic syndrome Some medicines Smoking deep vein ...

  19. Idiopathic Acquired Hemophilia A with Undetectable Factor VIII Inhibitor

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    Nicholas B. Abt

    2014-01-01

    Full Text Available Objective. We present the case of a 73-year-old female, with no family or personal history of a bleeding disorder, who had a classic presentation for acquired hemophilia A. Factor VIII activity was low but detectable and a factor VIII inhibitor was undetectable. Methods. The patient’s plasma was comprehensively studied to determine the cause of the acquired coagulopathy. Using the Nijmegen modification of the Bethesda assay, no factor VIII autoantibody was measureable despite varying the incubation time from 1 to 3 hours. Results. The aPTT was prolonged at 46.8 seconds, which did not correct in the 4 : 1 mix but did with 1 : 1 mix. Using a one stage factor VIII activity assay, the FVIII activity was 16% and chromogenic FVIII activity was also 16%. The patient was treated with recombinant FVII and transfusion, significantly reducing bleeding. Long-term therapy was initiated with cyclophosphamide and prednisone with normalization of FVIII activity. Conclusions. Physicians can be presented with the challenging clinical picture of an acquired factor VIII inhibitor without a detectable inhibitor by the Bethesda assay. Standard therapy for an acquired hemophilia A should be considered.

  20. Functional regions in coagulation factor VIII explored by mass spectrometry

    NARCIS (Netherlands)

    Bloem, E.

    2013-01-01

    The molecular mechanisms behind the function of factor VIII (FVIII) have remained poorly understood. FVIII acts in the blood coagulation cascade as cofactor for activated factor IX (FIXa) in the membrane bound activated factor X generating (FXase) complex. A functional absence in FVIII leads to the

  1. Collagen-bound von Willebrand factor has reduced affinity for factor VIII.

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    Bendetowicz, A V; Wise, R J; Gilbert, G E

    1999-04-30

    von Willebrand factor (vWf) is a multimeric adhesive glycoprotein that serves as a carrier for factor VIII in plasma. Although each vWf subunit displays a high affinity binding site for factor VIII in vitro, in plasma, only 2% of the vWf sites for factor VIII are occupied. We investigated whether interaction of plasma proteins with vWf or adhesion of vWf to collagen may alter the affinity or availability of factor VIII-binding sites on vWf. When vWf was immobilized on agarose-linked monoclonal antibody, factor VIII bound to vWf with high affinity, and neither the affinity nor binding site availability was influenced by the presence of 50% plasma. Therefore, plasma proteins do not alter the affinity or availability of factor VIII-binding sites. In contrast, when vWf was immobilized on agarose-linked collagen, its affinity for factor VIII was reduced 4-fold, with KD increasing from 0.9 to 3.8 nM. However, one factor VIII-binding site remained available on each vWf subunit. A comparable reduction in affinity for factor VIII was observed when vWf was a constituent of the subendothelial cell matrix and when it was bound to purified type VI collagen. In parallel with the decreased affinity for factor VIII, collagen-bound vWf displayed a 6-fold lower affinity for monoclonal antibody W5-6A, with an epitope composed of residues 78-96 within the factor VIII-binding motif of vWf. We conclude that collagen induces a conformational change within the factor VIII-binding motif of vWf that lowers the affinity for factor VIII.

  2. Isolation, subunit structure, and proteolytic modification of bovine factor VIII.

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    Legaz, M E; Weinstein, M J; Heldebrant, C M; Davie, E W

    1975-01-20

    A new method has been described for the isolation of factor VIII. The method results in a high yield of factor VIII that is homogeneous by several different criteria. The purified protein is very stable and is not dissociated in the presence of 1 M NaCl or 0.25 M CaCl2. The highly purified protein is readily activated and inactivated by various proteolytic enzymes, such as thrombin, plasmin, and trypsin. The molecular events that lead to the activation reaction, however, have not been established.

  3. Assessing patients' and caregivers' perspectives on stability of factor VIII products for haemophilia A

    DEFF Research Database (Denmark)

    DiBenedetti, D B; Coles, T M; Sharma, T

    2014-01-01

    Haemophilia A is a rare inherited bleeding disorder characterized by an inability of the blood to clot normally. Patients can experience spontaneous or trauma-induced joint and soft tissue bleeding and must keep coagulation factor VIII (FVIII) accessible at all times; thus, FVIII product storage ...... convenient and accessible FVIII products for patients in daily life and while travelling. In addition, the use of social media has potential value in recruiting this population....... and stability are critical. Our primary objective was to assess haemophilia A patients' and caregivers' experiences and preferences with FVIII product storage and stability. A secondary objective was to evaluate the use of the social media site Facebook in recruitment. In this cross-sectional study, 145 English...

  4. Recombinant B domain deleted porcine factor VIII for the treatment of bleeding episodes in adults with acquired hemophilia A.

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    Gomperts, Edward

    2015-08-01

    Hemophilia A is an inherited deficiency of clotting factor VIII (FVIII) often complicated by inhibitor development (CHAWI) in which neutralizing antibodies block the therapeutic benefit of replacement therapy. Inhibitors to FVIII can also be seen in an auto-immune disease known as acquired hemophilia A (AHA). 'Bypassing' therapies have been shown to provide hemostasis but dosing must be done empirically because current assays cannot measure objective markers of treatment efficacy and safety. A recombinant porcine sequence factor VIII (r-pFVIII) has been developed for the management of AHA. Preclinical, Phase I and Phase II clinical research studies in CHAWI subjects showed therapeutic potential and safety of this agent. A Phase II/III study in AHA with serious bleeding episodes shows a positive response in all subjects after administration. Based on current preclinical and clinical trial data, r-pFVIII should become the first line of treatment in the management of hemorrhage in patients with AHA.

  5. Factor VIII/V C-domain swaps reveal discrete C-domain roles in factor VIII function and intracellular trafficking.

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    Ebberink, Eduard H T M; Bouwens, Eveline A M; Bloem, Esther; Boon-Spijker, Mariëtte; van den Biggelaar, Maartje; Voorberg, Jan; Meijer, Alexander B; Mertens, Koen

    2017-04-01

    Factor VIII C-domains are believed to have specific functions in cofactor activity and in interactions with von Willebrand factor. We have previously shown that factor VIII is co-targeted with von Willebrand factor to the Weibel-Palade bodies in blood outgrowth endothelial cells, even when factor VIII carries mutations in the light chain that are associated with defective von Willebrand factor binding. In this study, we addressed the contribution of individual factor VIII C-domains in intracellular targeting, von Willebrand factor binding and cofactor activity by factor VIII/V C-domain swapping. Blood outgrowth endothelial cells were transduced with lentivirus encoding factor V, factor VIII or YFP-tagged C-domain chimeras, and examined by confocal microscopy. The same chimeras were produced in HEK293-cells for in vitro characterization and chemical foot-printing by mass spectrometry. In contrast to factor VIII, factor V did not target to Weibel-Palade bodies. The chimeras showed reduced Weibel-Palade body targeting, suggesting that this requires the factor VIII C1-C2 region. The factor VIII/V-C1 chimera did not bind von Willebrand factor and had reduced affinity for activated factor IX, whereas the factor VIII/V-C2 chimera showed a minor reduction in von Willebrand factor binding and normal interaction with activated factor IX. This suggests that mainly the C1-domain carries factor VIII-specific features in assembly with von Willebrand factor and activated factor IX. Foot-printing analysis of the chimeras revealed increased exposure of lysine residues in the A1/C2- and C1/C2-domain interface, suggesting increased C2-domain mobility and disruption of the natural C-domain tandem pair orientation. Apparently, this affects intracellular trafficking, but not extracellular function. Copyright© Ferrata Storti Foundation.

  6. Polymorphism of clotting factors in Hungarian patients with Raynaud's phenomenon.

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    Shemirani, Amir-Houshang; Szomják, Edit; Balogh, Emese; András, Csilla; Kovács, Dóra; Acs, Judit; Csiki, Zoltán

    2011-01-01

    Patients with primary Raynaud's phenomenon may have a genetically determined risk for clotting factors that predispose them to aberrant microvascular thrombosis. We investigated the prevalence of factor V substitution of G to A at position 1691 (FVLeiden), prothrombin G20210A, and methyltetrahydrofolate reductase C677T mutations in these patients. Two hundred (158 women, 42 men, mean age of 42.4 ± 13.7 years) consecutive patients with primary Raynaud's phenomenon and 200 age-sex-matched healthy controls of Hungarian origin were included in a case-control study. The prevalence of methyltetrahydrofolate reductase C677T homozygous among patients was significantly lower than in the control group (odds ratio 0.4, 95% confidence interval 0.2-0.9, P Raynaud's phenomenon and control subjects. FVLeiden, prothrombin G20210A, and polymorphism, prothrombin G20210A mutations have no apparent effect on the etiology of primary Raynaud's phenomenon.

  7. 42 CFR 410.63 - Hepatitis B vaccine and blood clotting factors: Conditions.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 2 2010-10-01 2010-10-01 false Hepatitis B vaccine and blood clotting factors... Other Health Services § 410.63 Hepatitis B vaccine and blood clotting factors: Conditions... under § 410.10, subject to the specified conditions: (a) Hepatitis B vaccine: Conditions. Effective...

  8. Evaluation of Factor VIII as a Risk Factor in Indian Patients with DVT.

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    Hazra, Darpanarayan; Sen, Indrani; Stephen, Edwin; Agarwal, Sunil; Nair, Sukesh Chandran; Mammen, Joy

    2015-01-01

    Introduction. Elevated factor VIII population in the Indian population has not been studied as a possible risk factor for deep vein thrombosis (DVT). High factor VIII level is considered a predisposing factor for DVT and its recurrence. However it is known to vary between populations and its exact role in the etiopathogenesis of thrombophilia remains unknown. Material and Methods. Factor VIII levels of patients with DVT who had undergone a prothrombotic workup as a part of their workup was compared to normal age matched controls in a 1 : 3 ratio. Results. There were 75 patients with DVT who had undergone a prothrombotic workup in the course of their treatment for lower limb DVT. In these, 64% had levels of factor VIII more than 150 as compared to 63% of normal controls (p > 0.05, not significant). Conclusion. Elevated factor VIII in the Indians may not be associated with the same thrombotic risk as seen in the West. We find a variation in the levels of factor VIII with a different "normal" than what is reported in other populations. This needs further study to elucidate the role of factor VIII in the evaluation and treatment of thrombophilia.

  9. Elevated factor VIII level and stroke in patients without traditional risk factors associated with cardiovascular diseases

    Directory of Open Access Journals (Sweden)

    Lasek-Bal A

    2013-06-01

    Full Text Available Anetta Lasek-Bal, Przemyslaw Puz, Zofia KazibutowskaStroke Unit, Department of Neurology, Medical University of Silesia, Professor Leszek Giec Upper Silesian Medical Centre, Katowice, PolandIntroduction: Hemostasis is affected by interactions between physiological processes, including those connected with the coagulation system, whose essence is converting fibrinogen into fibrin. The role of factor VIII (FVIII consists in activating factor X, which directly participates in the generation of thrombin, which is able to produce stable fibrin, which in turn forms blood clots. There are divergent opinions regarding the significance of high levels of FVIII in stroke pathogenesis.Aim: The aim of our study was to evaluate FVIII activity in individuals with cryptogenic stroke in order to determine a potential relationship between it and cerebral ischemia.Material and methods: Nine patients suffering with stroke were used in this study: six women and three men aged 49–63 years. In all of the patients, the presence of known and potential risk factors for stroke had been excluded during previous diagnostic procedures. These patients accounted for 1.2% of the 719 people who suffered a stroke and were hospitalized in 2011 at the Stroke Unit. FVIII activity was examined in each of the nine qualified subjects within 1–2 months of the occurrence of stroke (the first test and repeated (the second test in five patients with abnormal results obtained from the first examination.Results: Increased activity of FVIII was found in 5 out of 9 patients. In patients with abnormal results, elevated FVIII was found in follow-up examinations in the 8th–10th month following stroke. Hemodynamic abnormalities in carotid or cerebral artery (presence of thrombus were found in 3 of the 5 patients with increased FVIII levels. In the first 24 hours following stroke the neurological state of patients with abnormal FVIII was worse than individuals with normal FVIII activity. The

  10. Factor VIII therapy for hemophilia A: current and future issues.

    Science.gov (United States)

    Aledort, Louis; Ljung, Rolf; Mann, Kenneth; Pipe, Steven

    2014-06-01

    Hemophilia A is a congenital, recessive, X-linked bleeding disorder that is managed with infusions of plasma-derived or recombinant factor (F) VIII. The primary considerations in FVIII replacement therapy today are the: 1) immunogenicity of FVIII concentrates, 2) role of longer-acting FVIII products, 3) prophylactic use of FVIII in children and adults with severe hemophilia A, and 4) affordability and availability of FVIII products. Improving patient outcomes by increasing the use of FVIII prophylaxis, preventing or eliminating FVIII inhibitors, and expanding access to FVIII concentrates in developing countries are the major challenges confronting clinicians who care for patients with hemophilia A.

  11. Comparison of factor VIII transgenes bioengineered for improved expression in gene therapy of hemophilia A.

    Science.gov (United States)

    Dooriss, Kerry L; Denning, Gabriela; Gangadharan, Bagirath; Javazon, Elisabeth H; McCarty, David A; Spencer, H Trent; Doering, Christopher B

    2009-05-01

    Successful gene therapy of hemophilia A depends on the sustained expression of therapeutic levels of factor VIII (fVIII). Because of mRNA instability, interactions with resident endoplasmic reticulum (ER) chaperones, and the requirement for carbohydrate-facilitated transport from the ER to the Golgi apparatus, fVIII is expressed at much lower levels from mammalian cells than other proteins of similar size and complexity. A number of bioengineered forms of B domain-deleted (BDD) human fVIII have been generated and shown to have enhanced expression. Previously, we demonstrated that recombinant BDD porcine fVIII exhibits high-level expression due to specific sequence elements that increase biosynthesis via enhanced posttranslational transit through the secretory pathway. In the current study, high-expression recombinant fVIII constructs were compared directly in order to determine the relative expression of the various bioengineered fVIII transgenes. The data demonstrate that BDD porcine fVIII expression is superior to that of any of the human fVIII variant constructs tested. Mean fVIII expression of 18 units/10(6) cells/24 hr was observed from HEK-293 cells expressing a single copy of the porcine fVIII transgene, which was 36- to 225-fold greater than that of any human fVIII transgene tested. Furthermore, greater than 10-fold higher expression was observed in human cells transduced with BDD porcine fVIII versus BDD human fVIII-encoding lentiviral vectors, even at low proviral copy numbers, supporting its use over other human fVIII variants in future hemophilia A gene therapy clinical trials.

  12. Reduction of Factor VIII Inhibitor Titers During Immune Tolerance Induction With Recombinant Factor VIII-Fc Fusion Protein.

    Science.gov (United States)

    Groomes, Charles L; Gianferante, David M; Crouch, Gary D; Parekh, Dina S; Scott, David W; Lieuw, Kenneth

    2016-05-01

    The development of inhibitors toward factor VIII (FVIII) is a common and serious complication of hemophilia A (HA) therapy. Patients with hemophilia who develop inhibitors often undergo time- and resource-intensive immune tolerance induction (ITI) protocols. We report a 15-month-old male with severe HA and a high-titer inhibitor that occurred while receiving prophylactic treatment with recombinant FVIII (rFVIII), in whom significant inhibitor titer reduction was achieved with thrice weekly infusions of a new, prolonged half-life rFVIII-Fc fusion protein product (trade name Eloctate). Further studies are warranted to explore the potential of Eloctate in ITI protocols.

  13. von Willebrand factor binds to the surface of dendritic cells and modulates peptide presentation of factor VIII.

    Science.gov (United States)

    Sorvillo, Nicoletta; Hartholt, Robin B; Bloem, Esther; Sedek, Magdalena; ten Brinke, Anja; van der Zwaan, Carmen; van Alphen, Floris P; Meijer, Alexander B; Voorberg, Jan

    2016-03-01

    It has been proposed that von Willebrand factor might affect factor VIII immunogenicity by reducing factor VIII uptake by antigen presenting cells. Here we investigate the interaction of recombinant von Willebrand factor with immature monocyte-derived dendritic cells using flow cytometry and confocal microscopy. Surprisingly, von Willebrand factor was not internalized by immature dendritic cells, but remained bound to the cell surface. As von Willebrand factor reduces the uptake of factor VIII, we investigated the repertoire of factor VIII presented peptides when in complex with von Willebrand factor. Interestingly, factor VIII-derived peptides were still abundantly presented on major histocompatibility complex class II molecules, even though a reduction of factor VIII uptake by immature dendritic cells was observed. Inspection of peptide profiles from 5 different donors showed that different core factor VIII peptide sequences were presented upon incubation with factor VIII/von Willebrand factor complex when compared to factor VIII alone. No von Willebrand factor peptides were detected when immature dendritic cells were pulsed with different concentrations of von Willebrand factor, confirming lack of von Willebrand factor endocytosis. Several von Willebrand factor derived peptides were recovered when cells were pulsed with von Willebrand factor/factor VIII complex, suggesting that factor VIII promotes endocytosis of small amounts of von Willebrand factor by immature dendritic cells. Taken together, our results establish that von Willebrand factor is poorly internalized by immature dendritic cells. We also show that von Willebrand factor modulates the internalization and presentation of factor VIII-derived peptides on major histocompatibility complex class II.

  14. Enhancement of pig embryonic implants in factor VIII KO mice: a novel role for the coagulation cascade in organ size control.

    Directory of Open Access Journals (Sweden)

    Anna Aronovich

    Full Text Available Very little is known about the mechanisms that contribute to organ size differences between species. In the present study, we used a mouse model of embryonic pig tissue implantation to define the role of host Factor VIII in controlling the final size attained by the implant. We show here that pig embryonic spleen, pancreas, and liver all grow to an increased size in mice that are deficient in the Factor VIII clotting cascade. Similar results were obtained using the transplantation model after treatment with the low molecular weight heparin derivative Clexane which markedly enhanced transplant size. Likewise, enhanced size was found upon treatment with the direct thrombin inhibitor Dabigatran, suggesting that organ size regulation might be mediated by thrombin, downstream of Factor VIII. Considering that thrombin was shown to mediate various functions unrelated to blood clotting, either directly by cleavage of protease-activated receptors (PARs or indirectly by cleaving osteopontin (OPN on stroma cells, the role of PAR1 and PAR4 antagonists as well as treatment with cleaved form of OPN (tcOPN were tested. While the former was not found to have an impact on overgrowth of embryonic pig spleen implants, marked reduction of size was noted upon treatment with the (tcOPN. Collectively, our surprising set of observations suggests that factors of the coagulation cascade have a novel role in organ size control.

  15. Hemophilia A gene therapy via intraosseous delivery of factor VIII-lentiviral vectors.

    Science.gov (United States)

    Miao, Carol H

    2016-01-01

    Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, approximately 25 % of treated patients develop anti-factor VIII immune responses. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-factor VIII antibody formation is highly desired. Lentiviral vector (LV)-mediated gene transfer into hematopoietic stem cells (HSCs) results in stable integration of FVIII gene into the host genome, leading to persistent therapeutic effect. However, ex vivo HSC gene therapy requires pre-conditioning which is highly undesirable for hemophilia patients. The recently developed novel methodology of direct intraosseous (IO) delivery of LVs can efficiently transduce bone marrow cells, generating high levels of transgene expression in HSCs. IO delivery of E-F8-LV utilizing a ubiquitous EF1α promoter generated initially therapeutic levels of FVIII, however, robust anti-FVIII antibody responses ensued neutralized functional FVIII activity in the circulation. In contrast, a single IO delivery of G-FVIII-LV utilizing a megakaryocytic-specific GP1bα promoter achieved platelet-specific FVIII expression, leading to persistent, partial correction of HemA in treated animals. Most interestingly, comparable therapeutic benefit with G-F8-LV was obtained in HemA mice with pre-existing anti-FVIII inhibitors. Platelets is an ideal IO delivery vehicle since FVIII stored in α-granules of platelets is protected from high-titer anti-FVIII antibodies; and that even relatively small numbers of activated platelets that locally excrete FVIII may be sufficient to promote efficient clot formation during bleeding. Additionally, combination of pharmacological agents improved transduction of LVs and persistence of transduced cells and transgene expression. Overall, a single IO infusion of G-F8-LV can generate long-term stable

  16. The C1 and C2 domains of blood coagulation factor VIII mediate its endocytosis by dendritic cells

    Science.gov (United States)

    Gangadharan, Bagirath; Ing, Mathieu; Delignat, Sandrine; Peyron, Ivan; Teyssandier, Maud; Kaveri, Srinivas V.; Lacroix-Desmazes, Sébastien

    2017-01-01

    The development of inhibitory antibodies to therapeutic factor VIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-factor VIII immune response is factor VIII interaction with receptor(s) on antigen-presenting cells, followed by endocytosis and presentation to naïve CD4+ T cells. Recent studies indicate a role for the C1 domain in factor VIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic factor VIII uptake. Co-incubation of factor VIII with BO2C11, a monoclonal C2-specific immunoglobulin G, reduced factor VIII endocytosis by dendritic cells and presentation to CD4+ T cells, and diminished factor VIII immunogenicity in factor VIII-deficient mice. The mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent factor VIII immunogenicity in mice. BO2C11 prevents factor VIII binding to von Willebrand factor, thus potentially biasing factor VIII immunogenicity by perturbing its half-life. Interestingly, a factor VIIIY1680C mutant, that does not bind von Willebrand factor, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in factor VIII-deficient mice. Co-incubation of factor VIIIY1680C with BO2C11, however, resulted in decreased factor VIII immunogenicity in vivo. In addition, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of factor VIII are implicated in immunogenic factor VIII uptake, at least in vitro. Conversely, in vivo, the binding to endogenous von Willebrand factor masks the reducing effect of mutations in the C domains on factor VIII immunogenicity. PMID:27758819

  17. Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models

    Science.gov (United States)

    Siner, Joshua I.; Samelson-Jones, Benjamin J.; Crudele, Julie M.; French, Robert A.; Lee, Benjamin J.; Zhou, Shanzhen; Merricks, Elizabeth; Raymer, Robin; Camire, Rodney M.; Arruda, Valder R.

    2016-01-01

    Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This belief prompted the retention of the furin recognition motif (amino acids 1645–1648) in the design of B-domain–deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector–based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy. PMID:27734034

  18. Eradication of neutralizing antibodies to factor VIII in canine hemophilia A after liver gene therapy.

    Science.gov (United States)

    Finn, Jonathan D; Ozelo, Margareth C; Sabatino, Denise E; Franck, Helen W G; Merricks, Elizabeth P; Crudele, Julie M; Zhou, Shangzhen; Kazazian, Haig H; Lillicrap, David; Nichols, Timothy C; Arruda, Valder R

    2010-12-23

    Inhibitory antibodies to factor VIII (FVIII) are a major complication in the treatment of hemophilia A, affecting approximately 20% to 30% of patients. Current treatment for inhibitors is based on long-term, daily injections of large amounts of FVIII protein. Liver-directed gene therapy has been used to induce antigen-specific tolerance, but there are no data in hemophilic animals with pre-existing inhibitors. To determine whether sustained endogenous expression of FVIII could eradicate inhibitors, we injected adeno-associated viral vectors encoding canine FVIII (cFVIII) in 2 strains of inhibitor hemophilia A dogs. In 3 dogs, a transient increase in inhibitor titers (up to 7 Bethesda Units [BU]) at 2 weeks was followed by continuous decline to complete disappearance within 4-5 weeks. Subsequently, an increase in cFVIII levels (1.5%-8%), a shortening of clotting times, and a reduction (> 90%) of bleeding episodes were observed. Immune tolerance was confirmed by lack of antibody formation after repeated challenges with cFVIII protein and normal protein half-life. A fourth dog exhibited a strong early anamnestic response (216 BU), with slow decline to 0.8 BU and cFVIII antigen detection by 18 months after vector delivery. These data suggest that liver gene therapy has the potential to eradicate inhibitors and could improve the outcomes of hemophilia A patients.

  19. Factor VIII and von Willebrand factor changes during normal pregnancy and puerperium.

    Science.gov (United States)

    Sánchez-Luceros, Analía; Meschengieser, Susana S; Marchese, Carlos; Votta, Roberto; Casais, Patricia; Woods, Adriana I; Nadal, María V; Salviú, María J; Lazzari, María A

    2003-10-01

    Gestation is a challenge to haemostasis and it is associated with significant haemostatic changes. Several studies have evaluated von Willebrand factor in normal pregnancy, but none considered the personal history of bleeding. We studied a group of healthy non-bleeding women (184 pregnant, 64 puerperium, 37 non-pregnant) to evaluate normal ranges and their relationship to blood group and parity. The von Willebrand factor increased markedly from non-pregnant values up to the end of early puerperium (P < 0.0001), while factor VIII only showed a slight increase. Factor VIII and von Willebrand factor activity remained within the normal range for non-pregnant women. The return to non-pregnant factor levels occurred in late puerperium, later than previously reported. Only factor VIII was significantly lower in the O blood group (P = 0.035). As regards parity, there were no differences in factor VIII, von Willebrand factor antigen and von Willebrand factor ristocetin cofactor between primigravidae and multigravidae for any period studied (P = 0.888, 0.999, and 0.237, respectively). Our results provide reference ranges that may help to design a study in von Willebrand factor disease in pregnancy.

  20. Purification of coagulation factor VIII by immobilized metal affinity chromatography.

    Science.gov (United States)

    Rodrigues, Estela S; Verinaud, Claudia I; Oliveira, Douglas S; Raw, Isaías; Lopes, Alexandre P Y; Martins, Elizabeth A L; Cheng, Elisabeth

    2015-01-01

    Factor VIII (FVIII) is a glycoprotein that plays an essential role in blood coagulation cascade. Purification of plasma-derived coagulation FVIII by direct application of plasma to a chromatographic column is a method of choice. Anion exchange column is a very powerful method because FVIII is strongly adsorbed, resulting in good activity recovery and high purification factor. However, vitamin-K-dependent coagulation factors coelute with FVIII. In the present study, we report the separation of vitamin-K-dependent coagulation proteins from FVIII using immobilized metal affinity chromatography (IMAC) with Cu(2+) as the metal ligand. Plasma was directly loaded to a Q Sepharose Big Beads column, and FVIII was recovered with 65% activity and a purification factor of approximately 50 times. Then, the Q Sepharose eluate was applied to the IMAC-Cu(2+) column, and FVIII was eluted with 200 mM imidazole, with up to 85% recovery of activity. The mass recovery in this fraction was less than 10% of the applied mass of protein. Vitamin-K-dependent proteins elute with imidazole concentrations of lower than 60 mM. Because of the difference in affinity, FVIII could be completely separated from the vitamin-K-dependent proteins in the IMAC column.

  1. Danger signal-dependent activation of human dendritic cells by plasma-derived factor VIII products.

    Science.gov (United States)

    Miller, L; Weissmüller, S; Ringler, E; Crauwels, P; van Zandbergen, G; Seitz, R; Waibler, Z

    2015-08-01

    Treatment of haemophilia A by infusions of the clotting factor VIII (FVIII) results in the development of inhibitors/anti-drug antibodies in up to 25 % of patients. Mechanisms leading to immunogenicity of FVIII products are not yet fully understood. Amongst other factors, danger signals as elicited upon infection or surgery have been proposed to play a role. In the present study, we focused on effects of danger signals on maturation and activation of dendritic cells (DC) in the context of FVIII application. Human monocyte-derived DC were treated with FVIII alone, with a danger signal alone or a combination of both. By testing more than 60 different healthy donors, we show that FVIII and the bacterial danger signal lipopolysaccharide synergise in increasing DC activation, as characterised by increased expression of co-stimulatory molecules and secretion of pro-inflammatory cytokines. The degree and frequency of this synergistic activation correlate with CD86 expression levels on immature DC prior to stimulation. In our assay system, plasma-derived but not recombinant FVIII products activate human DC in a danger signal-dependent manner. Further tested danger signals, such as R848 also induced DC activation in combination with FVIII, albeit not in every tested donor. In our hands, human DC but not human B cells or macrophages could be activated by FVIII in a danger signal-dependent manner. Our results suggest that immunogenicity of FVIII is a result of multiple factors including the presence of danger, predisposition of the patient, and the choice of a FVIII product for treatment.

  2. Factor VIII and von Willebrand factor co-delivery by endothelial cells

    NARCIS (Netherlands)

    Bouwens, E.A.M.|info:eu-repo/dai/nl/314061894

    2011-01-01

    A defect in coagulation factor VIII (FVIII) results in the inherited bleeding disorder hemophilia A. Current treatment of hemophilia A is hampered by the need of frequent administration of costly FVIII products. Therefore gene therapy is an attractive alternative for protein replacement to treat

  3. Factor VIII and von Willebrand factor co-delivery by endothelial cells

    NARCIS (Netherlands)

    Bouwens, E.A.M.

    2011-01-01

    A defect in coagulation factor VIII (FVIII) results in the inherited bleeding disorder hemophilia A. Current treatment of hemophilia A is hampered by the need of frequent administration of costly FVIII products. Therefore gene therapy is an attractive alternative for protein replacement to treat hem

  4. piggyBac-mediated phenotypic correction of factor VIII deficiency

    Directory of Open Access Journals (Sweden)

    Janice M Staber

    2014-01-01

    Full Text Available Hemophilia A, caused by a deficiency in factor VIII (FVIII, is the most severe inherited bleeding disorder. Hemophilia A is an attractive gene therapy candidate because even small increases in FVIII levels will positively alter the phenotype. While several vectors are under investigation, gene addition from an integrated transgene offers the possibility of long term expression. We engineered the DNA transposon-based vector, piggyBac (PB, to carry a codon-optimized B-domain deleted human FVIII cDNA. Evaluation of gene transfer efficiency in FVIII null mice demonstrated that PB containing the FVIII cDNA, delivered via hydrodynamic injection to immunocompetent hemophilia mice, conferred persistent gene expression, attaining mean FVIII activity of approximately 60% with 3/19 developing inhibitors. In addition to efficacious expression, a goal of gene transfer-based therapies is to develop vectors with low toxicity. To assess endoplasmic reticulum stress in hepatocytes stably expressing the transgene, we evaluated levels of ER stress markers via qPCR and found no evidence of cell stress. To evaluate phenotypic correction, a tail clip assay performed at the end of the study revealed reduced blood loss. These data demonstrate that PB can be used to achieve sustained FVIII expression and long-term therapeutic benefit in a mouse model.

  5. Development and characterization of lipidic cochleate containing recombinant factor VIII

    Science.gov (United States)

    Miclea, Razvan D.; Varma, Prashant R.; Peng, Aaron; Balu-Iyer, Sathy V.

    2007-01-01

    Hemophilia A, a life threatening bleeding disorder is caused by deficiency of Factor VIII (FVIII). Replacement therapy using rFVIII is the first line therapy for hemophilia A. However, 15-30% of patients develop neutralizing antibody, mainly against the C2, A3 and A2 domains. It has been reported that PS-FVIII complex reduced total and neutralizing anti-rFVIII antibody titers in hemophilia A murine models. Here, we developed FVIII – containing cochleate cylinders, utilizing PS-Ca2+ interactions and characterized these particles for optimal in vivo properties using biophysical and biochemical techniques. Approximately 75% of the protein was associated with cochleate cylinders. Sandwich ELISA, acrylamide quenching and enzymatic digestion studies established that rFVIII was shielded from the bulk aqueous phase by the lipidic structures, possibly leading to improved in vivo stability. Freeze – thawing and rate limiting diffusion studies revealed that small cochleate cylinders with a particles size of 500 nm or less could be generated. The release kinetics and in vivo experiments suggested that there is slow and sustained release of FVIII from the complex upon systemic exposure. In vivo studies using tail clip method indicated that FVIII-cochleate complex is effective and protects hemophilic mice from bleeding. Based on these studies, we speculate that the molecular interaction between FVIII and PS may provide a basis for the design of novel FVIII lipidic structures for delivery applications. PMID:17936245

  6. A study of reported factor VIII use around the world.

    Science.gov (United States)

    Stonebraker, J S; Brooker, M; Amand, R E; Farrugia, A; Srivastava, A

    2010-01-01

    The effect of replacement therapy has significantly improved the morbidity and mortality of people with haemophilia A in high income countries, a recent socio-economic development as the availability of safe concentrates has been matched by a willingness for their provision through reimbursement. In the developing world, however, this state has not been achieved, primarily because of the low visibility of haemophilia coupled with its expense, leading to inadequate treatment with its sequelae of severe pain, joint deformities, arthropathy, disabilities, and even death in childhood or early adult life. The objective of this paper was to study the reported factor VIII (FVIII) use on a country-by-country basis. Data on the reported FVIII use for 104 countries were obtained from the Marketing Research Bureau, Inc. and the World Federation of Hemophilia. The results show that FVIII use varies considerably among countries, even among the wealthiest of countries. The use of FVIII concentrate increases as economic capacity increases; in addition, consumption of FVIII has been increasing at a greater rate in high income countries. Given these trends, there probably will be a global increase in FVIII concentrates usage. Such information is critical for national healthcare agencies to determine realistic budget priorities in planning for an increased allocation of resources required to improve the treatment of patients with haemophilia A. This information is also important for pharmaceutical manufacturers to adequately plan for increased production of FVIII concentrates.

  7. [Structure and function of the factor VIII/von Willebrand factor complex].

    Science.gov (United States)

    Müller, G

    1990-03-01

    In the blood plasma factor VIII is bound to the von Willebrand factor. The primary structure of the two proteins were clarified by gene clonation. Factor VIII descends from a precursor protein with 2,351 amino acids by splitting of 19 amino acid residues and is activated by partial proteolysis. In the blood coagulation factor VIII acts as co-factor for the activation of factor X by factor IX in the presence of phospholipids and Ca++ within the intrinsic coagulation system. The formation of the von Willebrand factor takes place by splitting of 22 and 741 amino acid residues, respectively, from pre-pro-von Willebrand factor via pro-von Willebrand factor. The subunits of the von Willebrand factor consist od 2,050 amino acid residues. In the blood plasma the von Willebrand factor is existing as a mixture of multimeres. Receptors of the von Willebrand factor on the thrombocytic membrane are the glycoproteins GPIb and GPIIb/GPIIIa, by means of which the adhesion of thrombocytes at the subendoethelium of the vascular wall and the aggregation of thrombocytes are mediated.

  8. Case of acquired hemophilia with factor VIII inhibitor in a mother and newborn.

    Science.gov (United States)

    Kotani, Yasushi; Shiota, Mitsuru; Umemoto, Masahiko; Koike, Eiji; Tsuritani, Mitsuhiro; Hoshiai, Hiroshi

    2011-08-01

    We report a mother and newborn in the puerperium with hemorrhage secondary to factor VIII inhibitor. A 31-year-old gravida 1 para 1 delivered at a local clinic with a massive postpartum hemorrhage. The activated partial thromboplastin time was prolonged and factor VIII inhibitor was detected. The persistent hemorrhage improved following treatment, including transfusion, steroid therapy, and bypass therapy with factor VII formulations. After hysteroscopic removal of the retained placenta, the hemorrhage decreased. The newborn developed significant swelling of the hands after routine blood sampling and factor VIII inhibitor was detected. The inhibitor disappeared without any special treatment in the 5th month postpartum in the mother and the 4th month postpartum in the newborn. Factor VIII inhibitor may be transferred via the placenta from the mother to the fetus. Therefore, the newborn should also be carefully observed in a case of massive hemorrhage after delivery.

  9. Honey Bee Venom (Apis mellifera Contains Anticoagulation Factors and Increases the Blood-clotting Time

    Directory of Open Access Journals (Sweden)

    Hossein Zolfagharian

    2015-12-01

    Full Text Available Objectives: Bee venom (BV is a complex mixture of proteins and contains proteins such as phospholipase and melittin, which have an effect on blood clotting and blood clots. The mechanism of action of honey bee venom (HBV, Apis mellifera on human plasma proteins and its anti-thrombotic effect were studied. The purpose of this study was to investigate the anti-coagulation effect of BV and its effects on blood coagulation and purification. Methods: Crude venom obtained from Apis mellifera was selected. The anti-coagulation factor of the crude venom from this species was purified by using gel filtration chromatography (sephadex G-50, and the molecular weights of the anti-coagulants in this venom estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. Blood samples were obtained from 10 rabbits, and the prothrombin time (PT and the partial thromboplastin time (PTT tests were conducted. The approximate lethal dose (LD values of BV were determined. Results: Crude BV increased the blood clotting time. For BV concentrations from 1 to 4 mg/mL, clotting was not observed even at more than 300 seconds, standard deviations (SDs = ± 0.71; however, clotting was observed in the control group 13.8 s, SDs = ± 0.52. Thus, BV can be considered as containing anti-coagulation factors. Crude BV is composed 4 protein bands with molecular weights of 3, 15, 20 and 41 kilodalton (kDa, respectively. The LD50 of the crude BV was found to be 177.8 μg/mouse. Conclusion: BV contains anti-coagulation factors. The fraction extracted from the Iranian bees contains proteins that are similar to anti-coagulation proteins, such as phospholipase A2 (PLA2 and melittin, and that can increase the blood clotting times in vitro.

  10. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... harming your baby. Jump To: Am I at Risk? The risk of developing a blood clot during pregnancy is ... prevent blood clots during pregnancy: Be aware of risk factors. Know your family history. Make sure your ...

  11. Blood Clotting and Pregnancy

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    Full Text Available ... pregnancy: Be aware of risk factors. Know your family history. Make sure your doctor knows about any ... blood clots or blood clotting disorders in your family. Remain active, with your doctor's approval. Be aware ...

  12. Blood Clotting and Pregnancy

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    Full Text Available ... blood clots A genetic predisposition to blood clots Obesity Prolonged immobility (e.g., bedrest, long distance travel) ... pregnancy: Be aware of risk factors. Know your family history. Make sure your doctor knows about any ...

  13. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... pregnancy: Be aware of risk factors. Know your family history. Make sure your doctor knows about any ... blood clots or blood clotting disorders in your family. Remain active, with your doctor's approval. Be aware ...

  14. A case of renal infarction associated with elevated factor VIII level.

    Science.gov (United States)

    Siddiqui, O Asif; Al-Absi, A; Showkat, A

    2011-09-01

    Elevated factor VIII level has recently been shown to be associated with increased risk of thrombosis. We report here a case of renal infarction in association with elevated factor VIII level. The patient presented with a three-day history of flank pain. Laboratory studies on presentation showed an elevated serum creatinine concentration and microscopic hematuria. He was found to have bilateral pulmonary emboli and left common femoral vein thrombosis; imaging studies showed evidence of renal arterial thrombosis with infarction. Hypercoagulability assessment showed an elevated factor VIII level. He was treated with heparin and warfarin with significant improvement in his renal function. Consideration should be given to measurement of factor VIII level as a part of the workup of unexplained thrombo-embolic events.

  15. Crystal Structure of Human Factor VIII: Implications for the Formation of the Factor IXa-Factor VIIIa Complex

    Energy Technology Data Exchange (ETDEWEB)

    Ngo, J.C.; Huang, M.; Roth, D.A.; Furie, B.C.; Furie, B. (Wyeth); (MBL)

    2008-06-03

    Factor VIII is a procofactor that plays a critical role in blood coagulation, and is missing or defective in hemophilia A. We determined the X-ray crystal structure of B domain-deleted human factor VIII. This protein is composed of five globular domains and contains one Ca{sup 2+} and two Cu{sup 2+} ions. The three homologous A domains form a triangular heterotrimer where the A1 and A3 domains serve as the base and interact with the C2 and C1 domains, respectively. The structurally homologous C1 and C2 domains reveal membrane binding features. Based on biochemical studies, a model of the factor IXa-factor VIIIa complex was constructed by in silico docking. Factor IXa wraps across the side of factor VIII, and an extended interface spans the factor VIII heavy and light chains. This model provides insight into the activation of factor VIII and the interaction of factor VIIIa with factor IXa on the membrane surface.

  16. Crystal Structure of Human Factor VIII: Implications for the Formation of the Factor IXa-Factor VIIIa Complex

    Energy Technology Data Exchange (ETDEWEB)

    Chi Ki Ngo,J.; Huang, M.; Roth, D.; Furie, B.; Furie, B.

    2008-01-01

    Factor VIII is a procofactor that plays a critical role in blood coagulation, and is missing or defective in hemophilia A. We determined the X-ray crystal structure of B domain-deleted human factor VIII. This protein is composed of five globular domains and contains one Ca(2+) and two Cu(2+) ions. The three homologous A domains form a triangular heterotrimer where the A1 and A3 domains serve as the base and interact with the C2 and C1 domains, respectively. The structurally homologous C1 and C2 domains reveal membrane binding features. Based on biochemical studies, a model of the factor IXa-factor VIIIa complex was constructed by in silico docking. Factor IXa wraps across the side of factor VIII, and an extended interface spans the factor VIII heavy and light chains. This model provides insight into the activation of factor VIII and the interaction of factor VIIIa with factor IXa on the membrane surface.

  17. Intraosseous delivery of lentiviral vectors targeting factor VIII expression in platelets corrects murine hemophilia A.

    Science.gov (United States)

    Wang, Xuefeng; Shin, Simon C; Chiang, Andy F J; Khan, Iram; Pan, Dao; Rawlings, David J; Miao, Carol H

    2015-04-01

    Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage(-)Sca1(+)c-Kit(+) hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency.

  18. Extrahepatic sources of factor VIII potentially contribute to the coagulation cascade correcting the bleeding phenotype of mice with hemophilia A.

    Science.gov (United States)

    Zanolini, Diego; Merlin, Simone; Feola, Maria; Ranaldo, Gabriella; Amoruso, Angela; Gaidano, Gianluca; Zaffaroni, Mauro; Ferrero, Alessandro; Brunelleschi, Sandra; Valente, Guido; Gupta, Sanjeev; Prat, Maria; Follenzi, Antonia

    2015-07-01

    A large fraction of factor VIII in blood originates from liver sinusoidal endothelial cells although extrahepatic sources also contribute to plasma factor VIII levels. Identification of cell-types other than endothelial cells with the capacity to synthesize and release factor VIII will be helpful for therapeutic approaches in hemophilia A. Recent cell therapy and bone marrow transplantation studies indicated that Küpffer cells, monocytes and mesenchymal stromal cells could synthesize factor VIII in sufficient amount to ameliorate the bleeding phenotype in hemophilic mice. To further establish the role of blood cells in expressing factor VIII, we studied various types of mouse and human hematopoietic cells. We identified factor VIII in cells isolated from peripheral and cord blood, as well as bone marrow. Co-staining for cell type-specific markers verified that factor VIII was expressed in monocytes, macrophages and megakaryocytes. We additionally verified that factor VIII was expressed in liver sinusoidal endothelial cells and endothelial cells elsewhere, e.g., in the spleen, lungs and kidneys. Factor VIII was well expressed in sinusoidal endothelial cells and Küpffer cells isolated from human liver, whereas by comparison isolated human hepatocytes expressed factor VIII at very low levels. After transplantation of CD34(+) human cord blood cells into NOD/SCIDγNull-hemophilia A mice, fluorescence activated cell sorting of peripheral blood showed >40% donor cells engrafted in the majority of mice. In these animals, plasma factor VIII activity 12 weeks after cell transplantation was up to 5% and nine of 12 mice survived after a tail clip-assay. In conclusion, hematopoietic cells, in addition to endothelial cells, express and secrete factor VIII: this information should offer further opportunities for understanding mechanisms of factor VIII synthesis and replenishment.

  19. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... prevent blood clots during pregnancy: Be aware of risk factors. Know your family history. Make sure your doctor knows about any ... blood clots or blood clotting disorders in your family. Remain active, with ... doctor about your risks and prevention. Depending on your condition, your OB- ...

  20. Relationship between Acquired Deficiency of Vitamin K-dependent Clotting Factors And Hemorrhage

    Institute of Scientific and Technical Information of China (English)

    杨锐; 张小平; 魏文宁; 洪梅; 杨焰; 胡豫

    2010-01-01

    This study examined the changes of activities of vitamin K-dependent clotting factors(VKDCF) under various pathological conditions and explored the relationship between acquired deficiency of VKDCFs and hemorrhage.Clinical data of 35 patients who were diagnosed as having acquired deficiency of VKDCF were retrospectively analyzed.Coagulation factors involved in the intrinsic and extrinsic pathways were detected in these patients and 41 control subjects.The results showed that the average activities of VKDCFs...

  1. Evaluation of the expression of VIII factor and VEGF in the regeneration of non-vital teeth in dogs using propolis

    Directory of Open Access Journals (Sweden)

    Mina Zarei

    2017-02-01

    Full Text Available Objective(s: The purpose of the present study was the immunohistochemical evaluation of VEGF and VII factors in dog’s teeth pulp revascularized with MTA and propolis. Materials and Methods: 144 mature and immature two rooted dog’s premolar canals were selected.  Pulp necrosis and infection were established after 2 weeks and the disinfection of the canals was done with copious NaOCl irrigation and triantibiotic mixture (ciprofloxacin, metronidazole, and minocycline for 3 weeks. Subsequently, the blood clot was evoked in the canal by periapical tissue irritation with a k-file. The samples were randomly divided into 6 experimental groups: propolis (groups 1, 2, MTA (groups 3, 4, and parafilm (groups 5, 6 in immature and mature teeth. The animals were sacrificed and samples were prepared for immunohistochemical evaluation of VEGF and the VIII factor. Results: Tissue regeneration was seen in 64.5% of MTA, 38% of propolis, and 0% of parafilm group samples. Expression of VEGF and VIII factor in the propolis group was more than the MTA group and it showed a reduction after 3 months in comparison to 1 month. VEGF and VIII factor were seen in stromal cells in addition to endothelial vessel cells. Overall, expression of angiogenic factors was more in the open apex teeth compared to close apex ones. Conclusion: According to the results of this study, propolis can induce the expression of VEGF and VIII factor in infected mature and immature dog’s teeth and is a suitable biomaterial for the revascularization technique.

  2. Evaluation of the expression of VIII factor and VEGF in the regeneration of non-vital teeth in dogs using propolis

    Science.gov (United States)

    Zarei, Mina; Jafarian, Amir Hossein; Harandi, Azadeh; Javidi, Maryam; Gharechahi, Maryam

    2017-01-01

    Objective(s): The purpose of the present study was the immunohistochemical evaluation of VEGF and VII factors in dog’s teeth pulp revascularized with MTA and propolis. Materials and Methods: 144 mature and immature two rooted dog’s premolar canals were selected. Pulp necrosis and infection were established after 2 weeks and the disinfection of the canals was done with copious NaOCl irrigation and triantibiotic mixture (ciprofloxacin, metronidazole, and minocycline) for 3 weeks. Subsequently, the blood clot was evoked in the canal by periapical tissue irritation with a k-file. The samples were randomly divided into 6 experimental groups: propolis (groups 1, 2), MTA (groups 3, 4), and parafilm (groups 5, 6) in immature and mature teeth. The animals were sacrificed and samples were prepared for immunohistochemical evaluation of VEGF and the VIII factor. Results: Tissue regeneration was seen in 64.5% of MTA, 38% of propolis, and 0% of parafilm group samples. Expression of VEGF and VIII factor in the propolis group was more than the MTA group and it showed a reduction after 3 months in comparison to 1 month. VEGF and VIII factor were seen in stromal cells in addition to endothelial vessel cells. Overall, expression of angiogenic factors was more in the open apex teeth compared to close apex ones. Conclusion: According to the results of this study, propolis can induce the expression of VEGF and VIII factor in infected mature and immature dog’s teeth and is a suitable biomaterial for the revascularization technique. PMID:28293394

  3. The F309S mutation increases factor VIII secretion in human cell line

    Directory of Open Access Journals (Sweden)

    Daianne Maciely Carvalho Fantacini

    2016-06-01

    Full Text Available ABSTRACT OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIΔB proteins in HEK 293 cells, but the same effect was not seen for FVIIIΔB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.

  4. Recurrent myocardial infarctions in a young football player secondary to thrombophilia, associated with elevated factor VIII activity.

    Science.gov (United States)

    Vacek, Thomas P; Yu, Shipeng; Rehman, Shahnaz; Grubb, Blair P; Kosinski, Daniel; Verghese, Cherian; Eltahawy, Ehab A; Shafiq, Qaiser

    2014-01-01

    Myocardial infarction (MI) due to coronary atherosclerosis in young adults is uncommon; rare causes such as cocaine abuse, arterial dissection, and thromboembolism should be considered. A 21-year-old football player, and otherwise healthy African American man, developed chest pain during exercise while bench-pressing 400 lbs. Acute MI was diagnosed based on physical examination, electrocardiography findings, and elevated cardiac enzymes. Coronary arteriography showed a thrombus occluding the proximal left anterior descending artery (LAD). Aggressive antiplatelet therapy with aspirin, clopidogrel, and eptifibatide was pursued, in addition to standard post-MI care. This led to the successful resolution of symptoms and dissolution of the thrombus, demonstrated by repeat coronary arteriography. Five months later, he presented with similar symptoms during exercise after lifting heavy weights, and was found to have another acute MI. Coronary arteriography again showed a thrombus occluding the LAD. No evidence of coronary artery dissection or vasospasm was found. Only mild atherosclerotic plaque burden was observed on both occasions by intravascular ultrasound. A bare metal stent was placed at the site as it was thought this site had acted as a nidus for small plaque rupture and thrombus formation. Elevated serum factor VIII activity at 205% (reference range 60%-140%) was found, a rare cause of hypercoagulability. Further workup revealed a patent foramen ovale during a Valsalva maneuver by transesophageal echocardiography. Both events occurred during weight lifting, which can transiently increase right heart pressure in a similar way to the Valsalva maneuver. In light of all the findings, we concluded that an exercise-related increase in factor VIII activity led to coronary arterial thrombosis in the presence of a small ruptured plaque. Alternatively, venous clots may have traversed the patent foramen ovale and occluded the LAD. In addition to continuing aggressive risk

  5. New polymorphic variants of human blood clotting factor IX

    Energy Technology Data Exchange (ETDEWEB)

    Surin, V.L.; Luk`yanenko, A.V.; Tagiev, A.F.; Smirnova, O.V. [Hematological Research Center, Moscow (Russian Federation); Plutalov, O.V.; Berlin, Yu.A. [Shemyakin Institute of Bioorganic Chemistry, Moscow (Russian Federation)

    1995-04-01

    The polymorphism of Alu-repeats, which are located in the introns of the human factor IX gene (copies 1-3), was studied. To identify polymorphic variants, direct sequencing of PCR products that contained appropriate repeats was used. In each case, 20 unrelated X chromosomes were studied. A polymorphic Dra I site was found near the 3{prime}-end of Alu copy 3 within the region of the polyA tract. A PCR-based testing system with internal control of restriction hydrolysis was suggested. Testing 81 unrelated X chromosomes revealed that the frequency of the polymorphic Dra I site is 0.23. Taq I polymorphism, which was revealed in Alu copy 4 of factor IX gene in our previous work, was found to be closely linked to Dra I polymorphism. Studies in linkage between different types of polymorphisms of the factor IX gene revealed the presence of a rare polymorphism in intron a that was located within the same minisatellite region as the known polymorphic insertion 50 bp/Dde I. However, the size of the insertion in our case was 26 bp. Only one polymorphic variant was found among over 150 unrelated X chromosomes derived from humans from Moscow and its vicinity. 10 refs., 4 figs., 1 tab.

  6. Recombinant factor VIII in the management of hemophilia A: current use and future promise

    Directory of Open Access Journals (Sweden)

    Jerry S Powell

    2009-05-01

    Full Text Available Jerry S PowellDivision of Hematology and Oncology, University of California Davis Cancer Center, Sacramento, CA, USAAbstract: Hemophilia A is a rare inherited bleeding disorder due to mutation of the gene that encodes the coagulation protein factor VIII. Historically, prior to the availability of treatment with factor VIII preparations, most boys died from uncontrolled bleeding, either spontaneous bleeding or after injury, before reaching 20 years of age. One of the most impressive triumphs of modern medicine is that with current recombinant factor VIII replacement therapy, a boy born in the 21st century with severe hemophilia A can anticipate a normal life expectancy with essentially no permanent complications from bleeding. For severe hemophilia A, current optimal treatment should have two goals: first, to provide sufficient factor VIII to prevent spontaneous bleeding, and second, to provide sufficient factor VIII to have normal coagulation function after any trauma. However, the replacement therapy requires tremendous resources for effective use, and remains extraordinarily expensive. Thus there are opportunities for further advances in therapy for hemophilia A. Two major concerns continue to trouble current optimal treatment approaches: some patients will develop neutralizing antibodies during the first 50 infusions of therapeutic factor VIII, and second, to administer therapeutic factor VIII every other day in young boys often requires placement of a central venous access device, and such use carries the life-threatening risks of infection and thrombosis. Because of the effectiveness of current therapy, any new developments in treatment will require significant concerns for safety, both immediate and in the long term. A number of research groups seek to prolong the biological efficacy of infused recombinant factor VIII. Currently, one such promising development is in the advanced stages of clinical trial. The goals will be to improve

  7. A micromethod for clotting tests and coagulation factor assays.

    Science.gov (United States)

    Exner, T; Margolis, J; Rickard, K A

    1980-01-01

    A simple microtechnique for carrying out partial thromboplastin time with kaolin tests with 2 microliter or less of test plasma is described. For single stage factor assays, less than 1 microliter of test solution may be used. Reagents and test plasma are loaded in sequence into a 10 microliter, long needle syringe and introduced into a micro test-tube immobilized in a water bath. The end-point is taken as a positive clearing of kaolin turbidity from the mixture while stirring. Correlation with normal techniques has been excellent.

  8. Hemodialysis in a patient with severe hemophilia A and factor VIII inhibitor.

    Science.gov (United States)

    Gopalakrishnan, Natarajan; Usha, Thiruvengadam; Thopalan, Balasubramaniyan; Dhanapriya, Jeyachandran; Dineshkumar, Thanigachalam; Thirumalvalavan, Kaliaperumal; Sakthirajan, Ramanathan

    2016-10-01

    Hemophilia A is a hereditary X-linked recessive disease caused by mutations in the gene encoding factor VIII (FVIII), occurring in 1 out of 10,000 persons. Life expectancy and quality of life have dramatically improved recently in patients with hemophilia. Chronic kidney disease and need for renal replacement therapy in these patients are rare. The development of inhibitors to FVIII is the most serious complication of hemophilia and makes treatment of bleeds very challenging. We describe here a 28-year-old male patient with severe hemophilia A with presence of factor VIII inhibitor, who had end stage renal disease. Central venous access device was inserted along with infusion of factor eight inhibitor bypass activity before and after the procedure. He is currently on thrice weekly hemodialysis and doing well for 6 months without bleeding episodes. To our knowledge, hemophilia A with factor VIII inhibitor managed with hemodialysis has not been reported so far. © 2016 International Society for Hemodialysis.

  9. The influence of various factors on milk clotting time

    Directory of Open Access Journals (Sweden)

    Jovanović Snežana T.

    2002-01-01

    Full Text Available The influence of pH (6.5 and 5.8, amount of added CaCl2 (0, 200 and 400 mg/l, coagulation temperature (30ºC and 35ºC and heat treatment of milk (65ºC/30 min and 87ºC/10 min on the rate of rennet induced milk coagulation (s were investigated. The time (s from rennet addition to onset of gelation (as indicated by the first visible floccules was measured. The milk samples heat-treated at 87ºC/10 min, with 400 mg/l added CaCl2, which were coagulated at 35ºC and pH 5.8, coagulated 23.28-fold faster than the same samples without added CaCl2, which were coagulated at 30ºC and pH 6.5. The results of investigations related to the influence of particular coagulation factors on the coagulation rate of heat-treated milk showed that at pH 6.5 the most pronounced influence was demonstrated by the amount of Ca2+ and temperature of coagulation. At pH 5.8, different amounts of Ca2 and used temperatures of coagulation did not influence coagulation rate regardless of the used heat treatment of milk. The influence of used heat treatment of milk was particularly pronounced during coagulation of samples without added CaCl2 that coagulated at 30ºC and pH 6.5. The used heat treatment of milk practically did not influence the milk coagulation rate at pH 5.8. The greatest influence on milk coagulation rate was showed by pH. This influence was the most marked in coagulation of samples in which the coaggregates were formed, regardless of the amount of added Ca2+ and used coagulation temperatures.

  10. Recurrent myocardial infarctions in a young football player secondary to thrombophilia, associated with elevated factor VIII activity

    Directory of Open Access Journals (Sweden)

    Vacek TP

    2014-10-01

    VIII activity led to coronary arterial thrombosis in the presence of a small ruptured plaque. Alternatively, venous clots may have traversed the patent foramen ovale and occluded the LAD. In addition to continuing aggressive risk factor modification, anticoagulation therapy with warfarin was initiated with close follow-up.Keywords: MI, factor VIII, exercise, myocardial infarction, young, football player

  11. Immunological abnormalities in haemophilia: are they caused by American factor VIII concentrate?

    Science.gov (United States)

    Froebel, K S; Madhok, R; Forbes, C D; Lennie, S E; Lowe, G D; Sturrock, R D

    1983-10-15

    Scottish patients with haemophilia, most of whom had received no American factor VIII concentrate for over two years, were found to have immunological abnormalities similar to those in their American counterparts--that is, a reduced proportion of T helper cells, an increased proportion of T suppressor cells, and a reduced response to concanavilin A. Factor VIII from both the United States and Scotland severely inhibited the in vitro lymphocyte response to mitogens in patients and controls. The American and Scottish concentrates could not be distinguished in terms of either patient usage or their effect in vitro. These results argue against a disease vector specific to American blood products.

  12. Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

    NARCIS (Netherlands)

    van den Biggelaar, M.; Bouwens, E.A.M.; Kootstra, N.A.; Hebbel, R.P.; Voorberg, J.; Mertens, K.

    2009-01-01

    Background Gene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this

  13. Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

    NARCIS (Netherlands)

    van den Biggelaar, M.; Bouwens, E.A.M.; Kootstra, N.A.; Hebbel, R.P.; Voorberg, J.; Mertens, K.

    2009-01-01

    Background Gene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this s

  14. Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

    NARCIS (Netherlands)

    van den Biggelaar, M.; Bouwens, E.A.M.; Kootstra, N.A.; Hebbel, R.P.; Voorberg, J.; Mertens, K.

    2009-01-01

    Background Gene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this s

  15. An Acquired Factor VIII Inhibitor in a Patient with HIV and HCV: A Case Presentation and Literature Review

    Directory of Open Access Journals (Sweden)

    S. B. Zeichner

    2013-01-01

    Full Text Available Introduction. Despite its low incidence, acquired factor VIII inhibitor is the most common autoantibody affecting the clotting cascade. The exact mechanism of acquisition remains unclear, but postpartum patients, those with autoimmune conditions or malignancies, and those with exposure to particular drugs appear most susceptible. There have been several case reports describing acquired FVIII inhibitors in patients receiving interferon alpha for HCV treatment and in patients being treated for HIV. To our knowledge, this is the first case of a patient with HCV and HIV who was not actively receiving treatment for either condition. Case Presentation. A 57-year-old Caucasian male with a history of HIV and HCV was admitted to our hospital for a several day history of progressively worsening right thigh bruising and generalized weakness. CTA of the abdominal arteries revealed large bilateral retroperitoneal hematomas. Laboratory studies revealed the presence of a high titer FVIII inhibitor. Conclusion. Our case of a very rare condition highlights the importance of recognizing and understanding the diagnosis of acquired FVIII inhibitor. Laboratory research and clinical data on the role of newer agents are needed in order to better characterize disease pathogenesis, disease associations, genetic markers, and optimal disease management.

  16. Safety and Efficacy of BAY 94-9027, a Prolonged-Half-Life Factor VIII

    DEFF Research Database (Denmark)

    Reding, M T; Ng, H J; Poulsen, Lone Hvitfeldt

    2016-01-01

    BACKGROUND: BAY 94-9027 is a B-domain-deleted prolonged-half-life recombinant factor VIII (FVIII) conjugates in a site-specific manner with polyethylene glycol. OBJECTIVE: Assess efficacy and safety of BAY 94-9027 for prophylaxis and treatment of bleeds in patients with severe hemophilia A PATIEN...

  17. Immune recognition and processing of blood coagulation factor VIII by antigen-presenting cells

    NARCIS (Netherlands)

    van Haren, S.D.

    2011-01-01

    Hemophilia A is an X chromosome-linked bleeding disorder caused by a reduction or com­plete absence of coagulation factor VIII (FVIII). The bleeding tendency in patients suffering from hemophilia A can be treated by regular intravenous administrations of FVIII. A severe complication that occurs in

  18. Binding of factor VIII to von willebrand factor is enabled by cleavage of the von Willebrand factor propeptide and enhanced by formation of disulfide-linked multimers.

    Science.gov (United States)

    Bendetowicz, A V; Morris, J A; Wise, R J; Gilbert, G E; Kaufman, R J

    1998-07-15

    von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

  19. Potentiation of thrombin generation in hemophilia A plasma by coagulation factor VIII and characterization of antibody-specific inhibition.

    Directory of Open Access Journals (Sweden)

    Bhavya S Doshi

    Full Text Available Development of inhibitory antibodies to coagulation factor VIII (fVIII is the primary obstacle to the treatment of hemophilia A in the developed world. This adverse reaction occurs in 20-30% of persons with severe hemophilia A treated with fVIII-replacement products and is characterized by the development of a humoral and neutralizing immune response to fVIII. Patients with inhibitory anti-fVIII antibodies are treated with bypassing agents including recombinant factor VIIa (rfVIIa. However, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Recently, we demonstrated that fVIII inhibitors display widely variable kinetics of inhibition that correlate with their respective target epitopes. Thus, it was hypothesized that for antibodies that display slow rates of inhibition, supplementation of rfVIIa with fVIII would result in improved thrombin generation and be predictive of clinical responses to this novel treatment regimen. In order to test this hypothesis, 10 murine monoclonal antibodies (MAbs with non-overlapping epitopes spanning fVIII, differential inhibition titers, and inhibition kinetics were studied using a thrombin generation assay. Of the 3 MAbs with high inhibitory titers, only the one with fast and complete (classically defined as "type I" kinetics displayed significant inhibition of thrombin generation with no improvement upon supplementation of rfVIIa with fVIII. The other two MAbs that displayed incomplete (classically defined as "type II" inhibition did not suppress the potentiation of thrombin generation by fVIII. All antibodies that did not completely inhibit fVIII activity demonstrated potentiation of thrombin generation by the addition of fVIII as compared to rfVIIa alone. In conclusion, fVIII alone or in combination with rfVIIa corrects the thrombin generation defect produced by the majority of anti-fVIII MAbs better than single agent rfVIIa. Therefore, combined fVIII

  20. An Evaluation of a Factor Xa-Based Clotting Time Test for Enoxaparin: A Proof-of-Concept Study.

    Science.gov (United States)

    Ng, Deborah P J; Duffull, Stephen B; Faed, James M; Isbister, Geoffrey K; Gulati, Abhishek

    2017-01-01

    A well-accepted test for monitoring anticoagulation by enoxaparin is not currently available. As inadequate dosing may result in thrombosis or bleeding, a clinical need exists for a suitable test. Previous in silico and in vitro studies have identified factor Xa as an appropriate activating agent, and the phospholipid Actin FS as a cofactor for a Xa clotting time (TenaCT) test. A proof-of-concept study was designed to (1) explore the reproducibility of the TenaCT test and (2) explore factors that could affect the performance of the test. In vitro clotting time tests were carried out using plasma from 20 healthy volunteers. The effect of enoxaparin was determined at concentrations of 0.25, 0.5, and 1.0 IU/mL. Clotting times for the volunteers were significantly prolonged with increasing enoxaparin concentrations. Clotting times were significantly shortened for frozen plasma samples. No significant differences in prolongation of clotting times were observed between male and female volunteers or between the 2 evaluated age groups. The clotting times were consistent between 2 separate occasions. The TenaCT test was able to distinguish between the subtherapeutic and therapeutic concentrations of enoxaparin. Plasma should not be frozen prior to performing the test, without defining a frozen plasma reference range. This study provided proof-of-concept for a Xa-based test that can detect enoxaparin dose effects, but additional studies are needed to further develop the test.

  1. Variation in baseline factor VIII concentration in a retrospective cohort of mild/moderate hemophilia A patients carrying identical F8 mutations

    NARCIS (Netherlands)

    Loomans, J.I.; Velzen, A.S. van; Eckhardt, C.L.; Peters, M.; Makipernaa, A.; Holmstrom, M.; Brons, P.P.T.; Dors, N.; Haya, S.; Voorberg, J.; Bom, J.G. Van Der; Fijnvandraat, K.

    2017-01-01

    Essentials Factor VIII levels vary in mild and moderate hemophilia A (MHA) patients with the same mutation. We aimed to estimate the variation and determinants of factor VIII levels among MHA patients. Age and genotype explain 59% of the observed inter-individual variation in factor VIII levels.

  2. Desenvolvimento de inibidores do fator VIII na hemofilia A Development of factor VIII inhibitors in hemophilia A

    Directory of Open Access Journals (Sweden)

    Daniel G. Chaves

    2009-01-01

    Full Text Available A hemofilia A é uma coagulopatia genética com herança recessiva ligada ao cromossomo X que afeta 1-2 a cada 10 mil indivíduos do sexo masculino nascidos vivos. Estes indivíduos têm baixas concentrações ou ausência do fator VIII (FVIII da coagulação no plasma e apresentam quadros hemorrágicos leves, moderados e graves, dependendo da atividade de FVIII circulante. Estes pacientes necessitam de constante reposição proteica e aproximadamente 30% deles desenvolvem aloanticorpos contra a proteína exógena. A síntese dos anticorpos anti-FVIII é iniciada quando o FVIII exógeno é endocitado por células apresentadoras de antígeno, degradado e apresentado às células T CD4+ na forma de peptídeos ligados a moléculas do complexo maior de histocompatibilidade (MHC de classe II. Alguns fatores de risco (paciente/tratamento podem ser relacionados ao desenvolvimento desta resposta imune. Neste contexto, as mutações no gene do FVIII e polimorfismos em genes envolvidos na resposta imune são candidatos moleculares como determinantes imunogenéticos na predisposição para o desenvolvimento de inibidores. Por não ser completamente entendido e controlado, o desenvolvimento desta resposta imune contra o FVIII constitui o maior problema decorrente do tratamento de indivíduos portadores de hemofilia A e faz-se necessária busca de opções que visem minimizar suas ações deletérias. Algumas alternativas de tratamento têm se mostrado eficazes no tratamento (anti-CD20, plasmaférese, concentrado de complexo protrombínico (PCCs, concentrado de complexo protrombínico ativado (APCCs, fator VII humano ativado, mas a retirada ou neutralização específica dos inibidores de FVIII ainda não foram alcançadas.Hemophilia A, which affects 1-2:10,000 live-born male neonates, is a genetic coagulopathy with recessive inheritance linked to the X chromosome. These individuals have low concentrations or no coagulation factor VIII (FVIII in the plasma

  3. Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A.

    Science.gov (United States)

    Johnston, J M; Denning, G; Doering, C B; Spencer, H T

    2013-06-01

    We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain-deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino-acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: (1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), (2) HIV versus SIV viral vector systems and (3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha, cytomegalovirus (CMV) or phosphoglycerate kinase promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.

  4. A population pharmacokinetic model for perioperative dosing of factor VIII in hemophilia A patients

    Science.gov (United States)

    Hazendonk, Hendrika; Fijnvandraat, Karin; Lock, Janske; Driessens, Mariëtte; van der Meer, Felix; Meijer, Karina; Kruip, Marieke; Gorkom, Britta Laros-van; Peters, Marjolein; de Wildt, Saskia; Leebeek, Frank; Cnossen, Marjon; Mathôt, Ron

    2016-01-01

    The role of pharmacokinetic-guided dosing of factor concentrates in hemophilia is currently a subject of debate and focuses on long-term prophylactic treatment. Few data are available on its impact in the perioperative period. In this study, a population pharmacokinetic model for currently registered factor VIII concentrates was developed for severe and moderate adult and pediatric hemophilia A patients (FVIII levels hemophilia A patients by Bayesian adaptive dosing. PMID:27390359

  5. Switching clotting factor concentrates: considerations in estimating the risk of immunogenicity.

    Science.gov (United States)

    Matino, D; Lillicrap, D; Astermark, J; Dolan, G; Kessler, C; Lambert, T; Makris, M; O'Donnell, J; Pipe, S; Santagostino, E; Saint-Remy, J-M; Schramm, W; Iorio, A

    2014-03-01

    The development of neutralizing antibodies to factor VIII (FVIII) is the most serious complication of therapy for haemophilia A. There is now excellent documentation that a large number of both genetic and environmental factors contribute to the risk of FVIII inhibitor incidence. One of the environmental factors that has been proposed as an influence on this complication is the occurrence of FVIII product switching. There are only a small number of clinical studies that have addressed this question, and thus, the amount of objective information available to assess this association is limited. In this review, in addition to summarizing past evidence pertinent to this subject, we present the results of a complementary strategy, a Delphi analysis, to add to the considerations of product switching and FVIII immunogenicity. With the imminent arrival in the clinic of several new FVIII products, the haemophilia community must be prepared to collect prospectively controlled data to better address this important management issue.

  6. Regulation of human clotting factor IX cDNA expression in transgenic mice

    Institute of Scientific and Technical Information of China (English)

    胡以平; 邱信芳; 薛京伦; 刘祖洞

    1995-01-01

    To study the expression of human dotting factor IX cDNA in transgenic mice,Which is an es-sential work on gene therapy for hemophilia B,3 recombinant constructions containing different lengths ofhuman dotting factor IX cDNA have been introduced into the cultured cells.All of the recombinant constructionswere found to he expressed well in vitro.They were then microinjected into the male pronudei of the fertilizedmouse eggs respectively for generating trahsgenic mice.Unfortunately,none of them was expressed in any transgenicmice.These results show that the expression of the human clotting factor IX cDNA in the transgenic mice canbe determined by cis regulatory element(s).As compared With the results from other related works,it is sug-gested that the cis regulatory element(s)is resided in the 5’-end non-coding region.

  7. Effect of transmembrane pressure on Factor VIII yield in ATF perfusion culture for the production of recombinant human Factor VIII co-expressed with von Willebrand factor.

    Science.gov (United States)

    Kim, Seung-Chul; An, Sora; Kim, Hyun-Ki; Park, Beom-Soo; Na, Kyu-Heum; Kim, Byung-Gee

    2016-10-01

    In this study, we evaluated three cell retention devices, an alternating tangential flow (ATF) system, a spin-filter, and a Centritech Lab III centrifuge, for the production of recombinant human Factor VIII co-expressed with von Willebrand factor. From the results, it was found that the FVIII activity in bioreactor was significantly higher in the ATF perfusion culture than two other perfusion cultures. Moreover, the FVIII activity yield was unexpectedly low in the ATF perfusion culture. We have, therefore, studied the reasons for this low FVIII activity yield. It was revealed that the inactivation and the surface adsorption of FVIII onto the harvest bag were not the main reasons for the low yield in the ATF perfusion culture. The FVIII activity yield was not increased by the use of a hollow fiber filter with 0.5 μm pore size instead of 0.2 μm pore size. Additionally, the retention of FVIII molecules by the hollow fiber filter was a dominant factor in the low FVIII activity yield in the ATF perfusion culture. We demonstrated that FVIII yield was significantly improved by controlling transmembrane pressure (TMP) across the hollow fiber filter membrane. Taken together, these results suggest that TMP control could be an efficient method for the enhancement of FVIII yield in an ATF perfusion culture.

  8. Effect of warfarin on the kinetics of the vitamin K-dependent clotting factors in rats.

    Science.gov (United States)

    Vainieri, H; Wingard, L B

    1977-05-01

    The objectives of this study were to compare the time course of activities and rates of synthesis of activities for the separate clotting factors II, VII, IX, and X and to relate the rate of synthesis of activity of each factor to the plasma concentration of warfarin in individual rats after acute and chronic dosing with warfarin. Sequences of blood samples were obtained from each rat for 50 to 70 hours after an acute dose of warfarin or for 120 hours after a chronic loading dose plus 12-hour maintenance doses of warfarin and assayed for factor activities and warfarin concentration. The half-lives for degradation of factor activities ranged from 2.6 to 9.0 hours for the four factors. During periods of changing warfarin concentration (acute dosing) factor VII and X activities and rates of synthesis of activity showed large rapid changes, while factors II and IX responded more slowly. As the warfarin concentration diminished, the factor X rate of synthesis of activity appeared to exceed predrug values in all rats. During chronic dosing with warfarin the factor II activity and rate of synthesis of activity was depressed the most. The percent depression of the rate of synthesis of activity for each factor was related linearly to the logarithm of the plasma concentration of warfarin for the range 0 to 80% depression with acute dosing. However, this relationship was not suitable to explain the apparent overshoot in factor X rate of synthesis of activity.

  9. The F7 gene and clotting factor VII levels: dissection of a human quantitative trait locus.

    Science.gov (United States)

    Soria, Jose Manuel; Almasy, Laura; Souto, Juan Carlos; Sabater-Lleal, Maria; Fontcuberta, Jordi; Blangero, John

    2005-10-01

    Localization of human quantitative trait loci (QTLs) is now routine. However, identifying their functional DNA variants is still a formidable challenge. We present a complete dissection of a human QTL using novel statistical techniques to infer the most likely functional polymorphisms of a QTL that influence plasma levels of clotting factor VII (FVII), a risk factor for cardiovascular disease. Resequencing of 15 kb in and around the F7 gene identified 49 polymorphisms, which were then genotyped in 398 people. Using a Bayesian quantitative trait nucleotide (BQTN) method, we identified four to seven functional variants that completely account for this QTL. These variants include both rare coding variants and more common, potentially regulatory polymorphisms in intronic and promoter regions.

  10. Molecular Analysis of Factor VIII and Factor IX Genes in Hemophilia Patients: Identification of Novel Mutations and Molecular Dynamics Studies

    Science.gov (United States)

    Al-Allaf, Faisal A.; Taher, Mohiuddin M.; Abduljaleel, Zainularifeen; Bouazzaoui, Abdellatif; Athar, Mohammed; Bogari, Neda M.; Abalkhail, Halah A.; Owaidah, Tarek MA.

    2017-01-01

    Background Hemophilias A and B are X-linked bleeding disorders caused by mutations in the factor VIII and factor IX genes, respectively. Our objective was to identify the spectrum of mutations of the factor VIII and factor IX genes in Saudi Arabian population and determine the genotype and phenotype correlations by molecular dynamics (MD) simulation. Methods For genotyping, blood samples from Saudi Arabian patients were collected, and the genomic DNA was amplified, and then sequenced by Sanger method. For molecular simulations, we have used softwares such as CHARMM (Chemistry at Harvard Macromolecular Mechanics; http://www.charmm-gui.org) and GROMACS. In addition, the secondary structure was determined based on the solvent accessibility for the confirmation of the protein stability at the site of mutation. Results Six mutations (three novel and three known) were identified in factor VIII gene, and six mutations (one novel and five known) were identified in factor IX gene. The factor VIII novel mutations identified were c.99G>T, p. (W33C) in exon 1, c.2138 DelA, p. (N713Tfs*9) in eon14, also a novel mutation at splicing acceptor site of exon 23 c.6430 - 1G>A. In factor IX, we found a novel mutation c.855G>C, p. (E285D) in exon 8. These novel mutations were not reported in any factor VIII or factor IX databases previously. The deleterious effects of these novel mutations were confirmed by PolyPhen2 and SIFT programs. Conclusion The protein functional and structural studies and the models built in this work would be appropriate for predicting the effects of deleterious amino acid substitutions causing these genetic disorders. These findings are useful for genetic counseling in the case of consanguineous marriages which is more common in the Saudi Arabia. PMID:28270892

  11. Treatment of radiation osteomyelitis of the mandible in a patient with acquired factor VIII inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Arimoto, Takamasa; Kishimoto, Hiromitsu; Matsumoto, Suwako; Kawanaka, Masao; Urabe, Masahiro; Yoshioka, Wataru [Hyogo Coll. of Medicine, Nishinomiya (Japan)

    1996-07-01

    A case of radiation osteomyelitis of mandible in a 56-year-old man with acquired factor VIII inhibitor is presented. He had undergone radiation therapy for malignant lymphoma of the tonsilar region seven years earlier, and also had received steroids to treat acquired factor VIII inhibitor for a year. On initial examination, he was given a diagnosis of phlegmon of the cheek caused by pericoronitis of the left lower wisdom tooth. To prevent the recurrence of inflammation, the wisdom tooth was extracted carefully, but radiation osteomyelitis occurred in association with a pathological fracture of the condylar head. After mini-pulse therapy with methylpredonisolone, segmental mandibulectomy and reconstruction with a titanium metal plate were performed. The postoperative course was satisfactory with no complications such as bleeding or infection. (author)

  12. Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Gouri Shankar Pandey

    2013-01-01

    Full Text Available Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs. An indirect intracellular staining (ICS method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.

  13. Construction of a mouse model of factor VIII deficiency by gene targeting

    Energy Technology Data Exchange (ETDEWEB)

    Bi, L.; Lawler, A.; Gearhart, J. [Univ. of Pennsylvania School of Medicine, Philadelphia, PA (United States)] [and others

    1994-09-01

    To develop a small animal model of hemophilia A for gene therapy experiments, we set out to construct a mouse model for factor VIII deficiency by gene targeting. First, we screened a mouse liver cDNA library using a human FVIII cDNA probe. We cloned a 2.6 Kb partial mouse factor VIII cDNA which extends from 800 base pairs of the 3{prime} end of exon 14 to the 5{prime} end of exon 26. A mouse genomic library made from strain 129 was then screened to obtain genomic fragments covering the exons desired for homologous recombination. Two genomic clones were obtained, and one covering exon 15 through 22 was used for gene targeting. To make gene targeting constructs, a 5.8 Kb genomic DNA fragment covering exons 15 to 19 of the mouse FVIII gene was subcloned, and the neo expression cassette was inserted into exons 16 and 17 separately by different strategies. These two constructs were named MFVIIIC-16 and MFVIIIC-17. The constructs were linearized and transfected into strain 129 mouse ES cells by electroporation. Factor VIII gene-knockout ES cell lines were selected by G-418 and screened by genomic Southern blots. Eight exon 16 targeted cell lines and five exon 17 targeted cell lines were obtained. Three cell lines from each construct were injected into blastocysts and surgically transferred into foster mothers. Multiple chimeric mice with 70-90% hair color derived from the ES-cell genotype were seen with both constructs. Germ line transmission of the ES-cell genotype has been obtained for the MFVIIIC-16 construct, and multiple hemophilia A carrier females have been identified. Factor VIII-deficient males will be conceived soon.

  14. Potential role of a new PEGylated recombinant factor VIII for hemophilia A

    OpenAIRE

    2016-01-01

    Tung Thanh Wynn,1 Burak Gumuscu,2,3 1Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Florida, Gainesville, FL, 2Pediatric Hematology-Oncology, Bon Secours Health System, St. Mary’s Hospital, Richmond, VA, 3Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Virginia, Charlottesville, VA, USA Abstract: Hemophilia A, a deficiency in the activity of coagulation factor (F) VIII, is an X-linked bleeding disorder with an ap...

  15. Immunogenicity and pharmacokinetic studies of recombinant Factor VIII containing lipid cochleates

    OpenAIRE

    Kosloski, Matthew P.; Peng, Aaron; Varma, Prashant R.; Fathallah, Anas M.; MICLEA, RAZVAN D.; Mager, Donald E.; Balu-Iyer, Sathy V.

    2010-01-01

    Replacement therapy using recombinant factor VIII (rFVIII) is currently the most common therapy for hemophilia A, a bleeding disorder caused by the deficiency of FVIII. However, 15–30% of patients develop inhibitory antibodies against administered rFVIII which complicates the therapy. Encapsulation or association of protein with lipidic structures can reduce this immune response. Previously, we developed and characterized rFVIII-containing phosphatidylserine (PS) cochleate cylinders using bio...

  16. HTLV-III seropositivity in European haemophiliacs exposed to Factor VIII concentrate imported from the USA.

    Science.gov (United States)

    Melbye, M; Froebel, K S; Madhok, R; Biggar, R J; Sarin, P S; Stenbjerg, S; Lowe, G D; Forbes, C D; Goedert, J J; Gallo, R C

    1984-12-22

    77 Scottish haemophiliacs and 22 Danish haemophiliacs were serologically tested for antibodies to human T-cell leukaemia virus III (HTLV-III). Since 1979 the Scottish patients had been treated largely with factor VIII concentrate produced in Scotland, whereas all but 2 of the Danish patients had received both locally prepared concentrate and commercial concentrate made from US donor material. 15.6% of Scottish and 59.1% of Danish haemophiliacs were antibody positive (p less than 0.001). None of 11 haemophiliacs not treated in the period 1979-84 was seropositive. 2 (6.7%) of 30 subjects who had been treated with locally produced concentrate only were antibody positive, compared with 23 (39.7%) of 58 subjects who had been treated with commercial concentrate. Among 52 users of both commercially and locally produced factor VIII concentrate, seropositivity was directly correlated with the consumption of commercial concentrate (p less than 0.001) but not locally produced material. These data indicate that European haemophiliacs were exposed to HTLV-III via some factor VIII concentrates obtained from the USA.

  17. Renal Vein Thrombosis in a Newborn With Abnormal Factor VIII Level: Clinical Case Report.

    Science.gov (United States)

    Szafranska, Agnieszka; Pajak, Agata; Kilis-Pstrusinska, Katarzyna; Królak-Olejnik, Barbara

    2015-08-01

    Renal vein thrombosis (RVT) in neonates is a rare condition of low mortality but significant morbidity due to renal impairment.We report the case of a male term newborn with left RVT and elevated serum factor VIII (FVIII).The main symptoms of the patient and the important clinical findings: prompt diagnosis of RVT was possible because the classic clinical presentation of macroscopic hematuria, thrombocytopenia, and palpable flank mass were present in this newborn infant.The main diagnoses: finally, the reason of RVT was established when the infant was 3 months of age: the increased level of FVIII was confirmed. We discuss the diagnosis, therapy, and outcome of the patient and compare with the literature.Therapeutics interventions: however, despite anticoagulant therapy the left kidney developed areas of scarring and then atrophy.Conclusions and outcomes: Prothrombotic defects should be considered in all patients with perinatal RVT. Elevated factor VIII as a reason of RVT in neonatal period is particularly rare. Given a poor renal outcome in children associated with elevated levels of factor VIII, consideration could be given to more aggressive antithrombotic therapy in such cases.

  18. Neonatal helper-dependent adenoviral vector gene therapy mediates correction of hemophilia A and tolerance to human factor VIII.

    Science.gov (United States)

    Hu, Chuhong; Cela, Racel G; Suzuki, Masataka; Lee, Brendan; Lipshutz, Gerald S

    2011-02-01

    Neonatal gene therapy is a promising strategy for treating a number of congenital diseases diagnosed shortly after birth as expression of therapeutic proteins during postnatal life may limit the pathologic consequences and result in a potential "cure." Hemophilia A is often complicated by the development of antibodies to recombinant protein resulting in treatment failure. Neonatal administration of vectors may avoid inhibitory antibody formation to factor VIII (FVIII) by taking advantage of immune immaturity. A helper-dependent adenoviral vector expressing human factor VIII was administered i.v. to neonatal hemophilia A knockout mice. Three days later, mice produced high levels of FVIII. Levels declined rapidly with animal growth to 5 wk of age with stable factor VIII expression thereafter to >1 y of age. Decline in factor VIII expression was not related to cell-mediated or humoral responses with lack of development of antibodies to capsid or human factor VIII proteins. Subsequent readministration and augmentation of expression was possible as operational tolerance was established to factor VIII without development of inhibitors; however, protective immunity to adenovirus remained.

  19. The effects of long-term malathion or diazinon ingestion on the activity of hepatic synthesized clotting factors.

    Science.gov (United States)

    Lox, C D; Davis, J R

    1983-12-01

    Female Sprague-Dawley rats were placed on a drinking solution of 1 ppm Diazinon or malathion dissolved in water for 6 months. At the conclusion of the experiment liver tissue and plasma were examined. Clotting studies included the prothrombin time (PT), partial thromboplastin time (APTT), fibrinogen, and factors II, V, VII, X, plus the platelet count. Hepatic morphology, basically hepatocyte degeneration, was altered by malathion while diazinon had no notable effect. Likewise, malathion prolonged both the PT and APTT, the only changes in clotting activity.

  20. Kinetic Model Facilitates Analysis of Fibrin Generation and Its Modulation by Clotting Factors: Implications for Hemostasis-Enhancing Therapies

    Science.gov (United States)

    2014-01-01

    facilitates analysis of fibrin generation and its modulation by clotting factors: implications for hemostasis-enhancing therapies† Alexander Y...Syst. Biol.Med., 2011, 3, 136–146. 62 M. Schneider, N. Brufatto, E. Neill and M. Nesheim, J. Biol. Chem., 2004, 279, 13340–13345. 63 J. H. Foley, P. F

  1. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... Find a Hematologist Clinical Trials Talking with Your Doctor Patient Group Links Advocacy Toolkit Home For Patients ... factors. Know your family history. Make sure your doctor knows about any history of blood clots or ...

  2. Blood Clots

    Science.gov (United States)

    ... and may include pain or cramping, swelling, tenderness, warmth to the touch and bluish- or red-colored ... caused the clot, and will also perform a physical examination. In an emergency situation where patients may ...

  3. Refreezing previously thawed fresh-frozen plasma. Stability of coagulation factors V and VIII:C.

    Science.gov (United States)

    Dzik, W H; Riibner, M A; Linehan, S K

    1989-09-01

    With the growth in autologous blood programs and the increased scrutiny of the indications for transfusion of fresh-frozen plasma (FFP), an increase has been seen in the number of occasions on which FFP was requested and thawed but then not transfused. The coagulation properties of FFP units that were refrozen and then rethawed were therefore studied. Fifty-eight units of plasma were studied, with each experimental unit of FFP paired with an identical control unit. Experimental units were frozen, stored at -65 degrees C, thawed, stored at 1 to 6 degrees C for various periods of time up to 24 hours, and then refrozen, stored at -65 degrees C, rethawed, and stored again in the refrigerator for up to 24 hours. Control units were frozen once at the time the experimental units were first frozen and thawed once at the time of the second thaw of the experimental units. Aliquots of plasma were sampled periodically and were later batch-tested for prothrombin time (PT), activated partial thromboplastin time (aPTT), and factor V and VIII:C activity. The results of coagulation testing of the twice-frozen plasmas were always within the normal range. There was a slight but statistically valid prolongation of the PT and aPTT and a decrease in the factor V and VIII:C levels for twice-frozen plasma compared with control plasma. The greatest decline occurred in the level of factor VIII:C. The measured deterioration in coagulation of twice-frozen FFP is unlikely to be of clinical importance. Refreezing FFP may eventually prove useful for rare donor, autologous, and massive transfusion programs.

  4. Analysis of inversions in the factor VIII gene in Spanish hemophilia A patients and families

    Energy Technology Data Exchange (ETDEWEB)

    Domenech, M.; Tizzano, E.; Baiget, M. [Hospital de Sant Pau, Barcelona (Spain); Altisent, C. [Hospital Vall d`Hebron, Barcelona (Spain)

    1994-09-01

    Intron 22 is the largest intron of the factor VIII gene and contains a CpG island from which two additional transcripts originate. One of these transcripts corresponds to the F8A gene which have telomeric extragenic copies in the X chromosome. An inversion involving homologous recombination between the intragenic and the distal or proximal copies of the F8A gene has been recently described as a common cause of severe hemophilia A (HA). We analyzed intron 22 rearrangements in 195 HA patients (123 familial and 72 sporadic cases). According to factor VIII levels, our sample was classified as severe in 114 cases, moderate in 29 cases and mild in 52 cases. An intron 22 (F8A) probe was hybridized to Southern blots of BcII digested DNA obtained from peripheral blood. A clear pattern of altered bands identifies distal or proximal inversions. We detected an abnormal pattern identifying an inversion in 49 (25%) of the analyzed cases. 43% of severe HA patients (49 cases) showed an inversion. As expected, no inversion was found in the moderate and mild group of patients. We found a high proportion (78%) of the distal rearrangement. From 49 identified inversions, 33 were found in familial cases (27%), while the remaining 15 were detected in sporadic patients (22%) in support that this mutational event occurs with a similar frequency in familial or sporadic cases. In addition, we detected a significant tendency of distal inversion to occur more frequently in familial cases than in sporadic cases. Inhibitor development to factor VIII was documented in approximately 1/3 of the patients with inversion. The identification of such a frequent molecular event in severe hemophilia A patients has been applied in our families to carrier and prenatal diagnosis, to determine the origin of the mutation in the sporadic cases and to detect the presence of germinal mosaicism.

  5. Characterization of Chinese Hamster Ovary Cells Producing Coagulation Factor VIII Using Multi-omics Tools

    DEFF Research Database (Denmark)

    Kaas, Christian Schrøder

    ,000 fold over the last couple of years due to the revolution of next-generation sequencing (NGS), has dramatically accelerated CHO-omics from virtually non-existent to a vibrant growing field. The aim of this thesis was to investigate the impact of coagulation factor VIII (FVIII) production in CHO cells...... for analysis and engineering of industrially relevant CHO cells. Full implementation of such tools for generating specifically engineered CHO production cell lines may allow significant cost-reductions in production of complex biopharmaceuticals such as FVIII....

  6. ADAMTS13 content in plasma-derived factor VIII/von Willebrand factor concentrates.

    Science.gov (United States)

    Peyvandi, Flora; Mannucci, Pier M; Valsecchi, Carla; Pontiggia, Silvia; Farina, Claudio; Retzios, Anastassios D

    2013-10-01

    Thrombotic thrombocytopenic purpura (TTP) is a microangiopathy syndrome caused by a congenital or acquired deficiency of ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor (VWF) and thus prevents the formation of platelet-rich thrombi in the microcirculation. TTP can be fatal if not appropriately and timely treated with the infusion of fresh frozen plasma (FFP) or exchange plasmapheresis, that reverse the process of microangiopathy by removing anti-ADAMTS13 autoantibodies and replacing functional ADAMTS13. The treatment of TTP with FFP is not free from risks and must be administered in hospitals or clinics, owing to the substantial amount of plasma volume infused or exchanged and the frequent need of catheter application. Moreover, most FFPs are not subjected to treatments to remove or inactivate blood-borne infectious agents. A number of recent reports indicate that certain plasma-derived VWF-factor VIII (FVIII) concentrates are clinically effective in the treatment of congenital TTP. In this study, we measured ADAMTS13 levels in various plasma-derived VWF-FVIII concentrates, showing that Koate(®) -DVI (Grifols), contained relatively high amounts of ADAMTS13 and that Alphanate(®) (Grifols) was the closest other product in terms of protease content. Koate(®) -DVI contains, on average (five lots tested), 0.091 ± 0.007 Units of ADAMTS13 activity per IU of FVIII. On the basis of this analysis and other reports of VWF-FVIII concentrate utilization in congenital TTP, potential dosing, and future clinical developments are discussed.

  7. The impact of von Willebrand factor on factor VIII memory immune responses.

    Science.gov (United States)

    Chen, Juan; Schroeder, Jocelyn A; Luo, Xiaofeng; Shi, Qizhen

    2017-08-22

    Immune tolerance induction (ITI) with aggressive infusion of factor VIII (FVIII) is the current strategy used to eradicate FVIII inhibitors and restore normal FVIII pharmacokinetics in inhibitor patients. Whether the use of FVIII products containing von Willebrand factor (VWF) will affect the efficacy of ITI is still controversial. In this study, we explored the impact of VWF on FVIII memory immune responses in hemophilia A (HA) mice. A T-cell proliferation assay and cytokine profile analysis were used to study FVIII-primed CD4(+) T cells. When CD4(+) T cells from primed FVIII(null) mice were restimulated with recombinant human FVIII (rhF8) plus recombinant human VWF (rhVWF) in vitro, the percentages of daughter CD4(+) T cells were significantly decreased compared with the groups cultured with rhF8 only. Levels of interferon-γ and interleukin 10 were significantly lower in the rhF8 plus rhVWF groups than in the rhF8 groups. When memory B-cell pools from primed FVIII(null) mice were cultured with rhF8 with or without rhVWF to induce differentiation of memory B cells into antibody-secreting cells (ASCs), the number of ASCs was significantly lower in the rhF8 plus VWF group than in the rhF8 group. When memory B-cell pools were transferred into NSGF8KO mice followed by rhF8 immunization with or without rhVWF, the titers of anti-F8 inhibitors and total immunoglobulin G were significantly higher in the rhF8 group than in the rhF8 plus rhVWF group, with an average difference of 2.23- and 2.04-fold. Together, our data demonstrate that VWF attenuates FVIII memory immune responses in HA mice.

  8. Efficient delivery of human clotting factor Ⅸ after injection of lentiviral vectors in utero

    Institute of Scientific and Technical Information of China (English)

    Xiao-guang CHEN; Huan-zhang ZHU; Ju-li GONG; Feng LI; Jing-lun XUE

    2004-01-01

    AIM: To explore gene transfer feasibility for human clotting factor IX (hFⅨ) mediated by recombinant lentivirus in utero. METHODS: ICR mice fetus at 17-19 d gestation were received lentiviral vectors carrying hFⅨ eDNA under the control of liver specific promoter by intrahepatic injection. The expression and distribution of hFⅨ cDNA and possible immune responses against the hFIX were assessed by ELISA, PCR, RT-PCR, and immunohistochemistry, respectively. RESULTS: The serum hFⅨ protein were detected at different time points in all newborn mice, the highest level of hFⅨ was 50 μg/L and lasted for more than 30 d. Anti-hⅨ antibody was not detected, hFIX cDNA was detected in liver, spleen, and heart. The expression of hFⅨ eDNA was only detected in liver. Besides, no germ line transmission was found at DNA and RNA levels, and no side effect associated with gene transfer was detected. CONCLUSION: The efficient delivery of hFⅨ can be achieved by prenatal gene transfer. It thus shows the feasibility of gene therapy for hemophilia in utero.

  9. Scanning electron microscopic and electrophoretic observations on barium sulphate used to absorb clotting factors.

    Science.gov (United States)

    Howell, R M; Deacon, S L

    1975-04-30

    Electron microscopy and particle electrophoresis were found to be complementary techniques with which to complete the physical data from an earlier study on barium sulphates used to absorb clotting factors from serum. The differences revealed by scanning electron microscopy (S. E. M) in the physical shape of low and high density grades of barium sulphate particles appear to be of greater significance than charge as expressed by electrophoretic mobility, in determining whether or not precursor or performed factor Xa is eluted. This conclusion was based on the finding that at pH values close to 7, where the adsorption from serum occurs, all samples with the exception of natural barytes were uncharged. However as the high-density, or soil-grade, was found by S. E. M. to consist of large solid crystals it was suggested that this shape might induce activiation of factor X as a result of partial denaturation and consequent unfolding of the adsorbed protein. In contrast, uptake of protein into the centre of the porous aggregates revealed by S. E. M. pictures of low-density or X-ray grade barium sulphate may afford protection against denaturation and exposure of the enzyme site. The porous nature of particles of low-density barium sulphate compared with the solid crystalline forms of other grades accounts not only for its lower bulk density but also for its greater surface/gram ratio which is reflected by an ability to adsorb more protein from serum. Neither technique produced evidence from any of the samples to indicate the presence of stabilising agents sometimes used to coat particles in barium meals.

  10. A single chain variant of factor VIII Fc fusion protein retains normal in vivo efficacy but exhibits altered in vitro activity.

    Directory of Open Access Journals (Sweden)

    Yang Buyue

    Full Text Available Recombinant factor VIII Fc (rFVIIIFc is a fusion protein consisting of a single B-domain-deleted (BDD FVIII linked recombinantly to the Fc domain of human IgG1 to extend half-life. To determine if rFVIIIFc could be further improved by maintaining the heavy and light chains within a contiguous single chain (SC, we evaluated the activity and function of SC rFVIIIFc, an isoform that is not processed at residue R1648. SC rFVIIIFc showed equivalent activity in a chromogenic assay compared to rFVIIIFc, but approximately 40% activity by the one-stage clotting assay in the presence of von Willebrand Factor (VWF, with full activity in the absence of VWF. Moreover, SC rFVIIIFc demonstrated markedly delayed thrombin-mediated release from VWF, but an activity similar to that of rFVIIIFc upon activation in FXa generation assays. Therefore, the apparent reduction in specific activity in the aPTT assay appears to be primarily due to delayed release of FVIII from VWF. To assess whether stability and activity of SC rFVIIIFc were affected in vivo, a tail vein transection model in Hemophilia A mice was utilized. The results demonstrated similar pharmacokinetic profiles and comparable efficacy for SC rFVIIIFc and rFVIIIFc. Thus, while the single chain configuration did not promote enhanced half-life, it reduced the rate of release of FVIII from VWF required for activation. This impaired release may underlie the observed reduction in the one-stage clotting assay, but does not appear to affect the physiological activity of SC rFVIIIFc.

  11. Many factor VIII products available in the treatment of hemophilia A: an embarrassment of riches?

    Science.gov (United States)

    Lieuw, Kenneth

    2017-01-01

    Hemophilia A (HA) is a common bleeding disorder caused by the deficiency of factor VIII (FVIII) with an incidence of ~1 in 5000 male births. Replacement of FVIII is necessary to prevent and treat bleeding episodes. However, with multiple new drugs in addition to old standards, choosing among the different FVIII treatment options is harder than ever. There are FVIII products that are plasma derived or recombinant, FVIII products designed to extend the half-life of FVIII, and the first single-chain FVIII product, recombinant factor VIII single chain (rFVIII-SC). As development of inhibitors to FVIII continues to be a major problem in the care of HA patients, recent studies showing lower rates of inhibitor development with plasma-derived FVIIII products versus recombinant FVIII products have made choosing among the many options now available even more complex. Although still unproven, extended half-life (EHL) products may provide the hope of decreased immunogenicity but need further testing in previously untreated patients (PUPs). This review highlights some of the differences between FVIII products currently available and hopefully assists the clinician to decide which FVIII product to choose for their patients.

  12. Factor V and VIII combined deficiency: clinical perioperative management for tonsillectomy in a child.

    Science.gov (United States)

    Lanchon, R; Robin, F; Brissaud, O; Marro, M; Nouette-Gaulain, K

    2014-03-01

    Combined factors V (FV) and VIII (FVIII) deficiency is a rarely seen hereditary coagulation disease. Experience of its management in surgery with a high-risk of bleeding is rare. The interest of this case report is to propose a strategy of perioperative management for such a deficit, but also to recall that a careful preoperative anesthetic evaluation with questioning and physical examination permits to detect unsuspected coagulation disorders and to schedule the preventive treatment. The protocol for the perioperative period consisted of the administration of desmopressin and fresh frozen plasma one hour before surgery. The administration of desmopressin was continued for 48hours. Fresh frozen plasma and tranexamic acid were administered during the first 9 postoperative days. A local bleeding occurred at 8 days (scab coming off) and required systematically a surgical hemostasis and an intensification of the therapeutic protocol. Recombinant plasmatic factor VIII was administered for 7 days together with a daily perfusion of fresh frozen plasma for a total treatment period of 14 days.

  13. A new recombinant factor VIII: from genetics to clinical use

    Directory of Open Access Journals (Sweden)

    Santagostino E

    2014-12-01

    Full Text Available Elena Santagostino Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy Abstract: Advances in recombinant technology and knowledge about coagulation factor VIII (FVIII are building a platform for new therapeutic options in patients with hemophilia A. The development of turoctocog alfa, a novel, high-purity, third-generation, B-domain truncated recombinant FVIII, has been produced and formulated without the use of animal-derived or human serum-derived components, in the wake of understanding of the new biochemical characteristics of FVIII, namely its protein structure, and glycosylation and sulfating patterns. Culture conditions and a five-step purification process have been developed to optimize the safety of turoctocog alfa. The results of two pilot clinical trials using turoctocog alfa confirmed high safety levels, with no patient developing inhibitors during the period of observation. The purpose of this review is to describe briefly the molecular and biological properties of turoctocog alfa, together with details of its clinical development, with emphasis on the needs of patients with hemophilia A. Keywords: hemophilia A, recombinant factor VIII, turoctocog alfa, purification, inhibitor, safety

  14. Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin

    Science.gov (United States)

    Reed, G. L.; Matsueda, G. R.; Haber, E.

    1992-01-01

    Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis.

  15. Investigation of the effect of kaolin and tissue factor-activated citrated whole blood, on clot forming variables, as evaluated by thromboelastograph

    DEFF Research Database (Denmark)

    Johansson, Per Ingemar; Bochsen, L.; Andersen, S.

    2008-01-01

    laboratory-based analysis, however, requires validation of the activators employed and the effect of storage of the WB sample in citrate before analysis. STUDY DESIGN AND METHODS: The effect of kaolin, tissue factor (TF) 1:17,000, or TF 1:42,500 on TEG clotting time (R), Angle (velocity of clot formation......), and maximum clot strength (amplitude [MA]) were evaluated, together with day-to-day variation, the coefficient of variance (CV%), and the effect of citrate storage time. RESULTS: Clot formation variables were equally affected by TF 1:17,000 and kaolin activation, whereas R was significantly longer when TF 1...... minutes were evaluated with kaolin as the activator. CONCLUSION: The TEG assays evaluated were reproducible and present with an acceptable CV% for routine clinical practice. Kaolin and TF 1:17,000 equally affected the clot formation variables. Storage of WB for up to 30 minutes in citrate did not, except...

  16. Anti-CD3 antibodies modulate anti-factor VIII immune responses in hemophilia A mice after factor VIII plasmid-mediated gene therapy.

    Science.gov (United States)

    Peng, Baowei; Ye, Peiqing; Rawlings, David J; Ochs, Hans D; Miao, Carol H

    2009-11-12

    One major obstacle in gene therapy is the generation of immune responses directed against transgene product. Five consecutive anti-CD3 treatments concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic levels of FVIII gene expression in treated hemophilia A mice. Repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4+ and CD8+ T cells, whereas increased transforming growth factor-beta levels in plasma and the frequency of both CD4+CD25+FoxP3+ and CD4+CD25-Foxp3+ regulatory T cells in the initial few weeks after treatment. Although prior depletion of CD4+CD25+ cells did not abrogate tolerance induction, adoptive transfer of CD4+ cells from tolerized mice at 6 weeks after treatment protected recipient mice from anti-FVIII immune responses. Anti-CD3-treated mice mounted immune responses against both T-dependent and T-independent neo-antigens, indicating that anti-CD3 did not hamper the immune systems in the long term. Concomitant FVIII plasmid + anti-CD3 treatment induced long-term tolerance specific to FVIII via a mechanism involving the increase in transforming growth factor-beta levels and the generation of adaptive FVIII-specific CD4+Foxp3+ regulatory T cells at the periphery. Furthermore, anti-CD3 can reduce the titers of preexisting anti-FVIII inhibitory antibodies in hemophilia A mice.

  17. Plasmin-induced procoagulant effects in the blood coagulation: a crucial role of coagulation factors V and VIII.

    Science.gov (United States)

    Ogiwara, Kenichi; Nogami, Keiji; Nishiya, Katsumi; Shima, Midori

    2010-09-01

    Plasminogen activators provide effective treatment for patients with acute myocardial infarction. However, paradoxical elevation of thrombin activity associated with failure of clot lysis and recurrent thrombosis has been reported. Generation of thrombin in these circumstances appears to be owing to plasmin (Plm)-induced activation of factor (F) XII. Plm catalyzes proteolysis of several coagulant factors, but the roles of these factors on Plm-mediated procoagulant activity remain to be determined. Recently developed global coagulation assays were used in this investigation. Rotational thromboelastometry using whole blood, clot waveform analysis and thrombin generation tests using plasma, showed that Plm (> or =125 nmol/l) shortened the clotting times in similar dose-dependent manners. In particular, the thrombin generation test, which was unaffected by products of fibrinolysis, revealed the enhanced coagulation with an approximately two-fold increase of peak level of thrombin generation. Studies using alpha2-antiplasmin-deficient plasma revealed that much lower dose of Plm (> or =16 nmol/l) actually contributed to enhancing thrombin generation. The shortening of clotting time could be observed even in the presence of corn trypsin inhibitor, supporting that Plm exerted the procoagulant activity independently of FXII. In addition, using specific coagulation-deficient plasmas, the clot waveform analysis showed that Plm did not shorten the clotting time in only FV-deficient or FVIII-deficient plasma in prothrombin time-based or activated partial thromboplastin time-based assay, respectively. Our results indicated that Plm did possess procoagulant activity in the blood coagulation, and this effect was likely attributed by multicoagulation factors, dependent on FV and/or FVIII.

  18. Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study

    Directory of Open Access Journals (Sweden)

    Venkateswarlu Divi

    2010-02-01

    Full Text Available Abstract Background Human blood coagulation factor VIII (fVIII is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3 that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa which serves as a critical co-factor for factor IXa (fIXa enzyme in the intrinsic coagulation pathway. Results In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 Å X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740 and A3-C1-C2 (Ser1669-Tyr2332 domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein. Conclusions MD simulations predicted that the acidic linker peptide (a1 between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569 region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1 undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the

  19. The course of preexistent immune abnormalities in HIV-negative hemophiliacs treated for 2 years with a monoclonal purified factor-VIII concentrate

    NARCIS (Netherlands)

    Smid, W. M.; van der Meer, J.; Smit, J. W.; Halie, M. R.

    1993-01-01

    The administration of factor VIII concentrates has been associated with immune abnormalities in patients with severe haemophilia A, even in the absence of HIV infection. The effects of a monoclonal purified factor VIII concentrate, Hemofil M (Baxter), on preexistent immune abnormalities were

  20. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... Blood Basics Blood Disorders Anemia Bleeding Disorders Blood Cancers Blood Clots Blood Clotting and Pregnancy Clots and ... Increased maternal age Other medical illness (e.g., cancer, infection) back to top How are Blood Clots ...

  1. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... Blood Basics Blood Disorders Anemia Bleeding Disorders Blood Cancers Blood Clots Blood Clotting and Pregnancy Clots and ... Increased maternal age Other medical illness (e.g., cancer, infection) back to top How are Blood Clots ...

  2. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... For Patients Blood Disorders Blood Clots Blood Clotting & Pregnancy If you are pregnant, or you have just ... The risk of developing a blood clot during pregnancy is increased by the following: Previous blood clots ...

  3. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... infection) back to top How are Blood Clots in Pregnant Women Treated? Typically, blood clots are treated ... history of blood clots or blood clotting disorders in your family. Remain active, with your doctor's approval. ...

  4. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... Blood Basics Blood Disorders Anemia Bleeding Disorders Blood Cancers Blood Clots Clots and Travel Blood Clotting and ... Increased maternal age Other medical illness (e.g., cancer, infection) back to top How are Blood Clots ...

  5. INSIGHT in risk factors and treatment of inhibitors in nonsevere hemophilia A

    NARCIS (Netherlands)

    van Velzen, A.S.

    2016-01-01

    Hemophilia A is an inherited X-linked bleeding disorder that occurs in male offspring of carrier females. In these individuals a mutation in the F8 gene, located on the X-chromosome, causes a deficiency of the factor VIII protein, clotting factor VIII. The worldwide prevalence of hemophilia is 1 in

  6. Evaluation of B&W UO2/ThO2 VIII experimental core: criticality and thermal disadvantage factor analysis

    Energy Technology Data Exchange (ETDEWEB)

    Carlo Parisi; Emanuele Negrenti

    2017-02-01

    In the framework of the OECD/NEA International Reactor Physics Experiment (IRPHE) Project, an evaluation of core VIII of the Babcock & Wilcox (B&W) Spectral Shift Control Reactor (SSCR) critical experiment program was performed. The SSCR concept, moderated and cooled by a variable mixture of heavy and light water, envisaged changing of the thermal neutron spectrum during the operation to encourage breeding and to sustain the core criticality. Core VIII contained 2188 fuel rods with 93% enriched UO2-ThO2 fuel in a moderator mixture of heavy and light water. The criticality experiment and measurements of the thermal disadvantage factor were evaluated.

  7. High-throughput proteomic characterization of plasma rich in growth factors (PRGF-Endoret)-derived fibrin clot interactome.

    Science.gov (United States)

    Anitua, Eduardo; Prado, Roberto; Azkargorta, Mikel; Rodriguez-Suárez, Eva; Iloro, Ibon; Casado-Vela, Juan; Elortza, Felix; Orive, Gorka

    2015-11-01

    Plasma rich in growth factors (PRGF®-Endoret®) is an autologous technology that contains a set of proteins specifically addressed to wound healing and tissue regeneration. The scaffold formed by using this technology is a clot mainly composed of fibrin protein, forming a three-dimensional (3D) macroscopic network. This biomaterial is easily obtained by biotechnological means from blood and can be used in a range of situations to help wound healing and tissue regeneration. Although the main constituent of this clot is the fibrin scaffold, little is known about other proteins interacting in this clot that may act as adjuvants in the healing process. The aim of this study was to characterize the proteins enclosed by PRGF-Endoret scaffold, using a double-proteomic approach that combines 1D-SDS-PAGE approach followed by LC-MS/MS, and 2-DE followed by MALDI-TOF/TOF. The results presented here provide a description of the catalogue of key proteins in close contact with the fibrin scaffold. The obtained lists of proteins were grouped into families and networks according to gene ontology. Taken together, an enrichment of both proteins and protein families specifically involved in tissue regeneration and wound healing has been found.

  8. Factor VIII brand and the incidence of factor VIII inhibitors in previously untreated UK children with severe hemophilia A, 2000-2011.

    Science.gov (United States)

    Collins, Peter W; Palmer, Benedict P; Chalmers, Elizabeth A; Hart, Daniel P; Liesner, Ri; Rangarajan, Savita; Talks, Katherine; Williams, Michael; Hay, Charles R M

    2014-11-27

    The effect of recombinant factor VIII (rFVIII) brand on inhibitor development was investigated in all 407 severe hemophilia A previously untreated patients born in the United Kingdom (UK) between 1 January 2000 and 31 December 2011. Eighty-eight (22%) had been in the RODIN study. Information was extracted from the National Haemophilia Database. Because exposure days (EDs) were not known for some patients, time from first treatment was used as a surrogate for rFVIII exposure. An inhibitor developed in 118 (29%) patients, 60 high and 58 low titer, after a median (interquartile range) of 7.8 (3.3-13.5) months from first exposure and 16 (9-30) EDs. Of 128 patients treated with Kogenate Bayer/Helixate NexGen, 45 (35.2%, 95% confidence interval [CI] 27.4-43.8) developed an inhibitor compared with 42/172 (24.4%, 95% CI 18.6% to 31.4%) with Advate (P = .04). The adjusted hazard ratio (HR) (95% CI) for Kogenate Bayer/Helixate NexGen compared with Advate was 2.14 (1.12-4.10) (P = .02) for high titer and 1.75 (1.11-2.76) (P = .02) for all inhibitors. When excluding UK-RODIN patients, the adjusted HR (95% CI) for high-titer inhibitors was 2.00 (0.93-4.34) (P = .08). ReFacto AF was associated with a higher incidence of all, but not high-titer, inhibitors than Advate. These results will help inform debate around the relative immunogenicity and use of rFVIII brands.

  9. Half-life extended factor VIII for the treatment of hemophilia A.

    Science.gov (United States)

    Tiede, A

    2015-06-01

    Prophylactic infusion of factor VIII (FVIII) prevents joint bleeding and other hemorrhages in patients with hemophilia A. Conventional FVIII concentrates have a short half-life, with an average of about 12 h in adults, ranging in individual patients between 6 and 24 h, and even shorter in younger children. Therefore, effective prophylaxis requires frequent intravenous injection, usually three times per week or every other day. Several technologies are currently under investigation to extend the half-life of FVIII, including Fc fusion (Eloctate, Elocta, efmoroctocog alfa), addition of polyethylene glycol (turoctocog alfa pegol [N8-GP], BAY 94-9027, BAX 855), and a single-chain construct (CSL627). This review summarizes characteristics of products in clinical development and discusses their potential benefits.

  10. Development of a peptidomimetic ligand for efficient isolation and purification of factor VIII via affinity chromatography.

    Science.gov (United States)

    Knör, Sebastian; Khrenov, Alexey V; Laufer, Burkhardt; Saenko, Evgueni L; Hauser, Charlotte A E; Kessler, Horst

    2007-09-06

    Hemophilia A, one of the most severe bleeding disorders, results from an inherited deficiency of factor VIII (FVIII) function. Treatment by injection of FVIII has been a common procedure for decades. Nevertheless, the production and purification of FVIII remains a challenging task. Current procedures using immunoaffinity chromatography are expensive and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified by use of octapeptide ligands, but their low protease-resistance limits their application. We here report the systematic rational and combinatorial optimization procedure that allowed us to transfer the octapeptide ligands into a small peptidomimetic. This compound is the smallest ligand known for separation of such a large protein (330 kDa). It not only binds and purifies FVIII with high efficiency but also is stable, protease-resistant, and cheap to produce in preparative scale. Hence it offers a valuable alternative to antibody-based purification procedures.

  11. Pharmacokinetics and dose requirements of factor VIII over the age range 3-74 years

    DEFF Research Database (Denmark)

    Björkman, Sven; Folkesson, Anna; Jönsson, Siv

    2009-01-01

    PURPOSE: The three aims of this investigation were (1) to develop a population pharmacokinetic (PK) model for factor VIII (FVIII) in haemophilia A patients, with estimates of inter-occasion and inter-individual variance, (2) to investigate whether appropriate dosing of FVIII for regular prophylaxis...... can be calculated according to patient characteristics, and (3) to present dosing recommendations for initiating prophylactic treatment. METHODS: A population PK model was developed using data from four PK studies on patients aged 7-74 years. The model was tested on sparse FVIII data from 42...... outpatient visits by haemophilia prophylaxis patients aged 3-66 years. Dose requirements for prophylaxis were calculated both according to the population model and from empirical Bayesian estimates of FVIII PK in the individual patients. RESULTS: The study data were well characterised by a two-compartment PK...

  12. Analysis of factor VIII gene inversions in 164 unrelated hemophilia A families

    Energy Technology Data Exchange (ETDEWEB)

    Vnencak-Jones, L.; Phillips, J.A. III; Janco, R.L. [Vanderbilt Univ. School of Medicine, Nashville, TN (United States)] [and others

    1994-09-01

    Hemophilia A is an X-linked recessive disease with variable phenotype and both heterogeneous and wide spread mutations in the factor VIII (F8) gene. As a result, diagnostic carrier or prenatal testing often relies upon laborious DNA linkage analysis. Recently, inversion mutations resulting from an intrachromosomal recombination between DNA sequences in one of two A genes {approximately}500 kb upstream from the F8 gene and a homologous A gene in intron 22 of the F8 gene were identified and found in 45% of severe hemophiliacs. We have analyzed banked DNA collected since 1986 from affected males or obligate carrier females representing 164 unrelated hemophilia A families. The disease was sporadic in 37%, familial in 54% and in 10% of families incomplete information was given. A unique deletion was identified in 1/164, a normal pattern was observed in 110/164 (67%), and 53/164 (32%) families had inversion mutations with 43/53 (81%) involving the distal A gene (R3 pattern) and 10/53 (19%) involving the proximal A gene (R2 pattern). While 19% of all rearrangements were R2, in 35 families with severe disease (< 1% VIII:C activity) all 16 rearrangements seen were R3. In 18 families with the R3 pattern and known activities, 16 (89%) had levels < 1%, with the remaining 2 families having {le} 2.4% activity. Further, 18 referrals specifically noted the production of inhibitors and 8/18 (45%) had the R3 pattern. Our findings demonstrate that the R3 inversion mutation patterns is (1) only seen with VIII:C activity levels of {le} 2.4%, (2) seen in 46% of families with severe hemophilia, (3) seen in 45% of hemophiliacs known to have inhibitors, (4) not correlated with sporadic or familial disease and (5) not in disequilibrium with the Bcl I or Taq I intron 18 or ST14 polymorphisms. Finally, in families positive for an inversion mutation, direct testing offers a highly accurate and less expensive alternative to DNA linkage analysis.

  13. Antidote strategies to reverse anticoagulation with TB-402, a long-acting partial inhibitor of factor VIII

    NARCIS (Netherlands)

    Tangelder, M.; Long, C.; Emmerechts, J.; Jacquemin, M.; Peerlinck, K.; Vanassche, T.; Glazer, S.; Giesen, P.; Hoylaerts, M.; Verhamme, P.

    2012-01-01

    . Background: TB-402 is a partially inhibiting antibody of factor VIII that is under development as a long-acting anticoagulant. Patients and Methods: The reversibility of FVIII inhibition by TB-402 was evaluated in vitro after spiking with recombinant human FVIII (rhFVIII), human plasma-derived FVI

  14. Evaluation of the biological differences of canine and human factor VIII in gene delivery: Implications in human hemophilia treatment

    Science.gov (United States)

    The canine is the most important large animal model for testing novel hemophilia A(HA) treatment. It is often necessary to use canine factor VIII (cFIII) gene or protein for the evaluation of HA treatment in the canine model. However, the different biological properties between cFVIII and human FVII...

  15. [Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli].

    Science.gov (United States)

    Zhu, Fuxiang; Liu, Zelong; Qu, Huige; Xin, Xiaolin; Dong, Hongxin; Liu, Xiangqin

    2009-07-01

    We studied the ligation of coagulation factor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220. Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of BDD-FVIII protein on SDS-PAGE gel. Western blotting using factor VIII specific antibodies confirmed that this protein band is BDD-FVIII produced by protein trans-splicing. It demonstrated that the heavy and light chains of BDD-FVIII can be efficiently ligated with the Ssp DnaB intein-mediated protein trans-splicing. These results provided evidence for encouraging our ongoing investigation with intein as a means in dual AAV vectors carrying the factor VIII gene to overcome the packaging size limitation of a single AAV vector in hemophilia A gene therapy.

  16. Coronary artery calcification in hemophilia A: No evidence for a protective effect of factor VIII deficiency on atherosclerosis

    NARCIS (Netherlands)

    Tuinenburg, A.; Rutten, A.; Kavousi, M.; Leebeek, F.W.G.; Ypma, P.F.; Laros-Van Gorkom, B.A.P.; Nijziel, M.R.; Kamphuisen, P.W.; Mauser-Bunschoten, E.P.; Roosendaal, G.; Biesma, D.H.; Van Der Lugt A., [No Value; Hofman, A.; Witteman, J.C.M.; Bots, M.L.; Schutgens, R.E.G.

    2011-01-01

    Mortality due to ischemic heart disease is lower in hemophilia patients when compared to the general male population. As coagulation plays a role in the inflammatory pathways involved in atherogenesis, we investigated whether the clotting factor deficiency protects hemophilia patients from developin

  17. Potential role of a new PEGylated recombinant factor VIII for hemophilia A

    Directory of Open Access Journals (Sweden)

    Wynn TT

    2016-06-01

    Full Text Available Tung Thanh Wynn,1 Burak Gumuscu,2,3 1Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Florida, Gainesville, FL, 2Pediatric Hematology-Oncology, Bon Secours Health System, St. Mary’s Hospital, Richmond, VA, 3Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Virginia, Charlottesville, VA, USA Abstract: Hemophilia A, a deficiency in the activity of coagulation factor (F VIII, is an X-linked bleeding disorder with an approximate incidence of one in 5,000 male infants. Bleeding-related complications often result in greater severity of disease, poor quality of life, surgical interventions for severe joint destruction, and shortened life span. With the availability of plasma-derived and recombinant FVIII products, the benefits of primary prophylaxis were demonstrated and is now the standard of care for patients with severe factor deficiencies. Current hemophilia research is focusing on the creation of new factor replacement therapies with longer half-lives; accessing alternative mechanisms to achieve desired hemostasis and enhance bypassing ­activity; and limiting the immunogenicity of the protein. PEGylation involves the covalent attachment of polyethylene glycol (PEG to a protein, peptide, or a small molecule drug. PEG effectively increases the molecular weight and size of the protein by creating a hydrophilic cloud around the molecule. This molecular change may reduce susceptibility of the molecule to proteolytic activity and degradation. It is also believed that PEGylation changes the surface charge of the protein that ultimately interferes with some receptor-mediated clearance processes. The half-life of PEGylated factor is more prolonged when compared to non-PEGylated full-length recombinant FVIII. The dawn of a new era in the care of hemophilia patients is upon us with the release of recombinant FVIII products with extended half-lives, and products with even more extended half

  18. Successful immune tolerance induction consisting of high-dose factor VIII rich in von Willebrand factor and pulsed intravenous immunoglobulin: a case report

    Directory of Open Access Journals (Sweden)

    Kubisz Peter

    2012-10-01

    Full Text Available Abstract Introduction The development of factor VIII inhibitors is a serious complication of replacement therapy in patients with congenital hemophilia A. Immune tolerance induction has been accepted as the only clinically proven treatment allowing antigen-specific tolerance to factor VIII. However, some of its issues, such as patient selection, timing, factor VIII dosing, use of immunosuppressive or immunomodulatory procedures, still remain the subject of debate. Case presentation A case of a 3-year-old Caucasian boy with severe congenital hemophilia A, intron 22 inversion of the F8 gene and high-titer inhibitor, who underwent an immune tolerance induction according to the modified Bonn regimen (high doses of plasma-derived factor VIII rich in von Willebrand factor and pulsed intravenous immunoglobulin is presented. The treatment lasted for 13 months and led to the eradication of inhibitor. Conclusion Addition of intravenous immunoglobulin did not negatively affect the course of immune tolerance induction and led to the rapid eradication of factor VIII inhibitor.

  19. Construction and high expression of retroviral vector with human clotting factor IX cDNA in vitro

    Institute of Scientific and Technical Information of China (English)

    卢大儒; 邱信芳; 郑冰; 邱晓赟; 薛京伦

    1995-01-01

    The construction of the high liter and highly expressed safety retroviral vector carrying human clotting factor IX cDNA is reported. Retroviral vectors LNCTX, LIXSN and LCTXSN, driven by hCMV, LTR and hCMV combined with LTR promoter respectively, were constructed, based on the retroviral vector LNL6, and transferred into packaging cell line PA317 with electroporalion. Human dolling factor IX was delected in the cultured cells transduced with LNCIX and LIXSN but not in the cells transduced with LCIXSN. The viral titer of PA317/LNC1X was 800000 CFU per mL. With ELISA detection, it was found that the cells transduced with this vector can express human clotting factor IX at the level of 3.3μg per 106 cells in 24 h in human fibrosarcoma cells HT-1080 and 2.5μg per 106 cells in 24 h in hemophilia B patients’ skin fibroblast HSF cells, and more than 80% of them were biologically active. The viral liter and expression of human FIX were increased, and the construction of retroviral vector backbone was improved

  20. Potential for cellular stress response to hepatic factor VIII expression from AAV vector

    Directory of Open Access Journals (Sweden)

    Irene Zolotukhin

    2016-01-01

    Full Text Available Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity.

  1. Potential for cellular stress response to hepatic factor VIII expression from AAV vector

    Science.gov (United States)

    Zolotukhin, Irene; Markusic, David M; Palaschak, Brett; Hoffman, Brad E; Srikanthan, Meera A; Herzog, Roland W

    2016-01-01

    Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity. PMID:27738644

  2. Continuous infusion of porcine factor VIII: stability, microbiological safety and clinical experience.

    Science.gov (United States)

    DiMichele, D M; Gorman, P O; Kasper, C K; Mannucci, P M; Santagostino, E; Hay, C R M

    2002-01-01

    Porcine factor VIII (pFVIII) is an effective haemostatic treatment for bleeding in selected patients with FVIII inhibitors. Its use is sometimes associated with a transient fall in platelet count and transfusion reactions, the risk of which may be related to the rate of administration. Theoretical considerations suggest that the administration of pFVIII by continuous infusion should be effective, and could have pharmacokinetic advantages that lead to an improvement in the side-effect profile. The results of a retrospective survey of continuous infusion of pFVIII with respect to clinical safety and efficacy are reported. Porcine FVIII stability and microbiological studies are included. It is concluded that pFVIII given by continuous infusion is safe and effective. The risk of transfusion reactions and fall in platelet count appears to be reduced, compared with bolus administration. Stability studies showed that pFVIII activity declined at room temperature, most rapidly in the dilute solution (5-10 U mL(-1)). More concentrated mixtures showed acceptable stability for up to 24 h using a variety of infusion devices. Various concentrations of pFVIII did not support the growth of Escherichia coli or Staphylococcus aureus. These observations suggest that the porcine factor is suitable for continuous infusion (CI).

  3. A population pharmacokinetic model for perioperative dosing of factor VIII in hemophilia A patients.

    Science.gov (United States)

    Hazendonk, Hendrika; Fijnvandraat, Karin; Lock, Janske; Driessens, Mariëtte; van der Meer, Felix; Meijer, Karina; Kruip, Marieke; Gorkom, Britta Laros-van; Peters, Marjolein; de Wildt, Saskia; Leebeek, Frank; Cnossen, Marjon; Mathôt, Ron

    2016-10-01

    The role of pharmacokinetic-guided dosing of factor concentrates in hemophilia is currently a subject of debate and focuses on long-term prophylactic treatment. Few data are available on its impact in the perioperative period. In this study, a population pharmacokinetic model for currently registered factor VIII concentrates was developed for severe and moderate adult and pediatric hemophilia A patients (FVIII levels modeling was performed using non-linear mixed-effects modeling. Population pharmacokinetic parameters were estimated in 75 adults undergoing 140 surgeries (median age: 48 years; median weight: 80 kg) and 44 children undergoing 58 surgeries (median age: 4.3 years; median weight: 18.5 kg). Pharmacokinetic profiles were best described by a two-compartment model. Typical values for clearance, intercompartment clearance, central and peripheral volume were 0.15 L/h/68 kg, 0.16 L/h/68 kg, 2.81 L/68 kg and 1.90 L/68 kg. Interpatient variability in clearance and central volume was 37% and 27%. Clearance decreased with increasing age (Pmodel describes the perioperative pharmacokinetics of various FVIII concentrates, allowing individualization of perioperative FVIII therapy for severe and moderate hemophilia A patients by Bayesian adaptive dosing. Copyright© Ferrata Storti Foundation.

  4. Immunogenicity and pharmacokinetic studies of recombinant factor VIII containing lipid cochleates.

    Science.gov (United States)

    Kosloski, Matthew P; Peng, Aaron; Varma, Prashant R; Fathallah, Anas M; Miclea, Razvan D; Mager, Donald E; Balu-iyer, Sathy V

    2011-05-01

    Replacement therapy using recombinant factor VIII (rFVIII) is currently the most common therapy for hemophilia A, a bleeding disorder caused by the deficiency of FVIII. However, 15-30% of patients develop inhibitory antibodies against administered rFVIII, which complicates the therapy. Encapsulation or association of protein with lipidic structures can reduce this immune response. Previous studies developed and characterized rFVIII-containing phosphatidylserine (PS) cochleate cylinders using biophysical techniques. It was hypothesized that these structures may provide a reduction in immunogenicity while avoiding the rapid clearance by the reticuloendothelial system (RES) previously observed with liposomal vesicles of similar composition. This study investigated in vivo behavior of the cochleates containing rFVIII including immunogenicity and pharmacokinetics in hemophilia A mice. The rFVIII-cochleate complex significantly reduced the level of inhibitory antibody developed against rFVIII following intravenous (i.v.) administration. Pharmacokinetic modeling allowed assessment of in vivo release kinetics. Cochleates acted as a delayed release delivery vehicle with an input peak of cochleates showed limited RES uptake and associated rFVIII displayed a similar disposition to the free protein upon release from the structure. Incomplete disassociation from the complex limits systemic availability of the protein. Further formulation efforts are warranted to regulate the rate and extent of release of rFVIII from cochleate complexes.

  5. Evolution of recombinant factor VIII safety: KOGENATE and Kogenate FS/Bayer.

    Science.gov (United States)

    Lusher, Jeanne M; Scharrer, Inge

    2009-11-01

    The use of factor VIII (FVIII) concentrates in the treatment of hemophilia A has raised important safety issues, historically of pathogen transmission and increasingly of inhibitor development to FVIII treatment. While manufacturing processes of current recombinant FVIII products have been shaped entirely around preventing pathogen transmission, the same modifications that afford a greater margin of safety could affect immunogenicity of the product, consequences of which could only be seen through long-term clinical experience. This review summarizes pathogen safety and inhibitor reports from clinical trials, post-marketing surveillance studies, and study reports on KOGENATE and its successor, Kogenate FS/Bayer. Although KOGENATE and Kogenate FS/Bayer are nearly identical products, subtle manufacturing improvements to address the need for greater margins of safety from a pathogen transmission perspective have also led to a potentially improved immunogenicity profile (15% in previously untreated/minimally treated patients with severe hemophilia A for Kogenate FS/Bayer). Notably, there has been no occurrence of pathogen contamination, and minimal de novo inhibitor formation in previously treated patients throughout the use of both products. Overall, KOGENATE and Kogenate FS/Bayer have a long history of safety in a variety of clinical settings, including treatment of bleeding, surgical management, and prophylaxis therapy.

  6. BAY 81-8973, a full-length recombinant factor VIII: manufacturing processes and product characteristics.

    Science.gov (United States)

    Garger, S; Severs, J; Regan, L; Hesslein, A; Ignowski, J; Wu, P; Long, E; Gupta, S; Liu, S; Wang, W

    2017-03-01

    BAY 81-8973 (Kovaltry(®) , Bayer, Berkeley, CA, USA) is an unmodified, full-length recombinant human factor VIII (FVIII) approved for prophylaxis and on-demand treatment of bleeding episodes in patients with haemophilia A. The BAY 81-8973 manufacturing process is based on the process used for sucrose-formulated recombinant FVIII (rFVIII-FS), with changes and enhancements made to improve production efficiency, further augment pathogen safety, and eliminate animal- and human-derived raw materials from the production processes. The baby hamster kidney cell line used for BAY 81-8973 was developed by introducing the gene for human heat shock protein 70 into the rFVIII-FS cell line, a change that improved cell line robustness and productivity. Pathogen safety was enhanced by including a 20-nm filtration step, which can remove viruses, transmissible spongiform encephalopathy agents and potential protein aggregates. No human- or animal-derived proteins are added to the cell culture process, purification or final formulation. The BAY 81-8973 manufacturing process results in a product of enhanced purity with a consistently high degree of sialylation of N-linked glycans on the molecular surface. The innovative manufacturing techniques used for BAY 81-8973 yield an effective rFVIII product with a favourable safety profile for treatment of haemophilia A. © 2016 Bayer. Haemophilia Published by John Wiley & Sons Ltd.

  7. Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

    DEFF Research Database (Denmark)

    Kolind, Mille Petersen; Nørby, Peder Lisby; Berchtold, Martin Werner

    2011-01-01

    and forms the tenase complex together with clotting Factor IX. In vitro, during serum free production of recombinant FVIII (rFVIII), production cells also expose PS, and since vWF is not present to hinder interaction of secreted rFVIII with PS, rFVIII is partly associated with the cell membrane...... of active membrane bound rFVIII to the culture medium. Moreover, the attachment of rFVIII to cell membranes of un-transfected HEK293 cells was studied in the presence of compounds that competes for interactions between rFVIII and PS. Competitive assays between iodinated rFVIII (¹²5I-rFVIII) and annexin V...... or ortho-phospho-L-serine (OPLS) demonstrated that annexin V and OPLS were able to reduce the membrane bound fraction of rFVIII by 70% and 30%, respectively. Finally, adding OPLS to CHO cells stably expressing FVIII increased the yield by 50%. Using this new knowledge, the recovery of rFVIII could...

  8. In vitro differentiation of mouse ES cells into hepatocytes with coagulation factors VIII and IX expression profiles

    Institute of Scientific and Technical Information of China (English)

    MENG; Ying; HUANG; Shaoliang; MIN; Jun; GUO; Zhongmin

    2006-01-01

    Coagulation factors II, V, VII, VIII, IX and X are produced by hepatocytes. So factors VIII and IX deficiencies, which result in hemophilia A and B, have the potential to respond to cellular re- placement therapy. Embryonic stem (ES) cells provide a unique source for therapeutic applications. Here, E14 mouse ES cells have been induced into hepatocytes in vitro. Morphology revealed that ES-derived hepatic-like cells were round or polyhedral shaped with distinct boundary of individual cells, and some arranged in trabeculae. These cells expressed endodermal- or liver-specific mRNA --transthyretin (TTR), α1-anti-trypsin (AAT), α-fetoprotein (AFP), albumin (ALB), glucose-6- phoshpatase (G6P) and tyrosine aminotransferase (TAT). Approximately (85.1(0.5)% of the ES-de- rived cells was stained positive green with ICG uptake. These cells were also stained magenta as a result of PAS reaction. In this paper, expression of coagulation factors VIII and IX mRNA in the ES-derived cells is documented. Therefore, ES cells might be developed as substitute donor cells for the therapy of coagulation factor deficiencies.

  9. [Detection of alloantibodies against Factor VIII in plasma of patients with hemophilia A and its relationship with Factor VIIIC domain].

    Science.gov (United States)

    Zhang, Lu-Lu; Yu, Zi-Qiang; Wan, Chu-Cheng; Zhang, Wei; Zhang, Zheng-Hua; Ruan, Chang-Geng

    2013-10-01

    This study was purposed to detect the alloantibodies against Factor VIII (FVIII) by ELISA for estimating the incidence of the alloantibodies against Factor VIII (FVIII) in patients with hemophilia A, and to investigate the relationship between factor VIIIC domain and alloantibodies. Total of 140 patients with hemophilia A and 80 normal controls were enrolled in this study, among them plasma FVIII level of 84 patients was less than 1%, plasma FVIII level of 34 patients was between 1% and 5%, and plasma FVIII level of 22 patients was more than 5%. All patients were treated with plasma-derived FVIII concentrate or plasma before. The ELISA plate was coated with McAb (SZ-132) against FVIII prepared in our laboratory, then human recombinant FVIII concentrates were applied. After incubation in room temperature for 2 hours, diluted plasma samples and HRP-conjugated goat anti-human IgG were added successively, finally A490 was recorded. The threshold of alloantibody of patient plasma was set as mean value>3 SD more than control. The plate was coated with antibody against His, then human recombinant FVIII-C1C2 prepared in our laboratory was added. After incubation in room temperature for 2 hours, diluted plasma samples and HRP-conjugated goat anti-human IgG were added successively, finally A490 were recorded. The threshold was set as the mean value>3 SD more than control. The results showed that the alloantibodies against FVIII were found in 56 patients (40%) by ELISA, and 82.1% (46/56) of this kind of alloantibody could interact with the C domain of FVIII. It is concluded that C domain of FVIII is one of the primary binding sites for the alloantibodies against FVIII in Chinese patients with hemophilia A.

  10. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... improve the diagnosis and treatment of acute myeloid leukemia (AML). 2018 Highlights of ASH Attend a meeting ... Blood Basics Blood Disorders Anemia Bleeding Disorders Blood Cancers Blood Clots Blood Clotting and Pregnancy Clots and ...

  11. Partial correction of a severe molecular defect in hemophilia A, because of errors during expression of the factor VIII gene

    Energy Technology Data Exchange (ETDEWEB)

    Young, M.; Antonarakis, S.E. [Univ. of Geneva (Switzerland); Inaba, Hiroshi [Tokyo Medical College (Japan)] [and others

    1997-03-01

    Although the molecular defect in patients in a Japanese family with mild to moderately severe hemophilia A was a deletion of a single nucleotide T within an A{sub 8}TA{sub 2} sequence of exon 14 of the factor VIII gene, the severity of the clinical phenotype did not correspond to that expected of a frameshift mutation. A small amount of functional factor VIII protein was detected in the patient`s plasma. Analysis of DNA and RNA molecules from normal and affected individuals and in vitro transcription/translation suggested a partial correction of the molecular defect, because of the following: (i) DNA replication/RNA transcription errors resulting in restoration of the reading frame and/or (ii) {open_quotes}ribosomal frameshifting{close_quotes} resulting in the production of normal factor VIII polypeptide and, thus, in a milder than expected hemophilia A. All of these mechanisms probably were promoted by the longer run of adenines, A{sub 10} instead of A{sub 8}TA{sub 2}, after the delT. Errors in the complex steps of gene expression therefore may partially correct a severe frameshift defect and ameliorate an expected severe phenotype. 36 refs., 6 figs.

  12. Investigation of the effect of kaolin and tissue-factor-activated citrated whole blood, on clot-forming variables, as evaluated by thromboelastography.

    Science.gov (United States)

    Johansson, Pär I; Bochsen, Louise; Andersen, Søren; Viuff, Dorthe

    2008-11-01

    The Thrombelastograph (TEG; Haemoscope Corp.) analyzes clot formation in whole blood (WB) and treatment based on this analysis has been shown to reduce transfusion requirements in liver and cardiac surgery when compared to conventional coagulation analysis. Implementing TEG as a routine laboratory-based analysis, however, requires validation of the activators employed and the effect of storage of the WB sample in citrate before analysis. The effect of kaolin, tissue factor (TF) 1:17,000, or TF 1:42,500 on TEG clotting time (R), Angle (velocity of clot formation), and maximum clot strength (amplitude [MA]) were evaluated, together with day-to-day variation, the coefficient of variance (CV%), and the effect of citrate storage time. Clot formation variables were equally affected by TF 1:17,000 and kaolin activation, whereas R was significantly longer when TF 1:42,500 was used. The CV for the different variables varied from 3 to 13 percent with no significant differences between assays. Storage of citrated WB significantly affected the TEG variables in a hypercoagulable direction. Only the R, however, was significantly affected (12%) when samples rested for 0 and 30 minutes were evaluated with kaolin as the activator. The TEG assays evaluated were reproducible and present with an acceptable CV% for routine clinical practice. Kaolin and TF 1:17,000 equally affected the clot formation variables. Storage of WB for up to 30 minutes in citrate did not, except for R, affect clot formation variables when kaolin was used as activator allowing for immediate analysis when the sample arrives in the laboratory.

  13. Factor VIII gene variants and inhibitor risk in African American hemophilia A patients.

    Science.gov (United States)

    Gunasekera, Devi; Ettinger, Ruth A; Nakaya Fletcher, Shelley; James, Eddie A; Liu, Maochang; Barrett, John C; Withycombe, Janice; Matthews, Dana C; Epstein, Melinda S; Hughes, Richard J; Pratt, Kathleen P

    2015-08-13

    African American hemophilia A (HA) patients experience a higher incidence of neutralizing anti-factor VIII (FVIII) antibodies ("inhibitors") vis-à-vis white patients. Nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in the F8 gene encoding FVIII-H484, FVIII-E1241, and FVIII-V2238 are more prevalent in African Americans. This study tested the hypothesis that immune responses to these sites provoke inhibitors. Blood samples were obtained from 174 African American and 198 white HA subjects and their F8 gene sequences determined. Major histocompatibility complex class II binding and T-cell recognition of polymorphic sequences were evaluated using quantitative binding assays and HLA-DRB1 tetramers. Peptides corresponding to 4 common ns-SNPs showed limited binding to 11 HLA-DRB1 proteins. CD4 T cells from 22 subjects treated with FVIII products having sequences at residues FVIII-484, 1241, and 2238 differing from those of putative proteins encoded by their F8 genes did not show high-avidity tetramer binding, whereas positive-control staining of tetanus-specific CD4 T cells was routinely successful. African Americans with an intron-22 inversion mutation showed a 2-3 times-higher inhibitor incidence than whites with the same mutation (odds ratio = 2.3 [1.1-5.0, P = .04]), but this did not correlate with any of the ns-SNPs. We conclude that immune responses to "sequence-mismatched" FVIII products are unlikely to contribute appreciably to the inhibitor incidence in African Americans.

  14. Application of a novel affinity adsorbent for the capture and purification of recombinant factor VIII compounds.

    Science.gov (United States)

    McCue, Justin T; Selvitelli, Keith; Walker, Joshua

    2009-11-06

    Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.

  15. Enhanced factor VIII heavy chain for gene therapy of hemophilia A.

    Science.gov (United States)

    Chen, Lingxia; Lu, Hui; Wang, Jinhui; Sarkar, Rita; Yang, Xiao; Wang, Hongli; High, Katherine A; Xiao, Weidong

    2009-03-01

    Hemophilia A gene therapy using recombinant adenovirus-associated virus (AAV) vectors has been hampered by the size of the factor VIII (FVIII) cDNA. Previously, splitting the FVIII coding sequence into a heavy-chain (HC) fragment and a light-chain (LC) fragment for dual recombinant AAV vector delivery has been successfully explored. However, the main disadvantage of this approach is a "chain imbalance" problem in which LC secretion is approximately 1-2 logs higher than that of HC, and therefore, the majority of protein synthesized is nonfunctional. To improve HC secretion, we constructed alternate FVIII HCs based on our observation that LC facilitates HC secretion. To our surprise, most of the new HC molecules exhibited enhanced expression over the traditional HC molecule (HC(745)). The optimized HC mutein, HC(HL), including additional acidic-region-3 (ar3) sequences, exhibited three- to fivefold higher activity in both enzyme-linked immunosorbent assay (ELISA) and activated partial thromboplastin time (aPTT) assay in in vitro testing. Further characterization suggested ar3 sequences increased HC secretion, rather than promoting HC synthesis. Intravenous delivery of AAV8-HC(HL)+AAV8-LC or AAV8-HC(745)+AAV8-LC achieved phenotypic correction in hemophilia A mice. Mice receiving AAV8-HC(HL)+AAV8-LC achieved three- to fourfold higher HC expression than AAV8-HC(745)+AAV8-LC, consistent with the FVIII functional assays. HC(HL) should be substituted for HC(745) in a dual AAV vector strategy due to its enhanced expression.

  16. The factor VIII Structure and Mutation Resource Site: HAMSTeRS version 4.

    Science.gov (United States)

    Kemball-Cook, G; Tuddenham, E G; Wacey, A I

    1998-01-01

    Since 1996 the HAMSTeRS (Haemophilia A Mutation, Search, Test and Resource Site) WWW site has provided an online resource for access to data on the molecular pathology of haemophilia A, replacing previous text editions of the Haemophilia A Database published in Nucleic Acids Research . This report describes the continued development of the site (version 4), and in particular the expansion of factor VIII (FVIII) structure-related features. Access to the mutation database itself, both for searching the listings and for submission of new mutations, is via custom-designed forms: more powerful Boolean searches of the point mutations in the database are also available. During 1997 a total of 22 novel missense mutations were reported, increasing the total number of unique variants now described to 252 (238 in exonic sequences and 14 at intronic splice junctions). Currently, a total of 586 individual reports with associated phenotypic data are available for searching by any category including phenotype. The FVIII structure section now includes a download of a FVIII A domain homology model in Protein Data Bank format and a multiple alignment of the FVIII amino-acid sequencies from four species (human, murine, porcine and canine) in addition to the virtual reality simulations, secondary structural data and FVIII animation already available. Finally, to aid navigation across this site, a clickable roadmap of the main features provides easy access to the page desired. Our intention is that continued development and updating of the site shall provide workers in the fields of molecular and structural biology with a one-stop resource site to facilitate FVIII research and education. The HAMSTeRS URL is http://europium.mrc.rpms.ac.uk

  17. Frequencies of VNTR and RFLP polymorphisms associated with factor VIII gene in Singapore

    Energy Technology Data Exchange (ETDEWEB)

    Fong, I.; Lai, P.S.; Ouah, T.C. [National Univ. of Singapore (Malaysia)] [and others

    1994-09-01

    The allelic frequency of any polymorphism within a population determines its usefulness for genetic counselling. This is important in populations of non-Caucasian origin as RFLPs may significantly differ among ethnic groups. We report a study of five intragenic polymorphisms in factor VIII gene carried out in Singapore. The three PCR-based RFLP markers studied were Intron 18/Bcl I, Intron 19/Hind III and Intron 22/Xba I. In an analysis of 148 unrelated normal X chromosomes, the allele frequencies were found to be A1 = 0.18, A2 = 0.82 (Bcl I RFLP), A1 = 0.80, A2 = 0.20 (Hind III RFLP) and A1 = 0.58, and A2 = 0.42 (Xba I RFLP). The heterozygosity rates of 74 females analyzed separately were 31%, 32% and 84.2%, respectively. Linkage disequilibrium was also observed to some degree between Bcl I and Hind III polymorphism in our population. We have also analyzed a sequence polymorphism in Intron 7 using hybridization with radioactive-labelled {sup 32}P allele-specific oligonucleotide probes. This polymorphism was not very polymorphic in our population with only 2% of 117 individuals analyzed being informative. However, the use of a hypervariable dinucleotide repeat sequence (VNTR) in Intron 13 showed that 25 of our of 27 (93%) females were heterozygous. Allele frequencies ranged from 1 to 55 %. We conclude that a viable strategy for molecular analysis of Hemophilia A families in our population should include the use of Intron 18/Bcl I and Intron 22/Xba I RFLP markers and the Intron 13 VNTR marker.

  18. Pharmacokinetic properties of BAY 81-8973, a full-length recombinant factor VIII.

    Science.gov (United States)

    Shah, A; Delesen, H; Garger, S; Lalezari, S

    2015-11-01

    BAY 81-8973 is a full-length recombinant factor VIII (FVIII) with the same primary amino acid sequence as sucrose-formulated recombinant FVIII (rFVIII-FS) but is produced with advanced manufacturing technologies. To analyse the pharmacokinetics (PK) of BAY 81-8973 after single and multiple dosing across different age and ethnic groups in the LEOPOLD clinical trial programme. The LEOPOLD trials enrolled patients with severe haemophilia A aged 12-65 years (LEOPOLD I and II) or ≤12 years (LEOPOLD Kids) with ≥150 (LEOPOLD I and II) or ≥50 (LEOPOLD Kids) exposure days to any FVIII product and no history of FVIII inhibitors. PK were assessed using chromogenic and one-stage assays (only chromogenic assay for LEOPOLD Kids) after a single 50-IU kg(-1) dose of BAY 81-8973 and, in a subset of patients in LEOPOLD I, after repeated dosing. Pharmacokinetic analyses were also performed based on age (18 to 65, 12 to <18, 6 to <12 and <6 years) and ethnicity (Asian and non-Asian). Pharmacokinetic assessments in the LEOPOLD I trial showed non-inferiority of BAY 81-8973 vs. rFVIII-FS. The PK of BAY 81-8973 were comparable after single and multiple dosing. Age-based analysis in the three trials showed that plasma concentrations were slightly lower for children, but similar for adolescents compared with adults. Pharmacokinetic results were similar in the different ethnic groups. Results of the LEOPOLD trials show that the BAY 81-8973 pharmacokinetic profile is non-inferior to rFVIII-FS. Similar BAY 81-8973 pharmacokinetic values were observed following single and repeated dosing and across ethnic groups. © 2015 John Wiley & Sons Ltd.

  19. Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery

    Directory of Open Access Journals (Sweden)

    Harrison C Brown

    2014-01-01

    Full Text Available Clinical data support the feasibility and safety of adeno-associated viral (AAV vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i the size of the factor VIII (fVIII transgene, (ii humoral immune responses to fVIII, (iii inefficient biosynthesis of human fVIII, and (iv AAV vector immunity. Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold. In this study, the utility of ET3 was assessed in the context of liver-directed, AAV-mediated gene transfer into hemophilia A mice. Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments. Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors. Therefore, ET3 appears to improve vector potency and mitigate at least one of the critical barriers to AAV-based clinical gene therapy for hemophilia A.

  20. Von Willebrand factor and coagulation factor VIII in Moyamoya disease associated with Graves' disease: A case report

    Science.gov (United States)

    Ren, Shou-Chen; Gao, Bao-Qin; Yang, Wei-Li; Feng, Wei-Xin; Xu, Jian; Li, Shao-Wu; Wang, Yong-Jun

    2016-01-01

    The present study reported the case of a Chinese boy who was diagnosed with Moyamoya disease (MMD) associated with Graves' disease (GD). An overactivation of von Willebrand factor (vWF) and coagulation factor VIII (FVIII) was identified in the plasma of the patient. Thiamazole and metoprolol treatment was thus administrated. After 2 months of treatment, the patient's thyroid function returned to normal and the neurological symptoms improved gradually. At the same time, the activities of vWF and FVIII were depressed. During the 20-month follow-up, information regarding the neurological symptoms, cerebrovascular imaging, thyroid function, thyroid autoantibodies and coagulation parameters was collected. High levels of thyroid autoantibodies persisted throughout the follow-up period, while other coagulation parameters remained in the normal range. In conclusion, considering the vital role of vWF and FVIII in vascular diseases, it is hypothesized that these two factors may serve an important role in the occurrence of GD associated with MMD. PMID:27882137

  1. Severe Hemophilia A in a Male Old English Sheep Dog with a C→T Transition that Created a Premature Stop Codon in Factor VIII

    Science.gov (United States)

    Lozier, Jay N; Kloos, Mark T; Merricks, Elizabeth P; Lemoine, Nathaly; Whitford, Margaret H; Raymer, Robin A; Bellinger, Dwight A; Nichols, Timothy C

    2016-01-01

    Animals with hemophilia are models for gene therapy, factor replacement, and inhibitor development in humans. We have actively sought dogs with severe hemophilia A that have novel factor VIII mutations unlike the previously described factor VIII intron 22 inversion. A male Old English Sheepdog with recurrent soft-tissue hemorrhage and hemarthrosis was diagnosed with severe hemophilia A (factor VIII activity less than 1% of normal). We purified genomic DNA from this dog and ruled out the common intron 22 inversion; we then sequenced all 26 exons. Comparing the results with the normal canine factor VIII sequence revealed a C→T transition in exon 12 of the factor VIII gene that created a premature stop codon at amino acid 577 in the A2 domain of the protein. In addition, 2 previously described polymorphisms that do not cause hemophilia were present at amino acids 909 and 1184. The hemophilia mutation creates a new TaqI site that facilitates rapid genotyping of affected offspring by PCR and restriction endonuclease analyses. This mutation is analogous to the previously described human factor VIII mutation at Arg583, which likewise is a CpG dinucleotide transition causing a premature stop codon in exon 12. Thus far, despite extensive treatment with factor VIII, this dog has not developed neutralizing antibodies (‘inhibitors’) to the protein. This novel mutation in a dog gives rise to severe hemophilia A analogous to a mutation seen in humans. This model will be useful for studies of the treatment of hemophilia. PMID:27780008

  2. Therapeutic and routine prophylactic properties of rFactor VIII Fc (efraloctocog alfa, Eloctate®) in hemophilia A

    Science.gov (United States)

    Chowdary, Pratima; Fosbury, Emma; Riddell, Anne; Mathias, Mary

    2016-01-01

    rFVIIIFc (efraloctocog alfa, Eloctate®) is an extended half-life (EHL) factor VIII licensed for use in patients with hemophilia A for prophylaxis and treatment of bleeding and surgical episodes. Pharmacokinetic studies in adults have shown a mean 1.5-fold increase in half-life compared to full-length factor VIII. When compared to adults, the half-life is decreased by 8% in adolescents between 12 and 17 years, by 18% in children 6 to <12 years, and by 33% in children between the ages of 2 and <6 years. There is a considerable interindividual variation in the prolongation of the half-life particularly in children and across the age groups, the range extending from no increase to a 2.5-fold increase. In addition to age, von willebrand factor (VWF) antigen level has demonstrated a significant impact on rFVIIIFc half-life, with higher VWF levels associated with greater prolongation of half-life. The pivotal and pediatric clinical trials have demonstrated the efficacy and safety of rFVIIIFc for use in regular prophylaxis and in management of bleeds and surgery. In these studies, just under half the participants showed a zero annualized bleed rate (ABR), and the median ABR (1.6 in the pivotal study for the individualized prophylaxis arm) showed a further decrease in the extension study. On average, the patients required fewer infusions (reduced by at least a third), and the mean weekly consumption seems to be in keeping with standard recombinant factor VIII. EHL rFVIIIFc has made decreased infusion frequency a possibility. However, the interindividual variability in dose and infusion frequency highlights the need for a personalized approach based on individual patient’s half-life and/or response to treatment. PMID:27695377

  3. Factor VIII alters tubular organization and functional properties of von Willebrand factor stored in Weibel-Palade bodies.

    Science.gov (United States)

    Bouwens, Eveline A M; Mourik, Marjon J; van den Biggelaar, Maartje; Eikenboom, Jeroen C J; Voorberg, Jan; Valentijn, Karine M; Mertens, Koen

    2011-11-24

    In endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative light-electron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultra-large VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIII-covered VWF strings, and impairs recruitment of platelets. Apparently, intra-cellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings.

  4. Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2

    Science.gov (United States)

    Zhang, Bin; McGee, Beth; Yamaoka, Jennifer S.; Guglielmone, Hugo; Downes, Katharine A.; Minoldo, Salvador; Jarchum, Gustavo; Peyvandi, Flora; de Bosch, Norma B.; Ruiz-Saez, Arlette; Chatelain, Bernard; Olpinski, Marian; Bockenstedt, Paula; Sperl, Wolfgang; Kaufman, Randal J.; Nichols, William C.; Tuddenham, Edward G. D.; Ginsburg, David

    2006-01-01

    Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and FVIII. PMID:16304051

  5. Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa

    Directory of Open Access Journals (Sweden)

    Sheffield William P

    2011-12-01

    Full Text Available Abstract Background The transglutaminase activated factor XIII (FXIIIa acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α2-antiplasmin (α2AP to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α2AP residues 13-42 linked to human serum albumin (HSA weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α2AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa. Results Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H6NQEQVSPLTLLAG4Y (designated XL1; H6DQMMLPWAVTLG4Y (XL2; H6WQHKIDLPYNGAG4Y (XL3; and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6. The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α2AP(13-42-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA most rapidly by FXIIIa, and was the most effective competitor of α2AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA. Conclusions Fusion protein XL5-HSA (DQMMLPWAVTLG4Y-HSAH6 was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in

  6. Human von Willebrand factor/factor VIII concentrates in the management of pediatric patients with von Willebrand disease/hemophilia A

    OpenAIRE

    Castaman G; Linari S

    2016-01-01

    Giancarlo Castaman, Silvia Linari Department of Oncology, Center for Bleeding Disorders, Careggi University Hospital, Florence, ItalyAbstract: Several plasma-derived intermediate and high-purity concentrates containing von Willebrand factor (VWF) and factor VIII (FVIII) are currently available. The main role of these products in the management of pediatric the population is represented by the replacement therapy in patients with severe or intermediate forms of von Willebrand disease, in whom ...

  7. Expression, purification, and partial in vitro characterization of biologically active human coagulation factor VIII light chain (A3-C1-C2) in Pichia pastoris.

    Science.gov (United States)

    A R, Sudheer Reddy; Satheeshkumar, Padikara Kutty; Vijayalakshmi, Mookambeswaran A

    2013-09-01

    Recombinant coagulation factor VIII (FVIII) expressed in mammalian expression systems is used extensively in the treatment of hemophilia A. It is reported that the heavy (A1-A2) and light chains (A3-C1-C2) of factor VIII purified from plasma regained the coagulation activity by dimerization in vitro. In this work, cDNA coding for the light chain of human coagulation factor VIII (FVIII-LC) was cloned into pPICZα-A expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae in order to express the protein with a native N-terminus. The methylotrophic yeast, Pichia pastoris X-33, was transformed with this cassette, and transformants were selected for production of human factor VIII light chain into culture media. SDS-PAGE and Western blot analysis confirmed the expression of factor VIII light chain protein. The expressed protein was purified to near homogeneity using histidine ligand affinity chromatography (2.342 mg/L). The biological activity of FVIII-LC was confirmed by analyzing the interaction between FVIII-LC and phospholipid vesicles. The data presented here indicate the possibilities of exploring cost-effective systems to express complex proteins of therapeutic value.

  8. Factors affecting the lung perfused blood volume in patients with intrapulmonary clots after anti-coagulation therapy

    Energy Technology Data Exchange (ETDEWEB)

    Okada, Munemasa, E-mail: radokada@yamaguchi-u.ac.jp [Department of Radiology, Yamaguchi University Graduate School of Medicine 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505 (Japan); Masuda, Yu [4th Grade of 6-year Medicine Doctor Program, Department of Medicine, Yamaguchi University Faculty of Medicine and Health Sciences 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505 (Japan); Nakashima, Yoshiteru [Department of Radiology, Yamaguchi Grand Medical Center, Oosaki 77, Hofu, Yamaguchi 747-8511 (Japan); Nomura, Takafumi; Nakao, Sei [Department of Radiology, Yamaguchi University Graduate School of Medicine 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505 (Japan); Suga, Kazuyoshi [Department of Radiology, St Hills Hospital, Imamurakita 3-7-18, Ube, Yamaguchi 755-0155 (Japan); Kido, Shoji [Computer-aided Diagnosis and Biomedical Imaging Research Biomedical Engineering, Applied Medical Engineering Science Graduate School of Medicine, Yamaguchi University, Tokiwadai 2-16-1, Ube, Yamaguchi 755-8611 (Japan); Matsunaga, Naofumi [Department of Radiology, Yamaguchi University Graduate School of Medicine 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505 (Japan)

    2015-08-15

    Highlights: • Dual-energy CT can provide morphological and functional lung images in the same examination. • The subsequent dual-energy CT demonstrates the increased whole lung perfused blood volume (V{sub 120}) despite the residual intrapulmonary clots after treatment in one examination. • The increased whole lung perfusion (V{sub 120}) and a decreased low perfusion volume (V{sub 5}) result in the improvement in the low perfusion rate (%V{sub 5}) in the patients with acute pulmonary embolism after treatment. - Abstract: Objectives: Factors affecting the improvement in the lung perfused blood volume (LPBV) were evaluated based on the presence of intrapulmonary clots (IPCs) after anti-coagulation therapy using 64-slice dual-energy CT. Materials and methods: 96 patients exhibiting venous thromboembolism underwent initial and repeated LPBV examinations between December 2008 and July 2014. Fifteen patients were excluded due to pulmonary comorbidities, and a total of 81 patients were included in this study. Acute pulmonary embolism (PE) was diagnosed in 46 of the patients (56.7%). LPBV images were three-dimensionally reconstructed with two threshold ranges: 1–120 HU (V{sub 120}) and 1–5 HU (V{sub 5}), and the relative value of V{sub 5} per V{sub 120} expressed as %V{sub 5}. These values were subsequently compared with indicators of the severity of PE, such as the D-dimer level, heart rate and CT measurements. This study was approved by the local ethics committee. Results: In patients with IPCs, the D-dimer, V{sub 5} and %V{sub 5}values were significantly larger (p ≤ 0.01) in the initial LPBV, although these differences disappeared in subsequent LPBV after treatment. The right ventricular (RV) diameter, RV/left ventricular (RV/LV) diameter ratio and %V{sub 5} values were also significantly reduced, whereas the V{sub 5} value did not significantly decrease (p = 0.07), but V{sub 120} value significantly increased (p < 0.001) after treatment. However, in

  9. Blood Clotting and Pregnancy

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    Full Text Available ... increased by the following: Previous blood clots A genetic predisposition to blood clots Obesity Prolonged immobility (e. ... you are interested in learning more about blood diseases and disorders, here are a few other resources ...

  10. Blood Clotting and Pregnancy

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    Full Text Available ... blood clots A genetic predisposition to blood clots Obesity Prolonged immobility (e.g., bedrest, long distance travel) ... If you find that you are interested in learning more about blood diseases and disorders, here are ...

  11. Recombinant factor VIII Fc (rFVIIIFc) fusion protein reduces immunogenicity and induces tolerance in hemophilia A mice.

    Science.gov (United States)

    Krishnamoorthy, Sriram; Liu, Tongyao; Drager, Douglas; Patarroyo-White, Susannah; Chhabra, Ekta Seth; Peters, Robert; Josephson, Neil; Lillicrap, David; Blumberg, Richard S; Pierce, Glenn F; Jiang, Haiyan

    2016-03-01

    Anti-factor VIII (FVIII) antibodies is a major complication of FVIII replacement therapy for hemophilia A. We investigated the immune response to recombinant human factor VIII Fc (rFVIIIFc) in comparison to BDD-rFVIII and full-length rFVIII (FL-rFVIII) in hemophilia A mice. Repeated administration of therapeutically relevant doses of rFVIIIFc in these mice resulted in significantly lower antibody responses to rFVIII compared to BDD-rFVIII and FL-rFVIII and reduced antibody production upon subsequent challenge with high doses of rFVIIIFc. The induction of a tolerogenic response by rFVIIIFc was associated with higher percentage of regulatory T-cells, a lower percentage of pro-inflammatory splenic T-cells, and up-regulation of tolerogenic cytokines and markers. Disruption of Fc interactions with either FcRn or Fcγ receptors diminished tolerance induction, suggesting the involvement of these pathways. These results indicate that rFVIIIFc reduces immunogenicity and imparts tolerance to rFVIII demonstrating that recombinant therapeutic proteins may be modified to influence immunogenicity and facilitate tolerance.

  12. Storage of factor VIII variants with impaired von Willebrand factor binding in Weibel-Palade bodies in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Maartje van den Biggelaar

    Full Text Available BACKGROUND: Point mutations resulting in reduced factor VIII (FVIII binding to von Willebrand factor (VWF are an important cause of mild/moderate hemophilia A. Treatment includes desmopressin infusion, which concomitantly increases VWF and FVIII plasma levels, apparently from storage pools containing both proteins. The source of these VWF/FVIII co-storage pools and the mechanism of granule biogenesis are not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: We studied intracellular trafficking of FVIII variants implicated in mild/moderate hemophilia A together with VWF in HEK293 cells and primary endothelial cells. The role of VWF binding was addressed using FVIII variants displaying reduced VWF interaction. Binding studies using purified FVIII proteins revealed moderate (Arg2150His, Del2201, Pro2300Ser to severe (Tyr1680Phe, Ser2119Tyr VWF binding defects. Expression studies in HEK293 cells and primary endothelial cells revealed that all FVIII variants were present within VWF-containing organelles. Quantitative studies showed that the relative amount of FVIII storage was independent of various mutations. Substantial amounts of FVIII variants are co-stored in VWF-containing storage organelles, presumably by virtue of their ability to interact with VWF at low pH. CONCLUSIONS: Our data suggest that the potential of FVIII co-storage with VWF is not affected in mild/moderate hemophilia A caused by reduced FVIII/VWF interaction in the circulation. These data support the hypothesis that Weibel-Palade bodies comprise the desmopressin-releasable FVIII storage pool in vivo.

  13. Expression of human clotting factor Ⅸ mediated by recombinant lentiviral vector in cultured cells and hemophilia B mice

    Institute of Scientific and Technical Information of China (English)

    ZHU Huanzhang; CHEN Xiaoguang; LI Feng; GONG Juli; XUE Jinglun

    2003-01-01

    To explore the expression of human clotting factor Ⅸ (hFⅨ) cDNA in vitro and the feasibility of gene therapy for hemophilia B mice mediated by recombinant lentiviral vector, a recombinant hFⅨ lentiviral vector driven by ubiquitin-C promoter, FUXW, and by ABP liver specific promoter, FAXW, was constructed respectively. Recombinant lentivirus was harvested from 293T cells by calcium phosphate-mediated transient cotransfection of three plasmids (transgene vector, CMVΔR8.2, VSV-G). hFⅨ expression was detected in supernatant of 293T, BHK and L-02 cells infected with FUXW virus, whereas higher expression of hFⅨ levels (630 ng/106 cells/48 h) was detected only in L-02 cells infected with FAXW virus. Serum hFⅨ antigen was detected in all hemophilia B mice treated with FAXW virus by tail vein injection, an efficiency level of hFⅨ was observed (45 ng/mL, approximately 1% of normal human levels), the expression lasted for more than 60 d. The results indicated that HIV-based lentiviral vectors offer a promising approach to the gene therapy of hemophilia B.

  14. Patient preference for needleless factor VIII reconstitution device: the Italian experience

    Directory of Open Access Journals (Sweden)

    Roberto Musso

    2010-07-01

    Full Text Available Roberto Musso1, Rita Santoro2, Antonio Coppola3, Maura Marcucci4, Gianluca Sottilotta5, Roberto Targhetta6, Ezio Zanon7, Massimo Franchini81Centro Emofilia, Istituto di Ematologia Università degli Studi di Catania, Ospedale Ferrarotto, Catania, Italy; 2Haemophilia Centre, Hemostasis and Thrombosis Service, Department of Haematology and Oncology, Azienda Ospedaliera “Pugliese-Ciaccio”, Catanzaro, Italy; 3Regional Reference Centre for Coagulation Disorders, Federico II University, Naples, Italy; 4Internal and Vascular Medicine, University of Perugia, Perugia, Italy; 5Hemophilia Centre, Hemostasis and Thrombosis Service, Azienda Ospedaliera “Bianchi-Melacrino-Morelli”, Reggio Calabria, Italy; 6Centro Emofilia Struttura Semplice Patologie della Coagulazione, Ospedale Microcitemico, Cagliari, Italy; 7Centro Emofilia, Clinica Medica II, Azienda Ospedaliera di Padova, Padova, Italy; 8Immunohaematology and Transfusion Centre, Department of Pathology and Laboratory Medicine, University Hospital of Parma, Parma, ItalyBackground: Needlestick injuries, mostly due to unsafe needle devices, are a frequent adverse event among health care workers and patients on chronic treatment, such as hemophiliacs. To improve the safety of these procedures, a needleless reconstitution system, Bio-Set® has been implemented for the sucrose-formulated recombinant factor VIII (rFVIII-FS Kogenate® Bayer (Bayer Healthcare, Berlin, Germany. The aim of this study was to collect patients’ satisfaction and safety data regarding the administration of rFVIII-FS with this new device.Methods: This was a multicenter, prospective, postmarketing surveillance study collecting data from seven Italian Haemophilia Centers within the framework of an international project involving patients from nine European countries. The patients were asked to fill out two preference questionnaires (one assessing the old method and one assessing the new method directly after the training and two

  15. Blood Clotting and Pregnancy

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    Full Text Available ... Blood: How I Treat A compendium of Blood articles updated to reflect the most recent scientific research View all publications For Patients Blood Basics Blood Disorders Anemia Bleeding Disorders Blood Cancers Blood Clots Blood Clotting and Pregnancy Clots and ...

  16. Blood Clotting and Pregnancy

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    Full Text Available ... blood clots A genetic predisposition to blood clots Obesity Prolonged immobility (e.g., bedrest, long distance travel) Multiple births Increased maternal age Other medical illness (e.g., cancer, infection) back to top How are Blood Clots ...

  17. Production of factor VIII by human liver sinusoidal endothelial cells transplanted in immunodeficient uPA mice.

    Directory of Open Access Journals (Sweden)

    Marina E Fomin

    Full Text Available Liver sinusoidal endothelial cells (LSECs form a semi-permeable barrier between parenchymal hepatocytes and the blood. LSECs participate in liver metabolism, clearance of pathological agents, immunological responses, architectural maintenance of the liver and synthesis of growth factors and cytokines. LSECs also play an important role in coagulation through the synthesis of Factor VIII (FVIII. Herein, we phenotypically define human LSECs isolated from fetal liver using flow cytometry and immunofluorescence microscopy. Isolated LSECs were cultured and shown to express endothelial markers and markers specific for the LSEC lineage. LSECs were also shown to engraft the liver when human fetal liver cells were transplanted into immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA transgene (uPA-NOG mice. Engrafted cells expressed human Factor VIII at levels approaching those found in human plasma. We also demonstrate engraftment of adult LSECs, as well as hepatocytes, transplanted into uPA-NOG mice. We propose that overexpression of uPA provides beneficial conditions for LSEC engraftment due to elevated expression of the angiogenic cytokine, vascular endothelial growth factor. This work provides a detailed characterization of human midgestation LSECs, thereby providing the means for their purification and culture based on their expression of CD14 and CD32 as well as a lack of CD45 expression. The uPA-NOG mouse is shown to be a permissive host for human LSECs and adult hepatocytes, but not fetal hepatoblasts. Thus, these mice provide a useful model system to study these cell types in vivo. Demonstration of human FVIII production by transplanted LSECs encourages further pursuit of LSEC transplantation as a cellular therapy for the treatment of hemophilia A.

  18. Zinc promotes clot stability by accelerating clot formation and modifying fibrin structure.

    Science.gov (United States)

    Henderson, Sara J; Xia, Jing; Wu, Huayin; Stafford, Alan R; Leslie, Beverly A; Fredenburgh, James C; Weitz, David A; Weitz, Jeffrey I

    2016-03-01

    Zinc released from activated platelets binds fibrin(ogen) and attenuates fibrinolysis. Although zinc also affects clot formation, the mechanism and consequences are poorly understood. To address these gaps, the effect of zinc on clot formation and structure was examined in the absence or presence of factor (F) XIII. Zinc accelerated a) plasma clotting by 1.4-fold, b) fibrinogen clotting by 3.5- and 2.3-fold in the absence or presence of FXIII, respectively, c) fragment X clotting by 1.3-fold, and d) polymerisation of fibrin monomers generated with thrombin or batroxobin by 2.5- and 1.8-fold, respectively. Whereas absorbance increased up to 3.3-fold when fibrinogen was clotted in the presence of zinc, absorbance of fragment X clots was unaffected by zinc, consistent with reports that zinc binds to the αC-domain of fibrin(ogen). Scanning electron microscopic analysis revealed a two-fold increase in fibre diameter in the presence of zinc and in permeability studies, zinc increased clot porosity by 30-fold with or without FXIII. Whereas FXIII increased clot stiffness from 128 ± 19 Pa to 415 ± 27 Pa in rheological analyses, zinc reduced clot stiffness by 10- and 8.5-fold in the absence and presence of FXIII, respectively. Clots formed in the presence of zinc were more stable and resisted rupture with or without FXIII. Therefore, zinc accelerates clotting and reduces fibrin clot stiffness in a FXIII-independent manner, suggesting that zinc may work in concert with FXIII to modulate clot strength and stability.

  19. Targeting factor VIII expression to platelets for hemophilia A gene therapy does not induce an apparent thrombotic risk in mice.

    Science.gov (United States)

    Baumgartner, C K; Mattson, J G; Weiler, H; Shi, Q; Montgomery, R R

    2017-01-01

    Essentials Platelet-Factor (F) VIII gene therapy is a promising treatment in hemophilia A. This study aims to evaluate if platelet-FVIII expression would increase the risk for thrombosis. Targeting FVIII expression to platelets does not induce or elevate thrombosis risk. Platelets expressing FVIII are neither hyper-activated nor hyper-responsive. Background Targeting factor (F) VIII expression to platelets is a promising gene therapy approach for hemophilia A, and is successful even in the presence of inhibitors. It is well known that platelets play important roles not only in hemostasis, but also in thrombosis and inflammation. Objective To evaluate whether platelet-FVIII expression might increase thrombotic risk and thereby compromise the safety of this approach. Methods In this study, platelet-FVIII-expressing transgenic mice were examined either in steady-state conditions or under prothrombotic conditions induced by inflammation or the FV Leiden mutation. Native whole blood thrombin generation assay, rotational thromboelastometry analysis and ferric chloride-induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with thrombosis risk, including D-dimer, thrombin-antithrombin complexes, fibrinogen, tissue fibrin deposition, platelet activation status and activatability, and platelet-leukocyte aggregates, were assessed. Results We generated a new line of transgenic mice that expressed 30-fold higher levels of platelet-expressed FVIII than are therapeutically required to restore hemostasis in hemophilic mice. Under both steady-state conditions and prothrombotic conditions induced by lipopolysaccharide-mediated inflammation or the FV Leiden mutation, supratherapeutic levels of platelet-expressed FVIII did not appear to be thrombogenic. Furthermore, FVIII-expressing platelets were neither hyperactivated nor hyperactivatable upon agonist activation. Conclusion We conclude that, in mice, more than 30-fold higher levels of

  20. Continuous infusion of porcine factor VIII in patients with haemophilia A and high-responding inhibitors: stability and clinical experience.

    Science.gov (United States)

    O'Gorman, P; Dimichele, D M; Kasper, C K; Mannucci, P M; Santagostini, E; Hay, C R

    2001-11-01

    A multicentre retrospective survey was conducted to assess the efficacy and side-effect profile of porcine factor VIII (pFVIII:C) given by continuous infusion (CI) to patients with congenital haemophilia A and inhibitors. Twenty-nine episodes in 18 patients were treated by CI of pFVIII:C. Efficacy was graded as good in 79% of infusions and fair in 17%. There was a failed response in only one episode. Fourteen percent of patients experienced transfusion reactions with bolus doses, but no reactions were observed in patients given CI. There were no severe reactions. All the reactions resolved following interruption of the infusion and administration of steroids. Premedication did not prevent reactions. In this series the median decrease in platelet count after bolus injection of pFVIII:C was -67 X 10(9) L(-1) compared with a median decrease of -2 x 109 L(-1) during the course of CI, thus, continuous infusion of pFVIII:C appears to have less effect on platelet count than bolus injection. An anamnestic response was associated with 77% of infusions. This high rate of anamnesis reflects patient selection, in that they were all known to have high-level high-responding FVIII inhibitors with cross-reactivity to pFVIII. After reconstitution, the pFVIII:C showed little loss in factor VIII activity in solution over a 24-h period. We conclude that pFVIII:C may be effectively administered by CI to patients with haemophilia A and high-responding FVIII inhibitors. CI is the probably the mode of administration of choice for intensive replacement therapy with pFVIII.

  1. EFFICACY AND SAFETY OF A MONOCLONAL PURIFIED FACTOR-VIII CONCENTRATE - 5-YEAR FOLLOW-UP IN PREVIOUSLY TREATED HIV-NEGATIVE HEMOPHILIACS

    NARCIS (Netherlands)

    SMID, WM; VANDERMEER, J; HALIE, MR

    1995-01-01

    The efficacy and safety of a monoclonal purified factor VIII concentrate (Hemofil M) were assessed in a historically controlled study in 22 HIV-negative patients with haemophilia A, previously treated with various concentrates. Data from 5 years of follow-up were compared with those from the precedi

  2. Efficacy and safety of a monoclonal purified factor-VIII concentrate - 5-year follow-up in previously treated HIV-negative hemophiliacs

    NARCIS (Netherlands)

    Smid, W. M.; van der Meer, J.; Halie, M. R.

    1995-01-01

    The efficacy and safety of a monoclonal purified factor VIII concentrate (Hemofil M) were assessed in a historically controlled study in 22 HIV-negative patients with haemophilia A, previously treated with various concentrates. Data from 5 years of follow-up were compared with those from the

  3. Blood Clotting and Pregnancy

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    Full Text Available ... just had a baby, you are at greater risk of developing a blood clot. Blood clots in pregnant women tend to form in the deep veins of the legs or in the pelvic area. This condition is known as deep vein thrombosis (DVT). Pulmonary embolism (PE) is a life-threatening ...

  4. Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.

    Science.gov (United States)

    Nguyen, G N; George, L A; Siner, J I; Davidson, R J; Zander, C B; Zheng, X L; Arruda, V R; Camire, R M; Sabatino, D E

    2017-01-01

    Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain-deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared with FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4-fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a

  5. Factors Associated with the Time of Admission among Notified Dengue Fever Cases in Region VIII Philippines from 2008 to 2014.

    Directory of Open Access Journals (Sweden)

    Jason Echavez Abello

    2016-10-01

    Full Text Available In cases of Dengue fever, late hospital admission can lead to treatment delay and even death. In order to improve early disease notification and management, it is essential to investigate the factors affecting the time of admission of Dengue cases. This study determined the factors associated with the time of admission among notified Dengue cases. The study covered the period between 2008 and 2014 in Region VIII, Philippines. The factors assessed were age, sex, hospital sector, hospital level, disease severity based on the 1997 WHO Dengue classification, and period of admission (distinguishing between the 2010 Dengue epidemic and non-epidemic time. We analysed secondary data from the surveillance of notified Dengue cases. We calculated the association through chi-square test, ordinal logistic regression and linear regression at p value < 0.05. The study included 16,357 admitted Dengue cases. The reported cases included a majority of children (70.09%, mild cases of the disease (64.00%, patients from the public sector (69.82%, and non-tertiary hospitals (62.76%. Only 1.40% of cases had a laboratory confirmation. The epidemic period in 2010 comprised 48.68% of all the admitted cases during this period. Late admission was more likely among adults than children (p<0.05. The severe type of the disease was more likely to be admitted late than the mild type (p<0.05. Late admission was also more likely in public hospitals than in private hospitals (p<0.05; and within tertiary level hospitals than non-tertiary hospitals (p<0.05. Late admission was more likely during the non-epidemic period than the 2010 epidemic period (p<0.05. A case fatality rate of 1 or greater was significantly associated with children, severe diseases, tertiary hospitals and public hospitals when admitted late (p<0.05. Data suggests that early admission among child cases was common in Region VIII. This behavior is encouraging, and should be continued. However, further study is needed

  6. Limit of detection and threshold for positivity of the Centers for Disease Control and Prevention assay for factor VIII inhibitors.

    Science.gov (United States)

    Miller, C H; Boylan, B; Shapiro, A D; Lentz, S R; Wicklund, B M

    2017-08-10

    Essentials Immunologic methods detect factor VIII (FVIII) antibodies in some inhibitor-negative specimens. Specimens were tested by modified Nijmegen-Bethesda assay (NBA) and fluorescence immunoassay. The NBA with preanalytical heat inactivation detects FVIII inhibitors down to 0.2 NBU. IgG4 frequency validates the established threshold for positivity of ≥ 0.5 NBU for this NBA. Background The Bethesda assay for measurement of factor VIII inhibitors called for quantification of positive inhibitors by using dilutions producing 25-75% residual activity (RA), corresponding to 0.4-2.0 Bethesda units, with the use of 'more sensitive methods' for samples with RA closer to 100% being recommended. The Nijmegen modification (Nijmegen-Bethesda assay [NBA]) changed the reagents used but not these calculations. Some specimens negative by the NBA have been shown to have FVIII antibodies detectable with sensitive immunologic methods. Objective To examine the performance at very low inhibitor titers of the Centers for Disease Control and Prevention (CDC)-modified NBA (CDC-NBA), which includes preanalytic heat inactivation to liberate bound anti-FVIII antibodies. Methods Specimens with known inhibitors were tested with the CDC-NBA. IgG4 anti-FVIII antibodies were measured by fluorescence immunoassay (FLI). Results Diluted inhibitors showed linearity below 0.4 Nijmegen-Bethesda units (NBU). With four statistical methods, the limit of detection of the CDC-NBA was determined to be 0.2 NBU. IgG4 anti-FVIII antibodies, which correlate most strongly with functional inhibitors, were present at rates above the background rate of healthy controls in specimens with titers ≥ 0.2 NBU and showed an increase in frequency from 14.3% at 0.4 NBU to 67% at the established threshold for positivity of 0.5 NBU. Conclusions The CDC-NBA can detect inhibitors down to 0.2 NBU. The FLI, which is more sensitive, demonstrates anti-FVIII IgG4 in some patients with negative (NBA, supporting the need for

  7. Phenotypic correction and stable expression of factor VIII in hemophilia A mice by embryonic stem cell therapy.

    Science.gov (United States)

    Wang, J J; Kuang, Y; Zhang, L L; Shen, C L; Wang, L; Lu, S Y; Lu, X B; Fei, J; Gu, M M; Wang, Z G

    2013-05-13

    Hereditary deficiency of factor VIII (FVIII) leads to hemophilia A, a severe X-linked bleeding disorder. Current therapies include fixed-dose FVIII prophylaxis, factor replacement therapy, and most recently, gene therapy. Prophylaxis and FVIII replacement therapies are limited by incomplete efficacy, high cost, restricted availability, and development of neutralizing antibodies in chronically treated individuals. Limited success has been obtained in preclinical trials using gene therapy for the treatment of hemophilia. Therefore, new options for therapy for hemophilia A are needed. We evaluated the potential of embryonic stem cells for correcting hemophilia A in mice. FVIII-deficient mouse blastocysts were collected and injected with mouse embryonic stem cells stably expressing green-fluorescent protein (GFP) and transferred to pseudopregnant recipient mice. Expression of FVIII was measured in the liver and plasma of the 5 chimeric mice that were produced. Three of these mice were GFP-positive at the age of 6 months. The plasma FVIII activity levels were equal to those of wild-type mice. These data demonstrate that embryonic stem cell transplantation at an early embryonic stage has potential as therapy for this progressively debilitating, life-threatening bleeding disorder.

  8. In vivo expansion of regulatory T cells with IL-2/IL-2 mAb complexes prevents anti-factor VIII immune responses in hemophilia A mice treated with factor VIII plasmid-mediated gene therapy.

    Science.gov (United States)

    Liu, Chao-Lien; Ye, Peiqing; Yen, Benjamin C; Miao, Carol H

    2011-08-01

    Generation of transgene-specific immune responses can constitute a major complication following gene therapy treatment. An in vivo approach to inducing selective expansion of Regulatory T (Treg) cells by injecting interleukin-2 (IL-2) mixed with a specific IL-2 monoclonal antibody (JES6-1) was adopted to modulate anti-factor VIII (anti-FVIII) immune responses. Three consecutive IL-2 complexes treatments combined with FVIII plasmid injection prevented anti-FVIII formation and achieved persistent, therapeutic-level of FVIII expression in hemophilia A (HemA) mice. The IL-2 complexes treatment expanded CD4(+)CD25(+)Foxp3(+) Treg cells five- to sevenfold on peak day, and they gradually returned to normal levels within 7-14 days without changing other lymphocyte populations. The transiently expanded Treg cells are highly activated and display suppressive function in vitro. Adoptive transfer of the expanded Treg cells protected recipient mice from generation of high-titer antibodies following FVIII plasmid challenge. Repeated plasmid transfer is applicable in tolerized mice without eliciting immune responses. Mice treated with IL-2 complexes mounted immune responses against both T-dependent and T-independent neoantigens, indicating that IL-2 complexes did not hamper the immune system for long. These results demonstrate the important role of Treg cells in suppressing anti-FVIII immune responses and the potential of developing Treg cell expansion therapies that induce long-term tolerance to FVIII.

  9. Unfavorably Altered Fibrin Clot Properties in Patients with Eosinophilic Granulomatosis with Polyangiitis (Churg-Strauss Syndrome: Association with Thrombin Generation and Eosinophilia.

    Directory of Open Access Journals (Sweden)

    Lucyna Mastalerz

    Full Text Available Given reports on the increased prevalence of thromboembolic incidents in patients with eosinophilic granulomatosis with polyangiitis (EGPA; Churg-Strauss syndrome, we investigated whether fibrin clot properties are unfavorably altered in EGPA.Ex vivo plasma fibrin clot characteristics, including clot permeability, turbidimetry and efficiency of fibrinolysis using two assays, were investigated in 34 consecutive patients with remission in EGPA according to the Birmingham Vasculitis Activity Score version 3 (23 female, 11 male, aged 48 (range, 21-80 years. The control group comprised 34 age- and sex- matched volunteers.Compared with controls, patients with EGPA were characterized by denser fiber clots (estimated pore size, Ks, 7.30±0.93 vs 10.14±1.07 10-9 cm2, faster fibrin polymerization (lag phase in a turbidimetric curve, 41.8±3.6 vs 47.4±2.9 s, thicker fibrin fibers (maximum absorbance, ΔAbs, 0.87±0.09 vs 0.72±0.07, higher maximum levels of D-dimer released from clots (DDmax 4.10±0.46 vs 3.54±0.35 mg/L, and prolonged clot lysis time (t50%; 9.50±1.45 vs 7.56±0.87 min; all p<0.0001. Scanning electron microscopy images confirmed denser plasma fibrin networks composed of thinner fibers formed in EGPA. Antineutrophil cytoplasmic antibody status and C-reactive protein did not affect clot variables. Multivariate analysis adjusted for fibrinogen showed that Ks was predicted by eosinophil count, peak thrombin generation, factor VIII, and soluble CD40 ligand, whereas eosinophil count, peak thrombin generation and antiplasmin predicted t50%.This study is the first to show that EGPA is associated with prothrombotic plasma fibrin clot phenotype, which may contribute to thromboembolic manifestations reported in this disease.

  10. Overexpression of factor VIII after AAV delivery is transiently associated with cellular stress in hemophilia A mice

    Directory of Open Access Journals (Sweden)

    Amy M Lange

    2016-01-01

    Full Text Available Factor VIII (FVIII is a large glycoprotein that is challenging to express both in vitro and in vivo. Several studies suggest that high levels of FVIII expression can lead to cellular stress. After gene transfer, transgene expression is restricted to a subset of cells and the increased FVIII load per cell may impact activation of the unfolded protein response. We sought to determine whether increased FVIII expression in mice after adeno-associated viral liver gene transfer would affect the unfolded protein response and/or immune response to the transgene. The FVIII gene was delivered as B-domain deleted single chain or two chain (light and heavy chains at a range of doses in hemophilia A mice. A correlation between FVIII expression and anti-FVIII antibody titers was observed. Analysis of key components of the unfolded protein response, binding immunoglobulin protein (BiP, and C/EBP homologous protein (CHOP, showed transient unfolded protein response activation in the single chain treated group expressing >200% of FVIII but not after two chain delivery. These studies suggest that supraphysiological single chain FVIII expression may increase the likelihood of a cellular stress response but does not alter liver function. These data are in agreement with the observed long-term expression of FVIII at therapeutic levels after adeno-associated viral delivery in hemophilia A dogs without evidence of cellular toxicity.

  11. A novel cell-sheet technology that achieves durable factor VIII delivery in a mouse model of hemophilia A.

    Science.gov (United States)

    Tatsumi, Kohei; Sugimoto, Mitsuhiko; Lillicrap, David; Shima, Midori; Ohashi, Kazuo; Okano, Teruo; Matsui, Hideto

    2013-01-01

    Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3-5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.

  12. A novel cell-sheet technology that achieves durable factor VIII delivery in a mouse model of hemophilia A.

    Directory of Open Access Journals (Sweden)

    Kohei Tatsumi

    Full Text Available Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs, we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3-5-fold higher expression of FVIII (up to 11% of normal in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.

  13. Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice.

    Science.gov (United States)

    Kren, Betsy T; Unger, Gretchen M; Sjeklocha, Lucas; Trossen, Alycia A; Korman, Vicci; Diethelm-Okita, Brenda M; Reding, Mark T; Steer, Clifford J

    2009-07-01

    Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type-specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain-deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.

  14. Molecular design and downstream processing of turoctocog alfa (NovoEight), a B-domain truncated factor VIII molecule.

    Science.gov (United States)

    Ahmadian, Haleh; Hansen, Ernst B; Faber, Johan H; Sejergaard, Lars; Karlsson, Johan; Bolt, Gert; Hansen, Jens J; Thim, Lars

    2016-07-01

    Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product.

  15. Overexpression of factor VIII after AAV delivery is transiently associated with cellular stress in hemophilia A mice

    Science.gov (United States)

    Lange, Amy M; Altynova, Ekaterina S; Nguyen, Giang N; Sabatino, Denise E

    2016-01-01

    Factor VIII (FVIII) is a large glycoprotein that is challenging to express both in vitro and in vivo. Several studies suggest that high levels of FVIII expression can lead to cellular stress. After gene transfer, transgene expression is restricted to a subset of cells and the increased FVIII load per cell may impact activation of the unfolded protein response. We sought to determine whether increased FVIII expression in mice after adeno-associated viral liver gene transfer would affect the unfolded protein response and/or immune response to the transgene. The FVIII gene was delivered as B-domain deleted single chain or two chain (light and heavy chains) at a range of doses in hemophilia A mice. A correlation between FVIII expression and anti-FVIII antibody titers was observed. Analysis of key components of the unfolded protein response, binding immunoglobulin protein (BiP), and C/EBP homologous protein (CHOP), showed transient unfolded protein response activation in the single chain treated group expressing >200% of FVIII but not after two chain delivery. These studies suggest that supraphysiological single chain FVIII expression may increase the likelihood of a cellular stress response but does not alter liver function. These data are in agreement with the observed long-term expression of FVIII at therapeutic levels after adeno-associated viral delivery in hemophilia A dogs without evidence of cellular toxicity. PMID:27738645

  16. Lineages of human T-cell clones, including T helper 17/T helper 1 cells, isolated at different stages of anti–factor VIII immune responses

    OpenAIRE

    Ettinger, Ruth A.; James, Eddie A.; Kwok, William W.; Arthur R Thompson; Pratt, Kathleen P.

    2009-01-01

    The development of neutralizing antibodies (inhibitors) after factor VIII (FVIII) infusions is a serious complication that affects approximately one-quarter of hemophilia A patients who have access to replacement therapy. To investigate the differentiation of naive T cells into FVIII-specific helper T cells that promote B-cell activation and antibody secretion, HLA-DRA-DRB1*0101-restricted T-cell clones that respond to a specific epitope in FVIII were isolated from a mild hemophilia A subject...

  17. Blood Clotting and Pregnancy

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    Full Text Available ... predisposition to blood clots Obesity Prolonged immobility (e.g., bedrest, long distance travel) Multiple births Increased maternal age Other medical illness (e.g., cancer, infection) back to top How are Blood ...

  18. Blood Clotting and Pregnancy

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    Full Text Available ... dangerous to your baby. Blood clots can form inside the placenta, cutting off blood flow and harming ... 0544 | Fax 202-776-0545 ASH Store ASH Job Center ASH Apps Share Your Idea Donate Research ...

  19. Blood Clotting and Pregnancy

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    Full Text Available ... harming your baby. Jump To: Am I at Risk? The risk of developing a blood clot during ... Advances The Hematologist ASH Clinical News ASH Self-Assessment Program Hematology , ASH Education Program About Awards Membership ...

  20. Blood Clotting and Pregnancy

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    Full Text Available ... education program designed to improve the diagnosis and treatment of acute myeloid leukemia (AML). 2017 ASH Meeting ... and increase research on the causes, prevention, and treatment. Blood clots are also potentially dangerous to your ...

  1. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... known as venous thromboembolism, are highly preventable (see prevention tips below). The U.S. Surgeon General has issued ... blood conditions and increase research on the causes, prevention, and treatment. Blood clots are also potentially dangerous ...

  2. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... genetic predisposition to blood clots Obesity Prolonged immobility (e.g., bedrest, long distance travel) Multiple births Increased maternal age Other medical illness (e.g., cancer, infection) back to top How are ...

  3. Blood Clotting and Pregnancy

    Medline Plus

    Full Text Available ... known as venous thromboembolism, are highly preventable (see prevention tips below). The U.S. Surgeon General has issued ... blood conditions and increase research on the causes, prevention, and treatment. Blood clots are also potentially dangerous ...

  4. Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity.

    Directory of Open Access Journals (Sweden)

    Zenjiro Sampei

    Full Text Available In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors. To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa and factor X (FX, mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910 will provide significant benefit for severe hemophilia A patients.

  5. Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings.

    Directory of Open Access Journals (Sweden)

    Nancy A Turner

    Full Text Available The cellular synthesis site and ensuing storage location for human factor VIII (FVIII, the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs and umbilical vein endothelial cells (HUVECs. Gene expression quantified by real time PCR revealed that levels of F8 and VWF are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

  6. Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings.

    Science.gov (United States)

    Turner, Nancy A; Moake, Joel L

    2015-01-01

    The cellular synthesis site and ensuing storage location for human factor VIII (FVIII), the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF) to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs) and umbilical vein endothelial cells (HUVECs). Gene expression quantified by real time PCR revealed that levels of F8 and VWF are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

  7. The prevalence of latex sensitisation and allergy and associated risk factors among healthcare workers using hypoallergenic latex gloves at King Edward VIII Hospital, KwaZulu-Natal South Africa: a cross-sectional study

    National Research Council Canada - National Science Library

    Phaswana, Shumani Makwarela; Naidoo, Saloshni

    2013-01-01

    The present study describes latex sensitisation and allergy prevalence and associated factors among healthcare workers using hypoallergenic latex gloves at King Edward VIII Hospital in KwaZulu-Natal, South Africa...

  8. Therapeutic levels of human factor VIII in mice implanted with encapsulated cells: potential for gene therapy of haemophilia A.

    Science.gov (United States)

    García-Martín, Carmen; Chuah, Marinee K L; Van Damme, An; Robinson, Kelly E; Vanzieleghem, Beatrijs; Saint-Remy, Jean-Marie; Gallardo, Dominique; Ofosu, Frederick A; Vandendriessche, Thierry; Hortelano, Gonzalo

    2002-01-01

    A gene therapy delivery system based on microcapsules enclosing recombinant cells engineered to secrete a therapeutic protein has been evaluated. The microcapsules are implanted intraperitoneally. In order to prevent cell immune rejection, cells are enclosed in non-antigenic biocompatible alginate microcapsules prior to their implantation into mice. It has been shown that encapsulated myoblasts can deliver therapeutic levels of Factor IX (FIX) in mice. The delivery of human Factor VIII (hFVIII) in mice using microcapsules was evaluated in this study. Mouse C2C12 myoblasts and canine MDCK epithelial kidney cells were transduced with MFG-FVIII (B-domain deleted) vector. Selected recombinant clones were enclosed in alginate microcapsules. Encapsulated recombinant clones were subsequently implanted intraperitoneally into C57BL/6 and immunodeficient SCID mice. Plasma of mice receiving C2C12 and encapsulated MDCK cells had transient therapeutic levels of FVIII in immunocompetent C57BL/6 mice (up to 20% and 7% of physiological levels, respectively). In addition, FVIII delivery in SCID mice was also transient, suggesting that a non-immune mechanism must have contributed to the decline of hFVIII in plasma. Quantitative RT-PCR analysis confirmed directly that the decline of hFVIII is due to a reduction in steady-state hFVIII mRNA, consistent with transcriptional repression. Furthermore, encapsulated cells retrieved from implanted mice were viable, but secreted FVIII ex vivo at three-fold lower levels than the pre-implantation levels. In addition, antibodies to hFVIII were detected in immunocompetent C57BL/6 mice. Implantable microcapsules can deliver therapeutic levels of FVIII in mice, suggesting the potential of this gene therapy approach for haemophilia A. The findings suggest vector down-regulation in vivo. Copyright 2002 John Wiley & Sons, Ltd.

  9. Endothelial cell processing and alternatively spliced transcripts of factor VIII: potential implications for coagulation cascades and pulmonary hypertension.

    Directory of Open Access Journals (Sweden)

    Claire L Shovlin

    Full Text Available BACKGROUND: Coagulation factor VIII (FVIII deficiency leads to haemophilia A. Conversely, elevated plasma levels are a strong predictor of recurrent venous thromboemboli and pulmonary hypertension phenotypes in which in situ thromboses are implicated. Extrahepatic sources of plasma FVIII are implicated, but have remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemistry of normal human lung tissue, and confocal microscopy, flow cytometry, and ELISA quantification of conditioned media from normal primary endothelial cells were used to examine endothelial expression of FVIII and coexpression with von Willebrand Factor (vWF, which protects secreted FVIII heavy chain from rapid proteloysis. FVIII transcripts predicted from database mining were identified by RT-PCR and sequencing. FVIII mAb-reactive material was demonstrated in CD31+ endothelial cells in normal human lung tissue, and in primary pulmonary artery, pulmonary microvascular, and dermal microvascular endothelial cells. In pulmonary endothelial cells, this protein occasionally colocalized with vWF, centered on Weibel Palade bodies. Pulmonary artery and pulmonary microvascular endothelial cells secreted low levels of FVIII and vWF to conditioned media, and demonstrated cell surface expression of FVIII and vWF Ab-reacting proteins compared to an isotype control. Four endothelial splice isoforms were identified. Two utilize transcription start sites in alternate 5' exons within the int22h-1 repeat responsible for intron 22 inversions in 40% of severe haemophiliacs. A reciprocal relationship between the presence of short isoforms and full-length FVIII transcript suggested potential splice-switching mechanisms. CONCLUSIONS/SIGNIFICANCE: The pulmonary endothelium is confirmed as a site of FVIII secretion, with evidence of synthesis, cell surface expression, and coexpression with vWF. There is complex alternate transcription initiation from the FVIII gene. These findings provide a

  10. A population pharmacokinetic model for perioperative dosing of factor VIII in hemophilia A patients

    NARCIS (Netherlands)

    Hazendonk, Hendrika; Fijnvandraat, Karin; Lock, Janske; Driessens, Mariette; van der Meer, Felix; Meijer, Karina; Kruip, Marieke; Laros-van Gorkom, Britta; Peters, Marjolein; de Wildt, Saskia; Leebeek, Frank; Cnossen, Marjon; Mathot, Ron

    2016-01-01

    The role of pharmacokinetic-guided dosing of factor concentrates in hemophilia is currently a subject of debate and focuses on long-term prophylactic treatment. Few data are available on its impact in the perioperative period. In this study, a population pharmacokinetic model for currently registere

  11. A population pharmacokinetic model for perioperative dosing of factor VIII in hemophilia A patients

    NARCIS (Netherlands)

    Hazendonk, Hendrika; Fijnvandraat, Karin; Lock, Janske; Driessens, Mariette; van der Meer, Felix; Meijer, Karina; Kruip, Marieke; Laros-van Gorkom, Britta; Peters, Marjolein; de Wildt, Saskia; Leebeek, Frank; Cnossen, Marjon; Mathot, Ron

    2016-01-01

    The role of pharmacokinetic-guided dosing of factor concentrates in hemophilia is currently a subject of debate and focuses on long-term prophylactic treatment. Few data are available on its impact in the perioperative period. In this study, a population pharmacokinetic model for currently

  12. Associations between high factor VIII and low free protein S levels with traditional arterial thrombotic risk factors and their risk on arterial thrombosis : Results from a retrospective family cohort study

    NARCIS (Netherlands)

    Mulder, Rene; van Schouwenburg, Inge M.; Mahmoodi, Bakhtawar K.; Veeger, Nic J. G. M.; Mulder, Andre B.; Middeldorp, Saskia; Kluin-Nelemans, Hanneke C.; Lijfering, Willem M.

    2010-01-01

    Introduction: Whether high factor (F)VIII and low free protein S levels are risk factors for arterial thrombosis is unclarified. Material and Methods: In a post-hoc analysis of a single-centre retrospective family cohort, we determined if these two proteins could increase the risk of arterial

  13. Factor VIII delivery devices in haemophilia A. Barriers and drivers for treatment adherence.

    Science.gov (United States)

    Fernández-Arias, Isabel; Kim, Hae Kyung

    2016-11-01

    Introducción y objetivo: Recabar la experiencia de pacientes con hemofilia A con sus dispositivos de reconstitucion de factor de coagulacion, barreras para la adherencia y determinar sus preferencias, presentando una nueva jeringa de doble camara (JDC). Método: Investigacion transversal mediante encuesta dirigida y sesion de prueba de la JDC. Resultados: Participaron 74 pacientes, el 50% en tratamiento con profilaxis, y 7 anos (mediana) con su tratamiento habitual (RIC 17,25). En la encuesta, la JDC recibio la mayor puntuacion (75/100, p probabilidad de uso en profilaxis (p probabilidad de favorecer la profilaxis.

  14. Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy.

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    Brandon K Sack

    Full Text Available The major complication in the treatment of hemophilia A is the development of neutralizing antibodies (inhibitors against factor VIII (FVIII. The current method for eradicating inhibitors, termed immune tolerance induction (ITI, is costly and protracted. Clinical protocols that prevent rather than treat inhibitors are not yet established. Liver-directed gene therapy hopes to achieve long-term correction of the disease while also inducing immune tolerance. We sought to investigate the use of adeno-associated viral (serotype 8 gene transfer to induce tolerance to human B domain deleted FVIII in hemophilia A mice. We administered an AAV8 vector with either human B domain deleted FVIII or a codon-optimized transgene, both under a liver-specific promoter to two strains of hemophilia A mice. Protein therapy or gene therapy was given either alone or in conjunction with anti-CD20 antibody-mediated B cell depletion. Gene therapy with a low-expressing vector resulted in sustained near-therapeutic expression. However, supplementary protein therapy revealed that gene transfer had sensitized mice to hFVIII in a high-responder strain but not in mice of a low-responding strain. This heightened response was ameliorated when gene therapy was delivered with anti-murine CD20 treatment. Transient B cell depletion prevented inhibitor formation in protein therapy, but failed to achieve a sustained hypo-responsiveness. Importantly, use of a codon-optimized hFVIII transgene resulted in sustained therapeutic expression and tolerance without a need for B cell depletion. Therefore, anti-CD20 may be beneficial in preventing vector-induced immune priming to FVIII, but higher levels of liver-restricted expression are preferred for tolerance.

  15. Characterization of Adeno-Associated Viral Vector-Mediated Human Factor VIII Gene Therapy in Hemophilia A Mice.

    Science.gov (United States)

    Greig, Jenny A; Wang, Qiang; Reicherter, Amanda L; Chen, Shu-Jen; Hanlon, Alexandra L; Tipper, Christopher H; Clark, K Reed; Wadsworth, Samuel; Wang, Lili; Wilson, James M

    2017-05-01

    Adeno-associated viral (AAV) vectors are promising vehicles for hemophilia gene therapy, with favorable clinical trial data seen in the treatment of hemophilia B. In an effort to optimize the expression of human coagulation factor VIII (hFVIII) for the treatment of hemophilia A, an extensive study was performed with numerous combinations of liver-specific promoter and enhancer elements with a codon-optimized hFVIII transgene. After generating 42 variants of three reduced-size promoters and three small enhancers, transgene cassettes were packaged within a single AAV capsid, AAVrh10, to eliminate performance differences due to the capsid type. Each hFVIII vector was administered to FVIII knockout (KO) mice at a dose of 10(10) genome copies (GC) per mouse. Criteria for distinguishing the performance of the different enhancer/promoter combinations were established prior to the initiation of the studies. These criteria included prominently the level of hFVIII activity (0.12-2.12 IU/mL) and the pattern of development of anti-hFVIII antibodies. In order to evaluate the impact of capsid on hFVIII expression and antibody formation, one of the enhancer and promoter combinations that exhibited high hFVIII immunogenicity was evaluated using AAV8, AAV9, AAVrh10, AAVhu37, and AAVrh64R1 capsids. The capsids subdivided into two groups: those that generated anti-hFVIII antibodies in ≤20% of mice (AAV8 and AAV9), and those that generated anti-hFVIII antibodies in >20% of mice (AAVrh10, AAVhu37, and AAVrh64R1). The results of this study, which entailed extensive vector optimization and in vivo testing, demonstrate the significant impact that transcriptional control elements and capsid can have on vector performance.

  16. The mesenchymal stem cells derived from transgenic mice carrying human coagulation factor VIII can correct phenotype in hemophilia A mice.

    Science.gov (United States)

    Wang, Qing; Gong, Xiuli; Gong, Zhijuan; Ren, Xiaoyie; Ren, Zhaorui; Huang, Shuzhen; Zeng, Yitao

    2013-12-20

    Hemophilia A (HA) is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. Previous studies showed that introduction of mesenchymal stem cells (MSCs) modified by FVIII-expressing retrovirus may result in phenotypic correction of HA animals. This study aimed at the investigation of an alternative gene therapy strategy that may lead to sustained FVIII transgene expression in HA mice. B-domain-deleted human FVIII (hFVIIIBD) vector was microinjected into single-cell embryos of wild-type mice to generate a transgenic mouse line, from which hFVIIIBD-MSCs were isolated, followed by transplantation into HA mice. RT-PCR and real-time PCR analysis demonstrated the expression of hFVIIIBD in multi-organs of recipient HA mice. Immunohistochemistry showed the presence of hFVIIIBD positive staining in multi-organs of recipient HA mice. ELISA indicated that plasma hFVIIIBD level in recipient mice reached its peak (77 ng/mL) at the 3rd week after implantation, and achieved sustained expression during the 5-week observation period. Plasma FVIII activities of recipient HA mice increased from 0% to 32% after hFVIIIBD-MSCs transplantation. APTT (activated partial thromboplastin time) value decreased in hFVIIIBD-MSCs transplanted HA mice compared with untreated HA mice (45.5 s vs. 91.3 s). Our study demonstrated an effective phenotypic correction in HA mice using genetically modified MSCs from hFVIIIBD transgenic mice. Copyright © 2013. Published by Elsevier Ltd.

  17. A sequence variation scan of the coagulation factor VIII (FVIII) structural gene and associations with plasma FVIII activity levels.

    Science.gov (United States)

    Viel, Kevin R; Machiah, Deepa K; Warren, Diane M; Khachidze, Manana; Buil, Alfonso; Fernstrom, Karl; Souto, Juan C; Peralta, Juan M; Smith, Todd; Blangero, John; Porter, Sandra; Warren, Stephen T; Fontcuberta, Jordi; Soria, Jose M; Flanders, W Dana; Almasy, Laura; Howard, Tom E

    2007-05-01

    Plasma factor VIII coagulant activity (FVIII:C) level is a highly heritable quantitative trait that is strongly correlated with thrombosis risk. Polymorphisms within only 1 gene, the ABO blood-group locus, have been unequivocally demonstrated to contribute to the broad population variability observed for this trait. Because less than 2.5% of the structural FVIII gene (F8) has been examined previously, we resequenced all known functional regions in 222 potentially distinct alleles from 137 unrelated nonhemophilic individuals representing 7 racial groups. Eighteen of the 47 variants identified, including 17 single-nucleotide polymorphisms (SNPs), were previously unknown. As the degree of linkage disequilibrium across F8 was weak overall, we used measured-genotype association analysis to evaluate the influence of each polymorphism on the FVIII:C levels in 398 subjects from 21 pedigrees known as the Genetic Analysis of Idiopathic Thrombophilia project (GAIT). Our results suggested that 92714C>G, a nonsynonymous SNP encoding the B-domain substitution D1241E, was significantly associated with FVIII:C level. After accounting for important covariates, including age and ABO genotype, the association persisted with each C-allele additively increasing the FVIII:C level by 14.3 IU dL(-1) (P = .016). Nevertheless, because the alleles of 56010G>A, a SNP within the 3' splice junction of intron 7, are strongly associated with 92714C>G in GAIT, additional studies are required to determine whether D1241E is itself a functional variant.

  18. Factor VIII (F8) inversions in severe hemophilia A: Male germ cell origin and diagnosis with RT-PCR

    Energy Technology Data Exchange (ETDEWEB)

    Antonarakis, S.E. [Geneva Medical School (Switzerland)]|[Johns Hopkins School of Medicine, Baltimore, MD (United States); Rossiter, J.P. [Johns Hopkins School of Medicine, Baltimore, MD (United States); Young, M. [Geneva Medical School (Switzerland)] [and others

    1994-09-01

    The Factor VIII (F8) gene, which is defective in hemophilia A, is located in the most telomeric megabase of Xq. Inversions due to intrachromosomal homologous recombination between mispaired copies of gene A located within intron 22 of the gene and about 500 kb telomeric to it account for nearly half of the cases of severe hemophilia A. We hypothesized that pairing of Xq with its homolog inhibits the inversion process, and that therefore the event originates predominantly in male germ cells. In all 21 informative cases in which the inversion originated in a maternal grandparent, DNA polymorphism analysis using markers within or very closely linked to F8, determined that it occurred in the male germline. In addition, all but one of 56 mothers of sporadic cases due to inversions were carriers. The data indicate that the F8 gene inversions leading to severe hemophilia A occur almost exclusively in male germ cells. The mean age of maternal grandfathers at the birth of their carrier daughters was 29.9 years (13 cases), i.e. not different from the mean paternal age in the general population, supporting the hypothesis that the inversions occur in meiosis. The inversions can be diagnosed by Southern blot analysis. For more rapid diagnosis we have used RT-PCR of RNA ectopically expressed in blood. Oligonucleotides were used to PCR amplify, after the initial RT reaction of RNA samples using random hexamers, either the normal transcript (F8 exons 21 to 24;312 bp product) or the novel abnormal transcript that is generated after the inversion. Both type 1 and 2 inversions can be recognized in affecteds and carriers by the presence of the diagnostic PcR product of 248 bp. Correct diagnoses were made in samples from 6 patients and 2 carriers with type 1 inversions, 2 patients and 2 carriers with type 2 inversions and 5 normal controls.

  19. Severe hemophilia A in a female by cryptic translocation: Order and orientation of factor VIII within Xq28

    Energy Technology Data Exchange (ETDEWEB)

    Migeon, B.R.; McGinniss, M.J.; Antonarakis, S.E.; Axelman, J.; Stasiowski, B.A.; Youssoufian, H.; Kearns, W.G.; Chung, A.; Pearson, P.L.; Kazazian, H.H. Jr. (Johns Hopkins Univ., Baltimore, MD (United States)); Muneer, R.S. (Univ. of Oklahoma, Norman (United States))

    1993-04-01

    The authors report studies of a female with severe hemophilia A resulting from a complex de novo translocation of chromosomes X and 17 (46,X,t(X; 17)). Somatic cell hybrids containing the normal X, the der(X), or the der(17) were analyzed for coagulation factor VIII (F8C) sequences using Southern blots and polymerase chain reaction. The normal X, always late replicating, contains a normal F8C gene, whereas the der(X) has no F8C sequences. The der(17) chromosome containing Xq24-Xq28 carries a functional G6PD locus and a deleted F8C allele that lacks exons 1--15. Also, it lacks the DXYS64-X locus, situated between the F8C locus and the Xq telomere. These results indicate that a cryptic breakpoint within Xq28 deleted the 5[prime] end of F8C, but left the more proximal G6PD locus intact on the der(17)chromosome. As the deleted segment includes the 5[prime] half of F8C as well as the subtelomeric DXYS64 locus, F8C must be oriented on the chromosome with its 5[prime] region closest to the telomere. Therefore, the order of these loci is Xcen-G6PD-3[prime]F8C-5[prime]F8C-DXYS64-Xqtel. The analysis of somatic cell hybrids has elucidated the true nature of the F8C mutation in the pro-band, revealing a more complex rearrangement (three chromosomes involved) than that expected from cytogenetic analysis, chromosome painting, and Southern blots. A 900-kb segment within Xq28 has been translocated to another autosome. Hemophilia A in this heterozygous female is due to the decapitation of the F8C gene on the der(17) and inactivation of the intact allele on the normal X. 27 refs., 5 figs., 1 tab.

  20. Joint bleeds increase the inhibitor response to human factor VIII in a rat model of severe haemophilia A.

    Science.gov (United States)

    Lövgren, K M; Søndergaard, H; Skov, S; Wiinberg, B

    2016-09-01

    The most serious complication in haemophilia A (HA) replacement therapy with coagulation factor VIII (FVIII) is neutralizing antibodies, i.e. inhibitors. It has been hypothesized that danger signals generated during a bleed might have an adjuvant effect on the immune response to FVIII in on-demand treatment, increasing the inhibitor risk. To compare the antibody response to treatment with recombinant human FVIII (rhFVIII) in relation to induced knee joint bleeds and treatment without concurrent bleeds in a HA rat model. HA rats were divided into two groups: one group (n = 10) receiving three needle induced knee joint bleeds 14 days apart and a control group (n = 9) receiving three sham procedures. Three hours after each injury/sham 50 IU kg(-1) rhFVIII was administrated intravenously. Subsequently, both groups continued rhFVIII treatment for another 9 weeks. Binding antibodies were analysed using an enzyme-linked immunosorbent assay and neutralizing antibodies using a Bethesda-like assay. Rats in the knee-bleed group developed a significantly faster inhibitor response and reached significantly higher inhibitor levels. In the knee-bleed group, 80% developed inhibitors vs. 33% in the control group, demonstrating a 2.4 times higher inhibitor risk when treating concurrent with bleeds. FVIII treatment in relation to a bleed potentiates inhibitor development compared to FVIII treatment alone in this HA rat, indicating that bleeding is a potential danger signal. Our results support the theory that FVIII replacement therapy concurrent with a bleeding episode increases the inhibitor risk, which to the best of our knowledge, has not been confirmed in an animal model before. © 2016 John Wiley & Sons Ltd.

  1. Antibody response to recombinant human coagulation factor VIII in a new rat model of severe hemophilia A.

    Science.gov (United States)

    Lövgren, K M; Søndergaard, H; Skov, S; Weldingh, K N; Tranholm, M; Wiinberg, B

    2016-04-01

    Neutralizing antibodies toward FVIII replacement therapy (inhibitors) are the most serious treatment-related complication in hemophilia A (HA). A rat model of severe HA (F8(-/-) ) has recently been developed, but an immunological characterization is needed to determine the value of using the model for research into inhibitor development. Characterize the antibody response towards recombinant human coagulation factor VIII (rhFVIII) in the HA rat, following a human prophylactic dosing regimen. Two identical studies were performed, which included a total of 17 homozygous HA rats (F8(-/-) , 0% FVIII activity), 12 heterozygous rats (F8(+/-) ), and 12 wild-type (F8(+/+) ) rats. All rats received intravenous injections of rhFVIII at 50 IU kg(-1) twice weekly for 4 weeks. Predosing blood samples were analyzed for binding and neutralizing anti-rhFVIII antibodies at weeks 1-7. In both studies, antibodies developed after 4-6 administrations of rhFVIII, and neutralizing antibodies reached levels similar to human patients (range 1-111 BU, median 6.0 BU) at the end of the study. There was no significant difference between the two studies or between genotypes in time to response or levels reached for binding and neutralizing antibodies. Interestingly, early spontaneous bleeds were associated with a faster antibody response. Following intravenous administration of human FVIII, according to a clinical prophylaxis regimen, a robust and reproducible antibody response is seen in this HA rat model, suggesting that the model is useful for intervention studies with the aim of suppressing, delaying, or preventing the inhibitor response. Also, bleeds seem to have an adjuvant effect on the immune response. © 2016 International Society on Thrombosis and Haemostasis.

  2. Plasma levels of plasminogen activator inhibitor type 1, factor VIII, prothrombin activation fragment 1+2, anticardiolipin, and antiprothrombin antibodies are risk factors for thrombosis in hemodialysis patients.

    Science.gov (United States)

    Molino, Daniela; De Santo, Natale G; Marotta, Rosa; Anastasio, Pietro; Mosavat, Mahrokh; De Lucia, Domenico

    2004-09-01

    Patients with end-stage renal disease are prone to hemorrhagic complications and simultaneously are at risk for a variety of thrombotic complications such as thrombosis of dialysis blood access, the subclavian vein, coronary arteries, cerebral vessel, and retinal veins, as well as priapism. The study was devised for the following purposes: (1) to identify the markers of thrombophilia in hemodialyzed patients, (2) to establish a role for antiphospholipid antibodies in thrombosis of the vascular access, (3) to characterize phospholipid antibodies in hemodialysis patients, and (4) to study the effects of dialysis on coagulation cascade. A group of 20 hemodialysis patients with no thrombotic complications (NTC) and 20 hemodialysis patients with thrombotic complications (TC) were studied along with 400 volunteer blood donors. Patients with systemic lupus erythematosus and those with nephrotic syndrome were excluded. All patients underwent a screening prothrombin time, activated partial thromboplastin time, fibrinogen (Fg), coagulation factors of the intrinsic and extrinsic pathways, antithrombin III (AT-III), protein C (PC), protein S (PS), resistance to activated protein C, prothrombin activation fragment 1+2 (F1+2), plasminogen, tissue type plasminogen activator (t-PA), plasminogen tissue activator inhibitor type-1 (PAI-1), anticardiolipin antibodies type M and G (ACA-IgM and ACA-IgG), lupus anticoagulant antibodies, and antiprothrombin antibodies type M and G (aPT-IgM and aPT-IgG). The study showed that PAI-1, F 1+2, factor VIII, ACA-IgM, and aPT-IgM levels were increased significantly over controls both in TC and NTC, however, they could distinguish patients with thrombotic complications from those without, being increased maximally in the former group. The novelty of the study is represented by the significant aPT increase that was observed in non-systemic lupus erythematosus hemodialysis patients, and particularly in those with thrombotic events. In addition

  3. The 1.7 A X-ray crystal structure of the porcine factor VIII C2 domain and binding analysis to anti-human C2 domain antibodies and phospholipid surfaces.

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    Caileen M Brison

    Full Text Available The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the "non-classical" antibody epitopes. Furthermore, antibody-binding results illustrate that the "classical" 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.

  4. BAY 81-8973, a full-length recombinant factor VIII: results from an International comparative laboratory field study.

    Science.gov (United States)

    Kitchen, S; Beckmann, H; Katterle, Y; Bruns, S; Tseneklidou-Stoeter, D; Maas Enriquez, M

    2016-05-01

    BAY 81-8973 is a full-length, unmodified, recombinant human factor VIII (FVIII) with the same primary amino acid sequence as sucrose-formulated recombinant FVIII but produced with certain more advanced manufacturing technologies. This global laboratory study evaluated variability in measurement of BAY 81-8973 using one-stage and chromogenic assays compared with antihaemophilic factor (recombinant) plasma/albumin-free method (rAHF-PFM; Advate(®) ) under assay conditions routinely used in clinical laboratories. BAY 81-8973 or rAHF-PFM was spiked into FVIII-deficient plasma at 0.043 (low), 0.375 (medium) and 0.865 (normal) IU mL(-1) . Participating laboratories analysed blinded samples and normal plasma in triplicate using their routine assay, reagents and standards. Results were analysed for intra- and interlaboratory variability. Forty-one laboratories in 11 countries participated in the study. One-stage assay and chromogenic assays were used by 40 and 10 laboratories, respectively; 9 laboratories used both assays. Intralaboratory variability was <11% for both assays and both products at all concentrations. Interlaboratory variability was highest at the low concentration in the chromogenic and one-stage assay for BAY 81-8973 (60.0% and 33.7%, respectively) and rAHF-PFM (51.0% and 30.8%) and was lowest at the normal concentration (BAY 81-8973, 5.4% and 14.0%; rAHF-PFM, 5.8% and 12.4%), which was similar to the plasma control (6.6% and 10.3%). The chromogenic:one-stage assay ratio ranged from 0.95 (low concentration) to 1.10 (normal concentration) for BAY 81-8973 and 0.96-1.18 for rAHF-PFM. BAY 81-8973 can be accurately measured in plasma using the one-stage and chromogenic assays routinely used in clinical laboratories without a product-specific standard. © 2016 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

  5. How Blood Clots

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    ... Japanese Espaniol Find information on medical topics, symptoms, drugs, procedures, news and more, written in everyday language. * This is ... Process How Blood Clots Resources In This Article Drugs Mentioned In This Article ... of the Blood (News) Is Binge-Watching Hazardous to Your Health? (News) ...

  6. Pathogen inactivation in fresh frozen plasma using riboflavin and ultraviolet light: Effects on plasma proteins and coagulation factor VIII

    Directory of Open Access Journals (Sweden)

    Stanojković Zoran

    2011-01-01

    Full Text Available Background/Aim. Riboflavin (vitamin B2 activated by ultraviolet (UV light, produces active oxygen which damages cell membrane and prevents replication of the carrier of diseases (viruses, bacteria, protozoa in all blood products. The aim of this study was to establish the influence of the process of photo inactivation in pathogens using riboflavin and UV rays on the concentration of coagulation factor VIII:C (FVIII:C and proteins in plasma that were treated before freezing. Methods. The examination included 20 units of plasma, separated from whole blood donated by voluntary blood donors around 6 hours from the moment of collection. The units were pooled and separated in to two groups: one consisted of 10 control units and the other of 10 experimental units. Experimental units of the plasma were treated by riboflavin (35 mL and UV rays (6.24 J/mL, 265-370 nm on Mirasol aparature (Caridian BCT Biotechnologies, USA in approximate duration of 6 minutes. Furthermore, 35 mL of saline solution was added to the control plasma. One sample for examining was taken from the control plasma (KG and two residual were taken from experimental plasma after the addition of riboflavin either before (EG1 or post illumination (EG2. Results. Comparing the mean values of FVIII:C (% we noticed statistically significantly higher level in the EG1 group than in the EG2 group (65.00 ± 4.52 vs 63.20 ± 4.73; t = 4.323, p = 0.002, while between the KG and experimental groups (EG1 and EG2 there was no statistically significant difference in the concentration of FVIII:C. There was a statistically significant decrease of albumin concentration (g/L in the EG2 group comparing to the KG (33.35 ± 0.94 vs 31.94 ± 0.84; t = 3.534, p = 0.002, but there was no mentioned difference in albumin concentration between the KG and the EG1, so as between the EG1 and the EG2. Conclusion. Plasma inactivated by riboflavin and UV rays (Mirasol PRT sistem, Caridian BCT, USA keeps all the

  7. Sustaining expression of B domain-deleted human factor VIII mediated by using lentiviral vectors in NOD/SCID mouse.

    Science.gov (United States)

    Li, Yan-Jie; Chen, Chong; Zeng, Ling-Yu; Cao, Jiang; Xu, Kai-Lin

    2012-06-01

    Recently, gene therapy has been become a promising approach to cure hemophilia A, a most common recessive bleeding disease. The aim of this study was to determine the perspective of lentiviral vector in hemophilia A gene therapy in vitro and in NOD/SCID mice. Lentivirus transfer vector pXZ9/BDDFVIII containing human B-domain-deleted Factor VIII-IRES-eGFP coding sequence and mock control pXZ9 were constructed. Lentivirus was prepared by co-transfecting 3 plasmids into 293FT cells. 293FT, HLF, human bone marrow mesenchymal stem cells and Chang-liver cells were transfected with the prepared virus. Coagulant activity of human FVIII, human FVIII antigen, human FVIII mRNA transcription and genomic integration were assayed by ELISA, one-step method, RT-PCR and PCR after infection. Lentiviral particles were concentrated by ultracentrifugation and NOD/SCID mice were transfected via portal vein injection. Human FVIII antigen in mouse blood plasma was analyzed by ELISA. eGFP expression was observed by fluorescent microscopy and human FVIII transcription in mouse liver was analyzed by RT-PCR at one month after transduction. The results showed that the high titer of recombinant virus was prepared and used to efficiently transduce the target cells in vitro. At 72 h after transfection, high levels of FVIII activity and FVIII antigen were detected. Human FVIII gene transcription could be detected in the liver of NOD/SCID mice received lentiviral particles carrying FVIII gene. Mouse hepatocytes were transfected with recombinant lentivirus efficiently in vivo. Human FVIII level in mouse blood plasma reached to (49 ± 6) mU, (54 ± 8) mU and (23 ± 4) mU at 72 h, one week and one month after transfection respectively. It is concluded that the lentiviral particles carrying BDDhFVIII gene can high efficiently transfect the target cells both in vitro and in vivo, and the transfected target cells can secrete hFVIII efficiently. The sustained expression of human FVIII in NOD/SCID mice is

  8. Preventing and Treating Blood Clots

    Science.gov (United States)

    ... of blood clots. Heparin is recommended to treat DVT and PE for the first five to ten days, as well as for preventing blood clots ... risk of bleeding. For patients who develop a deep vein thrombosis, and/or a ... blood clot prevention will be included in your overall treatment plan, ...

  9. Experimental arterial thrombosis in genetically or diet induced hyperlipidemia in rats--role of vitamin K-dependent clotting factors and prevention by low-intensity oral anticoagulation.

    Science.gov (United States)

    De Curtis, A; D'Adamo, M C; Amore, C; Polishchuck, R; Castelnuovo, A D; Donati, M B; Iacoviello, L

    2001-12-01

    To investigate the relationship among lipids, coagulation and thrombosis in the absence of atherosclerosis, spontaneous or dietary-induced hyperlipidemic (FHL) rats were studied. FHL showed higher levels of coagulation factors VII, IX, X, VIII and XII and a shortening of the occlusion time (OT) of an artificial arterial prosthesis as compared with normolipidemic (FNL) animals. Damage of abdominal aorta of FHL was followed by increased fibrin deposition in the vascular intima as compared to FNL. After 5 months of cholesterol-rich diet FNL showed increased cholesterol, triglycerides and factor II, VII, IX, X, XII levels. A significant shortening of the OT and increased fibrin deposition was also observed. Two-month diet withdrawal restored the initial condition. Warfarin treatment, at a dose decreasing vitamin K-dependent factor to levels found in FNL, prolonged the OT and reduced fibrin deposition, without modifying F XII or changing lipid profile. An increase in the activated form of F VII was observed. In contrast, no difference was found in F VII clearance. High lipid levels favour the process of thrombus formation by increasing the activation of vitamin K-dependent coagulation factors. Low-dose warfarin treatment reverts the prothrombotic effect of hyperlipidemia.

  10. Stable high-level expression of factor VIII in Chinese hamster ovary cells in improved elongation factor-1 alpha-based system.

    Science.gov (United States)

    Orlova, Nadezhda A; Kovnir, Sergey V; Gabibov, Alexandre G; Vorobiev, Ivan I

    2017-03-24

    Recombinant factor VIII (FVIII), used for haemophilia A therapy, is one of the most challenging among the therapeutic proteins produced in heterologous expression systems. Deletion variant of FVIII, in which the entire domain B is replaced by a short linker peptide, was approved for medical use. Efficacy and safety of this FVIII deletion variant are similar to full-length FVIII preparations while the level of production in CHO cells is substantially higher. Typical levels of productivity for CHO cell lines producing deletion variant FVIII-BDD SQ, described elsewhere, are 0.5-2 IU/ml, corresponding to the concentration of FVIII of about 0.2 μg/ml. Using standard vectors based on the cytomegalovirus promoter (CMV) and the dihydrofolate reductase cDNA we have previously obtained the cell line secreting 0.5 IU/ml of FVIII-BDD, which roughly corresponds to the previously published data. An expression system based on CHO genomic sequences including CHO-EEF1A promoter and Epstein-Barr virus terminal repeat fragment allowed us to achieve 80-fold increase in the production level as compared with the conventional expression system based on the CMV promoter. Immediately after the primary selection FVIII -producing cells secreted 5-10 IU/ml of FVIII-BDD, and after multi-stage methotrexate-driven amplification a stable clonal line 11A4H was selected, secreting 39 IU/ml of FVIII-BDD in the simple batch culturing conditions, which considerably exceeds known indicators for industrial producers of this protein. In contrast to other FVIII-BDD producing lines 11A4H accumulates low proportion of the secreted FVIII on the membrane. Its productivity may be further increased approximately two-fold by adding sodium butyrate and butylated hydroxyanisol to the culture medium. A five-stage purification process for the factor VIII was employed. It allowed isolation of the intact FVIII-BDD as was confirmed by mass spectrometry. Purified FVIII-BDD has a specific activity of 11,000

  11. A computer-based model to assess costs associated with the use of factor VIII and factor IX one-stage and chromogenic activity assays.

    Science.gov (United States)

    Kitchen, S; Blakemore, J; Friedman, K D; Hart, D P; Ko, R H; Perry, D; Platton, S; Tan-Castillo, D; Young, G; Luddington, R J

    2016-04-01

    Measurement of coagulation factor factor VIII (FVIII) and factor IX (FIX) activity can be associated with a high level of variability using one-stage assays based on activated partial thromboplastin time (APTT). Chromogenic assays show less variability, but are less commonly used in clinical laboratories. In addition, one-stage assay accuracy using certain reagent and instrument combinations is compromised by some modified recombinant factor concentrates. Reluctance among some in the hematology laboratory community to adopt the use of chromogenic assays may be partly attributable to lack of familiarity and perceived higher associated costs. To identify and characterize key cost parameters associated with one-stage APTT and chromogenic assays for FVIII and FIX activity using a computer-based cost analysis model. A cost model for FVIII and FIX chromogenic assays relative to APTT assays was generated using assumptions derived from interviews with hematologists and laboratory scientists, common clinical laboratory practise, manufacturer list prices and assay kit configurations. Key factors that contribute to costs are factor-deficient plasma and kit reagents for one-stage and chromogenic assays, respectively. The stability of chromogenic assay kit reagents also limits the cost efficiency compared with APTT testing. Costs for chromogenic assays might be reduced by 50-75% using batch testing, aliquoting and freezing of kit reagents. Both batch testing and aliquoting of chromogenic kit reagents might improve cost efficiency for FVIII and FIX chromogenic assays, but would require validation. Laboratory validation and regulatory approval as well as education and training in the use of chromogenic assays might facilitate wider adoption by clinical laboratories. © 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

  12. Estudo citofotométrico da expressão do marcador tumoral Fator VIII e fatores prognósticos no adenocarcinoma gástrico Cytophotometric study of the expression of the tumoral marker Factor VIII and prognostic factors in gastrci adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Mary Tossa Nakamura

    2007-12-01

    ção III ou IV apresentaram índice de marcagem maiores do que aqueles com Bormann I ou II, porém sem correlação com a profundidade de invasão tumoral, grau de diferenciação, envolvimento nodal e padrão histológico. CONCLUSÕES: O presente estudo identificou e marcou 95,24% das amostras para o Fator VIII. Em relação aos fatores prognósticos não houve correlação significativa exceto entre o Fator VIII e a classificação de Bormann no qual o tipo III ou IV foi maior que o tipo I ou II.INTRODUCTION: Regarding gastric cancer, the incidence, diagnosis and therapeutic options showed improvement in the last decades, but prognosis remains gloomy, specially due the fact that most patients, already diagnosed present advanced tumors, metastatic and not liable to be surgically resected. Molecular biology is an area in science, which can give the answer to many questions and current scientific facts show that the this should be through detection of tumoral markers. The great advances in informatics refined cell image analysis by image cytophotometry makes it possible to study cell proliferation and angiogenesis in various tumor processes using immunohistochemistry and several markers. At present, studies are conducted to demonstrate the prognostic value of their expressions, however, in gastric adenocarcinoma the results have been divergent and studies are scarce. AIM: To identify and quantify the expression of cell proliferation markers using Ki-67 and of angiogenesis with Factor VIII in gastric adenocarcinoma using cytophotometry, and compare their expressions with factors such as Bormanns´ classification, tumor invasion depth, degree of differentiation, nodal involvement, histologic pattern and age. METHODS: Twenty-one patients with gastric adenocarcinoma identified between 1998 and 2006 were studied. Ki-67 and Factor VIII expressions were performed using immunohistochemistry with clone MIB-1 primary antibodies, monoclonal for Ki-67 and policlonal for Factor VIII

  13. Effects of oral and intramuscular vitamin K prophylaxis on vitamin K1, PIVKA-II, and clotting factors in breast fed infants.

    Science.gov (United States)

    Cornelissen, E A; Kollée, L A; De Abreu, R A; van Baal, J M; Motohara, K; Verbruggen, B; Monnens, L A

    1992-10-01

    A randomised clinical trial was conducted to establish the effects of oral and intramuscular administration of vitamin K at birth on plasma concentrations of vitamin K1, proteins induced by vitamin K absence (PIVKA-II), and clotting factors. Two groups of about 165 healthy breast fed infants who received at random 1 mg vitamin K1 orally or intramuscularly after birth were studied at 2 weeks and 1 and 3 months of age. Although vitamin K1 concentrations were statistically significantly higher in the intramuscular group, blood coagulability, activities of factors VII and X and PIVKA-II concentrations did not reveal any difference between the two groups. At 2 weeks of age vitamin K1 concentrations were raised compared with reported unsupplemented concentrations and no PIVKA-II was detectable. At 3 months vitamin K1 concentrations were back at unsupplemented values and PIVKA-II was detectable in 11.5% of infants. Therefore, a repeated oral prophylaxis will be necessary to completely prevent (biochemical) vitamin K deficiency beyond the age of 1 month.

  14. Blood clotting reactions on nanoscale phospholipid bilayers.

    Science.gov (United States)

    Morrissey, James H; Pureza, Vincent; Davis-Harrison, Rebecca L; Sligar, Stephen G; Ohkubo, Y Zenmei; Tajkhorshid, Emad

    2008-01-01

    Blood clotting reactions, such as those catalyzed by the tissue factor:factor VIIa complex (TF:FVIIa), assemble on membrane surfaces containing anionic phospholipids such as phosphatidylserine (PS). In fact, membrane binding is critical for the function of most of the steps in the blood clotting cascade. In spite of this, our understanding of how the membrane contributes to catalysis, or even how these proteins interact with phospholipids, is incomplete. Making matters more complicated, membranes containing mixtures of PS and neutral phospholipids are known to spontaneously form PS-rich membrane microdomains in the presence of plasma concentrations of calcium ions, and it is likely that blood-clotting proteases such as TF:FVIIa partition into these PS-rich microdomains. Unfortunately, little is known about how membrane microdomain composition influences the activity of blood-clotting proteases, which is typically not under experimental control even in "simple" model membranes. Our laboratories have developed and applied new technologies for studying membrane proteins to gain insights into how blood-clotting protease-cofactor pairs assemble and function on membrane surfaces. This includes using a novel, nanoscale bilayer system (Nanodiscs) that permits assembling blood-clotting protease-cofactor pairs on stable bilayers containing from 65 to 250 phospholipid molecules per leaflet. We have used this system to investigate how local (nanometer-scale) changes in phospholipid bilayer composition modulate TF:FVIIa activity. We have also used detailed molecular-dynamics simulations of nanoscale bilayers to provide atomic-scale predictions of how the membrane-binding domain of factor VIIa interacts with PS in membranes.

  15. Immune regulatory gene polymorphisms as predisposing risk factors for the development of factor VIII inhibitors in Indian severe haemophilia A patients.

    Science.gov (United States)

    Pinto, P; Ghosh, K; Shetty, S

    2012-09-01

    Development of inhibitors to factor VIII, a serious complication of replacement therapy in haemophilia A patients, leads to increased bleeding, morbidity and mortality. There is no data on the risk factors for inhibitor development in Indian patients with severe haemophilia A. Our aim was to study the role of immune regulatory gene polymorphisms in the development of inhibitors. Fourteen immune regulatory gene polymorphisms (IL1β, IL4, IL10, TNFA and CTLA4) were analysed in 120 patients with severe haemophilia A, i.e. 50 inhibitor positive patients, and 70 inhibitor negative control patients, by PCR-RFLP, DNA sequencing and allele-specific PCRs. The IL10 promoter 'GCC' haplotypes overall (P: 0.002, OR: 3.452, 95% CI: 1.607-7.416), and 'GCC/ATA' (P: 0.011, OR: 3.492, 95% CI: 1.402-8.696) haplotype, associated with high and intermediate IL10 production, respectively, were significantly higher in inhibitor positive patients, whereas the 'non-GCC' haplotypes overall (P: 0.002,OR: 0.290, 95% CI 0.135-0.622) and 'ATA/ATA' haplotype (P: 0.025, OR: 0.278, 95% CI: 0.096-0.802), associated with low IL10 synthesis, were significantly higher among inhibitor negative patients. The TNFA rs1799724 C/T heterozygote prevalence was significantly higher in the inhibitor positive group (P: 0.021, OR: 3.190, 95% CI: 1.273-7.990), whereas the other polymorphisms showed no statistically significant association with the presence of inhibitors. Different immune regulatory gene polymorphisms play a significant role as possible risk factors for the development of inhibitors in severe haemophilia A patients. © 2012 Blackwell Publishing Ltd.

  16. Challenges of the management of severe hemophilia A with inhibitors: two case reports emphasizing the potential interest of a high-purity human Factor VIII/von Willebrand factor concentrate and individually tailored prophylaxis guided by thrombin-generation test.

    Science.gov (United States)

    Mathieu, Sophie; Crampe, Carine; Dargaud, Yesim; Lavigne-Lissalde, Géraldine; Escuriola-Ettingshausen, Carmen; Tardy, Brigitte; Meley, Roland; Thouvenin, Sandrine; Stephan, Jean L; Berger, Claire

    2015-12-01

    Severe hemophilia A is an X-linked bleeding disorder. Immune tolerance induction (ITI) is the best strategy of treatment when patients develop inhibitors. The objective is to illustrate the benefit of a high-purity human factor VIII/von Willebrand factor (VWF) concentrate (Octanate) in the management of ITI. We also wanted to raise the potential interest of laboratory assays such as thrombin-generation test (TGT) and epitope mapping. Two patients were treated during ITI, first with a recombinant FVIII and then with plasma-derived factor VIII without success, and, finally, with Octanate. Bypassing agents were used based on the results of TGT. Epitope mapping was performed during ITI therapy. These observations suggest the potential contribution of Octanate in the management of ITI in difficult cases. The use of bypassing agents can be necessary in prophylaxis or to treat bleedings, and may be guided by TGT results. Epitope mapping is used to describe the inhibitor. This article shows a decrease of the inhibitor directed against the C2 domain after initiation of Octanate. A high-purity human factor VIII/von Willebrand factor concentrate (Octanate) may be a valuable therapeutical option for ITI therapy. TGT and epitope mapping could be of help in the management of ITI.

  17. Combination of recombinant factor VIIa and fibrinogen corrects clot formation in primary immune thrombocytopenia at very low platelet counts

    DEFF Research Database (Denmark)

    Larsen, Ole H; Stentoft, Jesper; Radia, Deepti

    2013-01-01

    Haemostatic treatment modalities alternative to platelet transfusion are desirable to control serious acute bleeds in primary immune thrombocytopenia (ITP). This study challenged the hypothesis that recombinant activated factor VII (rFVIIa) combined with fibrinogen concentrate may correct whole b...

  18. Distinct roles of Ser-764 and Lys-773 at the N terminus of von Willebrand factor in complex assembly with coagulation factor VIII.

    Science.gov (United States)

    Castro-Núñez, Lydia; Bloem, Esther; Boon-Spijker, Mariëtte G; van der Zwaan, Carmen; van den Biggelaar, Maartje; Mertens, Koen; Meijer, Alexander B

    2013-01-04

    Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766-Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764-Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism.

  19. Patient preference and ease of use for different coagulation factor VIII reconstitution device scenarios: a cross-sectional survey in five European countries

    Directory of Open Access Journals (Sweden)

    Cimino E

    2014-12-01

    Full Text Available Ernesto Cimino,1 Silvia Linari,2 Mara Malerba,3 Susan Halimeh,4 Francesca Biondo,5 Martina Westfeld5 1Dipartimento Medicina Clinica e Sperimentale, Universita’ degli Studi di Napoli Federico II, Naples, Italy; 2Agenzia per l’ Emofilia, AOU Careggi di Firenze, Florence, Italy; 3Fondazione Cà Granda Ospedale Maggiore Policlinico, Centro Emofilia e Trombosi “A Bianchi Bonomi”, Milan, Italy; 4CRC Coagulation Research Centre GmbH, Duisburg, Germany; 5Pfizer Italia, Rome, Italy Introduction: Hemophilia A treatment involves replacing the deficient coagulation factor VIII. This process may involve multiple steps that might create a barrier to adherence. A new dual-chamber syringe (DCS; FuseNGo® was recently introduced with the aim of simplifying reconstitution. Aim: This study aimed to identify factors associated with adult patients’ preferences for different coagulation factor VIII reconstitution systems and to test ease of use and patient preference for the DCS. Methods: A cross-sectional survey of adults with hemophilia A in five European countries was conducted; a subset of subjects also participated in a practical testing session of the DCS. Results: Among the 299 survey participants, the device scenario requiring the least equipment and reconstitution steps (the DCS received a median preference rating of 71 out of 100 (0 being “the least desirable” and 100 “the most desirable” rating. This was significantly higher than the other scenarios (the next highest achieved a median of 50 points; P<0.001. Participants would be more likely to use this device prophylactically (P<0.001. Among the 98 participants who tested the DCS, 57% preferred this device over their current device, 26% preferred their current device, and 17% had no preference. The DCS was rated as easier to use than current treatment devices (median score 9/10 versus 7/10 for current treatment, P=0.001. Conclusion: The survey indicates that the prefilled DCS, Fuse

  20. Recombinant factor VIIa improves clot formation but not fibrolytic potential in patients with cirrhosis and during liver transplantation

    NARCIS (Netherlands)

    Lisman, T; Leebeek, FWG; Meijer, K; Van Der Meer, J; Nieuwenhuis, HK; De Groot, PG

    2002-01-01

    Cirrhosis is associated with a bleeding tendency, which is particularly pronounced during orthotopic liver transplantation (OLT). A novel approach to treating the bleeding diathesis of patients with cirrhosis is administration of recombinant factor VIIa (rFVIIa). This study examined whether the effi

  1. Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

    Directory of Open Access Journals (Sweden)

    Marcela Cristina Correa de Freitas

    2014-06-01

    Full Text Available OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO and baby hamster kidney cells (BHK. Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

  2. The intron-22-inverted F8 locus permits factor VIII synthesis: explanation for low inhibitor risk and a role for pharmacogenomics.

    Science.gov (United States)

    Sauna, Zuben E; Lozier, Jay N; Kasper, Carol K; Yanover, Chen; Nichols, Timothy; Howard, Tom E

    2015-01-01

    Intron-22-inversion patients express the entire Factor VIII (FVIII)-amino-acid sequence intracellularly as 2 non-secreted polypeptides and have a positive "intracellular (I)-FVIII-CRM" status. Mutations conferring a positive I-FVIII-CRM status are associated with low inhibitor risk and are pharmacogenetically relevant because inhibitor risk may be affected by the nature of the therapeutic FVIII-protein (tFVIII), the affinity of any tFVIII-derived foreign peptide (tFVIII-fp) for any HLA class-II isomer (HLA-II) comprising individual major histocompatibility complex (MHC) repertoires, and the stability of any tFVIII-fp/HLA-II complex. We hypothesize that mutations conferring a completely or substantially negative I-FVIII-CRM status are pharmacogenetically irrelevant because inhibitor risk is high with any tFVIII and individual MHC repertoire.

  3. A novel mutation (4040-4045 nt. delA in exon 14 of the factor VIII gene causing severe hemophilia A

    Directory of Open Access Journals (Sweden)

    Habib Onsori

    2011-01-01

    Full Text Available Hemophilia A is an X-linked congenital bleeding disorder caused by Factor VIII deficiency. Different mutations including point mutations, deletions, insertions and inversions have been reported in the FVIII gene, which cause hemophilia A. In the current study, with the use of conformational sensitive gel electrophoresis (CSGE analysis, we report a novel 1-nt deletion in the A6 sequence at codons 1328-1330 (4040-4045 nt delA occurring in exon 14 of the FVIII gene in a seven-year-old Iranian boy with severe hemophilia A. This mutation that causes frameshift and premature stop-codon at 1331 has not previously been reported in the F8 Hemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS database.

  4. Kaolin clotting time.

    Science.gov (United States)

    Radhakrishnan, Kottayam

    2013-01-01

    The kaolin clotting time (KCT) is a sensitive test used in the laboratory detection of lupus anticoagulants (LA) (Derksen and de Groot, Thromb Res 114:521-526, 2004). It is essentially an activated partial thromboplastin time (APTT) test with no added phospholipid. Kaolin acts as the activator in the KCT. In the absence of additional phospholipid reagent, the quality of the test sample is extremely important since the generation of thrombin completely depends on the presence of residual cell membranes and plasma lipids (Derksen and de Groot, Thromb Res 114:521-526, 2004). Since the test contains no exogenous phospholipid, a confirmatory test using excess phospholipid is required to confirm the presence of lupus anticoagulant in the sample (Court, Br J Biomed Sci 54:287-298, 1997).

  5. Treatment of Knee Osteochondral Lesions Using a Novel Clot of Autologous Plasma Rich in Growth Factors Mixed with Healthy Hyaline Cartilage Chips and Intra-Articular Injection of PRGF.

    Science.gov (United States)

    Cugat, Ramón; Alentorn-Geli, Eduard; Steinbacher, Gilbert; Álvarez-Díaz, Pedro; Cuscó, Xavier; Seijas, Roberto; Barastegui, David; Navarro, Jordi; Laiz, Patricia; García-Balletbó, Montserrat

    2017-01-01

    Knee cartilage or osteochondral lesions are common and challenging injuries. To date, most symptomatic lesions warrant surgical treatment. We present two cases of patients with knee osteochondral defects treated with a one-step surgical procedure consisting of an autologous-based matrix composed of healthy hyaline cartilage chips, mixed plasma poor-rich in platelets clot, and plasma rich in growth factors (PRGF). Both patients returned to playing soccer at the preinjury activity level and demonstrated excellent defect filling in both magnetic resonance imaging and second-look arthroscopy (in one of them). The use of a clot of autologous plasma poor in platelets with healthy hyaline cartilage chips and intra-articular injection of plasma rich in platelets is an effective, easy, and cheap option to treat knee cartilage injuries in young and athletic patients.

  6. Histotripsy Thrombolysis on Retracted Clots.

    Science.gov (United States)

    Zhang, Xi; Owens, Gabe E; Cain, Charles A; Gurm, Hitinder S; Macoskey, Jonathan; Xu, Zhen

    2016-08-01

    Retracted blood clots have been previously recognized to be more resistant to drug-based thrombolysis methods, even with ultrasound and microbubble enhancements. Microtripsy, a new histotripsy approach, has been investigated as a non-invasive, drug-free and image-guided method that uses ultrasound to break up clots with improved treatment accuracy and a lower risk of vessel damage compared with the traditional histotripsy thrombolysis approach. Unlike drug-mediated thrombolysis, which is dependent on the permeation of the thrombolytic agents into the clot, microtripsy controls acoustic cavitation to fractionate clots. We hypothesize that microtripsy thrombolysis is effective on retracted clots and that the treatment efficacy can be enhanced using strategies incorporating electronic focal steering. To test our hypothesis, retracted clots were prepared in vitro and the mechanical properties were quantitatively characterized. Microtripsy thrombolysis was applied on the retracted clots in an in vitro flow model using three different strategies: single-focus, electronically-steered multi-focus and dual-pass multi-focus. Results show that microtripsy was used to successfully generate a flow channel through the retracted clot and the flow was restored. The multi-focus and the dual-pass treatments incorporating the electronic focal steering significantly increased the recanalized flow channel size compared to the single-focus treatments. The dual-pass treatments achieved a restored flow rate up to 324 mL/min without cavitation contacting the vessel wall. The clot debris particles generated from microtripsy thrombolysis remained within the safe range. The results of this study show the potential of microtripsy thrombolysis for retracted clot recanalization with the enhancement of electronic focal steering. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  7. A study of gene transfer and expression of human clotting factor IX in Hemophilia B mice mediated by mini-adenoviral vector

    Institute of Scientific and Technical Information of China (English)

    高啸波; 叶晨波; 侍鼎; 陈立; 邱信芳; 薛京伦

    2003-01-01

    Vector Gti'IX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi'IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein coding sequences deleted. Mini-Ad packaging cell 293Cre4 was first transduced with Gti'IX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTi'IX were obtained. At the same time, previous normal adenoviral vector pAdSPi'IX containing viral genome and hFIX mini-gene was constructed, and then previous adenovirus (pre-Ad) AdSPi'IX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTi'IX was less than 0.8%. 3T3 cells were transfected with AdGTi'IX and AdSPi'IX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 μg /106@24 h and 1.6±0.3 μg/106@24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 1×1010pfu AdGTi'IX or AdSPi'IX. The highest expression level of hFIX in mouse plasma was 590 ng/mL and 690 ng/mL respectively, and the expression time lasted for 16 weeks and 9 weeks respectively. The bleeding time reduced from over 30 min to 7.5 min, and 5-min blood lost reduced from 430 μL to 60 μL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTi'IX was obviously weaker than that triggered by AdSPi'IX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector system prolonged the expression time of hFIX and reduced immune response, thus offering a promising result for further pre-clinical study.

  8. Efficacy and safety of rVIII-Singlechain : Results of a phase 1/3 multicenter clinical trial in severe hemophilia A

    NARCIS (Netherlands)

    Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V.; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N.; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I.; Fischer, Kathelijn; Gill, Joan C.; P'Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A.; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; Ledger, Katie St; Pabinger, Ingrid

    2016-01-01

    Recombinant VIII (rVIII)-SingleChain is a novel B-domain–truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the eff

  9. Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice.

    Science.gov (United States)

    Peng, Baowei; Ye, Peiqing; Blazar, Bruce R; Freeman, Gordon J; Rawlings, David J; Ochs, Hans D; Miao, Carol H

    2008-09-01

    Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid-treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS(+)CD4(+) T cells and activation of CD25(+)Foxp3(+) Tregs in the peripheral blood, spleen, and lymph nodes. CD4(+) T cells from anti-ICOS-treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-beta. Moreover, CD4(+)CD25(+) Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS-treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Phix 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

  10. Successful immune tolerance induction with low-dose coagulation factor VIII in a patient with hemophilia A from a developing country.

    Science.gov (United States)

    Ay, Yilmaz; Ersin, Toret; Yesim, Oymak; Hilkay, Karapinar Tuba; Dilek, Ince; Gulcihan, Ozek; Ahmet, Koc

    2016-09-01

    Inhibitor development is the most frequent and serious complication of the treatment in patients with hemophilia. Immune tolerance induction (ITI) is the only option of treatment for the eradication of factor VIII (FVIII) inhibitor. We would like to present our case with hemophilia whose FVIII inhibitor eradication was done by a low-dose ITI regimen. Our patient has been applied on-demand therapy until 8 years of age. Secondary prophylaxis was began because of having hemophilic arthropathy. A low titer of FVIII inhibitor (4.2 BU/ml) was detected in the fifth month of the prophylaxis. The peak inhibitor titer of patient was 4.6 BU/ml, and there was no decrease in inhibitor titer in the follow-up duration. The low-dose ITI (50 IU/kg, 3 days a week) was started. His inhibitor level was detected negative and the recovery test was ameliorated in the 15th of the ITI therapy. High-dose regimen ITI could not be given particularly in developing countries such as Turkey in view of the high cost of treatment. Patients who had good risk factors might be successfully treated by using low-dose ITI regimen as effective as high-dose ITI regimen.

  11. An apparatus for measuring the tensile strength of blood clots.

    Science.gov (United States)

    MACFARLANE, R G; TOMLINSON, A H

    1961-05-01

    An apparatus is described which uses the principle of the ballistic pendulum to measure the tensile strength of blood clots formed in special cuvettes. The method appears to have a reproducibility which would allow a study to be made of the factors influencing clot strength. A brief survey of the findings with normal blood and with blood from cases of thrombosis and haemophilia shows significant differences between the mean values for each group.

  12. Delivery of full-length factor VIII using a piggyBac transposon vector to correct a mouse model of hemophilia A.

    Directory of Open Access Journals (Sweden)

    Hideto Matsui

    Full Text Available Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice. piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.

  13. Endogenous factor VIII synthesis from the intron 22-inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A.

    Science.gov (United States)

    Pandey, Gouri Shankar; Yanover, Chen; Miller-Jenkins, Lisa M; Garfield, Susan; Cole, Shelley A; Curran, Joanne E; Moses, Eric K; Rydz, Natalia; Simhadri, Vijaya; Kimchi-Sarfaty, Chava; Lillicrap, David; Viel, Kevin R; Przytycka, Teresa M; Pierce, Glenn F; Howard, Tom E; Sauna, Zuben E

    2013-10-01

    Neutralizing antibodies (inhibitors) to replacement factor VIII (FVIII, either plasma derived or recombinant) impair the effective management of hemophilia A. Individuals with hemophilia A due to major deletions of the FVIII gene (F8) lack antigenically cross-reactive material in their plasma ("CRM-negative"), and the prevalence of inhibitors in these individuals may be as high as 90%. Conversely, individuals with hemophilia A caused by F8 missense mutations are CRM-positive, and their overall prevalence of inhibitors is hemophilia A) have been grouped with the former on the basis of their genetic defect and CRM-negative status. However, only ∼20% of these individuals develop inhibitors. Here we demonstrate that the levels of F8 mRNA and intracellular FVIII protein in B lymphoblastoid cells and liver biopsies from individuals with the intron 22 inversion are comparable to those in healthy controls. These results support the hypothesis that most individuals with the intron 22 inversion are tolerized to FVIII and thus do not develop inhibitors. Furthermore, we developed a new pharmacogenetic algorithm that permits the stratification of inhibitor risk for individuals and subpopulations by predicting the immunogenicity of replacement FVIII using, as input, the number of putative T cell epitopes in the infused protein and the competence of major histocompatibility complex class II molecules to present such epitopes. This algorithm showed statistically significant accuracy in predicting the presence of inhibitors in 25 unrelated individuals with the intron 22 inversion.

  14. Delivery of full-length factor VIII using a piggyBac transposon vector to correct a mouse model of hemophilia A.

    Science.gov (United States)

    Matsui, Hideto; Fujimoto, Naoko; Sasakawa, Noriko; Ohinata, Yasuhide; Shima, Midori; Yamanaka, Shinya; Sugimoto, Mitsuhiko; Hotta, Akitsu

    2014-01-01

    Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice. piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.

  15. The effect of factor VIII deficiencies and replacement and bypass therapies on thrombus formation under venous flow conditions in microfluidic and computational models.

    Directory of Open Access Journals (Sweden)

    Abimbola A Onasoga-Jarvis

    Full Text Available Clinical evidence suggests that individuals with factor VIII (FVIII deficiency (hemophilia A are protected against venous thrombosis, but treatment with recombinant proteins can increase their risk for thrombosis. In this study we examined the dynamics of thrombus formation in individuals with hemophilia A and their response to replacement and bypass therapies under venous flow conditions. Fibrin and platelet accumulation were measured in microfluidic flow assays on a TF-rich surface at a shear rate of 100 s⁻¹. Thrombin generation was calculated with a computational spatial-temporal model of thrombus formation. Mild FVIII deficiencies (5-30% normal levels could support fibrin fiber formation, while severe (1 nM, but too low to support fibrin formation (<10 nM. In the absence of platelets, fibrin formation was not supported even at normal FVIII levels, suggesting platelet adhesion is necessary for fibrin formation. Individuals treated by replacement therapy, recombinant FVIII, showed normalized fibrin formation. Individuals treated with bypass therapy, recombinant FVIIa, had a reduced lag time in fibrin formation, as well as elevated fibrin accumulation compared to healthy controls. Treatment of rFVIIa, but not rFVIII, resulted in significant changes in fibrin dynamics that could lead to a prothrombotic state.

  16. Early eradication of factor VIII inhibitor in patients with congenital hemophilia A by immune tolerance induction with a high dose of immunoglobulin.

    Science.gov (United States)

    Mizoguchi, Yoko; Furue, Aya; Kagawa, Reiko; Chijimatsu, Ikue; Tomioka, Keita; Shimomura, Maiko; Imanaka, Yusuke; Nishimura, Shiho; Saito, Satoshi; Miki, Mizuka; Ono, Atsushi; Konishi, Nakao; Kawaguchi, Hiroshi; Kobayashi, Masao

    2016-04-01

    The production of factor VIII (FVIII) inhibitory antibodies is a serious problem in patients with hemophilia A. Immune tolerance induction (ITI) is the only strategy proven to eradicate persistent inhibitors and has been shown to be successful in 70 % of patients with hemophilia A. However, a minority of hemophilia patients present life-long inhibitors. To eliminate such inhibitors, we designed an intravenous immunoglobulin (IVIG) strategy in combination with high dose recombinant FVIII for ITI in hemophilia A children with inhibitors. Four previously untreated patients produced inhibitors within 16 exposures to FVIII. The peak inhibitor titers in these patients ranged from 3 to 14 BU/mL. The patients received ITI combined with IVIG within 1.5 months after the inhibitors were detected. All patients showed a negative titer for inhibitors by 28 days, with no anamnestic responses. The recovery of FVIII in the plasma concentration was normalized within three months after initiation of ITI. An additional course of IVIG administration led to induction of complete tolerance by 20 months after initiation of ITI therapy in all patients. ITI treatment with high-dose FVIII combined with IVIG may be effective for the early elimination of inhibitors.

  17. The Factor VIII Mutation Database on the World Wide Web: the haemophilia A mutation, search, test and resource site. HAMSTeRS update (version 3.0).

    Science.gov (United States)

    Kemball-Cook, G; Tuddenham, E G

    1997-01-01

    The HAMSTeRS WWW site was set up in 1996 in order to facilitate easy access to, and aid understanding of, the causes of haemophilia A at the molecular level; previously, the first and second text editions of the database have been published in Nucleic Acids Research. This report describes the facilities originally available at the site and the recent additions which we have made to increase its usefulness to clinicians, the molecular genetics community and structural biologists interested in factor VIII. The database (version 3.0) has been completely updated with easy submission of point mutations, deletions and insertions via e-mail of custom-designed forms. The searching of point mutations in the database has been made simpler and more robust, with a concomitantly expanded real-time bioinformatic analysis of the database. A methods section devoted to mutation detection has been added, highlighting issues such as choice of technique and PCR primer sequences. Finally, a FVIII structure section gives access to 3D VRML (Virtual Reality Modelling Language) files for any user-definable residue in a FVIII A domain homology model based on the crystal structure of human caeruloplasmin, together with secondary structural data and a sound+video animation of the model. It is intended that the general availability of this model will assist both in interpretation of causative mutations and selection of candidate residues forin vitromutagenesis. The HAMSTeRS URL is http://europium.mrc.rpms.ac.uk.

  18. Elective surgery on factor VIII inhibitor patients using continuous infusion of recombinant activated factor VII: plasma factor VII activity of 10 IU/ml is associated with an increased incidence of bleeding.

    Science.gov (United States)

    Smith, M P; Ludlam, C A; Collins, P W; Hay, C R; Wilde, J T; Grigeri, A; Melsen, T; Savidge, G F

    2001-10-01

    We examined recombinant activated factor VII (rVIIa) administered by continuous infusion to eight patients with inhibitors to factor VIII, undergoing elective surgery. rVIIa was infused at a fixed rate of 16.5 microg/kg/h for a median of 13.5 days (range 1-26). There was effective haemostasis at this infusion rate in only one of two minor procedures and two of six major operations. Three patients experienced excessive bleeding despite plasma factor VII activity around 10 IU/ml. Serious bleeding occurred in two other patients caused by procedural errors unrelated to rVIIa and required re-operation. The median rVIIa clearance on day 1 was 57 ml/h/kg (range 18-100) and on day 3 was 100 ml/h/kg (range 61-200). Clearance on the final infusion day was not significantly different from day 3. The infusion did not induce pathological activation of the coagulation mechanism. The only thrombotic adverse events were two episodes of superficial thrombophlebitis of the infused vein in one subject. In conclusion, the 16.5 microg/kg/h infusion rate reliably achieves plasma factor VII activity levels of 10 IU/ml, but this level does not provide reliable haemostasis.

  19. Efficacy and safety of BAY 81-8973, a full-length recombinant factor VIII: results from the LEOPOLD I trial.

    Science.gov (United States)

    Saxena, K; Lalezari, S; Oldenburg, J; Tseneklidou-Stoeter, D; Beckmann, H; Yoon, M; Maas Enriquez, M

    2016-09-01

    BAY 81-8973 (Kovaltry(®) ) is a full-length, unmodified recombinant human factor VIII (FVIII) with the same amino acid sequence as sucrose-formulated recombinant FVIII and is produced using additional advanced manufacturing technologies. To demonstrate efficacy and safety of BAY 81-8973 for treatment of bleeds and as prophylaxis based on two different potency assignments. In LEOPOLD I (ClinicalTrials.gov identifier, NCT01029340), males aged 12-65 years with severe haemophilia A and ≥150 exposure days received BAY 81-8973 20-50 IU kg(-1) two or three times per week for 12 months. Potency was based on chromogenic substrate assay per European Pharmacopoeia and label adjusted to mimic one-stage assay potency. Patients were randomized for potency sequence and crossed over potency groups after 6 months, followed by an optional 12-month extension. Primary efficacy endpoint was annualized bleeding rate (ABR). Patients also received BAY 81-8973 during major surgeries. Sixty-two patients received BAY 81-8973 prophylaxis and were included in the analysis. Median ABR was 1.0 (quartile 1, 0; quartile 3, 5.1) without clinically relevant differences between potency periods. Median ABR was similar for twice-weekly vs. three times-weekly dosing (1.0 vs. 2.0). Haemostasis was maintained during 12 major surgeries. Treatment-related adverse event (AE) incidence was ≤7% overall; no patient developed inhibitors. One patient with risk factors for cardiovascular disease developed a myocardial infarction. BAY 81-8973 was efficacious in preventing and treating bleeding episodes, irrespective of the potency assignment method, with few treatment-related AEs. Caution should be used when treating older patients with cardiovascular risk factors. © 2016 Bayer. Haemophilia Published by John Wiley & Sons Ltd.

  20. Photoionization of Ar VIII

    Science.gov (United States)

    Liang, Liang; Jiang, Wen-xian; Zhou, Chao

    2017-01-01

    The photoionization cross section, energy levels and widths of 22 Rydberg series (in the autoionization region) for Na-like Ar VIII were investigated by using of R-matrix method. The relativistic distorted-wave method is used to calculate the radial functions, and QB method of Quigly-Berrington [Quigley et al. 1998] is employed to calculate the resonance levels and widths. We have identified the formant in the figure of the photoionization cross section.

  1. Influence of factor VIII level and its inhibitor titer on the therapeutic response to corticosteroids alone in the management of acquired hemophilia

    Science.gov (United States)

    Vautier, Mathieu; de Boysson, Hubert; Creveuil, Christian; Repesse, Yohan; Borel-Derlon, Annie; Troussard, Xavier; Damaj, Gandhi L.; Bienvenu, Boris; Gautier, Philippe; Aouba, Achille

    2016-01-01

    Abstract The treatment of acquired hemophilia (AH) involves discussing whether corticosteroids should be administered alone or combined with immunosuppressant drugs, which increase the risk of infection especially in elderly patients and/or those with autoimmunity or neoplastic diseases, who represent the target population of the disease. Prognostic factors highlighting adequate responses to corticosteroids alone must be identified for satisfactory clinical response and lower infectious risk. We aimed to evaluating the efficacy of corticosteroids alone in the management of AH depending on factor VIII (FVIII, ≥ or 20 Bethesda units per milliliter [BU/mL]) titer. We conducted a retrospective single-center study including 24 patients treated for AH with corticosteroids alone. Time to achieve partial remission (PR: absence of hemorrhage and FVIII levels >50 IU/dL) was significantly shorter in the FVIII ≥ 1 IU/dL group than in the FVIII  20 BU/mL group (15 [11–35] vs 41 [20–207] days, P = 0.003). In both subgroups, time to achieve complete remission (CR: negative INH and corticosteroids below 10 mg/d) was also significantly shorter than that observed in the opposite subgroups. INH titer, considered alone, did not affect the length of time to onset of PR or CR. CR and PR rates did not differ significantly depending on these variables. Our study suggests that in AH, patients with FVIII levels ≥1 IU/dL considered alone or combined with INH titer ≤20 BU/mL could be treated by corticosteroids alone, given that this subgroup of patients displayed faster therapeutic responses to this strategy. PMID:27902587

  2. [Relationship between factor VIII inhibitor development and polymorphisms of TNFα and CTLA-4 gene in Chinese Han patients with hemophilia A].

    Science.gov (United States)

    Zhang, Lu-lu; Yu, Zi-qiang; Zhang, Wei; Cao, Li-juan; Su, Jian; Bai, Xia; Ruan, Chang-geng

    2011-03-01

    To investigate the potential association between factor VIII inhibitor development and polymorphisms of tumor necrosis factor-α (TNF-α)-308 and cytotoxic T-lymphocyte associated protein-4 gene in Chinese Han patients with hemophilia A (HA). The single base change polymorphism in TNF-α and CTLA-4 gene was analyzed in 140 Chinese Han patients with hemophilia A who have been treated with plasma-derived FVIII concentrates and 108 normal controls by using PCR-restrictive fragment length polymorphism (RFLP). All of the HA patients' plasma samples were measured by modified-Nijmegen assay simultaneously. In HA patients, G/G genotype, G/A genotype and A/A genotype were detected in 118 (84.3%), 18 (12.8%) and 4 cases (2.9%) respectively; C/C genotype, C/T genotype and T/T genotype were detected in 108 (77.2%), 30 (21.4%) and 2 cases (1.4%) respectively. The difference in the genotype frequencies between HA patients and controls was nonsignificant (P > 0.05). Patients who were carriers of homozygotes for A allele had a higher risk of inhibitor development compared with those who were not (OR = 7.519, 95% CI = 3.168 - 17.844). Severe HA patients who were carriers of homozygotes for A allele had a higher risk of inhibitor development compared with those who were not (OR = 8.163, 95% CI = 2.521 - 26.434). There was no statistical difference in the risk of inhibitor development between the patients who were carriers or not (OR = 1.586, 95% CI = 0.729 - 3.450). TNF-α-308 gene polymorphism is significantly associated with inhibitor development in Chinese Han patients with severe hemophilia A. TNF-α gene may be a useful marker and potential modulator of the immune response to replacement therapy for hemophilia A patients.

  3. Six amino acid residues in a 1200 A2 interface mediate binding of factor VIII to an IgG4κ inhibitory antibody.

    Directory of Open Access Journals (Sweden)

    Jasper C Lin

    Full Text Available The development of neutralizing anti-factor VIII (FVIII antibodies complicates the treatment of many hemophilia A patients. The C-terminal C2 domain is a particularly antigenic FVIII region. A crystal structure of recombinant FVIII-C2 bound to an Fab fragment of the patient-derived monoclonal antibody BO2C11, which recognizes an immunodominant inhibitor epitope on FVIII and blocks its ability to bind von Willebrand factor (VWF and phospholipids, revealed that 15 amino acids in FVIII contact this antibody. Forty-three recombinant FVIII-C2 proteins, each with a surface-exposed side chain mutated to alanine or another residue, were generated, and surface plasmon resonance studies were carried out to evaluate effects of these substitutions on BO2C11/FVIII-C2 binding affinity. Thermodynamic analysis of experiments carried out at three temperatures indicated that one beta hairpin turn at the antigen-antibody interface (FVIII-F2196, N2198, M2199 and F2200 plus two non-contiguous arginines (FVIII-R2215 and R2220, contributed appreciably to the affinity. B-domain-deleted (BDD FVIII-F2196A, FVIII-F2196K and FVIII-M2199A were generated and characterized. Their pro-coagulant activities and binding to VWF were similar to those of WT-BDD-FVIII, and FVIII-F2196K avoided neutralization by BO2C11 and murine inhibitory mAb 1B5. This study suggests specific sites for amino acid substitutions to rationally design FVIII variants capable of evading immunodominant neutralizing anti-FVIII antibodies.

  4. Assessment of the frequency of regulatory T cells (CD4+CD25+CD127-) in children with hemophilia A: relation to factor VIII inhibitors and disease severity.

    Science.gov (United States)

    El-Asrar, Mohamed Abo; Hamed, Ahmed El-Saeed; Darwish, Yasser Wagih; Ismail, Eman Abdel Rahman; Ismail, Noha Ali

    2016-01-01

    A rapidly growing evidence showed that regulatory T cells (Tregs) play a crucial role in tolerance to coagulation factors and may be involved in the pathogenesis of inhibitor formation in patients with hemophilia. We determined the percentage of Tregs (CD4CD25CD127) in 45 children with hemophilia A compared with 45 healthy controls, and assessed their relation to the clinical characteristics of patients and factor VIII (FVIII) inhibitors. Patients were studied stressing on frequency of bleeding attacks, joint pain, history of viral hepatitis, and the received therapy (FVIII precipitate/cryotherapy). FVIII activity and FVIII inhibitors were assessed with flow cytometric analysis of CD4CD25CD127 Tregs. According to residual FVIII activity levels, 30 patients (66.7%) had mild/moderate hemophilia A, whereas 15 (33.3%) patients had severe hemophilia A. The frequency of Tregs was significantly lower among all patients with hemophilia A compared with controls (2.59 ± 1.1 versus 3.73 ± 1.12%; P = 0.002). Tregs were significantly decreased among patients with FVIII inhibitors compared with the inhibitor-negative group (P hemophilia A had lower Tregs levels than those without (P = 0.34 and P = 0.011, respectively). A significant positive correlation was found between the percentage of Tregs and FVIII among hemophilia A patients. ROC curve analysis revealed that the cut-off value of Tregs at 1.91% could differentiate patients with and without FVIII inhibitors, with a sensitivity of 100% and a specificity of 91.3%. We suggest that alteration in the frequency of Tregs in young patients with hemophilia A may contribute to inhibitor formation and disease severity.

  5. Seroprevalence of parvovirus B19, cytomegalovirus, hepatitis A virus and hepatitis E virus antibodies in haemophiliacs treated exclusively with clotting-factor concentrates considered safe against human immunodeficiency and hepatitis C viruses.

    Science.gov (United States)

    Flores, G; Juárez, J C; Montoro, J B; Tusell, J M; Altisent, C; Juste, C; Jardí, R

    1995-04-01

    Clotting-factor concentrates (CFC) are a potential source of transmission of blood-borne viruses. Newer physical and chemical methods (pasteurization, wet-heating, solvent/detergent treating) developed to inactivate viruses are effective against HIV, HBV and HCV. However, it is not clear if these methods protect against other pathogenic viruses such as parvovirus B19, cytomegalovirus (CMV), hepatitis A virus (HAV) and hepatitis E virus (HEV). To evaluate the safety of current CFC we have studied seroprevalence of parovirus B19, CMV, HAV and HEV antibodies in 22 HIV and HCV negative haemophiliacs who were treated exclusively with clotting-factor concentrates considered safe with respect to HIV and HCV transmission, 22 healthy individuals served as controls. Neither HAV nor HEV antibodies were detected in haemophiliacs or controls. Two controls and two haemophiliacs were seropositive for CMV. Five controls (32% prevalence) and 15 haemophiliacs (77%) were positive to parovirus B19. No statistical differences can be established for seropositivity with CMV, HAV and HEV between haemophilic patients and controls. In the case of parvovirus B19 the differences are statistically significant (P= 0.0128). The relative risk of parvovirus B19 is 2.4 in the case of haemophiliacs. CFC considered safe against HIV and HCV are not safe against parvovirus B19, although they seem to be safe against CMV, HAV and HEV.

  6. Experimental study of thawed cryoprecipitate clotting factor change%冷沉淀解冻后凝血因子变化的实验研究

    Institute of Scientific and Technical Information of China (English)

    梁其隆; 梁若鹄; 陈龙菊; 甘芳香; 符雪丽; 黄彦; 车火娇

    2012-01-01

    Objective To explore the changes of clotting factor activity of frozen cryoprecipitate after thawing in different temperature and time, which provided theory basis for clinical use of cryoprecipitate. Methods 12 samples of fresh frozen plasma were selected randomly, cryoprecipitate prepared by centrifugal method. Each bag of cryoprecipitate after mixing immediately to save tube distributed into multiple samples (2 mL/tube), frozen in -30X1. The sample was divided into two groups after melting at 37X1. One group was stored at 4X and the other was stored at 24X1. PT, APTT, TT, FIB, F II, F V, FVH, FVH, FIX and FXI were measured respectively at the time of 0, 3, 6, 12, 24, 36, 48 and 60 h. Results The changes of PT, APTT, TT, FIB, FV, FIX and FXI were not significant different (P > 0.05), after thawing cryoprecipitate stored at different temperatures. But significant changes were observed in F II , FVH and FVH (P < 0.01). Two groups APTT changes were observed over 48 h storage period at 4X and 24T! (P < 0.05). Both group activity of FVH were significantly reduced at 4X for 6 h and 24T! For 36 h (P < 0.01). FW reduced faster stored at 4X1 compared with 24T! Group. And other indicators changed no significant in both temperatures after 60 h. Conclusion Except for FW, the other Coagulation factors activity do not change significantly for thawed cryoprecipitate after stored 60 h. In the clinical infusion, thawed cryoprecipitate in 60 h could be used to infuse exception of FW.%目的 探讨冰冻冷沉淀解冻融化后,在不同温度、不同时间凝血因子活性的变化,为临床使用冷沉淀提供理论依据.方法 随机抽取12袋(200 mL/袋)新鲜冰冻血浆,用离心法制备冷沉淀12袋,每袋冷沉淀混匀后立即用保存管分装成多个样本(2 mL/管),速冻后-30℃保存.样本在37℃融化后分成两组,一组置于4℃保存,另一组置于24℃保存,两组分别于0、3、6、12、24、36、48、60 h进行PT、APTT、TT、FIB、FⅡ、F

  7. Novel, human cell line-derived recombinant factor VIII (Human-cl rhFVIII, Nuwiq(®) ) in children with severe haemophilia A: efficacy, safety and pharmacokinetics.

    Science.gov (United States)

    Klukowska, A; Szczepański, T; Vdovin, V; Knaub, S; Jansen, M; Liesner, R

    2015-09-14

    Nuwiq(®) (Human-cl rhFVIII) is a new-generation recombinant factor VIII (rFVIII) protein, without chemical modification or fusion to any other protein, produced in a human cell line. This prospective, open-label, multinational phase III study assessed the efficacy and safety of Human-cl rhFVIII in 59 previously treated patients (PTPs) with severe haemophilia A aged 2-12 years (2-5 [N = 29]; 6-12 [N = 30]) during standard prophylaxis (≥50 exposure days and ≥6 months). Efficacy in treating breakthrough bleeds and during surgical prophylaxis was also assessed. An initial pharmacokinetic assessment (N = 13 per age subgroup) demonstrated comparable results with the one-stage and chromogenic assays. Mean (SD) half-life was 11.9 (5.4) and 13.1 (2.6) hours in children aged 2-5 years and 6-12 years respectively (one-stage assay). Prophylactic efficacy, based on mean monthly bleeding rate, was 'excellent' or 'good' in 91.5% of children for all bleeds and in 96.6% of children for spontaneous bleeds. Mean (SD) annualized bleeding rate was 4.12 (5.22) [median 1.9] for all bleeds, 1.50 (3.32) [median 0] for spontaneous bleeds and 2.34 (3.54) [median 1.57] for traumatic bleeds. There were no major, life-threatening bleeds. Efficacy was 'excellent' or 'good' in the treatment of 82.4% of breakthrough bleeds. Overall efficacy during five major surgeries was rated as 'excellent'. There were no FVIII inhibitors or treatment-related serious adverse events. These results in paediatric PTPs indicate that Human-cl rhFVIII is effective for the prevention and treatment of bleeds. © 2015 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

  8. Anti-CD20 as the B cells targeting agent in the combined therapy to modulate anti-factor VIII immune responses in hemophilia A inhibitor mice

    Directory of Open Access Journals (Sweden)

    Chao Lien eLiu

    2014-01-01

    Full Text Available Neutralizing antibody formation against transgene products can represent a major complication following gene therapy with treatment of genetic diseases, such as hemophilia A. Although successful approaches have been developed to prevent the formation of anti-factor VIII (FVIII antibodies, innovative strategies to overcome pre-existing anti-FVIII immune responses in FVIII-primed subjects are still lacking. Anti-FVIII neutralizing antibodies circulate for long periods in part due to persistence of memory B cells. Anti-CD20 targets a variety of B cells (pre-B cells to mature/memory cells; therefore, we investigated the impact of B cell depletion on anti-FVIII immune responses in hemophilia A mice using anti-CD20 combined with regulatory T (Treg cell expansion using IL-2/IL-2mAb complexes plus rapamycin. We found that anti-CD20 alone can partially modulate anti-FVIII immune responses in both unprimed and FVIII-primed hemophilia A mice. Moreover, in mice treated with anti-CD20 + IL-2/IL-2mAb complexes + rapamycin + FVIII, anti-FVIII antibody titers were significantly reduced in comparison to mice treated with regimens targeting only B or T cells. In addition, titers remained low after a second challenge with FVIII plasmid . Treg cells and activation markers were transiently and significantly increased in the groups treated with IL-2/IL-2mAb complexes ; however,significant B cell depletion was obtained in anti-CD20-treated groups. Importantly, both FVIII-specific antibody-secreting cells and memory B cells were significantly reduced in mice treated with combination therapy. This study demonstrates that a combination regimen is highly promising as a treatment option for modulating anti-FVIII antibodies and facilitating induction of long-term tolerance to FVIII in hemophilia A mice.

  9. Lentivirus-mediated platelet gene therapy of murine hemophilia A with pre-existing anti-factor VIII immunity.

    Science.gov (United States)

    Kuether, E L; Schroeder, J A; Fahs, S A; Cooley, B C; Chen, Y; Montgomery, R R; Wilcox, D A; Shi, Q

    2012-08-01

    The development of inhibitory antibodies, referred to as inhibitors, against exogenous factor VIII in a significant subset of patients with hemophilia A remains a persistent challenge to the efficacy of protein replacement therapy. Our previous studies using the transgenic approach provided proof-of-principle that platelet-specific expression could be successful in treating hemophilia A in the presence of inhibitory antibodies. To investigate a clinically translatable approach for platelet gene therapy of hemophilia A with pre-existing inhibitors. Platelet FVIII expression in preimmunized FVIII(null) mice was introduced by transplantation of lentivirus-transduced bone marrow or enriched hematopoietic stem cells. FVIII expression was determined with a chromogenic assay. The transgene copy number per cell was quantitated with real-time PCR. Inhibitor titer was measured with the Bethesda assay. Phenotypic correction was assessed by the tail clipping assay and an electrolytically induced venous injury model. Integration sites were analyzed with linear amplification-mediated PCR. Therapeutic levels of platelet FVIII expression were sustained in the long term without evoking an anti-FVIII memory response in the transduced preimmunized recipients. The tail clip survival test and the electrolytic injury model confirmed that hemostasis was improved in the treated animals. Sequential bone marrow transplants showed sustained platelet FVIII expression resulting in phenotypic correction in preimmunized secondary and tertiary recipients. Lentivirus-mediated platelet-specific gene transfer improves hemostasis in mice with hemophilia A with pre-existing inhibitors, indicating that this approach may be a promising strategy for gene therapy of hemophilia A even in the high-risk setting of pre-existing inhibitory antibodies. © 2012 International Society on Thrombosis and Haemostasis.

  10. Genotyping of intron 22 inversion of factor VIII gene for diagnosis of hemophilia A by inverse-shifting polymerase chain reaction and capillary electrophoresis.

    Science.gov (United States)

    Pan, Tzu-Yu; Wang, Chun-Chi; Shih, Chi-Jen; Wu, Hui-Fen; Chiou, Shyh-Shin; Wu, Shou-Mei

    2014-09-01

    This is the first capillary electrophoresis (CE) analysis for diagnosis of hemophilia A (HA). The intron 22 inversion of factor VIII gene (F8) causes 40-50 % of severe bleeding disorder of HA in all human populations. Consequently, identification of the disease-causing mutations is becoming increasingly important for accurate genetic counseling and prenatal diagnosis. In this study, the key steps of inverse-shifting polymerase chain reaction (IS-PCR) and of short-end injection capillary electrophoresis were used for more specific and rapid genotyping of intron 22 inversion of F8. In IS-PCR, three specific primers were used to amplify 512-bp amplicon for wild type and 584-bp amplicon for patients with intron 22 inversion. The capillary gel electrophoresis (CGE) system was performed using 1× Tris-borate-EDTA (TBE) buffer containing 0.3 % (w/v) polyethylene oxide (PEO). The PCR amplicons were electrokinetically injected at 10 kV for 10 s at a temperature of 25 °C. The optimal short-end injection CGE was applied to detect the F8 gene of HA patients and carriers within 5 min. Intron 22 inversion was indeed found on some HA patients (13/35, 37.1 %). All genotyping results showed good agreement with DNA sequencing method and long-distance polymerase chain reaction (LD-PCR). The IS-PCR combined with short-end injection CGE method was feasible and efficient for intron 22 inversion screening of F8 in the HA populations.

  11. BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety.

    Science.gov (United States)

    Maas Enriquez, Monika; Thrift, John; Garger, Stephen; Katterle, Yvonne

    2016-11-01

    BAY 81-8973 is a full-length, unmodified recombinant human factor VIII (FVIII) approved for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as the currently marketed sucrose-formulated recombinant FVIII (rFVIII-FS) product and is produced using additional advanced manufacturing technologies. One of the key manufacturing advances for BAY 81-8973 is introduction of the gene for human heat shock protein 70 (HSP70) into the rFVIII-FS cell line. HSP70 facilitates proper folding of proteins, enhances cell survival by inhibiting apoptosis, and potentially impacts rFVIII glycosylation. HSP70 expression in the BAY 81-8973 cell line along with other manufacturing advances resulted in a higher-producing cell line and improvements in the pharmacokinetics of the final product as determined in clinical studies. HSP70 protein is not detected in the harvest or in the final BAY 81-8973 product. However, because this is a new process, clinical trial safety assessments included monitoring for anti-HSP70 antibodies. Most patients, across all age groups, had low levels of anti-HSP70 antibodies before exposure to the investigational product. During BAY 81-8973 treatment, 5% of patients had sporadic increases in anti-HSP70 antibody levels above a predefined threshold (cutoff value, 239 ng/mL). No clinical symptoms related to anti-HSP70 antibody development occurred. In conclusion, addition of HSP70 to the BAY 81-8973 cell line is an innovative technology for manufacturing rFVIII aimed at improving protein folding and expression. Improved pharmacokinetics and no effect on safety of BAY 81-8973 were observed in clinical trials in patients with hemophilia A. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Characterization of the anti-factor VIII immunoglobulin profile in patients with hemophilia A by use of a fluorescence-based immunoassay

    Science.gov (United States)

    Boylan, Brian; Rice, Anne S.; Dunn, Amy L.; Tarantino, Michael D.; Brettler, Doreen B.; Barrett, John C.; Miller, Connie H.

    2015-01-01

    Summary Background The development of neutralizing antibodies, referred to as inhibitors, against factor VIII (FVIII) is a major complication associated with FVIII infusion therapy for the treatment of hemophilia A (HA). Previous studies have shown that a subset of HA patients and a low percentage of healthy individuals harbor non-neutralizing anti-FVIII antibodies that do not elicit the clinical manifestations associated with inhibitor development. Objective Assess HA patients' anti-FVIII antibody profiles as potential predictors of clinical outcomes. Methods A fluorescence immunoassay (FLI) was used to detect anti-FVIII antibodies in 491 samples from 371 HA patients. Results Assessments of antibody profiles showed that the presence of anti-FVIII IgG1, IgG2, or IgG4 correlated qualitatively and quantitatively with the presence of a FVIII inhibitor as reported by the Nijmegen-Bethesda assay (NBA). Forty-eight patients with a negative inhibitor history contributed serial samples to the study, including seven patients who had negative NBA titers initially and later converted to NBA-positive. The FLI detected anti-FVIII IgG1 in five of those seven patients prior to their conversion to NBA-positive. Five of 15 serial-sample patients who had a negative inhibitor history and a positive anti-FVIII IgG1 later developed an inhibitor, compared to 2 of 33 patients with a negative inhibitor history without anti-FVIII IgG1. Conclusions These data provide a rationale for future studies designed both to monitor the dynamics of anti-FVIII antibody profiles in HA patients as a potential predictor of future inhibitor development and to assess the value of the anti-FVIII FLI as a supplement to traditional inhibitor testing. PMID:25354263

  13. The B-domain of factor VIII reduces cell membrane attachement to host cells in serum free conditions

    DEFF Research Database (Denmark)

    Kolind, Mille Petersen; Nørby, Peder Lisby; Flintegaard, Thomas Veje

    2010-01-01

    engineered extensively throughout the years to increase the low production yields that initially were obtained from mammalian cell cultures. The scope of this work was to investigate the interaction of rFVIII with the cell membrane surface of the producing cells in serum free medium. We wondered whether...... binding of rFVIII to the cell membrane could be a factor diminishing the production yield. We studied the contribution of the rFVIII B-domain to membrane attachment by transfecting several constructs containing increasing lengths of the B-domain into cells under serum free conditions. We found that 90......% of rFVIII is attached to the cell membrane of the producing cell when the rFVIII variant contains a short B-domain (21 aa). By increasing the length of the B-domain the membrane attached fraction can be reduced to 50% of the total expressed rFVIII. Further, our studies show that the N...

  14. Preventing Blood Clots After Orthopaedic Surgery

    Medline Plus

    Full Text Available ... blood clots during the first several weeks of recovery from surgery. Warning Signs of Blood Clots Pain ... herein. This information is provided as an educational service and is not intended to serve as medical ...

  15. Preventing Blood Clots After Orthopaedic Surgery

    Medline Plus

    Full Text Available ... Clots After Orthopaedic Surgery One of the major risks facing patients who undergo surgery is a complication ... on the legs and hip are especially at risk. A pulmonary embolism is a blood clot that ...

  16. Biological variation in tPA-induced plasma clot lysis time

    NARCIS (Netherlands)

    S. Talens (Simone); J.J.M.C. Malfliet (Joyce); G. Rudež (Goran); H.M.H. Spronk (Henri); N.A.H. Janssen (Nicole); P. Meijer (Piet); C. Kluft (Cornelius); M.P.M. de Maat (Moniek); D.C. Rijken (Dingeman)

    2012-01-01

    textabstractHypofibrinolysis is a risk factor for venous and arterial thrombosis, and can be assessed by using a turbidimetric tPA-induced clot lysis time (CLT) assay. Biological variation in clot lysis time may affect the interpretation and usefulness of CLT as a risk factor for thrombosis. Suffici

  17. Biological variation in tPA-induced plasma clot lysis time

    NARCIS (Netherlands)

    S. Talens (Simone); J.J.M.C. Malfliet (Joyce); G. Rudež (Goran); H.M.H. Spronk (Henri); N.A.H. Janssen (Nicole); P. Meijer (Piet); C. Kluft (Cornelius); M.P.M. de Maat (Moniek); D.C. Rijken (Dingeman)

    2012-01-01

    textabstractHypofibrinolysis is a risk factor for venous and arterial thrombosis, and can be assessed by using a turbidimetric tPA-induced clot lysis time (CLT) assay. Biological variation in clot lysis time may affect the interpretation and usefulness of CLT as a risk factor for thrombosis.

  18. Obtenção e caracterização de anticorpo monoclonal murino anti-fator VIII da coagulação sangüínea Attainment and characterization of murine monoclonal anti-factor VIII antibodies

    Directory of Open Access Journals (Sweden)

    Rosana Rossi-Ferreira

    2006-06-01

    Full Text Available Entre os avanços da engenharia celular e biotecnologia nas últimas décadas, destaca-se a produção de anticorpos monoclonais murinos (AcMm utilizados no aprimoramento diagnóstico nas rotinas laboratoriais. A produção de fator VIII de alta pureza sempre foi o desejo e a preocupação das indústrias de hemoderivados para tratamento de pacientes portadores de hemofilia A, porém este produto inexiste no Brasil, sendo necessária sua obtenção no mercado internacional a custos elevados. O trabalho tem por objetivo a produção de AcMm anti-fator VIII humano (FVIII H através da expansão dos clones e caracterização imunoquímica do anticorpo. Camundongos Balb/c foram imunizados com FVIII H purificado como também proveniente de crioprecipitado e as células esplênicas dos animais foram fusionadas com células mielomatosas murinas segundo o método descrito por Kohler e Milstein para produção de híbridos em cultura. Foram testados 1.983 híbridos dos quais 105 foram submetidos à clonagem. Destes, 39 obtiveram monoclonalidade e 7 destes clones foram caracterizados através de técnicas de immunoblotting. Foram submetidas à purificação por cromatografia três imunoglobulinas de diferentes classes pertencentes aos clones LAMB1-10A1A4, LAMB1-17A1A1 e LAMB1-24A2A1. A imunoglobulina purificada pertencente ao clone LAMB1-10A1A4 foi adsorvida em coluna de imunoafinidade para purificação de concentrado de FVIII proveniente de crioprecipitado plasmático.Among the advances in cellular engineering and biotechnology over the last decades, the production of murine monoclonal antibodies (AcMm, used to improve laboratory diagnoses, stands out. The production of very pure factor VIII has always been a concern of suppliers of blood products to treat patients with hemophilia A and this product is still not produced in Brazil. Hence, it can only be attained on the international market at a high cost. The aim of this work was to produce AcMm anti-factor

  19. Efficient transfer and expression of human clotting factor ⅨX cDNA in neonatal hemophilia B mice mediated by VSV-G pseudotyped retrovirus

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The feasibility of in vivo gene therapy for hemophilia B by VSV-G pseudotyped retroviral vector was introduced. The novel packaging cell line 293GPG was used to produce VSV-G/G1NaBAIX pseudotyped virus with the highest titers up to 8.5 × 10s cfu @ mL-1. In contrast to the conventional retrovirus, VSV-G pseudotyped virus was more resistant to inactivation by serum complements (P<0.001).Our results also demonstrated that VSV-G pseudotyped virus was more stable in neonatal mice serum than in adult mice serum (P<0.01). After intraperitoneal injection of different doses of virus, hFIX antigen was detected and lasted for more than 120 d, the highest level reached (72.5±6.1)ng@ mL-1. Moreover, the functional activity was improved to some extent in all hFIX-treated mice, the most remarkable improvement was observed in the mice treated with higher dose of virus whose clotting activity increased to (3.4 ±1.5) %and APTT (activated partial thromboplastin time) reducedto (43.2 ± 7.2) s. The anti-hFIX antibody was not detected by the method of Bethesda, no germ line transmission and any side effects associated with gene transfer were found. Our results indicated that neonatal gene therapy for hemophilia B mice by VSV-G pseudotyped retrovirus is promising.``

  20. Immune tolerance induced by platelet-targeted factor VIII gene therapy in hemophilia A mice is CD4 T cell mediated.

    Science.gov (United States)

    Chen, Y; Luo, X; Schroeder, J A; Chen, J; Baumgartner, C K; Hu, J; Shi, Q

    2017-10-01

    Essentials The immune response is a significant concern in gene therapy. Platelet-targeted gene therapy can restore hemostasis and induce immune tolerance. CD4 T cell compartment is tolerized after platelet gene therapy. Preconditioning regimen affects immune tolerance induction in platelet gene therapy. Background Immune responses are a major concern in gene therapy. Our previous studies demonstrated that platelet-targeted factor VIII (FVIII) (2bF8) gene therapy together with in vivo drug selection of transduced cells can rescue the bleeding diathesis and induce immune tolerance in FVIII(null) mice. Objective To investigate whether non-selectable 2bF8 lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and how immune tolerance is induced after 2bF8LV gene therapy. Methods Platelet-FVIII expression was introduced by 2bF8LV transduction and transplantation. FVIII assays and tail bleeding tests were used to confirm the success of platelet gene therapy. Animals were challenged with rhF8 to explore if immune tolerance was induced after gene therapy. Treg cell analysis, T-cell proliferation assay and memory B-cell-mediated ELISPOT assay were used to investigate the potential mechanisms of immune tolerance. Results We showed that platelet-FVIII expression was sustained and the bleeding diathesis was restored in FVIII(null) mice after 2bF8LV gene therapy. None of the transduced recipients developed anti-FVIII inhibitory antibodies in the groups preconditioned with 660 cGy irradiation or busulfan plus ATG treatment even after rhF8 challenge. Treg cells significantly increased in 2bF8LV-transduced recipients and the immune tolerance developed was transferable. CD4(+) T cells from treated animals failed to proliferate in response to rhF8 re-stimulation, but memory B cells could differentiate into antibody secreting cells in 2bF8LV-transduced recipients. Conclusion 2bF8LV gene transfer without in vivo selection of

  1. Clinical evaluation of recombinant factor VIII preparation (Kogenate) in previously treated patients with hemophilia A: descriptive meta-analysis of post-marketing study data.

    Science.gov (United States)

    Yoshioka, A; Fukutake, K; Takamatsu, J; Shirahata, A

    2006-08-01

    The safety and efficacy of Kogenate, a recombinant factor VIII (rFVIII) preparation for the treatment of bleeding episodes, were studied in a 123-patient meta-analysis population of previously treated patients (PTPs), including 15 enrolled in the registration Phase III trial (PTP-I group), 93 from the post-marketing special investigation (PTP-II group), and 15 from short-term special investigations in surgery or tooth extraction (SI group). These patients (82 severe, 31 moderate, 9 mild, and 1 unknown), aged 11 months to 72 years, were enrolled in 28 centers in Japan. Blood samples taken at the baseline and at 3, 6, 9, 12, 18, and 24 months after the introduction of Kogenate were evaluated for FVIII inhibitor antibodies, antibodies formed against trace proteins derived from the rFVIII production process, and for general changes in laboratory test results. Mean exposure to Kogenate was 1103 days in PTP-I, 86 days in PTP-II, 27 days in patients in surgery, and 2 days in patients with tooth extraction. Assessment of FVIII inhibitor activity was conducted in 115 of the 123 patients by means of the Bethesda assay. Twelve patients were found to have a low titer of FVIII inhibitor (0.5-3.0 BU/mL) prior to any administration of Kogenate, and 103 were inhibitor-negative at the baseline. Among this latter group, 3 patients (2.9%) tested inhibitor-positive, with titers ranging from 1.2 to 2.1 BU/mL, with 4 patients below 1.0 BU/mL. One patient in the 11 PTPs investigated (PTP-I) developed antibodies against baby hamster kidney protein and mouse immunoglobulin G, but these findings were transient and asymptomatic. Hemostasis was achieved (markedly effective or effective) in 3666 of the 3855 bleeding episodes (95.1%) observed in 108 patients. Only 1 infusion was necessary in 3790 (98.3%) of these episodes. These data indicate that Kogenate is safe and very effective for the treatment of bleeding in PTPs with hemophilia A.

  2. Characterization of the factor VIII defect in 147 patients with sporadic hemophilia A: Family studies indicate a mutation type-dependent sex ratio of mutation frequencies

    Energy Technology Data Exchange (ETDEWEB)

    Becker, J.; Schmidt, W.; Olek, K. [Univ. of Bonn (Germany)] [and others

    1996-04-01

    The clinical manifestation of hemophilia A is caused by a wide range of different mutations. In this study the factor VIII genes of 147 severe hemophilia A patients-all exclusively from sporadic families-were screened for mutations by use of the complete panel of modern DNA techniques. The pathogenous defect could be characterized in 126 patients (85.7%). Fifty-five patients (37.4%) showed a F8A-gene inversion, 47 (32.0%) a point mutation, 14 (9.5%) a small deletion, 8 (5.4%) a large deletion, and 2 (1.4%) a small insertion. Further, four (2.7%) mutations were localized but could not be sequenced yet. No mutation could be identified in 17 patients (11.6%). Sixteen (10.9%) of the P identified mutations occurred in the B domain. Four of these were located in an adenosine nucleotide stretch at codon 1192, indicating a mutation hotspot. Somatic mosaicisms were detected in 3 (3.9%) of 76 patients` mothers, comprising 3 of 16 de novo mutations in the patients` mothers. Investigation of family relatives allowed detection of a de novo mutation in 16 of 76 two-generation and 28 of 34 three-generation families. On the basis of these data, the male:female ratio of mutation frequencies (k) was estimated as k = 3.6. By use of the quotients of mutation origin in maternal grandfather to patient`s mother or to maternal grandmother, k was directly estimated as k = 15 and k = 7.5, respectively. Considering each mutation type separately, we revealed a mutation type-specific sex ratio of mutation frequencies. Point mutations showed a 5-to-10-fold-higher and inversions a >10-fold- higher mutation rate in male germ cells, whereas deletions showed a >5-fold-higher mutation rate in female germ cells. Consequently, and in accordance with the data of other diseases like Duchenne muscular dystrophy, our results indicate that at least for X-chromosomal disorders the male:female mutation rate of a disease is determined by its proportion of the different mutation types. 68 refs., 1 fig., 5 tabs.

  3. The impact of the centrifuge characteristics and centrifugation protocols on the cells, growth factors, and fibrin architecture of a leukocyte- and platelet-rich fibrin (L-PRF) clot and membrane.

    Science.gov (United States)

    Dohan Ehrenfest, David M; Pinto, Nelson R; Pereda, Andrea; Jiménez, Paula; Corso, Marco Del; Kang, Byung-Soo; Nally, Mauricio; Lanata, Nicole; Wang, Hom-Lay; Quirynen, Marc

    2017-04-24

    -PRF. All centrifugations were done using the original L-PRF centrifuge (Intra-Spin), as recommended by the two manufacturers. Half of the membranes were placed individually in culture media and transferred in a new tube at seven experimental times (up to 7 days). The releases of transforming growth factor β-1 (TGFβ-1), platelet derived growth factor AB (PDGF-AB), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2) were quantified using ELISA kits at these seven experimental times. The remaining membranes were used to evaluate the initial quantity of growth factors of the L-PRF and A-PRF membranes, through forcible extraction. Very significant differences in the level of vibrations at each rotational speed were observed between the four tested centrifuges. The original L-PRF centrifuge (Intra-Spin) was by far the most stable machine in all configurations and always remained under the threshold of resonance, unlike the three other tested machines. At the classical speed of production of L-PRF, the level of undesirable vibrations on the original centrifuge was between 4.5 and 6 times lower than with other centrifuges. Intra-Spin showed the lowest temperature of the tubes. A-PRF and Salvin were both associated with a significant increase in temperature in the tube. Intra-Spin produced the heaviest clot and quantity of exudate among the four techniques. A-PRF and LW produced much lighter, shorter and narrower clots and membranes than the two other centrifuges. Light microscopy analysis showed relatively similar features for all L-PRF types (concentration of cell bodies in the first half). However, SEM illustrated considerable differences between samples. The original Intra-Spin L-PRF showed a strongly polymerized thick fibrin matrix and all cells appeared alive with a normal shape, including the textured surface aspect of activated lymphocytes. The A-PRF, Salvin and LW PRF-like membranes presented a lightly polymerized slim fibrin gel and most

  4. First prospective report on immune tolerance in poor risk haemophilia A inhibitor patients with a single factor VIII/von Willebrand factor concentrate in an observational immune tolerance induction study.

    Science.gov (United States)

    Kreuz, W; Escuriola Ettingshausen, C; Vdovin, V; Zozulya, N; Plyushch, O; Svirin, P; Andreeva, T; Bubanská, E; Campos, M; Benedik-Dolničar, M; Jiménez-Yuste, V; Kitanovski, L; Klukowska, A; Momot, A; Osmulskaya, N; Prieto, M; Šalek, S Z; Velasco, F; Pavlova, A; Oldenburg, J; Knaub, S; Jansen, M; Belyanskaya, L; Walter, O

    2016-01-01

    Development of neutralizing inhibitors against factor VIII (FVIII) is a major complication of haemophilia A treatment. The ongoing, international, open-label, uncontrolled, observational immune tolerance induction (ObsITI) study evaluates ITI, the standard of care in patients with inhibitors. Forty-eight prospective patients in this interim analysis received a single plasma-derived, von Willebrand factor-stabilized, FVIII concentrate (pdFVIII/VWF) for ITI. According to recommended Bonn protocol, 'low responders' at ITI start (2 years since inhibitor diagnosis, inhibitor titre ≥10 BU at the start of ITI, or prior ITI failure). Nonetheless, 34 patients (70.8%) achieved complete success, 3 (6.3%) partial success, 1 (2.1%) partial response; ITI failed in 10 patients (20.8%), all with poor prognosis factors. All six low responders achieved complete success. ITI outcome was significantly associated with inhibitor titre level at ITI start (P = 0.0068), number of poor prognosis factors for ITI success (P = 0.0187), monthly bleeding rate during ITI (P = 0.0005) and peak inhibitor titre during ITI (P = 0.0007). Twenty-two of 35 high responder patients (62.9%) with ≥1 poor prognosis factor achieved complete success. Treatment with a single pdFVIII/VWF concentrate, mainly according to the Bonn protocol, resulted in a high ITI success rate in haemophilia A patients with inhibitors and poor prognosis for ITI success. © 2015 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

  5. A new device for measurement of fibrin clot lysis: application to the Euglobulin Clot Lysis Time

    Directory of Open Access Journals (Sweden)

    Brohée D

    2002-05-01

    Full Text Available Abstract Background Determination of clot lysis times on whole blood, diluted whole blood, plasma or plasma fraction has been used for many years to assess the overall activity of the fibrinolytic system. We designed a completely computerised semi-automatic 8-channel device for measurement and determination of fibrin clot lysis. The lysis time is evaluated by a mathematical analysis of the lysis curve and the results are expressed in minute (range: 5 to 9999. We have used this new device for Euglobulin Clot Lysis Time (ECLT determination, which is the most common test used in laboratories to estimate plasma fibrinolytic capacity. Results The correlation between ECLT and manual method is very tight : R = 0,99; p -6. The efficiency scores of the method are Conclusions The routine use of this new device could be useful in various situations such as assessment in atherosclerosis and arteriosclerosis associated diseases, coagulation survey of liver transplantations, cardiovascular surgery or pharmacological research. It has already provided highly promising results in preliminary studies on the relation between fibrinolysis and cardiovascular risk factors.

  6. A new device for measurement of fibrin clot lysis: application to the Euglobulin Clot Lysis Time

    Science.gov (United States)

    Boudjeltia, K Zouaoui; Cauchie, Ph; Remacle, Cl; Guillaume, M; Brohée, D; Hubert, JL; Vanhaeverbeek, M

    2002-01-01

    Background Determination of clot lysis times on whole blood, diluted whole blood, plasma or plasma fraction has been used for many years to assess the overall activity of the fibrinolytic system. We designed a completely computerised semi-automatic 8-channel device for measurement and determination of fibrin clot lysis. The lysis time is evaluated by a mathematical analysis of the lysis curve and the results are expressed in minute (range: 5 to 9999). We have used this new device for Euglobulin Clot Lysis Time (ECLT) determination, which is the most common test used in laboratories to estimate plasma fibrinolytic capacity. Results The correlation between ECLT and manual method is very tight : R = 0,99; p < 10-6. The efficiency scores of the method are <4% in intra-assay and <7% in inter-assay. It allows to achieve the tests on hyperlipaemic samples. This new device has been easily integrated in laboratory routine and allows to achieve several ECLT every day without disturbance of laboratory workflow. Conclusions The routine use of this new device could be useful in various situations such as assessment in atherosclerosis and arteriosclerosis associated diseases, coagulation survey of liver transplantations, cardiovascular surgery or pharmacological research. It has already provided highly promising results in preliminary studies on the relation between fibrinolysis and cardiovascular risk factors. PMID:11985782

  7. Fluid Mechanics of Blood Clot Formation.

    Science.gov (United States)

    Fogelson, Aaron L; Neeves, Keith B

    2015-01-01

    Intravascular blood clots form in an environment in which hydrodynamic forces dominate and in which fluid-mediated transport is the primary means of moving material. The clotting system has evolved to exploit fluid dynamic mechanisms and to overcome fluid dynamic challenges to ensure that clots that preserve vascular integrity can form over the wide range of flow conditions found in the circulation. Fluid-mediated interactions between the many large deformable red blood cells and the few small rigid platelets lead to high platelet concentrations near vessel walls where platelets contribute to clotting. Receptor-ligand pairs with diverse kinetic and mechanical characteristics work synergistically to arrest rapidly flowing cells on an injured vessel. Variations in hydrodynamic stresses switch on and off the function of key clotting polymers. Protein transport to, from, and within a developing clot determines whether and how fast it grows. We review ongoing experimental and modeling research to understand these and related phenomena.

  8. Polymorphisms in the F8 gene and MHC-II variants as risk factors for the development of inhibitory anti-factor VIII antibodies during the treatment of hemophilia a: a computational assessment.

    Directory of Open Access Journals (Sweden)

    Gouri Shankar Pandey

    Full Text Available The development of neutralizing anti-drug-antibodies to the Factor VIII protein-therapeutic is currently the most significant impediment to the effective management of hemophilia A. Common non-synonymous single nucleotide polymorphisms (ns-SNPs in the F8 gene occur as six haplotypes in the human population (denoted H1 to H6 of which H3 and H4 have been associated with an increased risk of developing anti-drug antibodies. There is evidence that CD4+ T-cell response is essential for the development of anti-drug antibodies and such a response requires the presentation of the peptides by the MHC-class-II (MHC-II molecules of the patient. We measured the binding and half-life of peptide-MHC-II complexes using synthetic peptides from regions of the Factor VIII protein where ns-SNPs occur and showed that these wild type peptides form stable complexes with six common MHC-II alleles, representing 46.5% of the North American population. Next, we compared the affinities computed by NetMHCIIpan, a neural network-based algorithm for MHC-II peptide binding prediction, to the experimentally measured values and concluded that these are in good agreement (area under the ROC-curve of 0.778 to 0.972 for the six MHC-II variants. Using a computational binding predictor, we were able to expand our analysis to (a include all wild type peptides spanning each polymorphic position; and (b consider more MHC-II variants, thus allowing for a better estimation of the risk for clinical manifestation of anti-drug antibodies in the entire population (or a specific sub-population. Analysis of these computational data confirmed that peptides which have the wild type sequence at positions where the polymorphisms associated with haplotypes H3, H4 and H5 occur bind MHC-II proteins significantly more than a negative control. Taken together, the experimental and computational results suggest that wild type peptides from polymorphic regions of FVIII constitute potential T-cell epitopes

  9. [Intein-mediated F309SfVIII ligation with enhanced secretion of its heavy chain.].

    Science.gov (United States)

    Zhu, Fu-Xiang; Liu, Ze-Long; Qu, Hui-Ge; Chi, Xiao-Yan

    2009-12-25

    Coagulation factor VIII (fVIII) is a secretion protein and plays a crucial role in the coagulation cascade. Hemophilia A resulted from deficiency of fVIII is the most common X-linked recessive bleeding disorder. Gene therapy is recognized as an attractive strategy for the eventual cure of this disease. However, the gene therapy is hampered by the big size of fVIII gene when using the most promising gene vectors, adeno-associated virus (AAV) vectors. In this study we explored the intein-mediated protein trans-splicing to deliver a Phe(309)-->Ser mutant full-length fVIII (F309SfVIII) gene by using a dual-vector system. An intein is a protein sequence embedded within a precursor protein and can excise itself through protein splicing. The F309SfVIII is proven to be beneficial to its secretion. The F309SfVIII gene was broken into heavy and light chains before Ser(1239) in B domain and fused with the coding sequences of Ssp DnaB intein respectively to construct a pair of plasmid vectors by inserting them into the pcDNA3.1 vectors. Forty-eight hours after co- or separate transfection of 293 cells, the co-transfected cell lysate showed an obvious ligated F309SfVIII protein band by Western blot with a polyclonal antibody against fVIII. The amounts of secreted F309SfVIII protein in culture supernatants and their bioactivities were (71+/-9) ng/mL and (0.38+/-0.09) IU/mL determined by ELISA and Coatest assay respectively. The supernatant from combined cells with separate transfections also displayed lower levels of F309SfVIII antigen and fVIII activity [(25+/-6) ng/mL and (0.12+/-0.05) IU/mL], indicating the F309SfVIII could be formed by splicing both before and after secretion. The content of F309SfVIII heavy chain protein from co-transfected cell supernatant was higher than that of intein-fused heavy chain transfection alone [(135+/-10) ng/mL vs (37+/-7) ng/mL, Pintein could be used as a technical strategy in a dual-vector system delivering F309SfVIII gene with improved

  10. Physiological levels of blood coagulation factors IX and X control coagulation kinetics in an in vitro model of circulating tissue factor

    Science.gov (United States)

    Tormoen, Garth W.; Khader, Ayesha; Gruber, András; McCarty, Owen J. T.

    2013-06-01

    Thrombosis significantly contributes to cancer morbidity and mortality. The mechanism behind thrombosis in cancer may be circulating tissue factor (TF), as levels of circulating TF are associated with thrombosis. However, circulating TF antigen level alone has failed to predict thrombosis in patients with cancer. We hypothesize that coagulation factor levels regulate the kinetics of circulating TF-induced thrombosis. Coagulation kinetics were measured as a function of individual coagulation factor levels and TF particle concentration. Clotting times increased when pooled plasma was mixed at or above a ratio of 4:6 with PBS. Clotting times increased when pooled plasma was mixed at or above a ratio of 8:2 with factor VII-depleted plasma, 7:3 with factor IX- or factor X-depleted plasmas, or 2:8 with factor II-, V- or VIII-depleted plasmas. Addition of coagulation factors VII, X, IX, V and II to depleted plasmas shortened clotting and enzyme initiation times, and increased enzyme generation rates in a concentration-dependent manner. Only additions of factors IX and X from low-normal to high-normal levels shortened clotting times and increased enzyme generation rates. Our results demonstrate that coagulation kinetics for TF particles are controlled by factor IX and X levels within the normal physiological range. We hypothesize that individual patient factor IX and X levels may be prognostic for susceptibility to circulating TF-induced thrombosis.

  11. Process development for human factor VIII purification by chromatography, the use of specific antibodies against fragments of the protein for evaluation of purity and stability during purification processes.

    OpenAIRE

    Daniela Jinzenji

    2008-01-01

    O fator VIII de coagulação (FVIII), recombinante ou purificado de plasma, é o biofármaco necessário para o tratamento da hemofília A, a doença hemorrágica mais freqüente em humanos. O método tradicional para a purificação de FVIII parte de crioprecipitado de plasma e precipitação alcoólica. No Instituto Butantan, foi proposto um método alternativo, utilizando somente cromatografia para esta purificação. Este projeto teve por objetivo comparar dois métodos cromatográficos de purificação do FVI...

  12. Photoacoustic monitoring of clot formation during surgery and tumor surgery

    Science.gov (United States)

    Juratli, Mazen A.; Galanzha, Ekaterina I.; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A.; Suen, James Y.; Zharov, Vladimir P.

    2013-03-01

    When a blood vessel is injured, the normal physiological response of the body is to form a clot (thrombus) to prevent blood loss. Alternatively, even without injury to the blood vessel, the pathological condition called thromboembolism may lead to the formation of circulating blood clots (CBCs), also called emboli, which can clog blood vessels throughout the body. Veins of the extremities (venous thromboembolism), lungs (pulmonary embolism ), brain (embolic stroke), heart (myocardial infarction), kidneys, and gastrointestinal tract are often affected. Emboli are also common complications of infection, inflammation, cancer, surgery, radiation and coronary artery bypass grafts. Despite the clear medical significance of CBCs, however, little progress has been made in the development of methods for real-time detection and identification of CBCs. To overcome these limitations, we developed a new modification of in vivo photoacoustic (PA) flow cytometry (PAFC) for real-time detection of white, red, and mixed clots through a transient decrease, increase or fluctuation of PA signal amplitude, respectively. In this work, using PAFC and mouse models, we present for the first time direct evidence that some medical procedures, such as conventional or cancer surgery may initiate the formation of CBCs. In conclusion, the PA diagnostic platform can be used in real-time to define risk factors for cardiovascular diseases, assist in the prognosis and potential prevention of stroke by using a well-timed therapy or as a clot count as a marker of therapy efficacy.

  13. A Serotype VIII Strain among Colonizing Group B Streptococcal Isolates in Boston, Massachusetts

    OpenAIRE

    Paoletti, Leanne J.; Bradford, Jessica; Paoletti, Lawrence C.

    1999-01-01

    Maternal colonization with group B Streptococcus (GBS) is a risk factor for neonatal GBS disease. Whereas serotypes Ia, Ib, II, III, and V are prevalent in the United States, types VI and VIII predominate in Japan. Recently, a serotype VIII strain was detected among 114 clinical GBS isolates from a Boston, Mass., hospital.

  14. A factor VIII-derived peptide enables von Willebrand factor (VWF)-binding of artificial platelet nanoconstructs without interfering with VWF-adhesion of natural platelets.

    Science.gov (United States)

    Haji-Valizadeh, Hassan; Modery-Pawlowski, Christa L; Sen Gupta, Anirban

    2014-05-01

    There is substantial clinical interest in synthetic platelet analogs for potential application in transfusion medicine. To this end, our research is focused on self-assembled peptide-lipid nanoconstructs that can undergo injury site-selective adhesion and subsequently promote site-directed active platelet aggregation, thus mimicking platelet's primary hemostatic actions. For injury site-selective adhesion, we have utilized a coagulation factor FVIII-derived VWF-binding peptide (VBP). FVIII binds to VWF's D'-D3 domain while natural platelet GPIbα binds to VWF's A1 domain. Therefore, we hypothesized that the VBP-decorated nanoconstructs will adhere to VWF without mutual competition with natural platelets. We further hypothesized that the adherent VBP-decorated constructs can enhance platelet aggregation when co-decorated with a fibrinogen-mimetic peptide (FMP). To test these hypotheses, we used glycocalicin to selectively block VWF's A1 domain and, using fluorescence microscopy, studied the binding of fluorescently labeled VBP-decorated nanoconstructs versus platelets to ristocetin-treated VWF. Subsequently, we co-decorated the nanoconstructs with VBP and FMP and incubated them with human platelets to study construct-mediated enhancement of platelet aggregation. Decoration with VBP resulted in substantial construct adhesion to ristocetin-treated VWF even if the A1-domain was blocked by glycocalicin. In comparison, such A1-blocking resulted in significant reduction of platelet adhesion. Without A1-blocking, the VBP-decorated constructs and natural platelets could adhere to VWF concomitantly. Furthermore, the constructs co-decorated with VBP and FMP enhanced active platelet aggregation. The results indicate significant promise in utilizing the FVIII-derived VBP in developing synthetic platelet analogs that do not interfere with VWF-binding of natural platelets but allow site-directed enhancement of platelet aggregation when combined with FMP.

  15. Blood clot detection using magnetic nanoparticles

    Science.gov (United States)

    Khurshid, Hafsa; Friedman, Bruce; Berwin, Brent; Shi, Yipeng; Ness, Dylan B.; Weaver, John B.

    2017-01-01

    Deep vein thrombosis, the development of blood clots in the peripheral veins, is a very serious, life threatening condition that is prevalent in the elderly. To deliver proper treatment that enhances the survival rate, it is very important to detect thrombi early and at the point of care. We explored the ability of magnetic particle spectroscopy (MSB) to detect thrombus via specific binding of aptamer functionalized magnetic nanoparticles with the blood clot. MSB uses the harmonics produced by nanoparticles in an alternating magnetic field to measure the rotational freedom and, therefore, the bound state of the nanoparticles. The nanoparticles’ relaxation time for Brownian rotation increases when bound [A.M. Rauwerdink and J. B. Weaver, Appl. Phys. Lett. 96, 1 (2010)]. The relaxation time can therefore be used to characterize the nanoparticle binding to thrombin in the blood clot. For longer relaxation times, the approach to saturation is more gradual reducing the higher harmonics and the harmonic ratio. The harmonic ratios of nanoparticles conjugated with anti-thrombin aptamers (ATP) decrease significantly over time with blood clot present in the sample medium, compared with nanoparticles without ATP. Moreover, the blood clot removed from the sample medium produced a significant MSB signal, indicating the nanoparticles are immobilized on the clot. Our results show that MSB could be a very useful non-invasive, quick tool to detect blood clots at the point of care so proper treatment can be used to reduce the risks inherent in deep vein thrombosis.

  16. A French National Survey on Clotting Disorders in Mastocytosis

    Science.gov (United States)

    Carvalhosa, Ana B.; Aouba, Achille; Damaj, Gandhi; Canioni, Danielle; Brouzes, Chantal; Gyan, Emmanuel; Durupt, Stéphane; Durieu, Isabelle; Cathebras, Pascal; Costédoat-Chalumeau, Nathalie; Launay, David; Pilmis, Benoit; Barete, Stephane; Frenzel, Laurent; Lortholary, Olivier; Hermine, Olivier; Hermans, Cedric; Chandesris, Marie-Olivia

    2015-01-01

    Abstract Mastocytosis is characterized by a clonal mast cell proliferation with organ infiltration and uncontrolled degranulation. Although not characteristic and poorly explained, some patients develop clotting abnormalities. We retrospectively identified patients with established diagnosis of mastocytosis and related clotting abnormalities (clinical and/or biological) using the national French Reference Centre for Mastocytosis database. From our cohort of 14 adult patients with clotting abnormalities (median age 46 years [range 26–75]), 4 had a presentation suggestive of a primary hemostasis disorder alone (by their symptoms and/or abnormal clotting tests [PFA, von Willebrand's disease [vWD] screening]) and 10 had a laboratory impairment of secondary hemostasis. Among these, 7 had bleeds characteristic of a coagulation cascade disorder (severe/life-threatening in 5 and mild in 2 patients). Clotting abnormalities were of variable severity, typically related to intense crisis of degranulation, such as anaphylactic reactions, and/or to severe organ infiltration by mast cells. Importantly, classical hemostatic management with platelet transfusion, fresh frozen plasma, or vitamin K infusions was unsuccessful, as opposed to the use of agents inhibiting mast cell activity, particularly steroids. This illustrates the crucial role of mast cell mediators such as tryptase and heparin, which interfere both with primary (mainly via inhibition of von Willebrand factor) and secondary hemostasis. There was interestingly an unusually high number of aggressive mastocytosis (particularly mast cell leukemia) and increased mortality in the group with secondary hemostasis disorders (n = 5, 36% of the whole cohort). Mast cell degranulation and/or high tumoral burden induce both specific biologic antiaggregant and anticoagulant states with a wide clinical spectrum ranging from asymptomatic to life-threatening bleeds. Hemostatic control is achieved by mast cell inhibitors such as

  17. Protein-phospholipid interactions in blood clotting.

    Science.gov (United States)

    Morrissey, James H; Davis-Harrison, Rebecca L; Tavoosi, Narjes; Ke, Ke; Pureza, Vincent; Boettcher, John M; Clay, Mary C; Rienstra, Chad M; Ohkubo, Y Zenmei; Pogorelov, Taras V; Tajkhorshid, Emad

    2010-04-01

    Most steps of the blood clotting cascade require the assembly of a serine protease with its specific regulatory protein on a suitable phospholipid bilayer. Unfortunately, the molecular details of how blood clotting proteins bind to membrane surfaces remain poorly understood, owing to a dearth of techniques for studying protein-membrane interactions at high resolution. Our laboratories are tackling this question using a combination of approaches, including nanoscale membrane bilayers, solid-state NMR, and large-scale molecular dynamics simulations. These studies are now providing structural insights at atomic resolution into clotting protein-membrane interactions. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  18. Synthetic Hormones and Clot Formation.

    Science.gov (United States)

    Swanepoel, Albe C; Visagie, Amcois; Pretorius, Etheresia

    2016-08-01

    Combined oral contraceptives (COCs), colloquially referred to as "the pill," have been regarded as a medical breakthrough, as they have improved the lives of countless women, from simplifying family planning to the treatment of acne, endometriosis, polycystic ovarian syndrome, and dysmenorrhea. Unfortunately, COC usage has been associated with an increased occurrence of venous thrombosis and therefore a systemic hypercoagulable state in susceptible females. Here we discuss the health risks of COC usage and use viscoelastic and morphological techniques to investigate the effect of different COC constituents on clot formation, particularly fibrin network packaging and whole blood viscoelasticity. Viscoelastic properties of whole blood showed gender-specific changes while morphological alterations were person-specific, regardless of gender. Using scanning electron microscopy and thromboelastography provides great insight regarding fibrin packaging and the development of a hypercoagulable state in high-risk individuals. We proposed a three-step approach where (1) an individual's coagulation profile baseline is determined, after which (2) the "ideal" combination of constituents is prescribed, and (3) the coagulation profile of the individual is monitored to assess possible risk of thrombosis. Only in following such an individualized patient-oriented approach will we be able to avoid the many health issues due to COC usage in susceptible females.

  19. Trp[superscript 2313]-His[superscript 2315] of Factor VIII C2 Domain Is Involved in Membrane Binding Structure of a Complex Between the C[subscript 2] Domain and an Inhibitor of Membrane Binding

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhuo; Lin, Lin; Yuan, Cai; Nicolaes, Gerry A.F.; Chen, Liqing; Meehan, Edward J.; Furie, Bruce; Furie, Barbara; Huang, Mingdong (Harvard-Med); (UAH); (Maastricht); (Chinese Aca. Sci.)

    2010-11-03

    Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 {angstrom}) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed {beta}-strand of the C2 domain, Trp{sup 2313}-His{sup 2315}. This result indicates that the Trp{sup 2313}-His{sup 2315} segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

  20. Preventing Blood Clots After Orthopaedic Surgery

    Medline Plus

    Full Text Available ... Surgeons. Many of the images included in this video are courtesy of Thinkstock ©2013. Warning Signs It is important to follow your doctor's instructions carefully to minimize the risk for blood clots ...

  1. Preventing Blood Clots After Orthopaedic Surgery

    Medline Plus

    Full Text Available ... of a blood clot within a deep vein. It commonly occurs in the thigh or calf. Deep ... breaks free and travels through the veins. If it reaches the lungs, it can block the flow ...

  2. Preventing Blood Clots After Orthopaedic Surgery

    Medline Plus

    Full Text Available ... recovery from surgery. Warning Signs of Blood Clots Pain in your calf and leg, unrelated to your ... of Pulmonary Embolism Sudden shortness of breath Chest pain, particularly with breathing Notify your doctor immediately if ...

  3. Preventing Blood Clots After Orthopaedic Surgery

    Medline Plus

    Full Text Available ... Patient Safety Patient Stories Resources Copyright 2017 American Academy of Orthopaedic Surgeons Preventing Blood Clots After Orthopaedic ... about DVT and its prevention. This video © American Academy of Orthopaedic Surgeons. Many of the images included ...

  4. Preventing Blood Clots After Orthopaedic Surgery

    Medline Plus

    Full Text Available ... Bones & Injuries Diseases & Conditions Arthritis Tumors Sports Injuries & Prevention Children Bone Health Health & Safety Treatment Treatments & Surgeries ... Your doctor will outline a program to help prevent the development of blood clots after your surgery. ...

  5. Preventing Blood Clots After Orthopaedic Surgery

    Medline Plus

    Full Text Available ... Bones & Injuries Diseases & Conditions Arthritis Tumors Sports Injuries & Prevention Children Bone Health Health & Safety Treatment Treatments & Surgeries ... Your doctor will outline a program to help prevent the development of blood clots after your surgery. ...

  6. Preventing Blood Clots After Orthopaedic Surgery

    Medline Plus

    Full Text Available ... major risks facing patients who undergo surgery is a complication called deep vein thrombosis. Deep vein thrombosis (DVT) is the formation of a blood clot within a deep vein. It commonly ...

  7. Preventing Blood Clots After Orthopaedic Surgery

    Medline Plus

    Full Text Available ... of a blood clot within a deep vein. It commonly occurs in the thigh or calf. Deep ... breaks free and travels through the veins. If it reaches the lungs, it can block the flow ...

  8. What Are Rare Clotting Factor Deficiencies?

    Science.gov (United States)

    ... Resource Annual Global Survey Treatment Guidelines Laboratory Manual Hemophilia in Pictures Young Voices Compendium of Assessment Tools Educational Games Video Library Find a Treatment Centre Haemophilia Journal ...

  9. Evaluation of ovine milk clotting aptitude

    Directory of Open Access Journals (Sweden)

    A. Caroli

    2010-01-01

    Full Text Available A comparative study of the lactodynamographic parameters was carried out on ovine milk. Besides evaluating the repeatability and reproducibility of the analytical method, the influence of some variables such as the genetic type (three breeds, the kind of milk (whole or skimmed, and its concentration after reconstitution (12g or 20g /100 ml was evaluated. The working plan involved 6 laboratories for the final statistic analyses, by the use of freeze-dried milk samples (adequately reconstituted on the basis of established methods from Sardinia, Comisana, and Massese ewes. All the considered variability factors showed a highly significant effect (P<0.001 on the lactodynamographic parameters considered. In particular, Massese ewe milk showed the shortest curd speed (k20 and the best coagulum strength (a30 and a45, although clotting time (CT was the highest one. The same trend was registered for skimmed milk and for the most concentrated one (20g. Repeatability values within laboratories were 96% and 97% for CT and k20, lowering for a30 e a45, (respectively 87% and 85%. Much lower coefficients were found for the among laboratories reproducibility, ranging from a maximum of 58% for CT to a minimum of 18% for k20. The wide variability observed indicates that lactodynamographic parameters are comparable only within the same lab. Further investigation is needed to compare different labs in order to obtain more homogeneous results.

  10. Problems concerning ovine milk clotting aptitude

    Directory of Open Access Journals (Sweden)

    M. Martini

    2003-01-01

    Full Text Available A comparative study of the lactodynamographic parameters was carried out on ovine milk. Besides evaluating the repeatability and reproducibility of the analytical method, the influence of some variables such as the genetic type (three breeds, the kind of milk (whole or skimmed, and its concentration after reconstitution (12g or 20g /100 ml was evaluated. The working plan involved 6 laboratories for the final statistic analyses, by the use of freeze-dried milk samples (adequately reconstituted on the basis of established methods from Sardinia, Comisana, and Massese ewes. All the considered variability factors showed a highly significant effect (P<0.001 on the lactodynamographic parameters considered. In particular, Massese ewe milk showed the shortest curd speed (k20 and the best coagulum strength (a30 and a45, although clotting time (CT was the highest one. The same trend was registered for skimmed milk and for the most concentrated one (20g. Repeatability values within laboratories were 96% and 97% for CT and k20, lowering for a30 e a45, (respectively 87% and 85%. Much lower coefficients were found for the among laboratories reproducibility, ranging from a maximum of 58% for CT to a minimum of 18% for k20. The wide variability observed indicates that lactodynamographic parameters are comparable only within the same lab. Further investigation is needed to compare different labs in order to obtain more homogeneous results.

  11. Clot dissolution is better with ultrasound assisted thrombolysis for fresh clots with higher cholesterol content

    Science.gov (United States)

    Zhou, Yufeng; Sharma, Vijay Kumar; Murugappan, Kanna Suresh; Ahmad, Aftab

    2012-11-01

    Tissue plasminogen activator (tPA) remains the only drug for recanalization in acute ischemic stroke, and the dose is determined by the patient's body-weight. Properties of the blood clot as well as ultrasound exposure might affect the thrombolysis outcome. In this study, clot was prepared by mixing horse blood with CaCl2 solution and cholesterin up to 1.0 mg/ml. To simulate the aging effect serum was replaced by fresh blood periodically. 225 IU/ml of tPA was used to initiate lysis. Clot was exposed to continuous 2 MHz transcranial Doppler ultrasound at acoustic intensity of 340 mW/cm2. The weight of the blood clot increased with its age (from 37.28±2.87 mg at 2 hrs to 51.56±5.34 mg at 10 hrs, p < 0.05). Although no difference between clot-cholesterol levels and thrombolysis with ultrasound or tPA alone was found, combination of these modalities induced significant lysis in the clots with cholesterol levels of more than 0.5 mg/ml (clot-weight reduced by 41.68±2.3%) as compared to clots with normal cholesterol (30.60±4.10%; p < 0.05). Altogether, sonothrombolysis seems to work better in fresh thrombi with high-cholesterol levels.

  12. An automated method for fibrin clot permeability assessment.

    Science.gov (United States)

    Ząbczyk, Michał; Piłat, Adam; Awsiuk, Magdalena; Undas, Anetta

    2015-01-01

    The fibrin clot permeability coefficient (Ks) is a useful measure of porosity of the fibrin network, which is determined by a number of genetic and environmental factors. Currently available methods to evaluate Ks are time-consuming, require constant supervision and provide only one parameter. We present an automated method in which drops are weighed individually, buffer is dosed by the pump and well defined clot washing is controlled by the software. The presence of a straight association between drop mass and their dripping time allows to shorten the measurement time twice. In 40 healthy individuals, Ks, the number of drops required to reach the plateau (DTP), the time to achieve the plateau (TTP) and the DTP/TTP ratio (DTR) were calculated. There was a positive association between Ks (r = 0.69, P Ks (r = -0.55, P Ks (r = 0.70, P < 0.0001 for the manual method and r = 0.76, P < 0.0001 for the automated method), fibrinogen (r = -0.58, P < 0.0001) and C-reactive protein (CRP) (r = -0.47, P < 0.01). The automated method might be a suitable tool for research and clinical use and may offer more additional parameters describing fibrin clot structure.

  13. Status and trend analysis of prophylactic usage of recombinant factor VIII in Chinese pediatric patients with hemophilia A: ReCare - a retrospective, phase IV, non-interventional study.

    Science.gov (United States)

    Li, Changgang; Zhang, Xinsheng; Zhao, Yongqiang; Wu, Runhui; Hu, Qun; Xu, Vicky; Sun, Jing; Yang, Renchi; Li, Xiaojing; Zhou, Rongfu; Lian, Shinmei; Gu, Jian; Wu, Junde; Hou, Qingsong

    2017-09-01

    No study has reported the status and chronological trend of prophylactic recombinant factor VIII (rFVIII) use in Chinese pediatric patients with hemophilia A (HA). We aimed to analyze the status and trend of rFVIII-containing prophylaxis in Chinese pediatric patients with HA. ReCARE (Retrospective study in Chinese pediatric hemophilia A patients with rFVIII contained REgular prophylaxis) was a retrospective study conducted in 12 hemophilia treatment centers across China. The trend of prophylaxis was evaluated by determining the mean duration of prophylaxis, mean injection frequency (per week), mean dose of each injection (IU/kg), mean total dose injected/week (IU) and proportion of rFVIII consumption relative to factor VIII (FVIII) consumption over the study period. We analyzed 183 male pediatric patients with HA (mean age, 7.1 ± 4.23 years), who received intermittent prophylaxis between 1 November 2007 and 31 May 2013. The mean duration of prophylaxis with rFVIII increased from 16.72 weeks in 2008 to 32.77 in 2012. Per injection dose of rFVIII increased significantly from 2008 to 2013 (25.89 to 28.31 IU/kg, p < .001). An increase was also reported in the mean total FVIII consumed (699.97 ± 173.25 IU in 2008 and 891.30 ± 730.341 in 2013) and mean proportion of rFVIII used (33.33 ± 57.73% in 2008 to 85.50 ± 29.077% in 2013). Our data revealed an overall improvement in treatment dosage and duration with an increase in the number of patients receiving prophylaxis. The total proportion of rFVIII also increased gradually indicating the development of economy and safety awareness. The trial is registered at ClinicalTrials.gov (CT.gov identifier: NCT02263066).

  14. Cavitation damage in blood clots under HIFU

    Science.gov (United States)

    Weiss, Hope; Ahadi, Golnaz; Hoelscher, Thilo; Szeri, Andrew

    2010-11-01

    High Intensity Focused Ultrasound (HIFU) has been shown to accelerate thrombolysis, the dissolution of blood clots, in vitro and in vivo, for treatment of ischemic stroke. Cavitation in sonothrombolysis is thought to play an important role, although the mechanisms are not fully understood. The damage to a blood clot associated with bubble collapses in a HIFU field is studied. The region of damage caused by a bubble collapse on the fibrin network of the blood clot exposed to HIFU is estimated, and compared with experimental assessment of the damage. The mechanical damage to the network caused by a bubble is probed using two independent approaches, a strain based method and an energy based method. Immunoflourescent fibrin staining is used to assess the region of damage experimentally.

  15. Alfonso VIII, Cruzada y Cristiandad = Alfonso VIII, Crusade and Christendom

    Directory of Open Access Journals (Sweden)

    Carlos de Ayala Martínez

    2016-06-01

    Full Text Available Lacruzada fue el cauce más directo para la integración de la Península Ibérica en el conjunto de la Cristiandad. Este hecho se pone especialmente de relieve cuando nos acercamos al reinado de Alfonso VIII de Castilla. El monarca, que casó con la hija del rey de Inglaterra, comprometió el matrimonio de su primogénita con el heredero del emperador alemán y casó a otra de sus hijas con el futuro rey de Francia, representa esa plena integración cuyo seguimiento es posible a través de diversos indicadores –políticos, ideológicos, culturales…-, que, en todo caso, refuerzan la imagen propagandística de un rey al servicio de la defensa de la Cristiandad.Crusading was the most effective method of integrating the Iberian Peninsula within Christendom. This fact is most evident when we consider the reign of Alfonso VIII of Castile. This monarch married the daughter of the king of England, arranged the marriage of his eldest daughter with the heir of the German Emperor and married another of his daughters with the future king of France. These actions convey the need for maximum integration which can be traced using several indicators –political, ideological, cultural...– that moreover reinforce the propaganda image of a king at the service and in defence of Christianity.

  16. In Vivo Quantification of Clot Formation in Extracorporeal Circuits

    CERN Document Server

    David, Omid

    2012-01-01

    Clot formation is a common complication in extracorporeal circuits. In this paper we describe a novel method for clot formation analysis using image processing. We assembled a closed extracorporeal circuit and circulated blood at varying speeds. Blood filters were placed in downstream of the flow, and clotting agents were added to the circuit. Digital images of the filter were subsequently taken, and image analysis was applied to calculate the density of the clot. Our results show a significant correlation between the cumulative size of the clots, the density measure of the clot based on image analysis, and flow duration in the system.

  17. Mechanical suction for clot evacuation: experience with "suction bridge" for safe and effective clot removal.

    Science.gov (United States)

    Goel, Apul; Dalela, Diwakar

    2015-05-01

    To present the experience with the use of a "suction bridge" for removal of bladder clots. In all patients presenting with bladder clots, mechanical suction was done using a "suction bridge". This bridge has a luer lock that is fixed to the cystoscope sheath, and the other end is connected to suction tube. The suction pressure was started at 250 mmHg and was increased up to 400 mmHg if needed. Twenty patients with a mean age of 59.4 years were included. The etiologies of bladder clots included bladder tumor in nine, benign prostate hyperplasia (BPH) in two, BPH with bladder stone in one, hematochyluria in three, and post-transurethral prostate resection in 10. Eighteen patients presented in clot retention. The estimated clot size ranged from 50 mL to more than 1 L. The mean duration for clot removal was 15 min (range 5-60). The procedure was successful in all patients. There was no bladder injury. The limitations include the small number of recruits, the non-randomized nature of study, and no control group for comparison. Mechanical suction is another safe, fast, and effective option of clot removal from the urinary bladder. The suction bridge is useful while using this method.

  18. Mechanical stability and fibrinolytic resistance of clots containing fibrin, DNA, and histones.

    Science.gov (United States)

    Longstaff, Colin; Varjú, Imre; Sótonyi, Péter; Szabó, László; Krumrey, Michael; Hoell, Armin; Bóta, Attila; Varga, Zoltán; Komorowicz, Erzsébet; Kolev, Krasimir

    2013-03-08

    Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones + DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nM dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator.

  19. Mechanical Stability and Fibrinolytic Resistance of Clots Containing Fibrin, DNA, and Histones*

    Science.gov (United States)

    Longstaff, Colin; Varjú, Imre; Sótonyi, Péter; Szabó, László; Krumrey, Michael; Hoell, Armin; Bóta, Attila; Varga, Zoltán; Komorowicz, Erzsébet; Kolev, Krasimir

    2013-01-01

    Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones + DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nm dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator. PMID:23293023

  20. Application and validation of the lattice Boltzmann method for modelling flow-related clotting.

    Science.gov (United States)

    Harrison, S E; Smith, S M; Bernsdorf, J; Hose, D R; Lawford, P V

    2007-01-01

    The purpose of this paper is to present a simple clotting model, based on residence time and shear stress distribution, that can simulate the deposition over time of enzyme-activated milk in an in vitro system. Results for the model are compared with experiments exhibiting clot deposition in the region of a sharp-edged stenosis. The milk experiments have been shown to be a valuable analogue for the experimental representation of flow-induced blood clotting, particularly in the context of separation of hydrodynamic from biochemical factors. The facility to predict the flow-induced clotting of the blood analogue, in which the chemistry reduces to what is effectively a zeroth order reaction, gives confidence in this physics-based approach to simulation of the final part of the coagulation cascade. This type of study is a necessary precursor to the development of a complex, multi-factorial, biochemical model of the process of thrombosis. In addition to the clotting simulations, comparisons are reported between the computed flow patterns prior to clot deposition and flow visualisation studies. Excellent agreement of hydrodynamic parameters is reported for a Reynolds number of 100, and qualitative agreement is seen for the complex, disturbed flow occurring at a physiologically relevant Reynolds number of 550. The explicit, time-stepping lattice Boltzmann approach may have particular merit for the transitional flow at this higher Reynolds number.

  1. Blood flow measurements and clot detection with nearinfrared spectroscopy

    OpenAIRE

    Rossow, MJ; Gatto, R.; D'amico, E.; Mantulin, WW; Gratton, E

    2006-01-01

    Detecting impeded blood flow and locating the clot causing it is a major challenge in neurosurgery. We propose an instrument that uses near-infrared spectroscopy to simultaneously detect clots and measure blood flow. © 2006 Optical Society of America.

  2. Procoagulant control strategies for the human blood clotting process.

    Science.gov (United States)

    Laurino, Marco; Menara, Tommaso; Stella, Alessandro; Betta, Monica; Landi, Alberto

    2015-08-01

    This paper describes the comparison between two drug control strategies to hemophilia A. To emulate blood clotting and the pathological condition of hemophilia, a mathematical model composed by 14 ordinary differential equations is considered. We adopt a variable structure non-linear PID approach and a Model Predictive Control in order to control the dosage of procoagulant factor used in the treatment of hemophiliac patient. The two control actions are sampled for a practical application. Finally, we discuss and compare the results of the two control approaches, introducing a suited control index (eINR).

  3. Identification of fibrin clot-bound plasma proteins

    NARCIS (Netherlands)

    S. Talens (Simone); F.W.G. Leebeek (Frank); J.A.A. Demmers (Jeroen); D.C. Rijken (Dingeman)

    2012-01-01

    textabstractSeveral proteins are known to bind to a fibrin network and to change clot properties or function. In this study we aimed to get an overview of fibrin clot-bound plasma proteins. A plasma clot was formed by adding thrombin, CaCl2 and aprotinin to citrated platelet-poor plasma and unbound

  4. Limitations of using synthetic blood clots for measuring in vitro clot capture efficiency of inferior vena cava filters

    Directory of Open Access Journals (Sweden)

    Robinson RA

    2013-05-01

    Full Text Available Ronald A Robinson, Luke H Herbertson, Srilekha Sarkar Das, Richard A Malinauskas, William F Pritchard, Laurence W GrossmanOffice of Science and Engineering Laboratories, Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, MD, USAAbstract: The purpose of this study was first to evaluate the clot capture efficiency and capture location of six currently-marketed vena cava filters in a physiological venous flow loop, using synthetic polyacrylamide hydrogel clots, which were intended to simulate actual blood clots. After observing a measured anomaly for one of the test filters, we redirected the focus of the study to identify the cause of poor clot capture performance for large synthetic hydrogel clots. We hypothesized that the uncharacteristic low clot capture efficiency observed when testing the outlying filter can be attributed to the inadvertent use of dense, stiff synthetic hydrogel clots, and not as a result of the filter design or filter orientation. To study this issue, sheep blood clots and polyacrylamide (PA synthetic clots were injected into a mock venous flow loop containing a clinical inferior vena cava (IVC filter, and their captures were observed. Testing was performed with clots of various diameters (3.2, 4.8, and 6.4 mm, length-to-diameter ratios (1:1, 3:1, 10:1, and stiffness. By adjusting the chemical formulation, PA clots were fabricated to be soft, moderately stiff, or stiff with elastic moduli of 805 ± 2, 1696 ± 10 and 3295 ± 37 Pa, respectively. In comparison, the elastic moduli for freshly prepared sheep blood clots were 1690 ± 360 Pa. The outlying filter had a design that was characterized by peripheral gaps (up to 14 mm between its wire struts. While a low clot capture rate was observed using large, stiff synthetic clots, the filter effectively captured similarly sized sheep blood clots and soft PA clots. Because the stiffer synthetic clots remained straight when approaching the

  5. Functional Role of Milk Fat Globule-Epidermal Growth Factor VIII in Macrophage-Mediated Inflammatory Responses and Inflammatory/Autoimmune Diseases

    OpenAIRE

    Young-Su Yi

    2016-01-01

    Inflammation involves a series of complex biological processes mediated by innate immunity for host defense against pathogen infection. Chronic inflammation is considered to be one of the major causes of serious diseases, including a number of autoimmune/inflammatory diseases, cancers, cardiovascular diseases, and neurological diseases. Milk fat globule-epidermal growth factor 8 (MFG-E8) is a secreted protein found in vertebrates and was initially discovered as a critical component of the mil...

  6. The Contribution of Pin End-Cup Interactions to Clot Strength Assessed with Thrombelastography.

    Science.gov (United States)

    Nielsen, Vance G

    2016-01-01

    Viscoelastic methods have been developed to assess the contribution of plasma proteins and platelets to coagulation in vitro to guide clinical transfusion therapy. One of the cardinal precepts of determining clot strength is making sure that the viscoelastic technique includes complete exposure of the plastic pin in the testing chamber with the fluid analyzed so as to assure maximal interaction of the cup wall with the pin surface. However, the various contributions of the pin surface area to final clot strength have not been investigated. That is, it is not clear what is more important in the in vitro determination of clot strength, the surface area shared between the cup and pin filled with fluid or the final viscoelastic resistance of the gel matrix formed. Thus, the purpose of this investigation was to determine the clot strength when only the tip of the pin was engaged with plasma thrombus and to compare these values with clot strength values obtained when the pin was completely in plasma. After determining the minimal amount of plasma required to cover a pin tip in a thrombelastographic system (30 μL), clot strength (elastic modulus, G) was determined in plasma samples of 30 or 360 μL final volume (n = 12 per condition) after tissue factor activation. The G value with 30 μL volume was 1057 ± 601 dynes/cm (mean ± SD; 95% confidence interval, 675-1439 dynes/cm), which was (P = 0.0015) smaller than the G value associated with 360-μL sample volumes, that was 1712 ± 48 dynes/cm (confidence interval, 1681-1742 dynes/cm). In conclusion, these data demonstrate that clot strength is not determined by a simple ratio of surface area of pin and cup to volume of sample, but rather strength is importantly influenced by the viscoelastic resistance of the fluid assessed.

  7. Brillouin spectroscopy of clotting dynamics in a model system

    Science.gov (United States)

    Bustamante-Lopez, Sandra C.; Traverso, Andrew J.; Yakovlev, Vladislav V.; Meissner, Kenith E.

    2016-02-01

    Keys to successful treatment of disease include early diagnosis and timely treatment. It is hypothesized that early clotting events may contribute to a pro-thrombotic state that exacerbates atherothrombotic vascular disease. Brillouin spectroscopy involves inelastic coupling of light with phonons and enables viscoelastic characterization of samples at the microscale. In this work, we apply Brillouin spectroscopy to a model fibrinogen-thrombin clotting system with the goal of measuring clotting dynamics at the microscale and providing characterization that is not possible with standard rheometric techniques. Here, the clotting dynamics of the model clotting system are measured at various fibrinogen and thrombin concentrations.

  8. 12 CFR 611.1137 - Title VIII service corporations.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Title VIII service corporations. 611.1137... Organizations § 611.1137 Title VIII service corporations. (a) What is a title VIII service corporation? A title VIII service corporation is a service corporation organized for the purpose of exercising...

  9. Prospective surveillance study of haemophilia A patients switching from moroctocog alfa or other factor VIII products to moroctocog alfa albumin-free cell culture (AF-CC) in usual care settings.

    Science.gov (United States)

    Parra Lopez, Rafael; Nemes, Laszlo; Jimenez-Yuste, Victor; Rusen, Luminita; Cid, Ana R; Charnigo, Robert J; Baumann, James A; Smith, Lynne; Korth-Bradley, Joan M; Rendo, Pablo

    2015-10-01

    This prospective, open-label, postauthorisation safety surveillance study assessed clinically significant inhibitor development in patients with severe haemophilia A transitioning from moroctocog alfa or other factor VIII (FVIII) replacement products to reformulated moroctocog alfa (AF-CC). Males aged ≥ 12 years with severe haemophilia A (FVIII:C) 150 exposure days (EDs) to recombinant or plasma-derived FVIII products, and no detectable inhibitor at screening were enrolled. Primary end point was the incidence of clinically significant FVIII inhibitor development. Secondary end points included annualised bleeding rate (ABR), less-than-expected therapeutic effect (LETE), and FVIII recovery. Patients were assigned to one of two cohorts based on whether they were transitioning to moroctocog alfa (AF-CC) from moroctocog alfa (cohort 1; n=146) or from another recombinant or plasma-derived FVIII product (cohort 2; n=62). Mean number of EDs on study was 94 (range, 1-139). Six positive FVIII inhibitor results, as determined by local laboratories, were reported in four patients; none were confirmed by a central laboratory, no inhibitor-related clinical manifestations were reported, and all anti-FVIII antibody assays were negative. Median ABRs were 23.4 and 3.4 in patients categorised at baseline as following on-demand and prophylactic regimens, respectively; 86.5% of bleeding episodes resolved after one infusion. LETE incidence was 0.06% and 0.19% in the on-demand and prophylaxis settings, respectively. FVIII recovery remained constant throughout the study. No new safety concerns were identified. This study found no increased risk of clinically significant FVIII inhibitor development in patients transitioning from moroctocog alfa or other FVIII replacement products to moroctocog alfa (AF-CC).

  10. Low risk of inhibitor formation in haemophilia A patients following en masse switch in treatment to a third generation full length plasma and albumin-free recombinant factor VIII product (ADVATE®).

    LENUS (Irish Health Repository)

    Bacon, C L

    2011-05-01

    Previous studies have suggested that development of inhibitors in previously treated patients (PTPs) may be attributable to a switch in factor VIII (FVIII) therapeutic product. Consequently, it is widely recognized that inhibitor development must be assessed in PTPs following the introduction of any new FVIII product. Following a national tender process in 2006, all patients with haemophilia A in Ireland changed their FVIII treatment product en masse to a plasma and albumin-free recombinant full-length FVIII product (ADVATE(®)). In this study, we retrospectively reviewed the case records of Irish PTPs to evaluate risk of inhibitor formation following this treatment switch. One hundred and thirteen patients participated in the study. Most patients (89%) had severe haemophilia. Only one of 96 patients with no inhibitor history developed an inhibitor. Prior to the switch in his recombinant FVIII (rFVIII) treatment of choice, this child had only experienced three exposure days (EDs). Consequently, in total he had only received 6 EDs when his inhibitor was first diagnosed. In keeping with this lack of de novo inhibitor development, we observed no evidence of any recurrent inhibitor formation in any of 16 patients with previously documented inhibitors. Similarly, following a previous en masse switch, we have previously reported that changing from a Chinese hamster ovary cell-produced to a baby hamster kidney cell-produced rFVIII was also associated with a low risk of inhibitor formation in PTPs. Our cumulative findings from these two studies clearly emphasizes that the risk of inhibitor development for PTPs following changes in commercial rFVIII product is low, at least in the Irish population.

  11. The local phospholipid environment modulates the activation of blood clotting.

    Science.gov (United States)

    Shaw, Andrew W; Pureza, Vincent S; Sligar, Stephen G; Morrissey, James H

    2007-03-02

    Examples abound of membrane-bound enzymes for which the local membrane environment plays an important role, including the ectoenzyme that triggers blood clotting, the plasma serine protease, factor VIIa, bound to the integral membrane protein, tissue factor. The activity of this enzyme complex is markedly influenced by lipid bilayer composition and further by tissue factor partitioning into membrane microdomains on some cell surfaces. Unfortunately, little is known about how membrane microdomain composition controls factor VIIa-tissue factor activity, as reactions catalyzed by membrane-tethered enzymes are typically studied under conditions in which the experimenter cannot control the composition of the membrane in the immediate vicinity of the enzyme. To overcome this problem, we used a nanoscale approach that afforded complete control over the membrane environment surrounding tissue factor by assembling the factor VIIa.tissue factor complex on stable bilayers containing 67 +/- 1 phospholipid molecules/leaflet (Nanodiscs). We investigated how local changes in phospholipid bilayer composition modulate the activity of the factor VIIa.tissue factor complex. We also addressed whether this enzyme requires a pool of membrane-bound protein substrate (factor X) for efficient catalysis, or alternatively if it could efficiently activate factor X, which binds directly to the membrane nanodomain adjacent to tissue factor. We have shown that full proteolytic activity of the factor VIIa.tissue factor complex requires extremely high local concentrations of anionic phospholipids and further that a large pool of membrane-bound factor X is not required to support sustained catalysis.

  12. Massive clot formation after tooth extraction

    Directory of Open Access Journals (Sweden)

    Santosh Hunasgi

    2015-01-01

    Full Text Available Oral surgical procedures mainly tooth extraction can be related with an extended hemorrhage owed to the nature of the process resulting in an "open wound." The attempt of this paper is to present a case of massive postoperative clot formation after tooth extraction and highlight on the oral complications of surgical procedures. A 32-year-old male patient reported to the Dental Clinic for evaluation and extraction of grossly decayed 46. Clinical evaluation of 46 revealed root stumps. Extraction of the root stumps was performed, and it was uneventful. Hemostasis was achieved and postsurgical instructions were specified to the patient. The patient reported to the clinic, the very subsequent morning with a criticism of bleeding at the extraction site. On clinical examination, bleeding was noted from the socket in relation to 46. To control bleeding, oral hemostatic drugs Revici - E (Ethamsylate 500 mg was prescribed and bleeding was stopped in 2 h. However, a massive clot was formed at the extraction site. Further, this clot resolved on its own in 1-week time. Despite the fact that dental extraction is considered to be a minor surgical procedure, some cases may present with life-threatening complications including hemorrhage. Vigilant and significant history taking, physical and dental examinations prior to dental procedures are a must to avoid intraoperative and postoperative complications.

  13. F8 gene mutation profile in Indian hemophilia A patients: Identification of 23 novel mutations and factor VIII inhibitor risk association.

    Science.gov (United States)

    Pinto, Patricia; Ghosh, Kanjaksha; Shetty, Shrimati

    2016-04-01

    'FVIII inhibitors', especially in severe hemophilia A (HA) patients, is a serious adverse effect that complicates their clinical management. Many genetic and non-genetic risk factors have been proposed for FVIII inhibitor development, diverse in different population groups. This is the first study in Indian hemophiliacs that analyzes inhibitor risk in relation to the complete F8 mutation profile, in a case-control study that included 145 Indian severe HA patients, i.e. 69 inhibitor positive (with 18 inhibitor concordant/discordant family members), and 58 inhibitor negative patients, after informed consent. While 53.54% (68/127) index cases were positive for intron 22 or intron 1 inversions, 55 causative F8 mutations were detected in the 59 inversion negative patients, of which 23 were novel mutations (in 24 patients) and 32 were reported earlier (in 35 patients). A higher incidence of mutations, in the C1 and C2 domains in inhibitor positive patients, and in the A1 domain in inhibitor negative patients was observed, though not significantly different. The study suggests that large F8 rearrangements (significantly higher in the inhibitor positive patients) pose the highest risk, while missense mutations (significantly higher in the inhibitor negative patients) pose the lowest risk of inhibitor development in Indian hemophilia A patients.

  14. Functional Role of Milk Fat Globule-Epidermal Growth Factor VIII in Macrophage-Mediated Inflammatory Responses and Inflammatory/Autoimmune Diseases

    Directory of Open Access Journals (Sweden)

    Young-Su Yi

    2016-01-01

    Full Text Available Inflammation involves a series of complex biological processes mediated by innate immunity for host defense against pathogen infection. Chronic inflammation is considered to be one of the major causes of serious diseases, including a number of autoimmune/inflammatory diseases, cancers, cardiovascular diseases, and neurological diseases. Milk fat globule-epidermal growth factor 8 (MFG-E8 is a secreted protein found in vertebrates and was initially discovered as a critical component of the milk fat globule. Previously, a number of studies have reported that MFG-E8 contributes to various biological functions including the phagocytic removal of damaged and apoptotic cells from tissues, the induction of VEGF-mediated neovascularization, the maintenance of intestinal epithelial homeostasis, and the promotion of mucosal healing. Recently, emerging studies have reported that MFG-E8 plays a role in inflammatory responses and inflammatory/autoimmune diseases. This review describes the characteristics of MFG-E8-mediated signaling pathways, summarizes recent findings supporting the roles of MFG-E8 in inflammatory responses and inflammatory/autoimmune diseases, and discusses MFG-E8 targeting as a potential therapeutic strategy for the development of anti-inflammatory/autoimmune disease drugs.

  15. Functional Role of Milk Fat Globule-Epidermal Growth Factor VIII in Macrophage-Mediated Inflammatory Responses and Inflammatory/Autoimmune Diseases.

    Science.gov (United States)

    Yi, Young-Su

    2016-01-01

    Inflammation involves a series of complex biological processes mediated by innate immunity for host defense against pathogen infection. Chronic inflammation is considered to be one of the major causes of serious diseases, including a number of autoimmune/inflammatory diseases, cancers, cardiovascular diseases, and neurological diseases. Milk fat globule-epidermal growth factor 8 (MFG-E8) is a secreted protein found in vertebrates and was initially discovered as a critical component of the milk fat globule. Previously, a number of studies have reported that MFG-E8 contributes to various biological functions including the phagocytic removal of damaged and apoptotic cells from tissues, the induction of VEGF-mediated neovascularization, the maintenance of intestinal epithelial homeostasis, and the promotion of mucosal healing. Recently, emerging studies have reported that MFG-E8 plays a role in inflammatory responses and inflammatory/autoimmune diseases. This review describes the characteristics of MFG-E8-mediated signaling pathways, summarizes recent findings supporting the roles of MFG-E8 in inflammatory responses and inflammatory/autoimmune diseases, and discusses MFG-E8 targeting as a potential therapeutic strategy for the development of anti-inflammatory/autoimmune disease drugs.

  16. Clotting of mammalian fibrinogens by papain: a re-examination.

    Science.gov (United States)

    Doolittle, Russell F

    2014-10-28

    Papain has long been known to cause the gelation of mammalian fibrinogens. It has also been reported that papain-fibrin is insoluble in dispersing solvents like strong urea or sodium bromide solutions, similar to what is observed with thrombin-generated clots in the presence of factor XIIIa and calcium. In those old studies, both the gelation and subsequent clot stabilization were attributed to papain, although the possibility that the second step might be due to contaminating factor XIII in fibrinogen preparations was considered. I have revisited this problem in light of knowledge acquired over the past half-century about thiol proteases like papain, which mostly cleave peptide bonds, and transglutaminases like factor XIIIa that catalyze the formation of ε-lysyl-γ-glutamyl cross-links. Recombinant fibrinogen, inherently free of factor XIII and other plasma proteins, formed a stable gel when treated with papain alone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the intermolecular cross-linking in papain-fibrin leads to γ-chain dimers, trimers, and tetramers, just as is the case with thrombin-factor XIIIa-stabilized fibrin. Mass spectrometry of bands excised from gels showed that the cross-linked material is quite different from what occurs with factor XIIIa, however. With papain, the cross-linking occurs between γ chains in neighboring protofibrils becoming covalently linked in a "head-to-tail" fashion by a transpeptidation reaction involving the α-amino group of γ-Tyr1 and a papain cleavage site at γ-Gly403 near the carboxy terminus, rather than by the (reciprocal) "tail-to-tail" manner that occurs with factor XIIIa and that depends on cross-links between γ-Lys406 and γ-Gln398.

  17. In black south africans from rural and urban communities, the 4G/5G PAI-1 polymorphism influences PAI-1 activity, but not plasma clot lysis time

    NARCIS (Netherlands)

    Z. de Lange (Zelda); D.C. Rijken (Dingeman); T. Hoekstra (Tiny); K.R. Conradie (Karin); J.C. Jerling (Johann); M. Pieters (Marlien)

    2013-01-01

    textabstractData on genetic and environmental factors influencing PAI-1 levels and their consequent effect on clot lysis in black African populations are limited. We identified polymorphisms in the promoter area of the PAI-1 gene and determined their influence on PAI-1act levels and plasma clot lysi

  18. Probing the coagulation pathway with aptamers identifies combinations that synergistically inhibit blood clot formation.

    Science.gov (United States)

    Bompiani, Kristin M; Lohrmann, Jens L; Pitoc, George A; Frederiksen, James W; Mackensen, George B; Sullenger, Bruce A

    2014-08-14

    Coordinated enzymatic reactions regulate blood clot generation. To explore the contributions of various coagulation enzymes in this process, we utilized a panel of aptamers against factors VIIa, IXa, Xa, and prothrombin. Each aptamer dose-dependently inhibited clot formation, yet none was able to completely impede this process in highly procoagulant settings. However, several combinations of two aptamers synergistically impaired clot formation. One extremely potent aptamer combination was able to maintain human blood fluidity even during extracorporeal circulation, a highly procoagulant setting encountered during cardiopulmonary bypass surgery. Moreover, this aptamer cocktail could be rapidly reversed with antidotes to restore normal hemostasis, indicating that even highly potent aptamer combinations can be rapidly controlled. These studies highlight the potential utility of using sets of aptamers to probe the functions of proteins in molecular pathways for research and therapeutic ends. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Addition of a sequence from α2-antiplasmin transforms human serum albumin into a blood clot component that speeds clot lysis

    Directory of Open Access Journals (Sweden)

    Gataiance Sharon

    2009-03-01

    Full Text Available Abstract Background The plasma protein α2-antiplasmin (α2AP is cross-linked to fibrin in blood clots by the transglutaminase factor XIIIa, and in that location retards clot lysis. Competition for this effect could be clinically useful in patients with thrombosis. We hypothesized that fusion of N-terminal portions of α2-antiplasmin to human serum albumin (HSA and production of the chimeric proteins in Pichia pastoris yeast would produce a stable and effective competitor protein. Results Fusion protein α2AP(13-42-HSA was efficiently secreted from transformed yeast and purified preparations contained within a mixed population the full-length intact form, while fusions with longer α2AP moieties were inefficiently secreted and/or degraded. The α2AP(13-42-HSA protein, but not recombinant HSA, was cross-linked to both chemical lysine donors and fibrin or fibrinogen by factor XIIIa, although with less rapid kinetics than native α2AP. Excess α2AP(13-42-HSA competed with α2AP for cross-linking to chemical lysine donors more effectively than a synthetic α2AP(13-42 peptide, and reduced the α2AP-dependent resistance to fibrinolysis of plasma clots equally effectively as the peptide. Native α2AP was found in in vivo clots in rabbits to a greater extent than α2AP(13-42, however. Conclusion In this first report of transfer of transglutamination substrate status from one plasma protein to another, fusion protein α2AP(13-42-HSA was shown to satisfy initial requirements for a long-lasting, well-tolerated competitive inhibitor of α2-antiplasmin predicted to act in a clot-localized manner.

  20. Impaired thrombin generation and fibrin clot formation in patients with dilutional coagulopathy during major surgery.

    NARCIS (Netherlands)

    Schols, S.E.; Lance, M.D.; Feijge, M.A.; Damoiseaux, J.; Marcus, M.A.; Hamulyak, K.; Cate, H. ten; Heemskerk, J.W.M.; Pampus, E.C.M. van

    2010-01-01

    Patients subjected to haemodilution during surgery are at increased risk of bleeding. We hypothesised that, in the acquired dilutional coagulopathy, insufficient haemostasis is due to either insufficient thrombin generation or insufficient fibrin clot formation. In tissue factor-activated plasmas fr

  1. Protein-membrane interactions: blood clotting on nanoscale bilayers.

    Science.gov (United States)

    Morrissey, J H; Pureza, V; Davis-Harrison, R L; Sligar, S G; Rienstra, C M; Kijac, A Z; Ohkubo, Y Z; Tajkhorshid, E

    2009-07-01

    The clotting cascade requires the assembly of protease-cofactor complexes on membranes with exposed anionic phospholipids. Despite their importance, protein-membrane interactions in clotting remain relatively poorly understood. Calcium ions are known to induce anionic phospholipids to cluster, and we propose that clotting proteins assemble preferentially on such anionic lipid-rich microdomains. Until recently, there was no way to control the partitioning of clotting proteins into or out of specific membrane microdomains, so experimenters only knew the average contributions of phospholipids to blood clotting. The development of nanoscale membrane bilayers (Nanodiscs) has now allowed us to probe, with nanometer resolution, how local variations in phospholipid composition regulate the activity of key protease-cofactor complexes in blood clotting. Furthermore, exciting new progress in solid-state NMR and large-scale molecular dynamics simulations allow structural insights into interactions between proteins and membrane surfaces with atomic resolution.

  2. Co-segregation of thrombophilic disorders in factor V Leiden carriers; the contributions of factor VIII, factor XI, thrombin activatable fibrinolysis inhibitor and lipoprotein(a) to the absolute risk of venous thromboembolism

    NARCIS (Netherlands)

    Libourel, EJ; Bank, [No Value; Meinardi, [No Value; Balje-Volkers, CP; Hamulyak, K; Middeldorp, S; Koopman, MMW; van Pampus, ECM; Prins, MH; Buller, HR; van der Meer, J

    2002-01-01

    Background and Objectives. The clinical expression of factor V Leiden varies widely within and between families and only a minority of carriers will ever develop venous thromboembolism. Co-segregation of thrombophilic disorders is a possible explanation, Our aim was to assess the contributions of

  3. Identification of Quantitative Trait Loci for Fibrin Clot Phenotypes. The EuroCLOT Study

    DEFF Research Database (Denmark)

    Williams, Frances M K; Carter, Angela M; Kato, Bernet;

    2009-01-01

    associated with fibrin phenotypes. METHODS AND RESULTS: 447 dizygotic (DZ) and 460 monozygotic (MZ) pairs of healthy UK white female twins and 199 DZ twin pairs from Denmark were studied. D-dimer, an indicator of fibrin turnover, was measured by ELISA and measures of clot formation, morphology, and lysis...

  4. Blood clot formation does not affect metastasis formation or tumor growth in a murine model of breast cancer.

    Directory of Open Access Journals (Sweden)

    Stephanie Rossnagl

    Full Text Available Cancer is associated with increased fracture risk, due either to metastasis or associated osteoporosis. After a fracture, blood clots form. Because proteins of the coagulation cascade and activated platelets promote cancer development, a fracture in patients with cancer often raises the question whether it is a pathologic fracture or whether the fracture itself might promote the formation of metastatic lesions. We therefore examined whether blood clot formation results in increased metastasis in a murine model of experimental breast cancer metastasis. For this purpose, a clot was surgically induced in the bone marrow of the left tibia of immundeficient mice. Either one minute prior to or five minutes after clot induction, human cancer cells were introduced in the circulation by intracardiac injection. The number of cancer cells that homed to the intervention site was determined by quantitative real-time PCR and flow cytometry. Metastasis formation and longitudinal growth were evaluated by bioluminescence imaging. The number of cancer cells that homed to the intervention site after 24 hours was similar to the number of cells in the opposite tibia that did not undergo clot induction. This effect was confirmed using two more cancer cell lines. Furthermore, no difference in the number of macroscopic lesions or their growth could be detected. In the control group 72% developed a lesion in the left tibia. In the experimental groups with clot formation 79% and 65% developed lesions in the left tibia (p = ns when comparing each experimental group with the controls. Survival was similar too. In summary, the growth factors accumulating in a clot/hematoma are neither enough to promote cancer cell homing nor support growth in an experimental model of breast cancer bone metastasis. This suggests that blood clot formation, as occurs in traumatic fractures, surgical interventions, and bruises, does not increase the risk of metastasis formation.

  5. Determining Human Clot Lysis Time (in vitro with Plasminogen/Plasmin from Four Species (Human, Bovine, Goat, and Swine

    Directory of Open Access Journals (Sweden)

    Omaira Cañas Bermúdez

    2015-05-01

    Full Text Available Cardiovascular disease is the leading cause of death worldwide, including failures in the plasminogen/plasmin system which is an important factor in poor lysis of blood clots. This article studies the fibrinolytic system in four species of mammals, and it identifies human plasminogen with highest thrombolysis efficiency. It examines plasminogen from four species (human, bovine, goat, and swine and identifies the most efficient one in human clot lysis in vitro. All plasminogens were identically purified by affinity chromatography. Human fibrinogen was purified by fractionation with ethanol. The purification of both plasminogen and fibrinogen was characterized by one-dimensional SDS-PAGE (10%. Human clot formation in vitro and its dissolution by plasminogen/plasmin consisted of determining lysis time from clot formation to its dilution. Purification of proteins showed greater than 95% purity, human plasminogen showed greater ability to lyse clot than animal plasminogen. The article concludes that human plasminogen/plasmin has the greatest catalysis and efficiency, as it dissolves human clot up to three times faster than that of irrational species.

  6. Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix.

    Science.gov (United States)

    Greiling, D; Clark, R A

    1997-04-01

    After injury, the wound space is filled with a fibrin/fibronectin clot containing growth factors released by platelets and monocytes. In response to these factors, fibroblasts migrate into the fibrin clot and contribute to the formation of granulation tissue. The functional mechanisms allowing fibroblasts to leave the collagenous matrix of normal connective tissue and invade the provisional matrix of the fibrin clot have not been fully defined. To investigate these mechanisms we established a new in vitro model which simulates specific aspects of early wound healing, that is, the migration of fibroblasts from a three-dimensional collagen matrix into a fibrin clot. This transmigration could be induced by physiological concentrations of platelet releasate or platelet-derived growth factor BB (PDGF-BB) in a concentration-dependent manner. At 24 hours irradiated fibroblasts invaded the fibrin gel almost as well as non-irradiated cells, indicating that transmigration was independent of proliferation. Plasminogen and its activators appear to be necessary for invasion of the fibrin clot since protease inhibitors decreased the amount of migration. These serine proteases, however, were not necessary for exit from the collagen gel as fibroblasts migrated out of the collagen gel onto a surface coated with fibrin fibrils even in the presence of inhibitors. Removal of fibronectin (FN) from either the collagen gel or the fibrin gel markedly decreased the number of migrating cells, suggesting that FN provides a conduit for transmigration. Cell movement in the in vitro model was inhibited by RGD peptide, and by monoclonal antibodies against the subunits of the alpha5 beta1 and alpha v beta3 integrin receptor. Thus, the functional requirements for fibroblast transmigration from collagen-rich to fibrin-rich matrices, such as occurs in early wound healing, have been partially defined using an in vitro paradigm of this important biologic process.

  7. THE ACTION OF CHLORINATED ANTISEPTICS ON BLOOD CLOT

    Science.gov (United States)

    Taylor, Herbert D.; Stebbins, Marianne G.

    1919-01-01

    This work demonstrates that the chlorinated antiseptics have no power to penetrate blood clots and destroy bacteria therein contained. Correspondingly, blood clots may protect virulent bacteria for a long period of time and the organisms properly planted will be able to proliferate in a normal manner. PMID:19868298

  8. Altered plasma fibrin clot properties in essential thrombocythemia.

    Science.gov (United States)

    Małecki, Rafał; Gacka, Małgorzata; Kuliszkiewicz-Janus, Małgorzata; Jakobsche-Policht, Urszula; Kwiatkowski, Jacek; Adamiec, Rajmund; Undas, Anetta

    2016-01-01

    Patients with increased thromboembolic risk tend to form denser fibrin clots which are relatively resistant to lysis. We sought to investigate whether essential thrombocythemia (ET) is associated with altered fibrin clot properties in plasma. Ex vivo plasma fibrin clot permeability coefficient (Ks), turbidimetry and clot lysis time (CLT) were measured in 43 consecutive patients with ET (platelet count from 245 to 991 × 10(3)/µL) and 50 control subjects matched for age, sex and comorbidities. Fibrinolysis proteins and inhibitors together with platelet activation markers were determined. Reduced Ks (-38%, p Ks inversely correlated with fibrinogen, PF4 and C-reactive protein. CLT positively correlated only with PAI-1. Patients with ET display prothrombotic plasma fibrin clot phenotype including impaired fibrinolysis, which represents a new prothrombotic mechanism in this disease.

  9. Identification of quantitative trait loci for fibrin clot phenotypes: The EuroCLOT study

    Science.gov (United States)

    Williams, Frances MK; Carter, Angela M; Kato, Bernet; Falchi, Mario; Bathum, Lise; Surdulescu, Gabriela; Kyvik, Kirsten Ohm; Palotie, Aarno; Spector, Tim D; Grant, Peter J

    2012-01-01

    Objectives Fibrin makes up the structural basis of an occlusive arterial thrombus and variability in fibrin phenotype relates to cardiovascular risk. The aims of the current study from the EU consortium EuroCLOT were to 1) determine the heritability of fibrin phenotypes and 2) identify QTLs associated with fibrin phenotypes. Methods 447 dizygotic (DZ) and 460 monozygotic (MZ) pairs of healthy UK Caucasian female twins and 199 DZ twin pairs from Denmark were studied. D-dimer, an indicator of fibrin turnover, was measured by ELISA and measures of clot formation, morphology and lysis were determined by turbidimetric assays. Heritability estimates and genome-wide linkage analysis were performed. Results Estimates of heritability for d-dimer and turbidometric variables were in the range 17 - 46%, with highest levels for maximal absorbance which provides an estimate of clot density. Genome-wide linkage analysis revealed 6 significant regions with LOD>3 on 5 chromosomes (5, 6, 9, 16 and 17). Conclusions The results indicate a significant genetic contribution to variability in fibrin phenotypes and highlight regions in the human genome which warrant further investigation in relation to ischaemic cardiovascular disorders and their therapy. PMID:19150881

  10. Removal of Chronic Intravascular Blood Clots using Liquid Plasma

    Science.gov (United States)

    Jung, Jae-Chul; Choi, Myeong; Koo, Il; Yu, Zengqi; Collins, George

    2011-10-01

    An electrical embolectomy device for removing chronic intravascular blood clots using liquid plasma under saline environment was demonstrated. We employed a proxy experimental blood clot model of deep vein thrombosis (DVT) and actual equine blood clot. Thermal damage to contiguous tissue and the collagen denaturing via the plasma irradiation were investigated by histological analysis using birefringence of the tissue and verified by FT-IR spectroscopic study, respectively, which showed the high removal rate up to 2 mm per minute at room temperature and small thermal damage less than 200 μm.

  11. Thrombin generation in Cushing's Syndrome: do the conventional clotting indices tell the whole truth?

    Science.gov (United States)

    Koutroumpi, S; Spiezia, L; Albiger, N; Barbot, M; Bon, M; Maggiolo, S; Gavasso, S; Simioni, P; Frigo, A; Mantero, F; Scaroni, C

    2014-02-01

    Cushing's Syndrome (CS) is associated with an increased mortality, where hypercoagulability seems to have a crucial role in both arterial and venous thrombosis. Parameters of in vitro thrombin generation (TG) such as lag time, peak thrombin and endogenous thrombin potential (ETP), that describe the time until thrombin burst, the peak amount of TG and the total amount of thrombin generated, respectively as well as classical clotting markers were evaluated in 33 CS patients compared to both a group of 28 patients matched for the features of Metabolic Syndrome (MetS) and 31 healthy individuals. CS and MetS patients had shorter lag time (p < 0.0001), higher peak and ETP (p < 0.0001) than healthy controls, though lag time was less shortened in CS (p < 0.0001) respect to MetS group. Prothrombin time (PT) was increased (p < 0.0001) in both CS and MetS patients, while partial thromboplastin time (PTT) was shorter (p < 0.0001) in CS compared to both MetS and healthy group (p < 0.0001). Factor VIII (FVIII), Antithrombin (AT), protein C and S were increased only in CS patients (p < 0.0001). lag time, AT and FVIII correlated to night salivary cortisol (r = + 0.59; p = 0.0005, r = + 0.40; p = 0.003, r = + 0.40; p = 0.04, respectively); PTT correlated inversely to urinary free cortisol (r = -0.45; p = 0.009). BMI correlated negatively to lag time (r = -0.40; p = 0.0001) and positively to peak and ETP (r = + 0.34; p = 0.001, r = + 0.28; p = 0.008, respectively). Obese and diabetic patients had shorter lag time (p = 0.0005; p = 0.0002, respectively), higher ETP (p = 0.0006; p = 0.007, respectively) and peak (p = 0.0003; p = 0.0005, respectively) as well as a more prolonged PT (p = 0.04; p = 0.009, respectively). Hypertensive individuals had higher ETP (p = 0.004), peak (p = 0.0008) and FVIII (p = 0.001). Our findings confirm a prothrombotic state in both CS and MetS patients, though lag time was less shortened in

  12. Drinking Peroxide as 'Natural' Cure Leads to Dangerous Blood Clots

    Science.gov (United States)

    ... https://medlineplus.gov/news/fullstory_163513.html Drinking Peroxide as 'Natural' Cure Leads to Dangerous Blood Clots ... 9, 2017 (HealthDay News) -- Ingesting high-concentration hydrogen peroxide as a "natural cure" or cleansing agent may ...

  13. Adenosine diphosphate-decorated chitosan nanoparticles shorten blood clotting times, influencing the structures and varying the mechanical properties of the clots.

    Science.gov (United States)

    Chung, Tze-Wen; Lin, Pei-Yi; Wang, Shoei-Shen; Chen, Yen-Fung

    2014-01-01

    Chitosan nanoparticles (NPs) decorated with adenosine diphosphate (ADP) (ANPs) or fibrinogen (FNPs) were used to fabricate hemostatic NPs that can shorten blood clotting time and prevent severe local hemorrhage. The structure and mechanical properties of the blood clot induced with ANP (clot/ANP) or FNP (clot/FNP) were also investigated. The NPs, ANPs, and FNPs, which had particle sizes of 245.1 ± 14.0, 251.0 ± 9.8, and 326.5 ± 14.5 nm and zeta potentials of 24.1 ± 0.5, 20.6 ± 1.9, and 15.3 ± 1.5 mV (n=4), respectively, were fabricated by ionic gelation and then decorated with ADP and fibrinogen. The zeta potentials and Fourier transform infrared (FTIR) spectroscopy of the NPs confirmed that their surfaces were successfully coated with ADP and fibrinogen. The scanning electron microscope (SEM) micrographs of the structure of the clot induced with "undecorated" chitosan NPs (clot/NP), clot/ANP, and clot/FNP (at 0.05 wt%) were different, after citrated bloods had been recalcified by a calcium chloride solution containing NPs, ANPs, or FNPs. This indicated that many NPs adhered on the membrane surfaces of red blood cells, that ANPs induced many platelet aggregates, and that FNPs were incorporated into the fibrin network in the clots. Measurements of the blood clotting times (Tc) of blood clot/NPs, clot/ANPs, and clot/FNPs, based on 90% of ultimate frequency shifts measured on a quartz crystal microbalance (QCM), were significantly (P<0.05) (n=4) shorter than that of a clot induced by a phosphate-buffered solution (PBS) (clot/PBS) (63.6% ± 3.1%, 48.3% ± 6.2%, and 63.2% ± 4.7%, respectively). The ΔF2 values in the spectra of frequency shifts associated with the propagation of fibrin networks in the clot/ANPs and clot/FNPs were significantly lower than those of clot/PBS. Interestingly, texture profile analysis of the compressional properties showed significantly lower hardness and compressibility in clot/NPs and clot/ANPs (P<0.05 or better) (n=4) compared with

  14. Weight reduction is associated with increased plasma fibrin clot lysis.

    Science.gov (United States)

    Brzezińska-Kolarz, Beata; Kolarz, Marek; Wałach, Angelika; Undas, Anetta

    2014-11-01

    Obesity is associated with an increased risk of vascular thrombotic events. We sought to investigate how obesity and weight loss affect plasma fibrin clot properties. A total of 29 obese patients were studied before and after 3-month low-fat diet. Plasma fibrin clot parameters, including fibrin clot permeation coefficient (Ks), the lag phase of the turbidity curve, clot lysis time (t 50%), maximum rate of increase in D-dimer levels, and maximum D-dimer concentrations, were determined. Low-fat diet resulted in the reduction of body weight (P < .0001), body mass index (P < .0001), fat mass (P < .0001), total cholesterol (P < .0001), low-density lipoprotein cholesterol (P = .0005), triglycerides (P = .008), and plasminogen activator inhibitor 1 (P = .02), but not in fibrinogen or C-reactive protein. The only change in fibrin clot variables was shorter t 50% (P = .02). Baseline t 50%, but not posttreatment, correlated with waist circumference (r = .44, p = .02). This study demonstrates that weight loss in obese people can increase the efficiency of fibrin clot lysis.

  15. Identification of fibrin clot-bound plasma proteins.

    Directory of Open Access Journals (Sweden)

    Simone Talens

    Full Text Available Several proteins are known to bind to a fibrin network and to change clot properties or function. In this study we aimed to get an overview of fibrin clot-bound plasma proteins. A plasma clot was formed by adding thrombin, CaCl(2 and aprotinin to citrated platelet-poor plasma and unbound proteins were washed away with Tris-buffered saline. Non-covalently bound proteins were extracted, separated with 2D gel electrophoresis and visualized with Sypro Ruby. Excised protein spots were analyzed with mass spectrometry. The identity of the proteins was verified by checking the mass of the protein, and, if necessary, by Western blot analysis. Next to established fibrin-binding proteins we identified several novel fibrin clot-bound plasma proteins, including α(2-macroglobulin, carboxypeptidase N, α(1-antitrypsin, haptoglobin, serum amyloid P, and the apolipoproteins A-I, E, J, and A-IV. The latter six proteins are associated with high-density lipoprotein particles. In addition we showed that high-density lipoprotein associated proteins were also present in fibrinogen preparations purified from plasma. Most plasma proteins in a fibrin clot can be classified into three groups according to either blood coagulation, protease inhibition or high-density lipoprotein metabolism. The presence of high-density lipoprotein in clots might point to a role in hemostasis.

  16. Isolation of Salmonella typhi from Standard Whole Blood Culture versus Blood-Clot Cultures

    Science.gov (United States)

    1988-12-01

    The use of 10% oxgall and bile broth medium, both supplemented with freshly prepared 100 u/ml streptokinase, for isolating Salmonella typhi by clot...significantly better rate of isolation than the clot culture methods. Keywords: Cultures biology; Clot cultures; Salmonella typhi ; Isolation of S. typhi; Whole blood culture; Blood-clot culture; Reprints.

  17. {sup 99m}Tc-NC100668, a new tracer for imaging venous thromboemboli: pre-clinical biodistribution and incorporation into plasma clots in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, David [Grove Centre, Research and Development, GE Healthcare Bio-Sciences, Little Chalfont (United Kingdom); Uppsala University Hospital, Institution of Oncology, Radiology and Clinical Immunology, Section of Radiology, Uppsala (Sweden); Lewis, Joanne; Battle, Mark; Lear, Rochelle; Farrar, Gill; Barnett, D.J.; Godden, Vanessa; Oliveira, Alexandra; Coombes, Catherine [Grove Centre, Research and Development, GE Healthcare Bio-Sciences, Little Chalfont (United Kingdom); Ahlstroem, Haakan [Uppsala University Hospital, Institution of Oncology, Radiology and Clinical Immunology, Section of Radiology, Uppsala (Sweden)

    2006-11-15

    {sup 99m}Tc-NC100668 is a new radiotracer being developed to aid the diagnosis of thromboembolism. The structure of NC100668 is similar to a region of human {alpha}{sub 2}-antiplasmin, which is a substrate for factor XIIIa (FXIIIa). The purpose of this study was to confirm the uptake of {sup 99m}Tc-NC100668 into forming plasma clot and to establish the biodistribution of {sup 99m}Tc-NC100668 in Wistar rats. The in vitro plasma clot uptake of {sup 99m}Tc-NC100668 and other compounds with known affinities to FXIIIa was measured using a plasma clot assay. The biodistribution and blood clot uptake of radioactivity of {sup 99m}Tc-NC100668 in normal Wistar rats and those bearing experimentally induced deep vein thrombi were investigated. The in vitro uptake of {sup 99m}Tc-NC100668 was greater than that for [{sup 14}C]dansyl cadaverine, a known substrate of FXIIIa in the plasma clot assay. The biodistribution of {sup 99m}Tc-NC100668 in male and female Wistar rats up to 24 h p.i. showed that radioactivity was rapidly excreted, predominantly into the urine, with very little background tissue retention. In vivo the uptake and retention of {sup 99m}Tc-NC100668 into the blood clot was greater than could be accounted for by non-specific accumulation of the radiotracer within the blood clot. {sup 99m}Tc-NC100668 was retained by plasma clots in vitro and blood clots in vivo. No significant tissue retention which could interfere with the ability to image thrombi in vivo was observed. This evidence suggests that {sup 99m}Tc-NC100668 might be useful in the detection of thromboembolism. (orig.)

  18. Purification and characterization of anti-clotting protein component (ACPF-7221) from venom of Agkistrodon acutus

    Institute of Scientific and Technical Information of China (English)

    RUI Jing; HUAI Jian-guo; ZHANG Ye; CHENG Dong-yun; PAN Xue-bing

    2009-01-01

    Background Snake venom contains a number of components with different pharmacological and biological activities, especially in cancer therapy, and has increasingly become a research focus. This study was designed to isolate and purify a novel anti-clotting protein component from the venom of Agkistrodon acutus, and to explore its physico-chemical properties and biological activity.Methods The venom of Agkistrodon was isolated and purified by ion-exchange chromatography on diethylaminoethyl (DEAE)-Sepharose Fast Flow, molecular sieve filtration through Sephadex G75, SP-Sepharose Fast Flow and molecular sieve filtration through Sephadex G50. We detected the activated partial thromboplastin time (APTT) of the eluant to select the anti-clotting protein component of interest. The molecular weight was determined by sodium dodecyl sulfate-polyacrylamid gel electrphoresis (SDS-PAGE) and liquid chromatography. Its protein content was detected by bicinchoninic acid (BCA).Results SDS-PAGE vertical gel electrophoresis showed that the anticoagulant factor is a tripolymer composed of three proteins whose molecular weights are 25 KDa, 30 KDa and 50 KDa. The factor contains about 65% percent protein.Conclusions A novel anti-clotting protein component was purified by ion-exchange chromatography and molecular sieve filtration from the venom of Agkistrodon acutus and was found to be composed of three kinds of proteins.

  19. Replacement of isoleucine-397 by threonine in the clotting proteinase factor IXa (Los Angeles and Long Beach variants) affects macromolecular catalysis but not L-tosylarginine methyl ester hydrolysis. Lack of correlation between the ox brain prothrombin time and the mutation site in the variant proteins.

    Science.gov (United States)

    Spitzer, S G; Warn-Cramer, B J; Kasper, C K; Bajaj, S P

    1990-01-01

    Previously, from the plasma of unrelated haemophilia-B patients, we isolated two non-functional Factor IX variants, namely Los Angeles (IXLA) and Long Beach (IXLB). Both variants could be cleaved to yield Factor IXa-like molecules, but were defective in catalysing the cleavage of Factor X (macromolecular substrate) and in binding to antithrombin III (macromolecular inhibitor). In the present study we have identified the mutation of IXLA by amplifying the exons (including flanking regions) as well as the 5' end of the gene by polymerase-chain-reaction (PCR) method and sequencing the amplified DNA by the dideoxy chain-termination method. Comparison of the normal IX and IXLA sequences revealed only one base substitution (T----C) in exon VIII of IXLA, with a predicted replacement of Ile-397 to Thr in the mature protein. This mutation is the same as found recently for IXLB. The observation that IXLB and IXLA have the same mutation is an unexpected finding, since, on the basis of their ox brain prothrombin time (PT, a test that measures the ability of the variant Factor IX molecules to inhibit the activation of Factor X by Factor VIIa-tissue factor complex), these variants have been classified into two different groups and were thought to be genetically different. Our observation thus suggests that the ox brain PT does not reflect the locus of mutation in the coding region of the variant molecules. However, our analysis suggests that the ox brain PT is related to Factor IX antigen concentration in the patient's plasma. Importantly, although the mutation in IXLA or IXLB protein is in the catalytic domain, purified IXaLA and IXaLB hydrolyse L-tosylarginine methyl ester at rates very similar to that of normal IXa. These data, in conjunction with our recent data on Factor IXBm Lake Elsinore (Ala-390----Val mutant), strengthen a conclusion that the peptide region containing residues 390-397 of normal Factor IXa plays an essential role in macromolecular substrate catalysis and

  20. Ex vivo effects of low-dose rivaroxaban on specific coagulation assays and coagulation factor activities in patients under real life conditions.

    Science.gov (United States)

    Mani, Helen; Hesse, Christian; Stratmann, Gertrud; Lindhoff-Last, Edelgard

    2013-01-01

    Global coagulation assays display variable effects at different concentrations of rivaroxaban. The aim of this study is to quantify the ex vivo effects of low-dose rivaroxaban on thrombophilia screening assays and coagulation factor activities based on the administration time, and to show how to mask possible interferences. Plasma samples from 40 patients receiving rivaroxaban 10 mg daily were investigated to measure activities of clotting factor II, V, VII, VIII, IX, XI, XII and XIII; protein C- and protein S-levels; lupus anticoagulants; anticardiolipin IgG and IgM; D-dimer, heparin-platelet factor 4 (HPF4) antibodies and screening tests for von Willebrand disease (VWD). Two hours after rivaroxaban administration, the activities of clotting factors were significantly decreased to different extents, except for factor XIII. Dilution of plasma samples resulted in neutralisation of these interferences. The chromogenic protein C activity assay was not affected by rivaroxaban. Depending on the timing of tablet intake in relation to blood sampling protein S activity was measured falsely high when a clotting assay was used. False-positive results for lupus anticoagulants were observed depending on the assay system used and the administration time of rivaroxaban. ELISA-based assays such as anticardiolipin IgG and IgM, D-dimer, HPF4-antibodies and the turbidimetric assays for VWD were not affected by rivaroxaban. Specific haemostasis clotting tests should be performed directly prior to rivaroxaban intake. Assay optimisation in the presence of rivaroxaban can be achieved by plasma dilution. Immunologic assays are not influenced by rivaroxaban, while chromogenic assays can be used, when they do not depend on factor Xa.

  1. Factor V deficiency

    Science.gov (United States)

    ... When certain blood clotting factors are low or missing, your blood does not clot properly. Factor V deficiency is rare. It may be caused by: A defective Factor V gene passed down through families (inherited) An antibody that interferes with normal Factor ...

  2. Understand Your Risk for Excessive Blood Clotting

    Science.gov (United States)

    ... such as car or plane trips Use of birth control pills or hormone replacement therapy Cancer Genetic Risk Factors ... Examples of medicines that may contain estrogen include birth control pills and hormone therapy. Other Factors Many other factors ...

  3. Novel mechanisms that regulate clot structure/function.

    Science.gov (United States)

    Ariëns, Robert A S

    2016-05-01

    The structure and function of the blood clot has been associated with altered risk of thrombosis. Dense fibrin structures with small pores increase the risk of thrombosis, and have major functional consequences by increasing the resistance to fibrinolysis and altering the visco-elastic properties of the clot. However, while the structural changes to the overall fibrin network have been extensively characterised, little is known regarding the intrafibrillar structure of fibrin, the way protofibrils are arranged inside the fibrin fibers and the functional consequences of this. This brief paper aims to review recent findings regarding novel mechanisms that regulate fibrin intrafibrillar structure, including the degree of protofibril packing, their functional consequences, and the effects of FXIII activation on clot structure and thrombosis. It is concluded that fibrin intrafibrillar structure represents a major novel mechanism that influences clot structure and stability. Future studies are required to investigate the role of fibrin intrafibrillar structure in the functional characteristics of the blood clot, and in diseases of bleeding and thrombosis.

  4. Highly stable plasminogen activator inhibitor type one (VLHL PAI-1) protects fibrin clots from tissue plasminogen activator-mediated fibrinolysis.

    Science.gov (United States)

    Jankun, Jerzy; Aleem, Ansari M; Selman, Steven H; Skrzypczak-Jankun, Ewa; Lysiak-Szydlowska, Wieslawa; Grafos, Nicholas; Fryer, Hugh J L; Greenfield, Robert S

    2007-11-01

    Plasminogen activator inhibitor-1 (PAI-1) is the major specific inhibitor of tissue-type plasminogen activator (tPA) which mediates fibrin clot lysis through activation of plasminogen. Wild-type-PAI-1 (wPAI-1) is rapidly converted to the latent form (half-life of approximately 2 h) and loses its ability to inhibit tPA. We developed a very long half-life PAI-1 (VLHL PAI-1), a recombinant protein with a half-life >700 h compared with wPAI-1. In this study, VLHL PAI-1 was assessed for its ability to inhibit clot lysis in vitro. Clot formation was initiated in normal plasma supplemented with tPA by the addition of either tissue factor or human recombinant FVIIa. Clot lysis time, monitored turbidimetrically in a microtiter plate reader, was determined at various concentrations of wPAI-1 and VLHL PAI-1. Both wPAI-1 and VLHL PAI-1 caused a significant increase in clot lysis time, although the latter was somewhat less effective at lower concentrations. The VLHL PAI-1, but not wPAI-1, maintained its anti-fibrinolytic activity after preincubation overnight at 37 degrees. These studies demonstrate that VLHL PAI-1 is an effective inhibitor of fibrin clot degradation. Due to the high stability of VLHL PAI-1 compared with wPAI-1, this novel inhibitor of tPA-mediated fibrinolysis may have therapeutic applications for treating surgical and trauma patients when used directly or in conjunction with the procoagulant recombinant FVIIa.

  5. Modification of fibrinogen with poly(ethylene glycol) and its effects on fibrin clot characteristics.

    Science.gov (United States)

    Barker, T H; Fuller, G M; Klinger, M M; Feldman, D S; Hagood, J S

    2001-09-15

    The suitability of existing topical fibrin glue preparations for tissue sealing or local drug delivery applications is greatly limited by their poor mechanical properties and the limited capacity of fibrinogen (Fgn) to actively bind growth factors or other therapeutic agents. Poly(ethylene glycol) (PEG) offers potential solutions to these problems by providing a mechanism for increasing the number of crosslinks between adjacent fibrin monomer molecules or for covalently crosslinking Fgn to therapeutic agents. The feasibility of this approach requires the full biological activity, or clottability, of PE glycolated Fgn. This study characterizes the clot characteristics of Fgn modified to varying degrees with monofunctional succinimidyl propionate PEG (5000 Da). The data indicate that, although thrombin clotting times are significantly altered, Fgn maintains 90% of its capacity to clot upon the addition of up to 5 PEG/Fgn. Further derivatization significantly decreases the Fgn clottability. The addition of up to 5 PEG/Fgn has little, if any, effect on the kinetics of degradation by plasmin. The results suggest that limited modification of Fgn with lysine-reactive PEG allows therapeutic enhancement of fibrin glues. Copyright 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 529--535, 2001

  6. Real-time monitoring of human blood clotting using a lateral excited film bulk acoustic resonator

    Science.gov (United States)

    Chen, Da; Wang, Jingjng; Wang, Peng; Guo, Qiuquan; Zhang, Zhen; Ma, Jilong

    2017-04-01

    Frequent assay of hemostatic status is an essential issue for the millions of patients using anticoagulant drugs. In this paper, we presented a micro-fabricated film bulk acoustic sensor for the real-time monitoring of blood clotting and the measurement of hemostatic parameters. The device was made of an Au/ZnO/Si3N4 film stack and excited by a lateral electric field. It operated under a shear mode resonance with the frequency of 1.42 GHz and had a quality factor of 342 in human blood. During the clotting process of blood, the resonant frequency decreased along with the change of blood viscosity and showed an apparent step-ladder curve, revealing the sequential clotting stages. An important hemostatic parameter, prothrombin time, was quantitatively determined from the frequency response for different dilutions of the blood samples. The effect of a typical anticoagulant drug (heparin) on the prothrombin time was exemplarily shown. The proposed sensor displayed a good consistency and clinical comparability with the standard coagulometric methods. Thanks to the availability of direct digital signals, excellent potentials of miniaturization and integration, the proposed sensor has promising application for point-of-care coagulation technologies.

  7. 病毒灭活新鲜冰冻血浆制备前后凝血因子变化研究%The research of clotting factor change before and after the preparation of fresh frozen plasma by virus inactivation

    Institute of Scientific and Technical Information of China (English)

    宋春明; 颜峰; 王岩; 王玥玲; 邓小湄; 林林; 刘奉莉

    2015-01-01

    Objective:Discussion of fresh frozen plasma before and after viral inactivation differences that exist between the clotting factor for clinical choose to use a different blood products provided basis.Methods:Random sample of 30 people Front and rear parts of all kinds of fresh frozen plasma viral inactivation of clotting factor [prothrombin time (PT), activated partial thromboplastin time (APTT), plasma fibrinogen (FIB), thrombin time (TT), coagulation factor Ⅷ (FⅧ)] were compared.Results:PT, FIB, TT had in Fresh frozen plasma viral inactivation after no significant change (P> 0.05), APTT, FⅧ activity levels significantly decreased (P <0.05).Conclusions:While some fresh frozen plasma coagulation factor levels after viral inactivation has decreased, but still higher than the national standard, and in the blood-borne virus inactivation off, the clinical transfusion safety can be guaranteed. we recommends that medical institutions based on clinical transfusion indications, advantages and disadvantages of two plasma reasonable infusion of both plasma infusion to achieve a reasonable, safe and effective purpose.%目的:探讨新鲜冰冻血浆病毒灭活前后凝血因子之间的存在的差别,为临床选择使用不同血浆制品提供依据。方法:随机抽取30人份新鲜冰冻血浆病毒灭活前后各类凝血因子[凝血酶原时间(PT)、活化部分凝血酶原时间(APTT)、血浆纤维蛋白原(FIB)、凝血酶时间(TT)、凝血因子Ⅷ(FⅧ)]进行对比。结果:新鲜冰冻血浆经病毒灭活后PT、FIB、TT无明显变化(P>0.05),APTT、FⅧ活性水平则显著降低(P<0.05)。结论:新鲜冰冻血浆尽管病毒灭活后部分凝血因子含量有所降低,但仍高于国家标准,而且在灭活掉经血传播病毒后,其临床输注安全性得以保障。建议医疗机构根据临床输血指征、两种血浆的各自优缺点,合理输注上述两种血浆,达到输注合

  8. Antihemophilic factor (recombinant plasma/albumin-free method for the management and prevention of bleeding episodes in patients with hemophilia A

    Directory of Open Access Journals (Sweden)

    Steven Pipe

    2009-02-01

    Full Text Available Steven PipeDepartment of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, USAAbstract: Hemophilia is a rare genetic bleeding disorder that, if not adequately controlled, is associated with life-threatening bleeding events and serious and costly complications, primarily from joint damage. The advent of effective clotting factor replacement therapy for patients with hemophilia is considered one of the foremost medical advances of the 20th century. The last 3 decades of experience in hemophilia care have witnessed the effectiveness of the care of patients with hemophilia within specialized comprehensive care centers, advances in factor replacement therapies, the benefits of prophylaxis over on-demand replacement therapy, and the role of aggressive management of joint disease to prevent dysfunction. Ongoing challenges, including the management of inhibitors to factor therapies and the consequences of thousands of patients with hemophilia becoming infected with human immunodeficiency virus and hepatitis C virus in the 1980s from contaminated plasma-derived factor concentrates, have highlighted the need for vigilance with respect to clotting factor product safety, access to care, and a full complement of choice of factor replacement therapies. Advate® (antihemophilic factor [recombinant] plasma/albumin-free method [rAHF-PFM] is the first recombinant factor VIII therapy manufactured without human or animal protein additives to eliminate the risk of pathogen transmission that could be carried by these additives. Preclinical studies established bioequivalence with recombinant antihemophilic factor (Recombinate®, a product with 16 years of clinical experience. Currently licensed in 44 countries worldwide, rAHF-PFM has over 7 years of clinical research within 5 global studies supporting its safety and efficacy in the treatment of patients with hemophilia A.Keywords: factor VIII, hemophilia A, recombinant proteins, clinical

  9. Unusual clotting dynamics of plasma supplemented with iron(III).

    Science.gov (United States)

    Jankun, Jerzy; Landeta, Philip; Pretorius, Etheresia; Skrzypczak-Jankun, Ewa; Lipinski, Bogusław

    2014-02-01

    Iron salts are used in the treatment of iron deficiency anemia. Diabetic patients are frequently anemic and treatment includes administration of iron. Anemic patients on hemodialysis are at an increased risk of thromboembolic coronary events associated with the formation of dense fibrin clots resistant to fibrinolysis. Moreover, in chronic kidney disease patients, high labile plasma iron levels associated with iron supplementation are involved in complications found in dialyzed patients such as myocardial infarction. The aim of the present study was to investigate whether iron treatment is involved in the formation of the fibrin clots. Clotting of citrated plasma supplemented with Fe(3+) was investigated by thromboelastometry and electron microscopy. The results revealed that iron modifies coagulation in a complex manner. FeCl(3) stock solution underwent gradual chemical modification during storage and altered the coagulation profile over 29 days, suggesting that Fe(3+) interacts with both proteins of the coagulation cascade as well as the hydrolytic Fe(3+) species. Iron extends clotting of plasma by interacting with proteins of the coagulation cascade. Fe(3+) and/or its hydrolytic species interact with fibrinogen and/or fibrin changing their morphology and properties. In general FeCl(3) weakens the fibrin clot while at the same time precipitating plasma proteins immediately after application. Fe(3+) or its derivatives induced the formation of insoluble coagulums in non-enzymatic reactions including albumin and transferrin. Iron plays a role in coagulation and can precipitate plasma proteins. The formation of coagulums resistant to lysis in non‑enzymatic reactions can increase the risk of thrombosis, and extending clotting of plasma can prolong bleeding.

  10. Evidence supporting the use of recombinant activated factor VII in congenital bleeding disorders

    Directory of Open Access Journals (Sweden)

    Pär I Johansson

    2010-06-01

    Full Text Available Pär I Johansson, Sisse R OstrowskiCapital Region Blood Bank, Section for Transfusion Medicine, Rigshospitalet, University of Copenhagen, Copenhagen, DenmarkBackground: Recombinant activated factor VII (rFVIIa, NovoSeven® was introduced in 1996 for the treatment of hemophilic patients with antibodies against coagulation factor VIII or IX.Objective: To review the evidence supporting the use of rFVIIa for the treatment of patients with congenital bleeding disorders.Patients and methods: English-language databases were searched in September 2009 for reports of randomized controlled trials (RCTs evaluating the ability of rFVIIa to restore hemostasis in patients with congenital bleeding disorders.Results: Eight RCTs involving 256 hemophilic patients with antibodies against coagulation factors, also known as inhibitors, were identified. The evidence supporting the use of rFVIIa in these patients was weak with regard to dose, clinical setting, mode of administration, efficacy, and adverse events, given the limited sample size of each RCT and the heterogeneity of the studies.Conclusion: The authors suggest that rFVIIa therapy in hemophilic patients with inhibitors should be based on the individual’s ability to generate thrombin and form a clot, and not on the patient’s weight alone. Therefore, assays for thrombin generation, such as whole-blood thromboelastography, have the potential to significantly improve the treatment of these patients.Keywords: hemophilia, inhibitors, coagulation factor VIII, coagulation factor IX, rFVIIa, NovoSeven, FEIBA, hemostasis, RCT

  11. Fibrin clot formation and lysis: basic mechanisms

    DEFF Research Database (Denmark)

    Sidelmann, JJ; Gram, J; Jespersen, J

    2000-01-01

    induced by coagulation factor XIII (FXIII), and the degradation of fibrinogen and fibrin mediated by plasmin and elastase. Furthermore, factors influencing fibrin structure and fibrin breakdown are addressed; in particular polymorphisms in the genes coding for fibrinogen and FXIII, but also the physical...

  12. An optical approach for non-invasive blood clot testing

    Science.gov (United States)

    Kalchenko, Vyacheslav; Brill, Alexander; Fine, Ilya; Harmelin, Alon

    2007-02-01

    Physiological blood coagulation is an essential biological process. Current tests for plasma coagulation (clotting) need to be performed ex vivo and require fresh blood sampling for every test. A recently published work describes a new, noninvasive, in vivo approach to assess blood coagulation status during mechanical occlusion1. For this purpose, we have tested this approach and applied a controlled laser beam to blood micro-vessels of the mouse ear during mechanical occlusion. Standard setup for intravital transillumination videomicroscopy and laser based imaging techniques were used for monitoring the blood clotting process. Temporal mechanical occlusion of blood vessels in the observed area was applied to ensure blood flow cessation. Subsequently, laser irradiation was used to induce vascular micro-injury. Changes in the vessel wall, as well as in the pattern of blood flow, predispose the area to vascular thrombosis, according to the paradigm of Virchow's triad. In our experiments, two elements of Virchow's triad were used to induce the process of clotting in vivo, and to assess it optically. We identified several parameters that can serve as markers of the blood clotting process in vivo. These include changes in light absorption in the area of illumination, as well as changes in the pattern of the red blood cells' micro-movement in the vessels where blood flow is completely arrested. Thus, our results indicate that blood coagulation status can be characterized by non-invasive, in vivo methodologies.

  13. Alignment of the Fibrin Network Within an Autologous Plasma Clot.

    Science.gov (United States)

    Gessmann, Jan; Seybold, Dominik; Peter, Elvira; Schildhauer, Thomas Armin; Köller, Manfred

    2016-01-01

    Autologous plasma clots with longitudinally aligned fibrin fibers could serve as a scaffold for longitudinal axonal regrowth in cases of traumatic peripheral nerve injuries. Three different techniques for assembling longitudinally oriented fibrin fibers during the fibrin polymerization process were investigated as follows: fiber alignment was induced by the application of either a magnetic field or-as a novel approach-electric field or by the induction of orientated flow. Fiber alignment was characterized by scanning electron microscopy analysis followed by image processing using fast Fourier transformation (FFT). Besides FFT output images, area xmin to xmax, as well as full width at half maximum (FWHM) of the FFT graph plot peaks, was calculated to determine the relative degree of fiber alignment. In addition, fluorescently labeled human fibrinogen and mesenchymal stem cells (MSCs) were used to visualize fibrin and cell orientation in aligned and nonaligned plasma clots. Varying degrees of fiber alignment were achieved by the three different methods, with the electric field application producing the highest degree of fiber alignment. The embedded MSCs showed a longitudinal orientation in the electric field-aligned plasma clots. The key feature of this study is the ability to produce autologous plasma clots with aligned fibrin fibers using physical techniques. This orientated internal structure of an autologous biomaterial is promising for distinct therapeutic applications, such as a guiding structure for cell migration and growth dynamics.

  14. 21 CFR 173.150 - Milk-clotting enzymes, microbial.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Milk-clotting enzymes, microbial. 173.150 Section 173.150 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD...

  15. Plasma carboxypeptidase U (CPU, CPB2, TAFIa) generation during in vitro clot lysis and its interplay between coagulation and fibrinolysis.

    Science.gov (United States)

    Leenaerts, Dorien; Aernouts, Jef; Van Der Veken, Pieter; Sim, Yani; Lambeir, Anne-Marie; Hendriks, Dirk

    2017-07-26

    Carboxypeptidase U (CPU, CPB2, TAFIa) is a basic carboxypeptidase that is able to attenuate fibrinolysis. The inactive precursor procarboxypeptidase U is converted to its active form by thrombin, the thrombin-thrombomodulin complex or plasmin. The aim of this study was to investigate and characterise the time course of CPU generation in healthy individuals. In plasma of 29 healthy volunteers, CPU generation was monitored during in vitro clot lysis. CPU activity was measured by means of an enzymatic assay that uses the specific substrate Bz-o-cyano-Phe-Arg. An algorithm was written to plot the CPU generation curve and calculate the parameters that define it. In all individuals, CPU generation was biphasic. Marked inter-individual differences were present and a reference range was determined. The endogenous CPU generation potential is the composite effect of multiple factors. With respect to the first CPU activity peak characteristics, we found correlations with baseline proCPU concentration, proCPU Thr325Ile polymorphism, time to clot initiation and the clot lysis time. The second CPU peak related with baseline proCPU levels and with the maximum turbidity of the clot lysis profile. In conclusion, our method offers a technique to determine the endogenous CPU generation potential of an individual. The parameters obtained by the method quantitatively describe the different mechanisms that influence CPU generation during the complex interplay between coagulation and fibrinolysis, which are in line with the threshold hypothesis.

  16. Measurement of Plasma Clotting Using Shear Horizontal Surface Acoustic Wave Sensor

    Science.gov (United States)

    Nagayama, Tatsuya; Kondoh, Jun; Oonishi, Tomoko; Hosokawa, Kazuya

    2013-07-01

    The monitoring of blood coagulation is important during operation. In this study, a shear horizontal surface acoustic wave (SH-SAW) sensor is applied to monitor plasma clotting. An SH-SAW sensor with a metallized surface for mechanical perturbation detection can detect plasma clotting. As plasma clotting is a gel formation reaction, the SH-SAW sensor detects viscoelastic property changes. On the other hand, an SH-SAW sensor with a free surface for electrical perturbation detection detects only the liquid mixing effect. No electrical property changes due to plasma clotting are obtained using this sensor. A planar electrochemical sensor is also used to monitor plasma clotting. In impedance spectral analysis, plasma clotting is measured. However, in the measurement of time responses, no differences between clotting and nonclotting are obtained. Therefore, the SH-SAW sensor is useful for monitoring plasma clotting.

  17. Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A

    Science.gov (United States)

    Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V.; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N.; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I.; Fischer, Kathelijn; Gill, Joan C.; P’Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A.; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; St. Ledger, Katie

    2016-01-01

    Recombinant VIII (rVIII)-SingleChain is a novel B-domain–truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927. PMID:27330001

  18. Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A.

    Science.gov (United States)

    Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I; Fischer, Kathelijn; Gill, Joan C; P'Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; St Ledger, Katie; Pabinger, Ingrid

    2016-08-04

    Recombinant VIII (rVIII)-SingleChain is a novel B-domain-truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927.

  19. Capture of lipopolysaccharide (endotoxin by the blood clot: a comparative study.

    Directory of Open Access Journals (Sweden)

    Margaret T Armstrong

    Full Text Available In vertebrates and arthropods, blood clotting involves the establishment of a plug of aggregated thrombocytes (the cellular clot and an extracellular fibrillar clot formed by the polymerization of the structural protein of the clot, which is fibrin in mammals, plasma lipoprotein in crustaceans, and coagulin in the horseshoe crab, Limulus polyphemus. Both elements of the clot function to staunch bleeding. Additionally, the extracellular clot functions as an agent of the innate immune system by providing a passive anti-microbial barrier and microbial entrapment device, which functions directly at the site of wounds to the integument. Here we show that, in addition to these passive functions in immunity, the plasma lipoprotein clot of lobster, the coagulin clot of Limulus, and both the platelet thrombus and the fibrin clot of mammals (human, mouse operate to capture lipopolysaccharide (LPS, endotoxin. The lipid A core of LPS is the principal agent of gram-negative septicemia, which is responsible for more than 100,000 human deaths annually in the United States and is similarly toxic to arthropods. Quantification using the Limulus Amebocyte Lysate (LAL test shows that clots capture significant quantities of LPS and fluorescent-labeled LPS can be seen by microscopy to decorate the clot fibrils. Thrombi generated in the living mouse accumulate LPS in vivo. It is suggested that capture of LPS released from gram-negative bacteria entrapped by the blood clot operates to protect against the disease that might be caused by its systemic dispersal.

  20. Activated protein C accelerates clot lysis by virtue of its coagulant activity

    NARCIS (Netherlands)

    Fouw, N.J. de; Tilburg, N.H. van; Haverkate, F.; Bertina, R.M.

    1993-01-01

    The effect of human activated protein C (APC) on t-PA dependent fibrinolysis was studied in vitro using plasma (and whole blood) clot lysis techniques. Clot lysis was monitored by measuring the release of soluble 125I-labelled fibrin degradation products from the clot over time. It was demonstrated

  1. Adenosine diphosphate-decorated chitosan nanoparticles shorten blood clotting times, influencing the structures and varying the mechanical properties of the clots

    Directory of Open Access Journals (Sweden)

    Chung TW

    2014-03-01

    Full Text Available Tze-Wen Chung,1,3 Pei-Yi Lin,2 Shoei-Shen Wang,2 Yen-Fung Chen31Department of Biomedical Engineering, National Yang-Ming University, 2Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan, Republic of China; 3Department of Chemical and Materials Engineering, National Yunlin University of Science and Technology, Yunlin, Taiwan, Republic of ChinaAbstract: Chitosan nanoparticles (NPs decorated with adenosine diphosphate (ADP (ANPs or fibrinogen (FNPs were used to fabricate hemostatic NPs that can shorten blood clotting time and prevent severe local hemorrhage. The structure and mechanical properties of the blood clot induced with ANP (clot/ANP or FNP (clot/FNP were also investigated. The NPs, ANPs, and FNPs, which had particle sizes of 245.1±14.0, 251.0±9.8, and 326.5±14.5 nm and zeta potentials of 24.1±0.5, 20.6±1.9, and 15.3±1.5 mV (n=4, respectively, were fabricated by ionic gelation and then decorated with ADP and fibrinogen. The zeta potentials and Fourier transform infrared (FTIR spectroscopy of the NPs confirmed that their surfaces were successfully coated with ADP and fibrinogen. The scanning electron microscope (SEM micrographs of the structure of the clot induced with "undecorated" chitosan NPs (clot/NP, clot/ANP, and clot/FNP (at 0.05 wt% were different, after citrated bloods had been recalcified by a calcium chloride solution containing NPs, ANPs, or FNPs. This indicated that many NPs adhered on the membrane surfaces of red blood cells, that ANPs induced many platelet aggregates, and that FNPs were incorporated into the fibrin network in the clots. Measurements of the blood clotting times (Tc of blood clot/NPs, clot/ANPs, and clot/FNPs, based on 90% of ultimate frequency shifts measured on a quartz crystal microbalance (QCM, were significantly (P<0.05 (n=4 shorter than that of a clot induced by a phosphate-buffered solution (PBS (clot/PBS (63.6%±3.1%, 48.3%±6.2%, and 63.2%±4.7%, respectively. The ∆F2

  2. Age relations of cardiovascular risk factors in a traditional Melanesian society: the Kitava Study.

    Science.gov (United States)

    Lindeberg, S; Berntorp, E; Nilsson-Ehle, P; Terént, A; Vessby, B

    1997-10-01

    This study examined cross-sectional age relations of blood pressure, anthropometric indexes, serum lipids, and hemostatic variables in 203 subsistence horticulturists aged 20-86 y in Kitava, Trobriand Islands, Papua New Guinea. The population is characterized by extreme leanness (despite food abundance), low blood pressure, low plasma plasminogen activator inhibitor 1 activity, and rarity of cardiovascular disease. Tubers, fruit, fish, and coconut are dietary staples whereas dairy products, refined fat and sugar, cereals, and alcohol are absent and salt intake is low. Although diastolic blood pressure was not associated with age in Kitavans, systolic blood pressure increased linearly after 50 y of age in both sexes. Body mass index decreased with age in both sexes. Serum total cholesterol, triacylglycerol, low-density-lipoprotein cholesterol, and apolipoprotein B increased in males between 20 and 50 y of age, whereas high-density-lipoprotein cholesterol and apolipoprotein A-I decreased. There were no significant differences in these indexes with age in the few females studied. A slight linear age-related increase of lipoprotein(a) was present in males. Plasma fibrinogen, factor VII clotting activity, factor VIII clotting activity, and von Willebrand factor antigen increased with age in both sexes but plasminogen activator inhibitor 1 activity did not. The modest or absent relations between the indexes measured and age are apparently important explanations of the virtual nonexistence of stroke and ischemic heart disease in Kitava.

  3. Prediction of recurrent venous thromboembolism by clot lysis time: a prospective cohort study.

    Directory of Open Access Journals (Sweden)

    Ludwig Traby

    Full Text Available Venous thromboembolism (VTE is a chronic disease, which tends to recur. Whether an abnormal fibrinolytic system is associated with an increased risk of VTE is unclear. We assessed the relationship between fibrinolytic capacity (reflected by clot lysis time [CLT] and risk of recurrent VTE. We followed 704 patients (378 women; mean age 48 yrs with a first unprovoked VTE for an average of 46 months after anticoagulation withdrawal. Patients with natural coagulation inhibitor deficiency, lupus anticoagulant, cancer, homozygosity for factor V Leiden or prothrombin mutation, or requirement for indefinite anticoagulation were excluded. Study endpoint was symptomatic recurrent VTE. For measurement of CLT, a tissue factor-induced clot was lysed by adding tissue-type plasminogen activator. Time between clot formation and lysis was determined by measuring the turbidity. 135 (19% patients had recurrent VTE. For each increase in CLT of 10 minutes, the crude relative risk (RR of recurrence was 1.13 (95% CI 1.02-1.25; p = 0.02 and was 1.08 (95% CI 0.98-1.20; p = 0.13 after adjustment for age and sex. For women only, the adjusted RR was 1.14 (95% CI, 0.91-1.42, p = 0.22 for each increase in CLT of 10 minutes. CLT values in the 4(th quartile of the female patient population, as compared to values in the 1(st quartile, conferred a risk of recurrence of 3.28 (95% CI, 1.07-10.05; p = 0.04. No association between CLT and recurrence risk was found in men. Hypofibrinolysis as assessed by CLT confers a moderate increase in the risk of recurrent VTE. A weak association between CLT and risk of recurrence was found in women only.

  4. Aggregation of Red Blood Cells: From Rouleaux to Clot Formation

    CERN Document Server

    Wagner, C; Svetina, S

    2013-01-01

    Red blood cells are known to form aggregates in the form of rouleaux. This aggregation process is believed to be reversible, but there is still no full understanding on the binding mechanism. There are at least two competing models, based either on bridging or on depletion. We review recent experimental results on the single cell level and theoretical analyses of the depletion model and of the influence of the cell shape on the binding strength. Another important aggregation mechanism is caused by activation of platelets. This leads to clot formation which is life saving in the case of wound healing but also a major cause of death in the case of a thrombus induced stroke. We review historical and recent results on the participation of red blood cells in clot formation.

  5. Selective light-triggered release of DNA from gold nanorods switches blood clotting on and off.

    Directory of Open Access Journals (Sweden)

    Helena de Puig

    Full Text Available Blood clotting is a precise cascade engineered to form a clot with temporal and spatial control. Current control of blood clotting is achieved predominantly by anticoagulants and thus inherently one-sided. Here we use a pair of nanorods (NRs to provide a two-way switch for the blood clotting cascade by utilizing their ability to selectively release species on their surface under two different laser excitations. We selectively trigger release of a thrombin binding aptamer from one nanorod, inhibiting blood clotting and resulting in increased clotting time. We then release the complementary DNA as an antidote from the other NR, reversing the effect of the aptamer and restoring blood clotting. Thus, the nanorod pair acts as an on/off switch. One challenge for nanobiotechnology is the bio-nano interface, where coronas of weakly adsorbed proteins can obscure biomolecular function. We exploit these adsorbed proteins to increase aptamer and antidote loading on the nanorods.

  6. Mesoscopic Modeling of Blood Clotting: Coagulation Cascade and Platelets Adhesion

    Science.gov (United States)

    Yazdani, Alireza; Li, Zhen; Karniadakis, George

    2015-11-01

    The process of clot formation and growth at a site on a blood vessel wall involve a number of multi-scale simultaneous processes including: multiple chemical reactions in the coagulation cascade, species transport and flow. To model these processes we have incorporated advection-diffusion-reaction (ADR) of multiple species into an extended version of Dissipative Particle Dynamics (DPD) method which is considered as a coarse-grained Molecular Dynamics method. At the continuum level this is equivalent to the Navier-Stokes equation plus one advection-diffusion equation for each specie. The chemistry of clot formation is now understood to be determined by mechanisms involving reactions among many species in dilute solution, where reaction rate constants and species diffusion coefficients in plasma are known. The role of blood particulates, i.e. red cells and platelets, in the clotting process is studied by including them separately and together in the simulations. An agonist-induced platelet activation mechanism is presented, while platelets adhesive dynamics based on a stochastic bond formation/dissociation process is included in the model.

  7. Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

    Directory of Open Access Journals (Sweden)

    Ana Rodrigues Duarte

    2009-02-01

    Full Text Available The partial characterization and purification of milk clotting enzyme obtained from the (root latex of Jacaratia corumbensis O. kuntze was studied, by fractional precipitation with ammonium sulphate and ion exchange chromatography. The ammonium sulphate precipitate showed five fractions (AS1- 0-20%; AS2 - 20-40%; AS3 - 40-60%; AS4 - 60-80%; AS5 - 80-100% and among the fractions obtained, the 40-60% fraction (AS3 showed the highest milk clotting activity with a purification factor of 1.2 fold in relation to the crude extract. This fraction when applied on Mono Q column yielded two protein peaks (p1 and p2, but p1 pool showed the best milk-clotting activity. The optimal pH for the crude and partially purified extract was 6.5 and 7.0, respectively. The maximum milk-clotting activity was at 55ºC for the both crude and partially purified extracts. The enzyme was inhibited by iodoacetic acid which suggested that this enzyme was a cysteine protease, with molecular weight of 33 kDa.A enzima coagulante de leite obtida de látex de raiz de Jacaratia corumbensis O. kuntze foi caracterizada parcialmente e purificada, por precipitação fracionária com sulfato de amônio e cromatografia de troca de íon. Foram utilizadas cinco frações de sulfato de amônio (AS1 - 0-20%; AS2 - 20-40%; AS3 - 40-60%; AS4 - 60-80%; AS5 - 80-100%, a fração 40-60% (AS3 mostrou alta atividade coagulante com um fator de purificação de 1,2 vezes em relação ao extrato bruto. Esta fração foi aplicada em coluna Mono Q obtendo dois picos de proteína (p1 e p2, o p1 mostrou melhor atividade coagulante. O pH ótimo para o extrato bruto e parcialmente purificado foi 6,5 e 7,0, respectivamente. A atividade coagulante foi atingida a 55ºC para ambos os extratos, bruto e parcialmente purificado. A enzima foi inibida por ácido iodoacético que sugere que esta enzima é uma cisteína protease, com peso molecular de 33 kDa.

  8. Inhibitor development in nonsevere hemophilia A

    OpenAIRE

    2014-01-01

    Hemophilia A is an X-linked inherited bleeding disorder that affects approximately 1 in 5000 male live births. It is caused by a deficient plasma level of clotting factor VIII and can be treated by the intravenous administration of factor VIII concentrates. A severe complication of the treatment with factor VIII concentrates is the development of inhibiting antibodies against factor VIII, also called inhibitors. Inhibitors challenge the treatment of hemophilia A as they inactivate factor VIII...

  9. PARTIAL PURIFICATION OF MILK-CLOTTING ENZYME FROM THE SEEDS OF MORINGA OLEIFERA

    Directory of Open Access Journals (Sweden)

    Amna E. Tajalsir

    2014-08-01

    Full Text Available The aim of the present study was to search for milk clotting substitute from different parts (flowers, seeds, stem, leaves, ripe and unripe fruits of Moringa oleifera. The samples were blended and extracted using different types of extracting solutions. The most reliable, quick and efficient enzyme extracting solution was found to be 5% NaCl in 100 mM sodium acetate buffer, pH 5.0, which was used throughout the study. The milk clotting activity was only observed in the seeds extract while the other parts were either deficient or has very low milk clotting activity. Thus, the moringa seeds were used as source of milk clotting enzyme. The extracted proteins were fractionated with ammonium sulfate at concentration of 20, 30, 40, 50 and 60 %. Highest milk clotting activity was observed in the 20 % fraction. This fraction was assumed to contain the clotting enzymes and characterized for its heating stability (30 – 90°C and optimum temperature (30 – 90°C. The results demonstrated that moringa seeds milk clotting enzyme is stable up to 50°C with an optimum milk clotting activity of 70°C. The high ratio of milk-clotting to proteolytic activity of the partially purified enzyme indicates the potential of this enzyme as suitable rennet substitute in dairy industry. However, further study is needed to completely purify and characterize this promising milk clotting enzyme from moringa seeds.

  10. Complementary effect of fibrinogen and rFVIIa on clotting ex vivo in Bernard-Soulier syndrome and combined use during three deliveries

    DEFF Research Database (Denmark)

    Palsson, Ragnar; Vidarsson, Brynjar; Gudmundsdottir, Brynja R

    2014-01-01

    coagulation profile of a pregnant woman with BSS before and after spiking ex vivo with different concentrations of recombinant activated factor VII (rFVIIa) and fibrinogen. As experiments suggested improved clotting with clinically applicable concentrations of both agents in a complementary manner...

  11. Influence of styptic fiber on clotting time in rabbits

    Institute of Scientific and Technical Information of China (English)

    LU Jun; JIANG Su-yun

    2008-01-01

    Objective To explore the influence of styptic fiber on clotting time in rabbits so as to provide experiment data for its development. Methods Onto 0.1 mL aliquots of citrated anti-coagulant rabbit blood placed in a surfacial plate 25 ul of 0.2 mol·L-1 CaCl2 solution was dropped, and mixed well with glass stirrer;the resulting mixture was immediately capped with a piece of styptic fiber (test product group) or absorptive gelatin sponge(positive control group) of 2 cm diameter. Then, the surficial plate was rinsed with 30ml of purified water at 5, 10, 20, 30, 40 and 50 min after capping; the rinsings were allowed to stand for 1 h and were subjected to OD determination at a wavelength of 541 nm. The above procedure was repeated twice, the average value of the twice experiments was taken for evaluation of the hemostatic effect of test product. For negative control group, all procedures except for capping were same as the test product group. The haemostatic effect was judged by percent OD relative to OD at 0 min in negative control group (OD 0 min) (OD 0 min was considered as 100% );if OD value at a time was less than 80% of OD 0 min, it should be designated as primary clotting time(PCT), less than 20 96 as complete clotting time(CCT). Results The measured PCT was 20min for both negative and positive control groups;CCT was 50, 30 and 5 min for negative control, positive control and test product groups, respectively, showing the test styptic fiber had a CCT 8 times shorter than untreated blood, 10 times shorter than negative control and 6 times shorter than positive control. Conclusions The test styptic fiber has powerful hemostatic effect.

  12. Travail et pouvoir d’agir d’Yves Clot

    Directory of Open Access Journals (Sweden)

    Jacques Leplat

    2008-11-01

    Full Text Available Yves Clot est titulaire de la Chaire de psychologie du travail du Conservatoire National des Arts et Métiers (CNAM au sein duquel il dirige l’équipe de clinique de l’activité. Cet ouvrage est le second qu’il publie dans cette collection, le premier « La fonction psychologique du travail » est paru en 1999. Entre les deux s’inscrivent un grand nombre de publications, dont beaucoup avec d’autres chercheurs, comme on pourra le constater en consultant la bibliographie de ce livre. Ce denier réun...

  13. The Effects of Morphine Sulfate on Agglutination, Clot Formation and Hemolysis in Packed Red Blood Cells

    Science.gov (United States)

    2007-11-02

    THE EFFECTS OF MORPHINE SULFATE ON AGGLUTINATION , CLOT FORMATION AND HEMOLYSIS IN PACKED RED BLOOD CELLS 6. AUTHOR(S) CAPT ESTAVILLO BRIAN K 7...ANSI Std8 239.18 Designed using Perform Pro, WHS/DIOR, Oct 94 THE EFFECTS OF MORPHINE SULFATE ON AGGLUTINATION , CLOT FORMATION AND HEMOLYSIS IN PACKED...that the use of any copyrighted material in the thesis entitled: "THE EFFECTS OF MORPHINE SULFATE ON AGGLUTINATION , CLOT FORMATION AND HEMOLYSIS IN

  14. An Antithrombin-Heparin Complex Increases the Anticoagulant Activity of Fibrin Clots

    Directory of Open Access Journals (Sweden)

    Lesley J. Smith

    2008-01-01

    Full Text Available Clotting blood contains fibrin-bound thrombin, which is a major source of procoagulant activity leading to clot extension and further activation of coagulation. When bound to fibrin, thrombin is protected from inhibition by antithrombin (AT + heparin but is neutralized when AT and heparin are covalently linked (ATH. Here, we report the surprising observation that, rather than yielding an inert complex, thrombin-ATH formation converts clots into anticoagulant surfaces that effectively catalyze inhibition of thrombin in the surrounding environment.

  15. Does prior administration of enoxaparin influence the effects of levobupivacaine on blood clotting? Assessment using the Thrombelastograph.

    LENUS (Irish Health Repository)

    Leonard, S A

    2012-02-03

    The low molecular weight heparin, enoxaparin (by inhibition of factors Xa and IIa) and amide local anaesthetics (by altering platelet function) exert anti-clotting effects. Although these agents are often used in combination during the perioperative period, their potential interactive effect on clotting has not been defined. Blood from 10 ASA I-II patients who received enoxaparin 0.5 mg kg(-1) s.c. was studied using a Thrombelastograph (TEG) either alone or in combination with levobupivacaine (2.5 mg ml(-1) or 2.5 microg ml(-1)) or saline (50% dilution). In blood from patients who had received enoxaparin 0.5 mg kg(-1) s.c. 12 h previously, levobupivacaine 2.5 mg ml(-1) (but not 2.5 microg ml(-1)) produced significant changes in TEG clotting parameters (mean (SD) 15.7 (4.8) mm, 29.6 (25.6) mm, 34.4 (14.6) mm, 34.3 (12.2) degrees compared with control values of 6.1 (1.3) mm, 2.5 (0.5) mm, 63.5 (6.4) mm and 74.1 (2.9) degrees for r, K, MA, and alpha angle respectively).

  16. Quantitative photoacoustic characterization of blood clot in blood: A mechanobiological assessment through spectral information

    Science.gov (United States)

    Biswas, Deblina; Vasudevan, Srivathsan; Chen, George C. K.; Sharma, Norman

    2017-02-01

    Formation of blood clots, called thrombus, can happen due to hyper-coagulation of blood. Thrombi, while moving through blood vessels can impede blood flow, an important criterion for many critical diseases like deep vein thrombosis and heart attacks. Understanding mechanical properties of clot formation is vital for assessment of severity of thrombosis and proper treatment. However, biomechanics of thrombus is less known to clinicians and not very well investigated. Photoacoustic (PA) spectral response, a non-invasive technique, is proposed to investigate the mechanism of formation of blood clots through elasticity and also differentiate clots from blood. Distinct shift (increase in frequency) of the PA response dominant frequency during clot formation is reported. In addition, quantitative differentiation of blood clots from blood has been achieved through parameters like dominant frequency and spectral energy of PA spectral response. Nearly twofold increases in dominant frequency in blood clots compared to blood were found in the PA spectral response. Significant changes in energy also help in quantitatively differentiating clots from blood, in the blood. Our results reveal that increase in density during clot formation is reflected in the PA spectral response, a significant step towards understanding the mechanobiology of thrombus formation. Hence, the proposed tool, in addition to detecting thrombus formation, could reveal mechanical properties of the sample through quantitative photoacoustic spectral parameters.

  17. Erythrocyte migration and gap formation in rabbit blood clots in vitro.

    Science.gov (United States)

    Ueki, T; Yazama, F; Horiuchi, T; Yamada, M

    2008-04-01

    Thrombolytic agents must be carried by the blood circulation to thrombi to exert their functions. Structural gaps exist between blood vessels and thrombi or in the area surrounding thrombi. Therefore, information about fundamental gap formation at thrombotic areas is critically important for thrombolytic therapy. We previously reported that t-PA accelerates the activities of bovine erythrocytes and hemoglobin (Hb) towards bovine plasminogen activation. Here, we examined gap generation by observing morphological changes during thrombolytic processes in rabbit blood clots deformation of erythrocytes from blood clots and Hb transfer from erythrocytes to serum in vitro. Rabbit venous blood samples (1 ml) were stored under sterile conditions in glass tubes at 37 degrees C for 2, 24, 48 h, 1, and 2 weeks. We examined clot diameter, erythrocyte diameter and number as well as Hb volume in the serum, as well as histological changes in the clots. The diameter of blood clots did not change until 2 weeks after sampling. Erythrocyte diameter decreased within 48 h and at 2 weeks after sampling at the clot surface (p erythrocytes in the serum started to increase starting from 24 h after sampling (p erythrocyte envelope became disrupted and cytoplasm started to flow through pores into the serum at 24 h. The results indicated that blood clots are reduced due to clot retraction, erythrocyte dissociation and cytoplasm leakage without a distinct fibrinolytic reaction. These results indicated that gaps start to form between 2 and 24 h after blood clotting.

  18. Seamless particle-based modeling of blood clotting

    Science.gov (United States)

    Yazdani, Alireza; Karniadakis, George

    2016-11-01

    We propose a new multiscale framework that seamlessly integrate four key components of blood clotting namely, blood rheology, cell mechanics, coagulation kinetics and transport of species and platelet adhesive dynamics. We use transport dissipative particle dynamics (tDPD) which is an extended form of original DPD as the base solver to model both blood flow and the reactive transport of chemical species in the coagulation cascade. Further, we use a coarse-grained representation of blood cell's membrane that accounts for its mechanics; both red blood cells and platelets are resolved at sub-cellular resolution, and stochastic bond formation/dissociation are included to account for platelet adhesive dynamics at the site of injury. Our results show good qualitative agreement with in vivo experiments. The numerical framework allows us to perform systematic analysis on different mechanisms of blood clotting. In addition, this new multiscale particle-based methodology can open new directions in addressing different biological processes from sub-cellular to macroscopic scales. NIH Grant No. U01HL116323.

  19. Calibrated automated thrombin generation measurement in clotting plasma.

    Science.gov (United States)

    Hemker, H Coenraad; Giesen, Peter; Al Dieri, Raed; Regnault, Véronique; de Smedt, Eric; Wagenvoord, Rob; Lecompte, Thomas; Béguin, Suzette

    2003-01-01

    Calibrated automated thrombography displays the concentration of thrombin in clotting plasma with or without platelets (platelet-rich plasma/platelet-poor plasma, PRP/PPP) in up to 48 samples by monitoring the splitting of a fluorogenic substrate and comparing it to a constant known thrombin activity in a parallel, non-clotting sample. Thus, the non-linearity of the reaction rate with thrombin concentration is compensated for, and adding an excess of substrate can be avoided. Standard conditions were established at which acceptable experimental variation accompanies sensitivity to pathological changes. The coefficients of variation of the surface under the curve (endogenous thrombin potential) are: within experiment approximately 3%; intra-individual: AVK, heparin(-likes), direct inhibitors]. In PRP, it is diminished in von Willebrand's disease, but it also shows the effect of platelet inhibitors (e.g. aspirin and abciximab). Addition of activated protein C (APC) or thrombomodulin inhibits thrombin generation and reflects disorders of the APC system (congenital and acquired resistance, deficiencies and lupus antibodies) independent of concomitant inhibition of the procoagulant pathway as for example by anticoagulants.

  20. The Effect of Cyanosis on Active Clotting Time During Diagnostic Catheterization

    Directory of Open Access Journals (Sweden)

    Akbar Molaei

    2012-09-01

    Full Text Available Introduction: Cardiac catheterization is a common procedure which needs a careful coagulation monitoring. In our study, we aimed to find factors influencing active clotting time (ACT following heparin therapy. Methods: ACT of 71 patients who were scheduled to undergo transcutaneous diagnostic catheterization and angiography were measured at baseline, 2 and 60 minutes after 50 IU/kg heparin loading. ACT in two groups of patients (cyanotic and non-cyanotic was compared. All data were analyzed with Wilcoxon, Mann-Whitney test and Pearson in SPSS 16, P value less than 0.05 was considered significant. Results: ACT following heparin at 2nd and 60th minutes was not significantly different in cyanotic and non-cyanotic groups. At 60th minute following heparin administration, ACT decreased more dramatically in older children. Conclusion: Cyanosis does not affect ACT measures following heparin treatment. Moreover, after 60 minutes, heparin efficacy (ACT values decreased more with increase in patients’ age.

  1. Altered fibrin clot structure/function in patients with antiphospholipid syndrome: association with thrombotic manifestation.

    Science.gov (United States)

    Celińska-Lowenhoff, M; Iwaniec, T; Padjas, A; Musiał, J; Undas, A

    2014-08-01

    We tested the hypothesis that plasma fibrin clot structure/function is unfavourably altered in patients with antiphospholipid syndrome (APS). Ex vivo plasma clot permeability, turbidity and susceptibility to lysis were determined in 126 consecutive patients with APS enrolled five months or more since thrombotic event vs 105 controls. Patients with both primary and secondary APS were characterised by 11% lower clot permeability (p<0.001), 4.8% shorter lag phase (p<0.001), 10% longer clot lysis time (p<0.001), and 4.7% higher maximum level of D-dimer released from clots (p=0.02) as compared to the controls. Scanning electron microscopy images confirmed denser fibrin networks composed of thinner fibres in APS. Clots from patients with "triple-antibody positivity" were formed after shorter lag phase (p=0.019) and were lysed at a slower rate (p=0.004) than in the remainder. Clots from APS patients who experienced stroke and/or myocardial infarction were 8% less permeable (p=0.01) and susceptible to lysis (10.4% longer clot lysis time [p=0.006] and 4.5% slower release of D-dimer from clots [p=0.01]) compared with those following venous thromboembolism alone. Multivariate analysis adjusted for potential confounders showed that in APS patients, lupus anticoagulant and "triple-positivity" were the independent predictors of clot permeability, while "triple-positivity" predicted lysis time. We conclude that APS is associated with prothrombotic plasma fibrin clot phenotype, with more pronounced abnormalities in arterial thrombosis. Molecular background for this novel prothrombotic mechanism in APS remains to be established.

  2. Igor Zabel 14. VIII 1958 - 23. VII 2005

    Index Scriptorium Estoniae

    2005-01-01

    Igor Zabel oli Ida-Euroopa tuntumaid kunstiteoreetikuid ja kuraatoreid. Töötas 1986. aastast Ljubljana moodsa kunsti muuseumi kuraatorina. 9. VIII avatakse seal viimane I. Zabeli kureeritud näitus "Territories, Identities, Nets-Slovene Art 1995-2005"

  3. Factor VIII deficiency does not protect against atherosclerosis

    NARCIS (Netherlands)

    Biere-Rafi, S.; Tuinenburg, A.; Haak, B.W.; Peters, M.; Huijgen, R.; de Groot, E.; Verhamme, P.; Peerlinck, K.; Visseren, F.L.J.; Kruip, M.J.H.A.; Laros-van Gorkom, B.A.P.; Gerdes, V.E.A.; Buller, H.R.; Schutgens, R.E.G.; Kamphuisen, P.W.

    2012-01-01

    Summary. Background: Hemophilia A patients have a lower cardiovascular mortality rate than the general population. Whether this protection is caused by hypocoagulability or decreased atherogenesis is unclear. Objectives: To evaluate atherosclerosis and endothelial function in hemophilia A patients w

  4. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be...

  5. Effects of aspirin on clot structure and fibrinolysis using a novel in vitro cellular system.

    Science.gov (United States)

    Ajjan, R A; Standeven, K F; Khanbhai, M; Phoenix, F; Gersh, K C; Weisel, J W; Kearney, M T; Ariëns, R A S; Grant, P J

    2009-05-01

    The purpose of this study was to investigate the direct effects of aspirin on fibrin structure/function. Chinese Hamster Ovary cell lines stably transfected with fibrinogen were grown in the absence (0) and presence of increasing concentrations of aspirin. Fibrinogen was purified from the media using affinity chromatography, and clots were made from recombinant protein. Mean final turbidity [OD(+/-SEM)] was 0.083(+/-0.03), 0.093(+/-0.002), 0.101(+/-0.005), and 0.125(+/-0.003) in clots made from 0, 1, 10, and 100 mg/L aspirin-treated fibrinogen, respectively (Paspirin respectively (Pstructure and increased fiber thickness of clots made from aspirin-treated fibrinogen, whereas rheometer studies showed a significant 30% reduction in clot rigidity. Fibrinolysis was quicker in clots made from aspirin-treated fibrinogen. Ex vivo studies in 3 normal volunteers given 150 mg aspirin daily for 1 week demonstrated similar changes in clot structure/function. Aspirin directly altered clot structure resulting in the formation of clots with thicker fibers and bigger pores, which are easier to lyse. This study clearly demonstrates an alternative mode of action for aspirin, which should be considered in studies evaluating the biochemical efficacy of this agent.

  6. Thermal Blood Clot Formation and use in Microfluidic Device Valving Applications

    Science.gov (United States)

    Tai, Yu-Chong (Inventor); Shi, Wendian (Inventor); Guo, Luke (Inventor)

    2014-01-01

    The present invention provides a method of forming a blood-clot microvalve by heating blood in a capillary tube of a microfluidic device. Also described are methods of modulating liquid flow in a capillary tube by forming and removing a blood-clot microvalve.

  7. Modelling of platelet-fibrin clot formation in flow with a DPD-PDE method.

    Science.gov (United States)

    Tosenberger, A; Ataullakhanov, F; Bessonov, N; Panteleev, M; Tokarev, A; Volpert, V

    2016-02-01

    The paper is devoted to mathematical modelling of clot growth in blood flow. Great complexity of the hemostatic system dictates the need of usage of the mathematical models to understand its functioning in the normal and especially in pathological situations. In this work we investigate the interaction of blood flow, platelet aggregation and plasma coagulation. We develop a hybrid DPD-PDE model where dissipative particle dynamics (DPD) is used to model plasma flow and platelets, while the regulatory network of plasma coagulation is described by a system of partial differential equations. Modelling results confirm the potency of the scenario of clot growth where at the first stage of clot formation platelets form an aggregate due to weak inter-platelet connections and then due to their activation. This enables the formation of the fibrin net in the centre of the platelet aggregate where the flow velocity is significantly reduced. The fibrin net reinforces the clot and allows its further growth. When the clot becomes sufficiently large, it stops growing due to the narrowed vessel and the increase of flow shear rate at the surface of the clot. Its outer part is detached by the flow revealing the inner part covered by fibrin. This fibrin cap does not allow new platelets to attach at the high shear rate, and the clot stops growing. Dependence of the final clot size on wall shear rate and on other parameters is studied.

  8. Integration of acoustic radiation force and optical imaging for blood plasma clot stiffness measurement.

    Science.gov (United States)

    Wang, Caroline W; Perez, Matthew J; Helmke, Brian P; Viola, Francesco; Lawrence, Michael B

    2015-01-01

    Despite the life-preserving function blood clotting serves in the body, inadequate or excessive blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between blood clot stiffness and vascular diseases underscores the importance of quantifying the magnitude and kinetics of blood's transformation from a fluid to a viscoelastic solid. To measure blood plasma clot stiffness, we have developed a method that uses ultrasound acoustic radiation force (ARF) to induce micron-scaled displacements (1-500 μm) on microbeads suspended in blood plasma. The displacements were detected by optical microscopy and took place within a micro-liter sized clot region formed within a larger volume (2 mL sample) to minimize container surface effects. Modulation of the ultrasound generated acoustic radiation force allowed stiffness measurements to be made in blood plasma from before its gel point to the stage where it was a fully developed viscoelastic solid. A 0.5 wt % agarose hydrogel was 9.8-fold stiffer than the plasma (platelet-rich) clot at 1 h post-kaolin stimulus. The acoustic radiation force microbead method was sensitive to the presence of platelets and strength of coagulation stimulus. Platelet depletion reduced clot stiffness 6.9 fold relative to platelet rich plasma. The sensitivity of acoustic radiation force based stiffness assessment may allow for studying platelet regulation of both incipient and mature clot mechanical properties.

  9. Growth of hydroxyapatite on physiologically clotted fibrin capped gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Sastry, T P; Sundaraseelan, J; Swarnalatha, K; Sobhana, S S Liji; Makheswari, M Uma; Sekar, S; Mandal, A B [Central Leather Research Institute, Chennai-600020, Tamilnadu (India)], E-mail: sastrytp@hotmail.com, E-mail: abmandal@hotmail.com

    2008-06-18

    The growth of hydroxyapatite (HAp) on physiologically clotted fibrin (PCF)-gold nanoparticles is presented for the first time by employing a wet precipitation method. Fourier transform infrared (FTIR) spectroscopy confirmed the characteristic functionalities of PCF and HAp in the PCF-Au-HAp nanocomposite. Scanning electron microscopy (SEM) images have shown cuboidal nanostructures having a size in the range of 70-300 nm of HAp, whereas 2-50 nm sized particles were visualized in high-resolution transmission electron microscopy (TEM). Energy-dispersive x-ray (EDX) and x-ray diffraction (XRD) studies have confirmed the presence of HAp. These results show that gold nanoparticles with PCF acted as a matrix for the growth of HAp, and that PCF-Au-HAp nanocomposite is expected to have better osteoinductive properties.

  10. MASP-1 Induced Clotting--The First Model of Prothrombin Activation by MASP-1.

    Directory of Open Access Journals (Sweden)

    Lorenz Jenny

    Full Text Available Mannan-binding lectin-associated serine protease-1 (MASP-1, a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity. Due to its interplay with several coagulation factors, it has the ability to induce fibrin clot formation independent of the usual coagulation activation pathways. We have recently shown that MASP-1 activates prothrombin and identified arginine (R 155, R271, and R393 as potential cleavage sites. FXa cleaves R320 instead of R393, and thrombin cleaves R155 and R284 in prothrombin. Here we have used three arginine-to-glutamine mutants of prothrombin, R271Q, R320Q, R393Q and the serine-to-alanine active site mutant S525A to investigate in detail the mechanism of MASP-1 mediated prothrombin activation. Prothrombin wildtype and mutants were digested with MASP-1 and the cleavage products were analysed by SDS-PAGE and N-terminal sequencing. A functional clotting assay was performed by thrombelastography. We have found that MASP-1 activates prothrombin via two simultaneous pathways, either cleaving at R271 or R393 first. Both pathways result in the formation of several active alternative thrombin species. Functional studies confirmed that both R393 and R320 are required for prothrombin activation by MASP-1, whereas R155 is not considered to be an important cleavage site in this process. In conclusion, we have described for the first time a detailed model of prothrombin activation by MASP-1.

  11. Genetics Home Reference: factor V Leiden thrombophilia

    Science.gov (United States)

    ... Conditions factor V Leiden thrombophilia factor V Leiden thrombophilia Enable Javascript to view the expand/collapse boxes. ... Open All Close All Description Factor V Leiden thrombophilia is an inherited disorder of blood clotting . Factor ...

  12. Random Forests Are Able to Identify Differences in Clotting Dynamics from Kinetic Models of Thrombin Generation.

    Directory of Open Access Journals (Sweden)

    Jayavel Arumugam

    Full Text Available Current methods for distinguishing acute coronary syndromes such as heart attack from stable coronary artery disease, based on the kinetics of thrombin formation, have been limited to evaluating sensitivity of well-established chemical species (e.g., thrombin using simple quantifiers of their concentration profiles (e.g., maximum level of thrombin concentration, area under the thrombin concentration versus time curve. In order to get an improved classifier, we use a 34-protein factor clotting cascade model and convert the simulation data into a high-dimensional representation (about 19000 features using a piecewise cubic polynomial fit. Then, we systematically find plausible assays to effectively gauge changes in acute coronary syndrome/coronary artery disease populations by introducing a statistical learning technique called Random Forests. We find that differences associated with acute coronary syndromes emerge in combinations of a handful of features. For instance, concentrations of 3 chemical species, namely, active alpha-thrombin, tissue factor-factor VIIa-factor Xa ternary complex, and intrinsic tenase complex with factor X, at specific time windows, could be used to classify acute coronary syndromes to an accuracy of about 87.2%. Such a combination could be used to efficiently assay the coagulation system.

  13. Factor V Leiden

    Science.gov (United States)

    ... increase your chance of developing abnormal blood clots (thrombophilia), usually in your veins. Most people with factor ... sharp tools. References Bauer KA. Management of inherited thrombophilia. http://www.uptodate.com/home. Accessed June 6, ...

  14. Activity of recombinant factor VIIa under different conditions in vitro

    DEFF Research Database (Denmark)

    Bladbjerg, Else-Marie; Jespersen, Jørgen

    2008-01-01

    , but no effects on clotting time indicating that haemodilution does not affect clot formation, but the clot formed at high haemodilution may not be so firm. In conclusion, the activity of recombinant activated factor VII was affected in vitro by pH, temperature, and haemodilution. Additional studies are necessary...... investigated the in-vitro effects of pH, temperature, and haemodilution on the activity of recombinant activated factor VII. Samples from eight healthy volunteers were spiked with recombinant activated factor VII (final concentration 1.7 microg/ml) and adjusted to pH 6.0, 6.5, 7.0, and 7.4 or analysed at 30...... activity in plasma. Significant effects of pH were observed for clotting time, clot formation time, maximum clot firmness, and factor VII coagulant activity in the direction of longer clot formation times and less firm clots with decreasing pH. Temperature had significant effects on clotting time, clot...

  15. New study on the XeVIII spectrum

    Energy Technology Data Exchange (ETDEWEB)

    Gallardo, M. [Centro de Investigaciones Opticas (CIOp) Casilla de Correo 124, 1900 La Plata (Argentina)]. E-mail: gallardom@ciop.unlp.edu.ar; Raineri, M. [Centro de Investigaciones Opticas (CIOp) Casilla de Correo 124, 1900 La Plata (Argentina)]. E-mail: monicar@ciop.unlp.edu.ar; Giuliani, M. [Dto. de Fisica, Fac. de C. Exactas y Naturales, Universidad Nacional de Mar del Plata Funes 3350 (7600) Mar del Plata (Argentina); Lagorio, C. [Dto. de Fisica, Fac. de C. Exactas y Naturales, Universidad Nacional de Mar del Plata Funes 3350 (7600) Mar del Plata (Argentina); Padilla, S. [Dto. de Fisica, Fac. de C. Exactas y Naturales, Universidad Nacional de Mar del Plata Funes 3350 (7600) Mar del Plata (Argentina); Sarmiento, R. [Dto. De Fisica, Universidad del Atlantico, A.A 1890 Barranquilla (Colombia); Reyna Almandos, J.G. [Centro de Investigaciones Opticas (CIOp) Casilla de Correo 124, 1900 La Plata (Argentina)]. E-mail: jreyna@ciop.unlp.edu.ar

    2005-10-15

    Capillary light sources were used to observe the spectrum of seven-times-ionized xenon, XeVIII, in the 300-6000 A wavelength range. Twenty-eight energy levels belonging to the 5s-9s, 5d-9d, 5g-9g, 7i-10i, 9l, 5p-9p, 4f-9f, 6h-9h and 8k-10k configurations were adjusted and 38 new spectral lines were classified. The XeVIII ionization energy was determined with improved accuracy using the polarization model. The analysis was supported by Hartree-Fock calculations. The weighted oscillator strengths for all the observed spectral lines were also calculated considering the fitted values for the energy parameters.

  16. Fibrin clot properties and haemostatic function in men and women with type 1 diabetes.

    Science.gov (United States)

    Tehrani, Sara; Jörneskog, Gun; Ågren, Anna; Lins, Per-Eric; Wallén, Håkan; Antovic, Aleksandra

    2015-02-01

    The increased risk of vascular complications in type 1 diabetes may in part be explained by changes in haemostatic function. In the present study, we investigated the fibrin clot properties in patients with type 1 diabetes in relation to sex and microvascular complications. The study included 236 patients (107 women) aged between 20-70 years and without any history of cardiovascular disease. Fibrin clot properties, assessed by determination of the permeability coefficient (Ks) and turbidimetric clotting and lysis assays, did not differ between men and women. Compared with men, women had worse glycaemic control as well as higher levels of prothrombin fragment 1+2 and peak thrombin generation in vitro, indicating increased thrombin generation both in vivo and in vitro. Subgroup analyses of patients younger than 30 years revealed less permeable fibrin clots and prolonged lysis time in females compared with age-matched men. Patients with microvascular complications had higher fibrinogen concentrations and denser and less permeable fibrin clots. Thus, we conclude that in vitro fibrin clot properties in patients with type 1 diabetes without cardiovascular disease are not different between the sexes, but associate with prevalence of microvascular complications. Tighter fibrin clot formation in younger women, as suggested by our results, may affect their future cardiovascular risk and should be investigated in a larger population.

  17. Zwitterionic Group VIII transition metal initiators supported by olefin ligands

    Science.gov (United States)

    Bazan, Guillermo C.; Chen, Yaofeng

    2011-10-25

    A zwitterionic Group VIII transition metal complex containing the simple and relatively small 3-(arylimino)-but-1-en-2-olato ligand that catalyzes the formation of polypropylene and high molecular weight polyethylene. A novel feature of this catalyst is that the active species is stabilized by a chelated olefin adduct. The present invention also provides methods of polymerizing olefin monomers using zwitterionic catalysts, particularly polypropylene and high molecular weight polyethylene.

  18. Cationic PAMAM dendrimers aggressively initiate blood clot formation.

    Science.gov (United States)

    Jones, Clinton F; Campbell, Robert A; Brooks, Amanda E; Assemi, Shoeleh; Tadjiki, Soheyl; Thiagarajan, Giridhar; Mulcock, Cheyanne; Weyrich, Andrew S; Brooks, Benjamin D; Ghandehari, Hamidreza; Grainger, David W

    2012-11-27

    Poly(amidoamine) (PAMAM) dendrimers are increasingly studied as model nanoparticles for a variety of biomedical applications, notably in systemic administrations. However, with respect to blood-contacting applications, amine-terminated dendrimers have recently been shown to activate platelets and cause a fatal, disseminated intravascular coagulation (DIC)-like condition in mice and rats. We here demonstrate that, upon addition to blood, cationic G7 PAMAM dendrimers induce fibrinogen aggregation, which may contribute to the in vivo DIC-like phenomenon. We demonstrate that amine-terminated dendrimers act directly on fibrinogen in a thrombin-independent manner to generate dense, high-molecular-weight fibrinogen aggregates with minimal fibrin fibril formation. In addition, we hypothesize this clot-like behavior is likely mediated by electrostatic interactions between the densely charged cationic dendrimer surface and negatively charged fibrinogen domains. Interestingly, cationic dendrimers also induced aggregation of albumin, suggesting that many negatively charged blood proteins may be affected by cationic dendrimers. To investigate this further, zebrafish embryos were employed to more specifically determine the speed of this phenomenon and the pathway- and dose-dependency of the resulting vascular occlusion phenotype. These novel findings show that G7 PAMAM dendrimers significantly and adversely impact many blood components to produce rapid coagulation and strongly suggest that these effects are independent of classic coagulation mechanisms. These results also strongly suggest the need to fully characterize amine-terminated PAMAM dendrimers in regard to their adverse effects on both coagulation and platelets, which may contribute to blood toxicity.

  19. Ehlers-Danlos Syndrome Type VIII: A Rare Cause of Leg Ulcers in Young Patients

    Science.gov (United States)

    Lucas, Antoine; Piérard, Gérald E.; Hermanns-Lê, Trinh; De Paepe, Anne; Dupuy, Alain

    2013-01-01

    Ehlers-Danlos syndrome type VIII (EDS-VIII) is a very rare autosomal dominant disease characterized by early-onset periodontitis associated with features of Ehlers-Danlos syndrome. We report a 32-year-old man whose chronic leg ulcer led to the diagnosis of EDS-VIII. He had severe periodontitis with complete loss of permanent teeth and skin fragility with thin skin, atrophic scars, and brownish atrophic pretibial plaques. Leg ulcer is not a prominent feature of EDS-VIII. We suggest adding EDS-VIII to the list of rare diseases accounting for chronic leg ulcers, if this case report prompts others to report leg ulcers associated with EDS-VIII. PMID:24198978

  20. Too Many Stroke Victims Don't Get Clot-Busting Drug

    Science.gov (United States)

    ... medlineplus.gov/news/fullstory_163756.html Too Many Stroke Victims Don't Get Clot-Busting Drug: Study ... 23, 2017 THURSDAY, Feb. 23, 2017 (HealthDay News) -- Stroke victims can be saved through the timely use ...

  1. Imaging and Elastometry of Blood Clots Using Magnetomotive Optical Coherence Tomography and Labeled Platelets

    Science.gov (United States)

    Oldenburg, Amy L.; Wu, Gongting; Spivak, Dmitry; Tsui, Frank; Wolberg, Alisa S.; Fischer, Thomas H.

    2013-01-01

    Improved methods for imaging and assessment of vascular defects are needed for directing treatment of cardiovascular pathologies. In this paper, we employ magnetomotive optical coherence tomography (MMOCT) as a platform both to detect and to measure the elasticity of blood clots. Detection is enabled through the use of rehydrated, lyophilized platelets loaded with superparamagnetic iron oxides (SPIO-RL platelets) that are functional infusion agents that adhere to sites of vascular endothelial damage. Evidence suggests that the sensitivity for detection is improved over threefold by magnetic interactions between SPIOs inside RL platelets. Using the same MMOCT system, we show how elastometry of simulated clots, using resonant acoustic spectroscopy, is correlated with the fibrin content of the clot. Both methods are based upon magnetic actuation and phase-sensitive optical monitoring of nanoscale displacements using MMOCT, underscoring its utility as a broad-based platform to detect and measure the molecular structure and composition of blood clots. PMID:23833549

  2. [Results of fibrin clot application for acceleration of regeneration of the damaged mandible in experiment].

    Science.gov (United States)

    Maĭborodin, I V; Kolesnikov, I S; Shevela, A I; Sheplev, B V; Drovosekov, M N; Toder, M S

    2011-01-01

    The processes of regeneration of the damaged rat bottom jaw bone after application of enriched thrombocytes a fibrin clot were studied by morphological and radiovisiographic methods. At a natural course of regeneration the artificial aperture of bone was filled with blood and there the blood clot was formed. After 1 week the separate bone islets of a young tissue occurred in bone defect. In 2-3 weeks the aperture in a bottom jaw bone was completely closed by a young bone tissue. After operation with filling of bone bottom jaw defect by fibrin clot there was no formation of a blood clot. Already after 1 week the bone tissue defect was filled by the merged islets of again generated bone. By second week after fibrin use the further formation of bone tissue in defect and formation of a bone callosity was noted.

  3. C-Section Raises Risk of Blood Clots After Childbirth: Review

    Science.gov (United States)

    ... page: https://medlineplus.gov/news/fullstory_161301.html C-Section Raises Risk of Blood Clots After Childbirth: ... international studies found that women who had a C-section were four times more likely to develop ...

  4. Antenatal glucocorticoids attenuate activation of the inflammatory reaction and clotting in preterm lambs

    NARCIS (Netherlands)

    Jaarsma, AS; Geven, WB; van Oeveren, W; Bambang-Oetomo, S

    2004-01-01

    Recently we have shown that activation of inflammatory reaction and clotting can be found immediately after delivery in preterm lambs ventilated for respiratory distress syndrome (RDS). To investigate whether antenatal glucocorticoids would attenuate postnatal activation of the inflammatory reaction

  5. 40 CFR Appendix Viii to Part 600 - Fuel Economy Label Formats

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 29 2010-07-01 2010-07-01 false Fuel Economy Label Formats VIII... POLICY FUEL ECONOMY AND CARBON-RELATED EXHAUST EMISSIONS OF MOTOR VEHICLES Pt. 600, App. VIII Appendix VIII to Part 600—Fuel Economy Label Formats EC01MY92.117 EC01MY92.118 EC01MY92.119 EC01MY92.120...

  6. Localization of Short-Chain Polyphosphate Enhances its Ability to Clot Flowing Blood Plasma

    Science.gov (United States)

    Yeon, Ju Hun; Mazinani, Nima; Schlappi, Travis S.; Chan, Karen Y. T.; Baylis, James R.; Smith, Stephanie A.; Donovan, Alexander J.; Kudela, Damien; Stucky, Galen D.; Liu, Ying; Morrissey, James H.; Kastrup, Christian J.

    2017-02-01

    Short-chain polyphosphate (polyP) is released from platelets upon platelet activation, but it is not clear if it contributes to thrombosis. PolyP has increased propensity to clot blood with increased polymer length and when localized onto particles, but it is unknown whether spatial localization of short-chain polyP can accelerate clotting of flowing blood. Here, numerical simulations predicted the effect of localization of polyP on clotting under flow, and this was tested in vitro using microfluidics. Synthetic polyP was more effective at triggering clotting of flowing blood plasma when localized on a surface than when solubilized in solution or when localized as nanoparticles, accelerating clotting at 10–200 fold lower concentrations, particularly at low to sub-physiological shear rates typical of where thrombosis occurs in large veins or valves. Thus, sub-micromolar concentrations of short-chain polyP can accelerate clotting of flowing blood plasma under flow at low to sub-physiological shear rates. However, a physiological mechanism for the localization of polyP to platelet or vascular surfaces remains unknown.

  7. Partial purification of new milk-clotting enzyme produced by Nocardiopsis sp.

    Science.gov (United States)

    Cavalcanti, M T H; Teixeira, M F S; Lima Filho, J L; Porto, A L F

    2004-05-01

    Numerous attempts have been made to replace calf rennet with other milk clotting proteases because of limited supply and increasingly high prices. The aim of this work was to investigate the characteristic of the milk-clotting enzyme from Nocardiopsis sp. The partial purification extract was obtained by fractional precipitation with ammonium sulphate. Of the fractions obtained by precipitation, 40-60% possessed the milk-clotting activity (156.25 U/mg). The chromatography of 40-100% ammonium sulphate fraction in DEAE-cellulose yielded four fractions (F4, F5, F6, F7) with milk-clotting activity. The F5 yielded the best milk-clotting activity (20 U/ml). Both crude and partially purified extract were active at the range pH 4.5-11.0, however, optimum activity was displayed at pH 11.0 and pH 7.5, respectively. The milk-clotting activity was highest at 55 degrees C for both crude and partially purified extract. The crude and partial purification extract were inactivated at 65 and 75 degrees C after 30 min.

  8. Acute toxicity of diphacinone in Northern bobwhite: Effects on survival and blood clotting

    Science.gov (United States)

    Rattner, Barnett A.; Horak, Katherine E.; Warner, Sarah E.; Johnston, John J.

    2010-01-01

    The anticoagulant rodenticide diphacinone was slightly toxic (acute oral LD50 2014 mg/kg) to Northern bobwhite (Colinus virginianus) in a 14-day acute toxicity trial. Precise and sensitive assays of blood clotting (prothrombin time, Russell?s Viper venom time, and thrombin clotting time) were adapted for use in quail, and this combination of assays is recommended to measure the effects of anticoagulant rodenticides. A single oral sublethal dose of diphacinone (434 mg/kg body weight) prolonged clotting time at 48 h post-dose compared to controls. At 783 mg/kg (approximate LD02), clotting time was prolonged at both 24 and 48 h post-dose. Prolongation of in vitro clotting time reflects impaired coagulation complex activity, and was detected before overt signs of toxicity were apparent at the greatest dosages (2868 and 3666 mg/kg) in the acute toxicity trial. These clotting time assays and toxicity data will assist in the development of a pharmacodynamic model to predict toxicity, and also facilitate rodenticide hazard and risk assessments in avian species.

  9. A Fictitious Domain Method for Resolving the Interaction of Blood Flow with Clot Growth

    Science.gov (United States)

    Mukherjee, Debanjan; Shadden, Shawn

    2016-11-01

    Thrombosis and thrombo-embolism cause a range of diseases including heart attack and stroke. Closer understanding of clot and blood flow mechanics provides valuable insights on the etiology, diagnosis, and treatment of thrombotic diseases. Such mechanics are complicated, however, by the discrete and multi-scale phenomena underlying thrombosis, and the complex interactions of unsteady, pulsatile hemodynamics with a clot of arbitrary shape and microstructure. We have developed a computational technique, based on a fictitious domain based finite element method, to study these interactions. The method can resolve arbitrary clot geometries, and dynamically couple fluid flow with static or growing clot boundaries. Macroscopic thrombus-hemodynamics interactions were investigated within idealized vessel geometries representative of the common carotid artery, with realistic unsteady flow profiles as inputs. The method was also employed successfully to resolve micro-scale interactions using a model driven by in-vivo morphology data. The results provide insights into the flow structures and hemodynamic loading around an arbitrarily grown clot at arterial length-scales, as well as flow and transport within the interstices of platelet aggregates composing the clot. The work was supported by AHA Award No: 16POST27500023.

  10. New photoacoustic platform for early detection of circulating clots to prevent stroke and other fatal thromboembolic complications (Conference Presentation)

    Science.gov (United States)

    Carey, Kai A.

    2017-03-01

    Nearly 800,000 people in the U.S. experience an incident of stroke each year; 80% of these are first time occurrences and 87% are ischemic in nature. Someone dies of a stroke every few minutes in the U.S. but despite its prevalence there have been minimal advances in the early detection and screening of thromboembolic events, especially during patient post-operative periods or in genetically predisposed individuals. Environmental or genetic factors may disrupt the balance between coagulation and lysis of micro-thrombi in circulation and increase the risk of stroke. We introduced here a novel in vivo multicolor negative-contrast photoacoustic (PA) flow cytometry (PAFC ) platform with many innovations including customized high pulse repetition rate 1064 laser from IPG Photonics Corporation, powerful laser diode array, multichannel optical schematic, and time-resolved recording system. Using animal models, we verified the potential of this technology to detect small clots in relatively large vessels in vivo. If future clinical trials using a cost-effective, easy-to-use, safe, watch-like, wearable PA probe are successful, PAFC could provide breakthroughs in early monitoring of the growth in size and number of small clots that may predict and potentially prevent fatal thromboembolic complications. We also believe that this technology could be utilized to assess therapeutic benefits of anticoagulants and develop more efficient dosage in treatments by analyzing changes in the composition and frequency of micro-thrombi

  11. OPTIMIZATION OF MILK-CLOTTING PROTEASE PRODUCTION BY A LOCAL ISOLATE OF ASPERGILLUS NIGER FFB1 IN SOLID-STATE FERMENTATION

    Directory of Open Access Journals (Sweden)

    Souhila Bensmail

    2015-04-01

    Full Text Available The need to surmount the limitation of obtaining rennin, has been actively pushed researches to find new substitutes that present high milk-clotting activity which enables the production of high yields of cheese. In this study, the production of extracellular milk-clotting protease by locally isolated fungal specie, Aspergillus niger FFB1 under solid-state fermentation (SSF using cheep agro-industrial byproduct (wheat bran was optimized. The effects of several physicochemical and environmental factors were investigated to select the optimal conditions that ensure the best milk-clotting activity by application of "One-factor-at-a-time" method. A trial of cheese production using the crude extract was also carried out. The maximum enzyme activity (830 SU/g bran with a ratio MCA/PA of 4.25 was obtained under the optimum conditions of temperature (30°C, spores concentration (106 spores/mL, incubation time (72 hours, and moisture content of solid substrate (39.2% adjusted suitably with mineral solution (Czapek-Dox of pH 4.

  12. Optimization of the production and characterization of milk clotting enzymes by Bacillus subtilis natto.

    Science.gov (United States)

    Wu, Fang-Chen; Chang, Chen-Wei; Shih, Ing-Lung

    2013-12-01

    Suitable medium for production of milk clotting enzyme (MCE) by Bacillus subtilis (natto) Takahashi in submerged liquid-state fermentation was screened, the nutrient factors affecting MCE production was optimized by response surface methodology. The MCE production by B. subtilis (natto) Takahashi was increased significantly by 428% in the optimal medium developed. The MCE was filtered and concentrated by ultrafiltration. The retentate after tandem filtration carried out with the combined membranes of MWCO 50kDa and 5 kDa showed two major bands between 25kDa and 30kDa on SDS-PAGE, and the MCA and MCA/PA improved significantly in comparison with those in the initial broth. The crude enzyme thus obtained showed MCA and MCA/PA ratio of 48,000 SU/g and 6,400, which are commensurate with those (MCA 26,667 SU/g and MCA/PA 6,667) of the commercial rennet. It had optimal pH and temperature at pH 6 and 60°C, and showed excellent pH and thermal stability.

  13. Prolonged clot lysis time increases the risk of a first but not recurrent venous thrombosis.

    Science.gov (United States)

    Karasu, Alev; Baglin, Trevor P; Luddington, Roger; Baglin, Caroline A; van Hylckama Vlieg, Astrid

    2016-03-01

    The role of the fibrinolytic system in the development of venous thrombosis (VT) is unclear. We studied the risk of first and recurrent VT associated with reduced fibrinolysis, as measured by clot lysis time (CLT). We also studied the relationship between CLT and thrombin generation to determine if any relationship between CLT and VT was affected by thrombin generation. Analyses were performed in the Thrombophilia Hypercoagulability Environmental risk for Venous Thromboembolism Study, a two-centre population-based case-control study, including 579 patients and 338 controls, with patients followed from the event to determine incidence of recurrent VT. Hypofibrinolysis was associated with a 1·8-fold increased risk of a first VT [95% confidence interval (CI) 1·2-2·7]. Adjustment for sex, age, study location and Endogenous Thrombin Potential (ETP) did not change the result. The risk of VT was 2·9-fold increased when the 90th percentiles of prolonged CLT and high ETP were combined, with the highest risk for unprovoked first events (Odds Ratio = 4·2, 95% CI 1·3-13·5). In the follow-up study the Hazard Ratio for a recurrent VT associated with hypofibrinolysis was 1·5 (95% CI 0·9-2·6). A weak dose response effect was observed in relation to prolongation of CLT and recurrent VT. Although hypofibrinolysis constitutes a risk factor for a first VT, an association with recurrence is, at best, weak.

  14. Effects of calcium-modified titanium implant surfaces on platelet activation, clot formation, and osseointegration.

    Science.gov (United States)

    Anitua, Eduardo; Prado, Roberto; Orive, Gorka; Tejero, Ricardo

    2015-03-01

    The clinical success of load bearing dental and orthopedic implants relies on adequate osseointegration. Because of its favorable properties, titanium is generally considered as the material of choice. Following implant placement, titanium surfaces establish an ionic equilibrium with the surrounding tissues in which calcium plays major roles. Calcium is a cofactor of the coagulation cascade that mediates plasma protein adsorption and intervenes in a number of other intra and extracellular processes relevant for bone regeneration. In this study, titanium surfaces were modified with calcium ions (Ca(2+) surfaces) and their responses to in vitro and in vivo models were analyzed. Unlike unmodified surfaces, Ca(2+) surfaces were superhydrophilic and induced surface clot formation, platelet adsorption and activation when exposed to blood plasma. Interestingly, in vivo osseointegration using a peri-implant gap model in rabbit demonstrated that Ca(2+) surfaces significantly improved peri-implant bone volume and density at 2 weeks and bone implant contact at 8 weeks as compared to the unmodified controls. The combination of Ca(2+) surfaces with plasma rich in growth factors produced significantly more bone contact already at 2 weeks of implantation. These findings suggest the importance of the provisional matrix formation on tissue integration and highlight the clinical potential of Ca(2+) titanium surfaces as efficient stimulators of implant osseointegration.

  15. Diagnostic difficulties of factor XI deficiencies: interferences' assay or real deficit?

    Science.gov (United States)

    Gaymard, Alexandre; Nougier, Christophe

    2016-06-01

    Madam P, 77 years old, consulted in the hemostasis department after a coagulation anomaly was discovered during her preoperative test for a total hip prosthesis. After confirmation of a persistent and increased aPTT, additional tests were performed and showed the presence of antiphospholipid antibodies. Factor VIII level could be corrected after the plasma dilution to 1/40(th). But successive dilutions were not enough to obtain a correct factor IX (FIX) and factor XI (FXI) level. FIX level was obtained by chromogenic method in order to avoid the interferences caused by the antibodies. Finally, despite the change of reagents and dilutions up to 1/160(th), the FXI level couldn't be determined. Despite these results and those of the thrombin generation assay, the surgery was successfully done without specific treatment thanks to the absence of hemorrhagic history. This observation highlights the diagnostic and monitoring difficulties for uncommon clotting factor deficit. The development of interference free test could increase the support for these patients.

  16. Diagnostic value of blood clot core during endobronchial ultrasound-guided transbronchial needle aspirate.

    Science.gov (United States)

    Amin, Emily N; Russell, Christopher D; Shilo, Konstantin; Islam, Shaheen; Wood, Karen L

    2013-06-01

    Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is being increasingly used in the sampling of pulmonary masses and mediastinal lymphadenopathy. The blood clot core (BCC) often obtained during EBUS-TBNA may not be a true core and therefore may not be submitted for histological analysis. The frequency in which the blood clot core is positive in patients with negative cytology undergoing EBUS-TBNA is not known. The purpose of this study was to evaluate the diagnostic role of the blood clot core obtained during EBUS-TBNA. An Institutional Review Board-approved retrospective chart review was performed from January through September 2011 for all patients who underwent EBUS-TBNA at The Ohio State University. The data collection included cytology and histology results for each procedure. Blood clot cores obtained from the EBUS-TBNA needle were sent in formalin for histological examination. Seventy patients underwent EBUS-TBNA and 51 (72.8 %) patients had procedures that yielded a BCC for histology and aspirate for cytology. Forty-nine percent of patients with a BCC were diagnosed with malignancy. Of those with a BCC obtained, five (9.8 %) patients diagnosed with malignancy were done so based only on the results of blood clot core alone with negative cytology. Blood clot cores obtained at EBUS-TBNA contain diagnostic material and should be subjected histopathological examination. When blood clot cores are sent for analysis, there is the potential to spare up to 10 % of patients more invasive diagnostic biopsy procedures.

  17. Effectiveness of Mind Mapping in English Teaching among VIII Standard Students

    Science.gov (United States)

    Hallen, D.; Sangeetha, N.

    2015-01-01

    The aim of the study is to find out the effectiveness of mind mapping technique over conventional method in teaching English at high school level (VIII), in terms of Control and Experimental group. The sample of the study comprised, 60 VIII Standard students in Tiruchendur Taluk. Mind Maps and Achievement Test (Pretest & Posttest) were…

  18. Shrimp Alpha-2-Macroglobulin Prevents the Bacterial Escape by Inhibiting Fibrinolysis of Blood Clots

    Science.gov (United States)

    Chaikeeratisak, Vorrapon; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

    2012-01-01

    Proteomic analysis of the hemocytic proteins of Penaeus monodon (Pm) has previously shown that alpha-2-macroglobulin (A2M) was among the proteins that showed substantially altered expression levels upon Vibrio harveyi infection. Therefore, in this study its potentially important role in the response of shrimp to bacterial infection was further characterized. The yeast two-hybrid system revealed that the receptor binding domain of PmA2M interacted with the carboxyl-terminus of one or both of the transglutaminase type II isoforms, which are key enzymes involved in the shrimp clotting system. In accord with this, PmA2M was found to be localized on the extracellular blood clots and to colocalize with clottable proteins. RNA interference (RNAi)-mediated knockdown of A2M transcript levels reduced the PmA2M transcript levels (∼94%) and significantly reduced the bacterial seizing ability of the clotting system, resulting in an up to 3.3-fold higher number of V. harveyi that systemically disseminated into the circulatory system at 5 min post-infection before subsequent clearance by the immune system. Furthermore, an appearance of PmA2M depleted clots in the presence of V. harveyi strikingly demonstrated fibrinolysis zones surrounding the bacteria. This study provides the first evidence of the vital role of PmA2M in enhancing bacterial sequestration by protecting blood clots against fibrinolysis. PMID:23082160

  19. Blood clot initiation by mesocellular foams: dependence on nanopore size and enzyme immobilization.

    Science.gov (United States)

    Baker, Sarah E; Sawvel, April M; Fan, Jie; Shi, Qihui; Strandwitz, Nicholas; Stucky, Galen D

    2008-12-16

    Porous silica materials are attractive for hemorrhage control because of their blood clot promoting surface chemistry, the wide variety of surface topologies and porous structures that can be created, and the potential ability to achieve high loading of therapeutic proteins within the silica support. We show that silica cell-window size variation in the nanometers to tens of nanometers range greatly affects the rate at which blood clots are formed in human plasma, indicating that window sizes in this size range directly impact the accessibility and diffusion of clotting-promoting proteins to and from the interior surfaces and pore volume of mesocellular foams (MCFs). These studies point toward a critical window size at which the clotting speed is minimized and serve as a model for the design of more effective wound-dressing materials. We demonstrate that the clotting times of plasma exposed to MCF materials are dramatically reduced by immobilizing thrombin in the pores of the MCF, validating the utility of enzyme-immobilized mesoporous silicas in biomedical applications.

  20. Effect of thiol derivatives on mixed mucus and blood clots in vitro.

    Science.gov (United States)

    Risack, L E; Vandevelde, M E; Gobert, J G

    1978-01-01

    The disintegrating effect of three reducing thiol derivatives: [sodium mercaptoethane sulphonate (Mesna), N-acetyl-L-cysteine (NAC) and dithio-1,4-threitol (DTT)] was investigated in vitro upon blood clots formed in the absence or in the presence of tracheobronchial secretions and compared with the effect of iso-osmotic saline solution. The amounts of haemoglobin released from the clots after 30 min incubation and the initial rates of haemoglobin release were compared for the different products at different concentrations. All three reducing agents showed some ability to disintegrate mixed clots to an extent depending on their concentration. After 30 min incubation, statistical analysis showed a highly significant difference in favour of Mesna at the three concentrations used, i.e. 0.1, 1.0 and 10 mmol/1. The initial rate of haemoglobin release in presence of Mesna was at all concentrations significantly higher than that of NAC or DTT. The effects on normal blood clots were much less pronounced. The effectiveness of Mesna in splitting up mixed blood and mucus clots in the management of patients who had inhaled blood is discussed.

  1. A Serpin Released by an Entomopathogen Impairs Clot Formation in Insect Defense System

    Science.gov (United States)

    Hao, YouJin; Balasubramanian, Natesan; Jing, Yingjun; Montiel, Rafael; Faria, Tiago Q.; Brito, Rui M.; Simões, Nelson

    2013-01-01

    Steinernema carpocapsae is an entomopathogenic nematode widely used for the control of insect pests due to its virulence, which is mainly attributed to the ability the parasitic stage has to overcome insect defences. To identify the mechanisms underlying such a characteristic, we studied a novel serpin-like inhibitor (sc-srp-6) that was detected in a transcriptome analysis. Recombinant Sc-SRP-6 produced in Escherichia coli had a native fold of serpins belonging to the α-1-peptidase family and exhibited inhibitory activity against trypsin and α-chymotrypsin with Ki of 0.42×10−7 M and 1.22×10−7 M, respectively. Functional analysis revealed that Sc-SRP-6 inhibits insect digestive enzymes, thus preventing the hydrolysis of ingested particles. Moreover, Sc-SRP-6 impaired the formation of hard clots at the injury site, a major insect defence mechanism against invasive pathogens. Sc-SRP-6 does not prevent the formation of clot fibres and the activation of prophenoloxidases but impairs the incorporation of the melanin into the clot. Binding assays showed a complex formation between Sc-SRP-6 and three proteins in the hemolymph of lepidopteran required for clotting, apolipophorin, hexamerin and trypsin-like, although the catalytic inhibition occurred exclusively in trypsin-like. This data allowed the conclusion that Sc-SRP-6 promotes nematode virulence by inhibiting insect gut juices and by impairing immune clot reaction. PMID:23874900

  2. In vivo ultrasound visualization of non-occlusive blood clots with thrombin-sensitive contrast agents.

    Science.gov (United States)

    Nakatsuka, Matthew A; Barback, Christopher V; Fitch, Kirsten R; Farwell, Alexander R; Esener, Sadik C; Mattrey, Robert F; Cha, Jennifer N; Goodwin, Andrew P

    2013-12-01

    The use of microbubbles as ultrasound contrast agents is one of the primary methods to diagnose deep venous thrombosis. However, current microbubble imaging strategies require either a clot sufficiently large to produce a circulation filling defect or a clot with sufficient vascularization to allow for targeted accumulation of contrast agents. Previously, we reported the design of a microbubble formulation that modulated its ability to generate ultrasound contrast from interaction with thrombin through incorporation of aptamer-containing DNA crosslinks in the encapsulating shell, enabling the measurement of a local chemical environment by changes in acoustic activity. However, this contrast agent lacked sufficient stability and lifetime in blood to be used as a diagnostic tool. Here we describe a PEG-stabilized, thrombin-activated microbubble (PSTA-MB) with sufficient stability to be used in vivo in circulation with no change in biomarker sensitivity. In the presence of actively clotting blood, PSTA-MBs showed a 5-fold increase in acoustic activity. Specificity for the presence of thrombin and stability under constant shear flow were demonstrated in a home-built in vitro model. Finally, PSTA-MBs were able to detect the presence of an active clot within the vena cava of a rabbit sufficiently small as to not be visible by current non-specific contrast agents. By activating in non-occlusive environments, these contrast agents will be able to detect clots not diagnosable by current contrast agents. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Measurement of the ultrasonic attenuation coefficient of human blood plasma during clotting in the frequency range of 8 to 22 MHz.

    Science.gov (United States)

    Calor-Filho, Marcos Muniz; Machado, João Carlos

    2006-07-01

    The blood coagulation mechanism consists of a series of concatenated chemical reactions, governed by the coagulation factors present in the blood plasma, after the activation of the clot mechanism. The last reaction corresponds to the fibrinogen conversion into fibrin, followed by the fibrin polymerisation and production of a stable fibrin network. During the clotting process, there is a sol-gel transformation of the medium. The subject of the present paper is the measurement of the ultrasonic attenuation coefficient for human blood plasma during the coagulation process, in the frequency range of 8 to 22 MHz. The clot was obtained after the procedure to measure the prothrombin time (approximately 12 s): mixing 150 microL of reconstituted lyophilised normal plasma with 300 microL of reconstituted lyophilised thromboplastin immersed in a water bath with the temperature controlled at 36.5 degrees C. The attenuation coefficient for pure plasma remained constant within the measurement period of 10 s and at frequencies of 8, 9, 10, 15, 20, 21 and 22 MHz. On the other hand, there is a detectable time-decay of the attenuation coefficient for samples of plasma going through the coagulation process and at frequencies of 8, 9, 10 and 15 MHz. The time-decay becomes less and less detectable as the frequency increases and it becomes completely undetectable at 20, 21 and 22 MHz.

  4. Effect of carryover of clot activators on coagulation tests during phlebotomy.

    Science.gov (United States)

    Fukugawa, Yoko; Ohnishi, Hiroaki; Ishii, Takahiro; Tanouchi, Ayako; Sano, Junko; Miyawaki, Haruko; Kishino, Tomonori; Ohtsuka, Kouki; Yoshino, Hideaki; Watanabe, Takashi

    2012-06-01

    We investigated the effect of clot activators carried over from the serum tube on major coagulation tests during phlebotomy. First, blood specimens from 30 normal subjects were mixed with small amounts of fluid containing clot activators, and their effects on various coagulation tests were determined. Only the value of fibrin monomer complex displayed a remarkable change when thrombin-containing fluid was added to the blood specimens. Subsequently, 100 paired blood specimens (taken from 75 healthy volunteers and 25 patients taking warfarin) were collected in coagulation tubes before and after the serum tube using standard phlebotomy procedures. Various coagulation tests were performed to determine the effect of contamination of thrombin-containing blood on coagulation parameters. Differences between the 2 tubes were minimal but significant for some of the coagulation tests. Therefore, we conclude that the effect of clot activators in the serum tube on coagulation tests is minimal when standard phlebotomy procedures are used.

  5. A turbidimetric assay for the measurement of clotting times of procoagulant venoms in plasma.

    Science.gov (United States)

    O'Leary, Margaret A; Isbister, Geoffrey K

    2010-01-01

    Assessment of the procoagulant effect of snake venoms is important for understanding their effects. The aim of this study was to develop a simple automated method to measure clotting times to assess procoagulant venoms. A turbidimetric assay was developed which monitors changes in optical density when plasma and venom are mixed. Plasma was added simultaneously to venom solutions in a 96 well microtitre plate. After mixing, the optical density at 340 nm was monitored in a microplate reader every 30 s over 30 min. The clotting time was defined as the lag time until the absorbance sharply increased. The turbidimetric method was compared to manual measurement of the clotting time defined as the time when a strand of fibrin can be drawn out of the mixture. The two methods were done simultaneously, with the same venom and plasma, and compared by plotting the manual versus turbidimetric clotting times. Within-day and between-day runs were done and the coefficient of variation (CV) was calculated. Plots comparing manual clotting times to the lag time in the turbidimetric assay showed good correlation between the two methods for brown snake (Pseudonaja textilis) venom, including 24 determinations in triplicate over six days for seven different venom concentrations. Good correlation was also found for four other venoms: tiger snake (Notechis scutatus), Carpet viper (Echis carinatus), Russell's viper (Daboia russelii) and Malaysian pit piper (Calloselasma rhodostoma). Between-day CV was in the range 10-20% for both methods, while within-day CV <10%. The turbidimetric assay appears to be a simple and convenient automated method for the measurement of clotting times to assess the effects of procoagulant venoms. Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.

  6. Surgery as a risk factor for inhibitor development in haemophilia A : A systematic review

    NARCIS (Netherlands)

    Eckhardt, C.; Van Der Bom, J.; Van Der Naald, M.; Peters, M.; Kamphuisen, P.; Fijnvandraat, K.

    2010-01-01

    Introduction: Surgery is a risk factor for the development of factor VIII inhibiting antibodies (inhibitors) in haemophilia A. This may be due to the combination of danger signal release resulting from tissue damage and intensive exposure to factor VIII (FVIII) concentrate. The purpose of this syste

  7. Surgery as a risk factor for inhibitor development in haemophilia A : A systematic review

    NARCIS (Netherlands)

    Eckhardt, C.; Van Der Bom, J.; Van Der Naald, M.; Peters, M.; Kamphuisen, P.; Fijnvandraat, K.

    2010-01-01

    Introduction: Surgery is a risk factor for the development of factor VIII inhibiting antibodies (inhibitors) in haemophilia A. This may be due to the combination of danger signal release resulting from tissue damage and intensive exposure to factor VIII (FVIII) concentrate. The purpose of this syste

  8. High-Quality and -Quantity DNA Extraction from Frozen Archival Blood Clots for Genotyping of Single-Nucleotide Polymorphisms

    DEFF Research Database (Denmark)

    Bank, Steffen; Nexø, Bjørn Andersen; Andersen, Vibeke

    2013-01-01

    Background: The recovery of biological samples for genetic epidemiological studies can be cumbersome. Blood clots are routinely collected for serological examinations. However, the extraction of DNA from blood clots can be difficult and often results in low yields. Aim: The aim was to compare...

  9. Clot resolution after 3 weeks of anticoagulant treatment of pulmonary embolism: Comparison of computed tomography and perfusion scintigraphy

    NARCIS (Netherlands)

    Van Es, J.; Douma, Renee; Kamphuisen, P.W.; Gerdes, V.E.A.; Verhamme, P.; Wells, P.S.; Bounameaux, H.; Lensing, A.W.A.; Büller, H.R.

    2013-01-01

    Introduction: Little is known about the natural history of clot resolution in the initial weeks of anticoagulant therapy in patients with acute pulmonary embolism (PE). Aim: To assess clot resolution of acute PE with either computed tomography pulmonary angiography (CT-scan) or perfusion

  10. Clot resolution after 3 weeks of anticoagulant treatment for pulmonary embolism : comparison of computed tomography and perfusion scintigraphy

    NARCIS (Netherlands)

    van Es, J.; Douma, R. A.; Kamphuisen, P. W.; Gerdes, V. E. A.; Verhamme, P.; Wells, P. S.; Bounameaux, H.; Lensing, A. W. A.; Bueller, H. R.

    2013-01-01

    Introduction Little is known about the natural history of clot resolution in the initial weeks of anticoagulant therapy in patients with acute pulmonary embolism (PE). Clot resolution of acute PE was assessed with either computed tomography pulmonary angiography scan (CT-scan) or perfusion scintigra

  11. The activated clotting time in cardiac surgery : Should Celite or kaolin be used?

    NARCIS (Netherlands)

    De Vries, Adrianus J.; Lansink-Hartgring, Annemieke Oude; Fernhout, Freek-Jan; Huet, Rolf C. G.; van den Heuvel, Edwin R.

    OBJECTIVES: Both kaolin- and Celite-activated clotting times (ACT) are used to guide anticoagulation during cardiopulmonary bypass. It is unknown whether these methods lead to similar management procedures for anticoagulation in patients and are thus interchangeable in terms of bias, precision and

  12. Characterization of partially purified milk-clotting enzyme from sunflower (Helianthus annuus) seeds.

    Science.gov (United States)

    Nasr, Assia I A M; Mohamed Ahmed, Isam A; Hamid, Omer I A

    2016-09-01

    This study was aimed to extract milk-clotting enzyme from sunflower seeds and to determine its potentiality for manufacturing white soft cheese from cows and goats milk. The seeds were blended and extracted using two types of buffers and milk-clotting and proteolytic activities were evaluated. The enzyme was partially purified using ammonium sulfate fractionation techniques. Results indicated that sunflower seeds extracted with 5% NaCl in 50 mmol/L acetate buffer, pH 5.0, had the highest milk-clotting activity (MCA) and lowest coagulation time compared to that extracted with only acetate buffer (pH 5.0). Ammonium sulfate at 30-50% saturation purified the enzyme to 4.3 folds with MCA of 241.0 U/mL and final enzyme yield of 10.9%. The partially purified enzyme was characterized by SDS-PAGE that showed two bands with molecular weight of 120 and 62 kDa. When compared with other plant enzymes, the partially purified sunflower enzyme was found to have higher milk-clotting activity and lower proteolytic activity. Also, both milk sources and enzyme types significantly affected the cheese yield and curd formation time. The cheese made from cow milk using sunflower enzyme had higher yield compared to that obtained using commercial rennet, whereas the opposite was observed when using goat milk.

  13. Using hydrogen peroxide as a bladder irrigation solution for clot evacuation

    Directory of Open Access Journals (Sweden)

    Mahdi Bagheri

    2017-03-01

    Full Text Available Gross hematuria or macroscopic hematuria is a high risk urologic condition that might occur in different settings. In the case of continued gross hematuria, blood clot size may grow and lead to complete obstruction of urinary outflow. Placement of three-way catheter, continuous bladder irrigation with normal saline, and cystoscopy are conventional treatments. Here we introduce a case with urinary obstruction who did not respond to conventional therapies. A subject of Hodgkin lymphoma with urinary obstruction caused by heavy gross hematuria was presented to emergency department. Three-way catheter was inserted to facilitate urination. However, there was no urinary drainage and bladder was distended. Consequently, 100 ml solution of hydrogen peroxide 0.15% was prepared and administered into the bladder to irrigate and evacuate the clots. A single intravesical infusion of hydrogen peroxide rapidly resolved urinary obstruction and improved patient distress. After administration of hydrogen peroxide solution, blood clots and bloody urine were evacuated successfully. These findings suggest that an intravesical injection of hydrogen peroxide can induce dissolution of blood clots and may be a simple and efficient therapy for urinary obstruction due to gross hematuria.

  14. Thrombin-Accelerated Quick Clotting Serum Tubes: An Evaluation with 22 Common Biochemical Analytes

    Directory of Open Access Journals (Sweden)

    Wai-Yoong Ng

    2013-01-01

    Full Text Available Clot activator serum tubes have significantly improved turnaround times for result reporting compared to plain tubes. With increasing workload and service performance expectations confronting clinical laboratories with high-volume testing and with particular emphasis on critical analytes, attention has focussed on preanalytical variables that can be improved. We carried out a field study on the test performance of BD vacutainer rapid serum tubes (RSTs compared to current institutional issued BD vacutainer serum separator tubes (SSTs in its test result comparability, clotting time, and stability on serum storage. Data from the study population (n=160 of patients attending outpatient clinics and healthy subjects showed that results for renal, liver, lipids, cardiac, thyroid, and prostate biochemical markers were comparable between RSTs and SSTs. Clotting times of the RSTs were verified to be quick with a median time of 2.05 min. Analyte stability on serum storage at 4°C showed no statistically significant deterioration except for bicarbonate, electrolytes, and albumin over a period of 4 days. In conclusion, RSTs offered savings in the time required for the clotting process of serum specimens. This should translate to further trimming of the whole process from blood collection to result reporting without too much sacrifice on test accuracy and performance compared to the current widely used SSTs in most clinical laboratories.

  15. Staphylococcus chromogenes, a Coagulase-Negative Staphylococcus Species That Can Clot Plasma.

    Science.gov (United States)

    Dos Santos, Danielle Cabral; Lange, Carla Christine; Avellar-Costa, Pedro; Dos Santos, Katia Regina Netto; Brito, Maria Aparecida Vasconcelos Paiva; Giambiagi-deMarval, Marcia

    2016-05-01

    Staphylococcus chromogenes is one of the main coagulase-negative staphylococci isolated from mastitis of dairy cows. We describe S. chromogenes isolates that can clot plasma. Since the main pathogen causing mastitis is coagulase-positive Staphylococcus aureus, the coagulase-positive phenotype of S. chromogenes described here can easily lead to misidentification. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  16. 21 CFR 864.7140 - Activated whole blood clotting time tests.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Activated whole blood clotting time tests. 864.7140 Section 864.7140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class II...

  17. Alteration of blood clot structures by interleukin-1 beta in association with bone defects healing

    Science.gov (United States)

    Wang, Xin; Friis, Thor E.; Masci, Paul P.; Crawford, Ross W.; Liao, Wenbo; Xiao, Yin

    2016-01-01

    The quality of hematomas are crucial for successful early bone defect healing, as the structure of fibrin clots can significantly influence the infiltration of cells, necessary for bone regeneration, from adjacent tissues into the fibrin network. This study investigated if there were structural differences between hematomas from normal and delayed healing bone defects and whether such differences were linked to changes in the expression of IL-1β. Using a bone defect model in rats, we found that the hematomas in the delayed healing model had thinner fibers and denser clot structures. Moreover, IL-1β protein levels were significantly higher in the delayed healing hematomas. The effects of IL-1β on the structural properties of human whole blood clots were evaluated by thrombelastograph (TEG), scanning electronic microscopy (SEM), compressive study, and thrombolytic assays. S-nitrosoglutathione (GSNO) was applied to modulate de novo hematoma structure and the impact on bone healing was evaluated in the delayed healing model. We found that GSNO produced more porous hematomas with thicker fibers and resulted in significantly enhanced bone healing. This study demonstrated that IL-1β and GSNO had opposing effects on clot architecture, the structure of which plays a pivotal role in early bone healing. PMID:27767056

  18. Ultrasonographic imaging of abomasal milk clotting and abomasal diameter in healthy and diarrheic calves.

    Science.gov (United States)

    Kirchner, Daniela; Schwedhelm, Lea; Wenge, Julia; Steinhöfel, Ilka; Heinrich, Christian; Coenen, Manfred; Bachmann, Lisa

    2015-11-01

    In case of diarrhea calves are treated with oral rehydration solutions (ORS), which are known to increase abomasal pH and inhibit milk clotting in vitro. Nevertheless, recent studies have shown that ORS with HCO3(-) ≤ 62 mmol/L do not interfere with abomasal milk clotting in healthy calves. However, in diarrheic calves, feeding ORS and milk simultaneously may disturb abomasal curd formation and exacerbate diarrhea due to faster abomasal passage of ingesta. Therefore, the aim of the present study was to ultrasonographically examine abomasal milk clotting and diameter after feeding milk and milk replacer (MR) with and without ORS to healthy and diarrheic calves. Abomasal curd formation and diameter in healthy and diarrheic calves were ultrasonographically imaged before and after feeding milk, MR and ORS prepared in milk or MR. Feeding mixtures of milk or MR with ORS did not cause any remarkable differences in the ultrasonographic images of abomasal content. Moreover, abomasal milk clotting was not disturbed due to diarrhea. Statistically significant differences of abomasal diameter after feeding between healthy and diarrheic calves indicated that abomasal emptying is delayed in diarrheic calves. Hence, further studies are needed to determine reasons for decelerated abomasal passage in calves suffering from diarrhea.

  19. Ischemic Stroke Is Associated with the ABO Locus: The EuroCLOT Study

    Science.gov (United States)

    Williams, Frances M K; Carter, Angela M; Hysi, Pirro G; Surdulescu, Gabriela; Hodgkiss, Dylan; Soranzo, Nicole; Traylor, Matthew; Bevan, Steve; Dichgans, Martin; Rothwell, Peter M W; Sudlow, Cathie; Farrall, Martin; Silander, Kaisa; Kaunisto, Mari; Wagner, Peter; Saarela, Olli; Kuulasmaa, Kari; Virtamo, Jarmo; Salomaa, Veikko; Amouyel, Philippe; Arveiler, Dominique; Ferrieres, Jean; Wiklund, Per-Gunnar; Arfan Ikram, M; Hofman, Albert; Boncoraglio, Giorgio B; Parati, Eugenio A; Helgadottir, Anna; Gretarsdottir, Solveig; Thorsteinsdottir, Unnur; Thorleifsson, Gudmar; Stefansson, Kari; Seshadri, Sudha; DeStefano, Anita; Gschwendtner, Andreas; Psaty, Bruce; Longstreth, Will; Mitchell, Braxton D; Cheng, Yu-Ching; Clarke, Robert; Ferrario, Marco; Bis, Joshua C; Levi, Christopher; Attia, John; Holliday, Elizabeth G; Scott, Rodney J; Fornage, Myriam; Sharma, Pankaj; Furie, Karen L; Rosand, Jonathan; Nalls, Mike; Meschia, James; Mosely, Thomas H; Evans, Alun; Palotie, Aarno; Markus, Hugh S; Grant, Peter J; Spector, Tim D

    2013-01-01

    Objective End-stage coagulation and the structure/function of fibrin are implicated in the pathogenesis of ischemic stroke. We explored whether genetic variants associated with end-stage coagulation in healthy REFVIDunteers account for the genetic predisposition to ischemic stroke and examined their influence on stroke subtype. Methods Common genetic variants identified through genome-wide association studies of coagulation factors and fibrin structure/function in healthy twins (n = 2,100, Stage 1) were examined in ischemic stroke (n = 4,200 cases) using 2 independent samples of European ancestry (Stage 2). A third clinical collection having stroke subtyping (total 8,900 cases, 55,000 controls) was used for replication (Stage 3). Results Stage 1 identified 524 single nucleotide polymorphisms (SNPs) from 23 linkage disequilibrium blocks having significant association (p < 5 × 10–8) with 1 or more coagulation/fibrin phenotypes. The most striking associations included SNP rs5985 with factor XIII activity (p = 2.6 × 10–186), rs10665 with FVII (p = 2.4 × 10–47), and rs505922 in the ABO gene with both von Willebrand factor (p = 4.7 × 10–57) and factor VIII (p = 1.2 × 10–36). In Stage 2, the 23 independent SNPs were examined in stroke cases/noncases using MOnica Risk, Genetics, Archiving and Monograph (MORGAM) and Wellcome Trust Case Control Consortium 2 collections. SNP rs505922 was nominally associated with ischemic stroke (odds ratio = 0.94, 95% confidence interval = 0.88–0.99, p = 0.023). Independent replication in Meta-Stroke confirmed the rs505922 association with stroke, beta (standard error, SE) = 0.066 (0.02), p = 0.001, a finding specific to large-vessel and cardioembolic stroke (p = 0.001 and p = < 0.001, respectively) but not seen with small-vessel stroke (p = 0.811). Interpretation ABO gene variants are associated with large-vessel and cardioembolic stroke but not small-vessel disease. This work sheds light on the different pathogenic

  20. Fibrin clot structure and platelet aggregation in patients with aspirin treatment failure.

    Directory of Open Access Journals (Sweden)

    Søs Neergaard-Petersen

    Full Text Available BACKGROUND: Aspirin is a cornerstone in prevention of cardiovascular events and modulates both platelet aggregation and fibrin clot formation. Some patients experience cardiovascular events whilst on aspirin, often termed aspirin treatment failure (ATF. This study evaluated both platelet aggregation and fibrin clot structure in patients with ATF. METHODS: We included 177 stable coronary artery disease patients on aspirin monotherapy. Among these, 116 (66% had ATF defined as myocardial infarction (MI whilst on aspirin. Platelet aggregation was assessed by Multiplate® aggregometry and VerifyNow®, whereas turbidimetric assays and scanning electron microscopy were employed to study fibrin clot characteristics. RESULTS: Enhanced platelet aggregation was observed in patients with ATF compared with non-MI patients following stimulation with arachidonic acid 1.0 mM (median 161 (IQR 95; 222 vs. 97 (60; 1776 AU*min, p = 0.005 and collagen 1.0 µg/mL (293 (198; 427 vs. 220 (165; 370 AU*min, p = 0.03. Similarly, clot maximum absorbance, a measure of fibrin network density, was increased in patients with ATF (0.48 (0.41; 0.52 vs. 0.42 (0.38; 0.50, p = 0.02, and this was associated with thinner fibres (mean ± SD: 119.7±27.5 vs. 127.8±31.1 nm, p = 0.003 and prolonged lysis time (552 (498; 756 vs. 519 (468; 633 seconds; p = 0.02. Patients with ATF also had increased levels of C-reactive protein (CRP (1.34 (0.48; 2.94 and 0.88 (0.32; 1.77 mg/L, p = 0.01 compared with the non-MI group. Clot maximum absorbance correlated with platelet aggregation (r = 0.31-0.35, p-values<0.001 and CRP levels (r = 0.60, p<0.001. CONCLUSIONS: Patients with aspirin treatment failure showed increased platelet aggregation and altered clot structure with impaired fibrinolysis compared with stable CAD patients without previous MI. These findings suggest that an increased risk of aspirin treatment failure may be identified by measuring both platelet

  1. Fibrin clot structure and platelet aggregation in patients with aspirin treatment failure.

    Science.gov (United States)

    Neergaard-Petersen, Søs; Ajjan, Ramzi; Hvas, Anne-Mette; Hess, Katharina; Larsen, Sanne Bøjet; Kristensen, Steen Dalby; Grove, Erik Lerkevang

    2013-01-01

    Aspirin is a cornerstone in prevention of cardiovascular events and modulates both platelet aggregation and fibrin clot formation. Some patients experience cardiovascular events whilst on aspirin, often termed aspirin treatment failure (ATF). This study evaluated both platelet aggregation and fibrin clot structure in patients with ATF. We included 177 stable coronary artery disease patients on aspirin monotherapy. Among these, 116 (66%) had ATF defined as myocardial infarction (MI) whilst on aspirin. Platelet aggregation was assessed by Multiplate® aggregometry and VerifyNow®, whereas turbidimetric assays and scanning electron microscopy were employed to study fibrin clot characteristics. Enhanced platelet aggregation was observed in patients with ATF compared with non-MI patients following stimulation with arachidonic acid 1.0 mM (median 161 (IQR 95; 222) vs. 97 (60; 1776) AU*min, p = 0.005) and collagen 1.0 µg/mL (293 (198; 427) vs. 220 (165; 370) AU*min, p = 0.03). Similarly, clot maximum absorbance, a measure of fibrin network density, was increased in patients with ATF (0.48 (0.41; 0.52) vs. 0.42 (0.38; 0.50), p = 0.02), and this was associated with thinner fibres (mean ± SD: 119.7±27.5 vs. 127.8±31.1 nm, p = 0.003) and prolonged lysis time (552 (498; 756) vs. 519 (468; 633) seconds; p = 0.02). Patients with ATF also had increased levels of C-reactive protein (CRP) (1.34 (0.48; 2.94) and 0.88 (0.32; 1.77) mg/L, p = 0.01) compared with the non-MI group. Clot maximum absorbance correlated with platelet aggregation (r = 0.31-0.35, p-valuesaspirin treatment failure showed increased platelet aggregation and altered clot structure with impaired fibrinolysis compared with stable CAD patients without previous MI. These findings suggest that an increased risk of aspirin treatment failure may be identified by measuring both platelet function and fibrin clot structure.

  2. Solar Wind Charge Exchange and O VIII emission toward MBM 12

    Science.gov (United States)

    Edgar, Richard J.; Wargelin, B. J.; Raymond, J. C.; Slavin, J. D.; Smith, R. K.; Kharchenko, V.

    2006-09-01

    A Chandra observation toward MBM-12, a nearby (MBM 12 during the Chandra observation. The Solar Wind Charge Exchange mechanism can account for essentially all of the O VIII emission seen in this observation.

  3. Correlation of fibrinogen level and absorbance change in both PT and APTT clotting curves on BCSXP

    Institute of Scientific and Technical Information of China (English)

    Xiaojie Zhang; Bing Bai

    2008-01-01

    Objective: To investigate the correlation of fibrinogen level and absorbance change in both PT and APTT clotting curves on BCSXP Analyzer. Methods:A serial of standard fibrinogen and 250 patient plasma samples with different qualities(normal, hemolysis,icterus, and lipemia) were run on BCSXP for assays PT, APTT and Fibrinogen. The absorbance change(DeltaA) from baseline to plateau in clotting curve was retrieved and analyzed on its correlation with the Fibrinogen result. Influence of plasma quality and PT/APTT result on this correlation was also studied respectively. Results:Both PT-DeltaA and APTT-DeltaA showed good linear regression with fibrinogen level in the sample, with Re close to 0.90 in both standard and patient samples. Hemolysis(H), itcterus(1) and lipemia(L) of the sample with valid clotting curves were found to have no significant difference in this correlation from normal(N) sample(R2: 0.83H, 0.92I 0.81L and 0.79N in PT; 0.89H, 0.95I, 0.91L and 0.89N in APTT) in either PT or APTT curve. PT or APTT result also has little impact on this correlation(0.71 in range 7 ~ 10 sec, 0.56 in10 ~ 20 sec, and 0.70 in 20 sec~; R2 in APTT: 0.88 in 20~30 sec,0.92 in 30~40 sec, and 0.95 in 40 sec~). Conclusion:The absorbance change in either PT or APTT clotting curve correlates well with the fibrinogen level in plasma, which is independent of plasma quality PT or APTT results. The absorbance change can be used as an alternative way to roughly estimate fibrinogen level in either PT or APTT clotting curve when the result of clauss-based fibrinogen measurement is not available.

  4. Hemophilia A

    Science.gov (United States)

    Factor VIII deficiency; Classic hemophilia; Bleeding disorder - hemophilia A ... When you bleed, a series of reactions take place in the body that helps blood clots form. This process is called the coagulation ...

  5. Fractal dimension (df) as a new structural biomarker of clot microstructure in different stages of lung cancer.

    Science.gov (United States)

    Davies, Nia Anne; Harrison, Nicholas Kim; Morris, Roger H Keith; Noble, Simon; Lawrence, Matthew James; D'Silva, Lindsay Antonio; Broome, Laura; Brown, Martin Rowan; Hawkins, Karl M; Williams, Phylip Rhodri; Davidson, Simon; Evans, Phillip Adrian

    2015-11-25

    Venous thromboembolism (VTE) is common in cancer patients, and is the second commonest cause of death associated with the disease. Patients with chronic inflammation, such as cancer, have been shown to have pathological clot structures with modulated mechanical properties. Fractal dimension (df) is a new technique which has been shown to act as a marker of the microstructure and mechanical properties of blood clots, and can be performed more readily than current methods such as scanning electron microscopy (SEM). We measured df in 87 consecutive patients with newly diagnosed lung cancer prior to treatment and 47 matched-controls. Mean group values were compared for all patients with lung cancer vs controls and for limited disease vs extensive disease. Results were compared with conventional markers of coagulation, fibrinolysis and SEM images. Significantly higher values of df were observed in lung cancer patients compared with controls and patients with extensive disease had higher values than those with limited disease (p< 0.05), whilst conventional markers failed to distinguish between these groups. The relationship between df of the incipient clot and mature clot microstructure was confirmed by SEM and computational modelling: higher df was associated with highly dense clots formed of smaller fibrin fibres in lung cancer patients compared to controls. This study demonstrates that df is a sensitive technique which quantifies the structure and mechanical properties of blood clots in patients with lung cancer. Our data suggests that df has the potential to identify patients with an abnormal clot microstructure and greatest VTE risk.

  6. Large Bladder Clot-An Unusual Presentation of Neonatal Bilateral Renal Vein Thrombosis-Case Report and Review of Literature.

    Science.gov (United States)

    Bandari, Jathin; Dangle, Pankaj P; Tennyson, Lauren E; Correa, Andres F; Cannon, Glenn M

    2015-10-01

    A 1-day-old boy born at 37 weeks gestation presented with hematuria, thrombocytopenia, and palpable irregular right flank mass. Renal ultrasound demonstrated large clot within the bladder, bilateral kidney masses with loss of corticomedullary differentiation, and reversal of diastolic flow. The patient was diagnosed with bilateral renal vein thrombosis and was managed conservatively. There was complete resolution of the bladder clot with restoration of corticomedullary differentiation bilaterally. We report the first case of renal vein thrombosis associated with a large bladder clot in a neonate.

  7. Implications of an updated ultraviolet background for the ionization mechanisms of intervening Ne VIII absorbers

    Science.gov (United States)

    Hussain, Tanvir; Khaire, Vikram; Srianand, Raghunathan; Muzahid, Sowgat; Pathak, Amit

    2017-04-01

    Ne VIII absorbers seen in QSO spectra are useful tracers of warm ionized gas, when collisional ionization is the dominant ionization process. While photoionization by the ultraviolet background (UVB) is a viable option, it tends to predict large line-of-sight thickness for the absorbing gas. Here, we study the implications of the recently updated UVB at low z to understand the ionization mechanisms of intervening Ne VIII absorbers. With the updated UVB, one typically needs higher density and metallicity to reproduce the observed ionic column densities under photoionization. Both reduce the inferred line-of-sight thicknesses of the absorbers. We find a critical density of ≥5 × 10-5 cm-3 above which the observed N({Ne VIII})/N({O VI}) can be reproduced by pure collisional processes. If the gas is of near solar metallicity (as measured for the low ions) then the cooling time-scales will be small (<108 yrs). Therefore, a continuous injection of heat is required in order to enhance the detectability of the collisionally ionized gas. Using photoionization models we find that in almost all Ne VIII systems the inferred low ion metallicity is near solar or supersolar. If we assume the Ne VIII phase to have similar metallicities then photoionization can reproduce the observed N({Ne VIII})/N({O VI}) without the line-of-sight thickness being unreasonably large and avoids cooling issues related to the collisional ionization at these metallicities. However, the indication of broad Lyα absorption in a couple of systems, if true, suggests that the Ne VIII phase is distinct from the low ion phase having much lower metallicity.

  8. Blood-clotting-inspired reversible polymer-colloid composite assembly in flow

    Science.gov (United States)

    Chen, Hsieh; Fallah, Mohammad A.; Huck, Volker; Angerer, Jennifer I.; Reininger, Armin J.; Schneider, Stefan W.; Schneider, Matthias F.; Alexander-Katz, Alfredo

    2013-01-01

    Blood clotting is a process by which a haemostatic plug is assembled at the site of injury. The formation of such a plug, which is essentially a (bio)polymer-colloid composite, is believed to be driven by shear flow in its initial phase, and contrary to our intuition, its assembly is enhanced under stronger flowing conditions. Here, inspired by blood clotting, we show that polymer-colloid composite assembly in shear flow is a universal process that can be tailored to obtain different types of aggregates including loose and dense aggregates, as well as hydrodynamically induced ‘log’-type aggregates. The process is highly controllable and reversible, depending mostly on the shear rate and the strength of the polymer-colloidbinding potential. Our results have important implications for the assembly of polymer-colloid composites, an important challenge of immense technological relevance. Furthermore, flow-driven reversible composite formation represents a new paradigm in non-equilibrium self-assembly.

  9. Hepatic veins as a site of clot formation following liver resection

    Institute of Scientific and Technical Information of China (English)

    Emmanuel Buc; Safi Dokmak; Magaly Zappa; Marie Helene Denninger; Dominique Charles Valla; Jacques Belghiti; Olivier Farges

    2011-01-01

    Pulmonary embolism occurs more frequently after hepatectomy than previously thought but is infrequently associated with peripheral deep vein thrombosis. In thispaper, we report 2 cases of postoperative hepatic vein thrombosis after liver resection. Both patients had undergone major hepatectomy of a non-cirrhotic liver largely exposing the middle hepatic vein. Clots were incidentally found in the middle hepatic vein 4 and 17 d after surgery despite routine systemic thrombo-prophylaxis with low molecular weight heparin. Coagulation of the transitionplan in a context of mutation of the prothrombin gene and inflammation induced biloma were the likely predisposing conditions. Clots disappeared following curative anticoagulation. We conclude that thrombosis of hepatic veins may occur after liver resection and is a potential source of pulmonary embolism.

  10. Real-time visual/near-infrared analysis of milk-clotting parameters for industrial applications.

    Science.gov (United States)

    Leitner, G; Merin, U; Lemberskiy-Kuzin, L; Bezman, D; Katz, G

    2012-07-01

    The economical profitability of the dairy industry is based on the quality of the bulk milk collected in the farms, therefore it was based on the herd level rather than on the individual animals at real time. Udder infection and stage of lactation are directly related to the quality of milk produced on the herd level. However, improvement of milk quality requires testing each animal's milk separately and continuously. Recently, it was postulated that online equipment can estimate milk quality according to its clotting parameters, and thus result in better economical return for cheese making. This study further investigated the potential application of the AfiLab™ equipment to provide real-time analysis of milk-clotting parameters for cheese manufacture and cheese yield on quarter (1018) and individual cow (277) levels. Days in milk, lactose, log SCC and udder infection were found to have a significant effect on curd firmness and cheese properties and yield. The results clearly indicate that: (a) the parameter Afi-CF determined with the AfiLab™ is suitable for assessing milk quality for its clotting parameters, a value which is not provided by merely measuring fat and protein content on the gland and the cow levels; (b) bacterial type is the single major cause of reduced milk quality, with variations depending on the bacterial species; and (c) early and late lactation also had negative effects on milk-clotting parameters. Cheese made from the various milk samples that were determined by the Afilab™ to be of higher quality for cheese making resulted in higher yield and better texture, which were related mainly to the bacterial species and stage of lactation.

  11. Possibilities of the Videothoracoscopy for the Post-Traumatic Clotted Hemothorax

    OpenAIRE

    Shavkat I. Karimov; Ulugbek B. Berkinov; Erjan R. Fayzullaev; Yuliya V. Khamidova

    2014-01-01

    Background. Among the complications of blunt thoracic trauma, hemothorax is the most common and serious problem of modern thoracic surgery. The frequency of this complication according to some authors, varies from 26% to 80% and is not connnected with nature of the trauma and patient's age. 804 patients with the chest injuries treated between 2006 and 2012 at the 2nd clinic Tashkent Medical Academy. 179 of patients had penetrating stab wounds, and 625 - blunt. The clotted hemothorax was d...

  12. Large Right Ventricular Clot in Pulmonary Atresia With Intact Ventricular Septum: In Defense of Biventricular Approach.

    Science.gov (United States)

    Dutta, Nilanjan; Ghosh, Rajarshi; Awasthy, Neeraj; Iyer, Parvathi U; Girotra, Sumir; Iyer, Krishna S

    2016-09-01

    Thrombus formation within the right ventricle (RV) in the setting of pulmonary atresia with intact ventricular septum (PAIVS) is not a very common occurrence and can be catastrophic. We present the case of a seven-month-old child with PAIVS and RV clot who successfully underwent biventricular repair. We discuss the interesting case and the rationale for management by means of biventricular repair over single ventricle repair when feasible in such a setting.

  13. Specialized proresolving lipid mediators in patients with coronary artery disease and their potential for clot remodeling.

    Science.gov (United States)

    Elajami, Tarec K; Colas, Romain A; Dalli, Jesmond; Chiang, Nan; Serhan, Charles N; Welty, Francine K

    2016-08-01

    Inflammation in arterial walls leads to coronary artery disease (CAD). Because specialized proresolving lipid mediators (SPMs; lipoxins, resolvins, and protectins) stimulate resolution of inflammation in animal models, we tested whether n-3 fatty acids impact SPM profiles in patients with CAD and promote clot remodeling. Six patients with stable CAD were randomly assigned to either treatment with daily 3.36 g Lovaza for 1 yr or without. Targeted lipid mediator-metabololipidomics showed that both groups had absence of resolvin D1 (RvD1), RvD2, RvD3, RvD5 and resolvin E1-all of which are present in healthy patients. Those not taking Lovaza had an absence of aspirin-triggered resolvin D3 (AT-RvD3) and aspirin-triggered lipoxin B4 (AT-LXB4). Lovaza treatment restored AT-RvD3 and AT-LXB4 and gave levels of RvD6 and aspirin-triggered protectin D1 (AT-PD1) twice as high (resolvin E2 ∼5 fold) as well as lower prostaglandins. Principal component analysis indicated positive relationships for patients with CAD who were receiving Lovaza with increased AT-RvD3, RvD6, AT-PD1, and AT-LXB4 SPMs identified in Lovaza-treated patients with CAD enhanced ∼50% at 1 nM macrophage uptake of blood clots. These results indicate that patients with CAD have lower levels and/or absence of specific SPMs that were restored with Lovaza; these SPMs promote macrophage phagocytosis of blood clots. Together, they suggest that low vascular SPMs may enable progression of chronic vascular inflammation predisposing to coronary atherosclerosis and to thrombosis.-Elajami, T. K., Colas, R. A., Dalli, J., Chiang, N., Serhan, C. N., Welty, F. K. Specialized proresolving lipid mediators in patients with coronary artery disease and their potential for clot remodeling.

  14. Suction Curettage for Removal of Retained Intrathoracic Blood Clots and Pleural Lesions

    OpenAIRE

    Redan, Jay Alan; Palmer, Meade Todd; Tylutki, Francis John

    1999-01-01

    Objective: To develop a thoracoscopic technique for correcting and/or removing an intrathoracic disease process using our existing operating room equipment and without a “small thoracotomy.” Methods and Procedures: Fifty-eight patients from October 1994 to April 1998 were prospectively studied. All were undergoing procedures involving the removal of a suspected benign (or infectious) pleural process or a retained blood clot. Three or four thoracic ports were used in all cases. Straight and cu...

  15. Adhesins of Leptospira interrogans Mediate the Interaction to Fibrinogen and Inhibit Fibrin Clot Formation In Vitro

    Science.gov (United States)

    Oliveira, Rosane; Domingos, Renan F.; Siqueira, Gabriela H.; Fernandes, Luis G.; Souza, Natalie M.; Vieira, Monica L.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Nascimento, Ana L. T. O.

    2013-01-01

    We report in this work that Leptospira strains, virulent L. interrogans serovar Copenhageni, attenuated L. interrogans serovar Copenhageni and saprophytic L. biflexa serovar Patoc are capable of binding fibrinogen (Fg). The interaction of leptospires with Fg inhibits thrombin- induced fibrin clot formation that may affect the haemostatic equilibrium. Additionally, we show that plasminogen (PLG)/plasmin (PLA) generation on the surface of Leptospira causes degradation of human Fg. The data suggest that PLA-coated leptospires were capable to employ their proteolytic activity to decrease one substrate of the coagulation cascade. We also present six leptospiral adhesins and PLG- interacting proteins, rLIC12238, Lsa33, Lsa30, OmpL1, rLIC11360 and rLIC11975, as novel Fg-binding proteins. The recombinant proteins interact with Fg in a dose-dependent and saturable fashion when increasing protein concentration was set to react to a fix human Fg concentration. The calculated dissociation equilibrium constants (KD) of these reactions ranged from 733.3±276.8 to 128±89.9 nM for rLIC12238 and Lsa33, respectively. The interaction of recombinant proteins with human Fg resulted in inhibition of fibrin clot by thrombin-catalyzed reaction, suggesting that these versatile proteins could mediate Fg interaction in Leptospira. Our data reveal for the first time the inhibition of fibrin clot by Leptospira spp. and presents adhesins that could mediate these interactions. Decreasing fibrin clot would cause an imbalance of the coagulation cascade that may facilitate bleeding and help bacteria dissemination PMID:24009788

  16. SMTP (Stachybotrys microspora triprenyl phenol enhances clot clearance in a pulmonary embolism model in rats

    Directory of Open Access Journals (Sweden)

    Hu Weimin

    2012-01-01

    Full Text Available Abstract Background Stachybotrys microspora triprenyl phenols (SMTPs are a novel family of small molecules that enhance both activation and fibrin-binding of plasminogen. While their effects on fibrinolysis have been characterized in vitro, little is known about their activity in vivo with respect to plasminogen activation and blood clot clearance. Results To select a potent SMTP congener for the evaluation of its action in vitro and in vivo, we tested several SMTP congeners with distinct structural properties for their effects on plasminogen activation. As a result, SMTP-7 (orniplabin was found to have distinguished activity. Several lines of biochemical evidence supported the idea that SMTP-7 acted as a plasminogen modulator. SMTP-7 elevated plasma level of plasmin-α2-antiplasmin complex, an index of plasmin formation in vivo, 1.5-fold in mice after the intravenous injections at doses of 5 and 10 mg kg-1. In a rat pulmonary embolism model, SMTP-7 (5 mg kg-1 enhanced the rate of clot clearance ~3-fold in the absence of exogenous plasminogen activator. Clot clearance was enhanced further by 5 mg kg-1 of SMTP-7 in combination with single-chain urokinase-type plasminogen activator. Conclusions Our results show that SMTP-7 is a superior plasminogen modulator among the SMTP family compounds and suggest that the agent enhances plasmin generation in vivo, leading to clearance of thrombi in a model of pulmonary embolism.

  17. Evaluation of the effects of levobupivacaine on clotting and fibrinolysis using thromboelastography.

    LENUS (Irish Health Repository)

    Leonard, S A

    2012-02-03

    Amide local anaesthetics inhibit platelet function. We hypothesized that residual anaesthetic in the epidural space could decrease efficacy of an epidural blood patch in preventing postdural puncture headache. Levobupivacaine has recently been approved for epidural anaesthesia. Its effects on coagulation have not previously been studied. The aim of this study was to determine the effects of levobupivacaine on clotting using thromboelastography. Ten ASA Class I volunteers were studied. Venous blood samples were analysed using a Haemoscope 2000D TEG analyser. Whole blood, a 50% saline control and two levobupivacaine solutions (2.5 mg mL(-1) and 2.5 microg mL(-1) in blood) were compared. The former reproduces that produced in the epidural space by blood (20 mL for an epidural blood patch) and levobupivacaine 0.5% (20 mL). The latter approximates plasma concentrations following epidural injection of levobupivacaine 0.5% (20 mL). P < 0.05 was considered significant. Maximum amplitude (MA), a measure of clot strength, is decreased by levobupivacaine 2.5 mg mL(-1). Levobupivacaine 2.5 mg mL(-1) decreases clot strength and may reduce efficacy of a prophylactic epidural blood patch.

  18. The etiology of inhibitor development in children with severe hemophilia A

    NARCIS (Netherlands)

    Gouw, S.C.

    2011-01-01

    Patients with severe hemophilia, a deficiency of functional clotting factor VIII, typically suffer from joint and muscle bleedings spontaneously or after minor trauma. The bleeding tendency can be effectively corrected by intravenous substitution of factor VIII products. However, about 25% of

  19. Inhibitor development in nonsevere hemophilia A

    NARCIS (Netherlands)

    Eckhardt, C.L.

    2014-01-01

    Hemophilia A is an X-linked inherited bleeding disorder that affects approximately 1 in 5000 male live births. It is caused by a deficient plasma level of clotting factor VIII and can be treated by the intravenous administration of factor VIII concentrates. A severe complication of the treatment wit

  20. Co-morbidity in adult haemophilia patients

    NARCIS (Netherlands)

    Fransen van de Putte, D.E.

    2012-01-01

    Haemophilia is an X-linked inherited bleeding disorder, caused by a deficiency of clotting factor VIII or IX. Due to the availability of treatment with clotting factor concentrates, life expectancy of haemophilia patients is now approaching that of the general population. Haemophilia patients are th

  1. Ultrastructural characteristics of fibrin clots from canine and feline platelet concentrates activated with calcium gluconate or calcium gluconate plus batroxobin

    OpenAIRE

    Silva, Raúl F; Carmona, Jorge U; Rezende, Cleuza MF

    2013-01-01

    Background The aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots obtained from canine and feline platelet concentrates (PC) that had been activated with calcium gluconate (CG) or CG plus batroxobin (CGB). Platelets from fibrin clots were classified according their morphological changes. The area of the intercellular space (μm2), the area of the fibrin fibers (μm2), and the width of the fibrin fibers (μm) were determined for ...

  2. Kinetics of Formations of Ag Clots on AgBr Microcrystals at The Normal Laser Hershel Effect

    Directory of Open Access Journals (Sweden)

    A.B. Piven

    2011-01-01

    Full Text Available It is established that clots of silver have the amorphous structure at the normal laser Hershel effect after exposure of the photographic layer SP–1 (photosensitivity 6 standart units with low time texpos = 10 – 5 sec, and at high exposure times (texpos = 0.5 sec they have the crystal structure. The energy of the crystallization of Ag clots equals Wmin = 10 – 8 J.

  3. Homocysteine influences blood clot properties alone and in combination with total fibrinogen but not with fibrinogen γ' in Africans.

    Science.gov (United States)

    Nienaber-Rousseau, Cornelie; de Lange, Zelda; Pieters, Marlien

    2015-06-01

    Simultaneously increased fibrinogen and homocysteine (Hcy) in blood are believed to elevate the risk of cardiovascular disease mortality. However, the pathophysiological mechanisms involved are unknown. We sought to determine whether Hcy or its genetic determinants influence blood clot properties alone or in combination with fibrinogen. In addition, we investigated, for the first time, the gamma prime (γ') isoform of fibrinogen with Hcy in relation to clot architecture and lysis. Single-nucleotide polymorphisms, Hcy and hemostatic variables, including clot lysis, determined with a global fibrinolytic assay [giving lag time, slope, maximum absorbance and clot lysis time (CLT)], were measured in 1867 healthy black South Africans and cross-sectionally analyzed. Increasing Hcy did not affect fiber cross-sectional area (maximum absorbance). However, it decreased the time needed to initiate the coagulation cascade and for fibrin fibers to grow (lag time), it increased the tempo of lateral aggregation (slope) and reduced CLT. None of the single-nucleotide polymorphisms measured had effects on clot properties. Combined effects were observed between Hcy and total fibrinogen in predicting CLT. Fibrinogen γ', which affected markers of the fibrinolytic assay, did not have conjoint effects with Hcy. We believe that there is value in recognizing the combined effects of Hcy and fibrinogen, but not its γ' isoform in relation to clot structure and lysis. The enhanced fibrinolysis rate observed in patients with low fibrinogen and high Hcy may have adverse consequences for health if it disturbs hemostasis and results in a bleeding tendency.

  4. Utility of single-energy and dual-energy computed tomography in clot characterization: An in-vitro study.

    Science.gov (United States)

    Brinjikji, Waleed; Michalak, Gregory; Kadirvel, Ramanathan; Dai, Daying; Gilvarry, Michael; Duffy, Sharon; Kallmes, David F; McCollough, Cynthia; Leng, Shuai

    2017-06-01

    Background and purpose Because computed tomography (CT) is the most commonly used imaging modality for the evaluation of acute ischemic stroke patients, developing CT-based techniques for improving clot characterization could prove useful. The purpose of this in-vitro study was to determine which single-energy or dual-energy CT techniques provided optimum discrimination between red blood cell (RBC) and fibrin-rich clots. Materials and methods Seven clot types with varying fibrin and RBC densities were made (90% RBC, 99% RBC, 63% RBC, 36% RBC, 18% RBC and 0% RBC with high and low fibrin density) and their composition was verified histologically. Ten of each clot type were created and scanned with a second generation dual source scanner using three single (80 kV, 100 kV, 120 kV) and two dual-energy protocols (80/Sn 140 kV and 100/Sn 140 kV). A region of interest (ROI) was placed over each clot and mean attenuation was measured. Receiver operating characteristic curves were calculated at each energy level to determine the accuracy at differentiating RBC-rich clots from fibrin-rich clots. Results Clot attenuation increased with RBC content at all energy levels. Single-energy at 80 kV and 120 kV and dual-energy 80/Sn 140 kV protocols allowed for distinguishing between all clot types, with the exception of 36% RBC and 18% RBC. On receiver operating characteristic curve analysis, the 80/Sn 140 kV dual-energy protocol had the highest area under the curve for distinguishing between fibrin-rich and RBC-rich clots (area under the curve 0.99). Conclusions Dual-energy CT with 80/Sn 140 kV had the highest accuracy for differentiating RBC-rich and fibrin-rich in-vitro thrombi. Further studies are needed to study the utility of non-contrast dual-energy CT in thrombus characterization in acute ischemic stroke.

  5. Drevnerusskij pamflet na Mihaila VIII Paleologa v svete germenevtičeskogo analiza

    Directory of Open Access Journals (Sweden)

    Demincev Mihail

    2015-01-01

    Full Text Available In the article the perception of the image of Michael VIII Palaeologus by literate citizens of Tsardom of Russia is considered. The study is based on analysis of writings that date back to XVII century. The study focuses on identifying the symbols and latent ideas, that point to a semantic connection of the studied writings with The Old Testament, Apocalypse and some antique texts. In the end of the article the authjr makes a conclusion, that the perception of the image of Michael VIII Palaeologus in Tsardom of Russia was negative.

  6. What Is Combined Deficiency of Vitamin K-Dependent Clotting Factors?

    Science.gov (United States)

    ... Resource Annual Global Survey Treatment Guidelines Laboratory Manual Hemophilia in Pictures Young Voices Compendium of Assessment Tools Educational Games Video Library Find a Treatment Centre Haemophilia Journal ...

  7. Computational Study of Thrombus Formation and Clotting Factor Effects under Venous Flow Conditions

    Science.gov (United States)

    2016-04-26

    each has its limitations. The recent emergence of systems biology has suggested a way to circumvent some of the lim- itations of traditional...when geometries for large blood vessels (e.g., coronary arteries) are involved (37). These considerations reinforced our decision to simplify the...bleeding. Vox Sang. 94:221–226. 11. Karlsson, M., L. Ternström,., A. Jeppsson. 2009. Prophylactic fibrin- ogen infusion reduces bleeding after coronary

  8. Blood Clots

    Science.gov (United States)

    ... York, N.Y.: McGraw-Hill Education; 2015. http://accessmedicine.com. Accessed April 18, 2016. Understand your risk ... York, N.Y.: McGraw-Hill Education; 2015. http://accessmedicine.com. Accessed April 18, 2016. Papadakis MA, et ...

  9. Blood Clots

    Science.gov (United States)

    ... of ASH ASH Meeting on Hematologic Malignancies Consultative Hematology Course ASH Meeting on Lymphoma Biology ASH Workshop on Genome Editing Publications Blood The Hematologist ASH Clinical News ASH Self-Assessment Program Hematology , ASH Education Program About Awards Membership ASH Foundation ...

  10. Factor V Leiden Mutation and PT 20210 Mutation Test

    Science.gov (United States)

    ... is suspected that a person has an inherited risk factor for blood clots, for example, when an individual: Has a first deep venous thrombosis (DVT) or venous thromboembolism (VTE) before age 50 ...

  11. Comparative evaluation of Bacillus licheniformis 5A5 and Aloe variegata milk-clotting enzymes

    Directory of Open Access Journals (Sweden)

    S. A. Ahmed

    2012-03-01

    Full Text Available The properties of a milk clotting enzyme (MCE produced by bacteria (Bacillus licheniformis 5A5 were investigated and compared to those of rennet extracted from a plant (Aloe variegata. Production of MCE by B. licheniformis 5A5 was better in static than in shaken cultures. Maximum activity (98.3 and 160.3 U/ml of clotting was obtained at 75ºC and 80ºC with bacterial and plant rennet, respectively. In the absence of substrate, the clotting activity of Aloe MCE was found to be less sensitive to heat inactivation up to 80ºC for 75 min, retaining 63.8% of its activity, while bacterial MCE was completely inhibited. CaCl2 stimulated milk clotting activity (MCA up to 2% and 1.5% for bacterial and plant enzymes. NaCl inhibited MCA for both enzymes, even at low concentration (1%. Plant MCE was more sensitive to NaCl at 3% concentration it retained 30.2% of its activity, whereas bacterial MCE retained 64.1%. Increasing skim milk concentration caused a significant increase in MCA up to 6% for both enzymes. Mn2+ stimulated the activity of bacterial and plant enzymes to 158.6 and 177.9%, respectively. EDTA and PMSF increased the activity of plant MCE by 34.4 and 41.1%, respectively, which is higher than those for the bacterial MCE (19.1 and 20.9%. Some natural materials activated MCE, the highest activation of bacterial MCE (128.1% was obtained in the presence of Fenugreek (with acid extraction. However Lupine Giza 1 (with neutral extraction gave the highest activation of plant MCE (137.9%. All extracts from Neem plant increased MCA at range from 105.6% to 136.4%. Plant MCE exhibited much better stability when stored at room temperature (25-30ºC for 30 days, retaining 51.2% of its activity. Bacterial MCE was highly stabile when stored under freezing (-18ºC, retaining 100% of its activity after 30 days. Moreover, bacterial MCE was highly tolerant to repeated freezing and thawing without loss of activity for 8 months.

  12. Abolished ventilation and perfusion of lung caused by blood clot in the left main bronchus

    DEFF Research Database (Denmark)

    Afzelius, P; Bergmann, A; Henriksen, J H

    2015-01-01

    /Q) scintigraphy with single-photon emission CT (SPECT)/CT. V/Q SPECT/CT demonstrated abolished ventilation due to obstruction of the left main bronchus and markedly reduced perfusion of the entire left lung, a condition that was completely reversed after removal of a blood clot. We present the first pictorially......It is generally assumed that the lungs possess arterial autoregulation associated with bronchial obstruction. A patient with pneumonia and congestive heart failure unexpectedly developed frequent haemoptysis. High-resolution CT and diagnostic CT were performed as well as ventilation/perfusion (V...

  13. Changes to the structure of blood clots formed in the presence of fine particulate matter

    Energy Technology Data Exchange (ETDEWEB)

    Metassan, Sofian; Routledge, Michael N [Molecular Epidemiology Unit, Leeds Institute for Health, Genetics and Therapeutics, LIGHT Laboratories, University of Leeds, Leeds LS2 9JT (United Kingdom); Ariens, Robert A S; Scott, D Julian, E-mail: umphsp@leeds.ac.u [Cardiovascular and Diabetes Research Division, Leeds Institute for Health, Genetics and Therapeutics, The LIGHT Laboratories, University of Leeds, Leeds LS2 9JT (United Kingdom)

    2009-02-01

    Both long-term and short-term exposure (one to two hours) to particulate matter are associated with morbidity and mortality caused by cardiovascular diseases. The underlying mechanisms leading to cardiovascular events are unclear, however, changes to blood coagulability upon exposure to ultrafine particulate matter (UFPM, the smallest of which can enter the circulation) is a plausible mechanism. Objectives: This study aims to investigate the direct effects of particulate matter on fibrin polymerization, lateral aggregation and the formation of fibrin network structure. Methods: Standard Urban Particulate Matter (PM) was suspended in Tris buffer centrifuged and filtered with <200nm filter to obtain ultrafine PM or their water-soluble components. Purified normal fibrinogen was made to clot by adding thrombin and calcium chloride in the presence of varying concentrations of PM. Permeation properties (Darcy constant [Ks]) and turbidity of clots were measured to investigate the effects on flow-rate, pore size, and fibrin polymerization. In addition, confocal microscopy was performed to study detailed clot structure. Results: Total PM increased the Ks of clots in a dose dependant manner (Ks = 4.4, 6.9 and 13.2 x 10-9 cm2 for 0, 50 and 100 |ag/ml total PM concentrations, respectively). Filtered PM also produced a significant increase in Ks at PM concentration of 17 |ag/ml. Final turbidity measurements at 20min were obtained for varying concentrations of PM. Maximum optical density (OD) for 1 mg/ml fibrinogen at 0, 50, 100 and 200 |ag/ml total PM concentrations were 0.39, 0.42, 0.45 and 0.46, respectively. The maximum OD for 0, 17, 34 and 68 |ag/ml filtered PM concentrations were 0.39, 0.42 0.47 and 0.51, respectively, suggesting an increase in fibre diameter with increasing particulate concentration. The lag phase was significantly shorter and the rate of polymerisation was significantly faster in the presence of 68 |ag/ml filtered PM. Confocal microscopy results showed

  14. Quark matter coupled to domain walls in Bianchi types II, VIII and IX Universes

    Indian Academy of Sciences (India)

    S D Katore; M M Sancheti; S P Hatkar

    2014-10-01

    In this study of Bianchi types II, VIII and IX Universes, quark matter coupled to domain walls in the context of general relativity are explored. To obtain deterministic solution of the Einstein’s field equations, various techniques are adopted. The features of the obtained solution are discussed.

  15. Challenges and Opportunities of Information Technology in the 90s. Track VIII: Managing Distributed Computing Services.

    Science.gov (United States)

    CAUSE, Boulder, CO.

    Six papers from the 1990 CAUSE Conference Track VIII: Managing Distributed Computing are presented. Authors discuss the challenges and opportunities involved in providing user managers with direct access to institutional databases to support their decision making and planning activities. Papers and their authors are as follows: "Rendering an…

  16. Aspectos literarios de la obra de don Joan de Castellanos : Capitulo VIII

    Directory of Open Access Journals (Sweden)

    Mario Germán Romero

    1966-03-01

    Full Text Available Capítulo VIII. Los discursos de Jiménez de Quesada. Toda historia de la literatura colombiana comienza con el nombre del licenciado don Gonzalo Jiménez de Quesada. Que era un letrado, no puede ponerse en duda. Los escritos que de él conocemos justifican plenamente el título.

  17. 7 CFR 1901.203 - Title VIII of the Civil Rights Act of 1968.

    Science.gov (United States)

    2010-01-01

    ..., rental, or financing of housing contained in title VIII apply to: (1) All dwellings financed by loans... equal housing opportunity throughout the nation. We encourage and support an affirmative advertising and... Public Law 103-354 programs and referral to the Department of Justice for suit by the United States...

  18. Alkali treatment of microrough titanium surfaces affects macrophage/monocyte adhesion, platelet activation and architecture of blood clot formation

    Directory of Open Access Journals (Sweden)

    V Milleret

    2011-05-01

    Full Text Available Titanium implants are most commonly used for bone augmentation and replacement due to their favorable osseointegration properties. Here, hyperhydrophilic sand-blasted and acid-etched (SBA titanium surfaces were produced by alkali treatment and their responses to partially heparinized whole human blood were analyzed. Blood clot formation, platelet activation and activation of the complement system was analyzed revealing that exposure time between blood and the material surface is crucial as increasing exposure time results in higher amount of activated platelets, more blood clots formed and stronger complement activation. In contrast, the number of macrophages/monocytes found on alkali-treated surfaces was significantly reduced as compared to untreated SBA Ti surfaces. Interestingly, when comparing untreated to modified SBA Ti surfaces very different blood clots formed on their surfaces. On untreated Ti surfaces blood clots remain thin (below 15 mm, patchy and non-structured lacking large fibrin fiber networks whereas blood clots on differentiated surfaces assemble in an organized and layered architecture of more than 30 mm thickness. Close to the material surface most nucleated cells adhere, above large amounts of non-nucleated platelets remain entrapped within a dense fibrin fiber network providing a continuous cover of the entire surface. These findings might indicate that, combined with findings of previous in vivo studies demonstrating that alkali-treated SBA Ti surfaces perform better in terms of osseointegration, a continuous and structured layer of blood components on the blood-facing surface supports later tissue integration of an endosseous implant.

  19. Detection of two intervening Ne viii absorbers probing warm gas at z ˜ 0.6

    Science.gov (United States)

    Pachat, Sachin; Narayanan, Anand; Khaire, Vikram; Savage, Blair D.; Muzahid, Sowgat; Wakker, Bart P.

    2017-10-01

    We report on the detection of two Ne viii absorbers, at z = 0.619 07 and 0.570 52 in the Hubble Space Telescope/Cosmic Origins Spectrograph spectrum of background quasars SDSS J080908.13 + 461925.6 and SBS 1122 + 594, respectively. The Ne viii 770 line is at ∼3σ significance. In both instances, the Ne viii is found to be tracing gas with T ≳ 105 K, predominantly collisionally ionized, with moderate densities of n_{H} ≲ 10^{-4} cm-3, sub-solar metallicities and total hydrogen column densities of N(H) ≳ 1019 cm-2. In the z = 0.619 07 absorber, the low, intermediate ions and O vi are consistent with origin in photoionized gas, with the O vi potentially having some contribution from the warm collisional phase traced by Ne viii. The z = 0.570 52 system has H I absorption in at least three kinematically distinct components, with one of them having b({H I}) = 49 {± } 11 km s-1. The intermediate-ionization lines, O vi and Ne viii, are coincident in velocity with this component. Their different line widths suggest warm temperatures of T = (0.5-1.5) × 105 K. Both absorbers are residing in regions where there are several luminous (≳L★) galaxies. The absorber at z = 0.570 52 is within the virial radius of a 2.6L★ galaxy, possibly associated with shock-heated circumgalactic material.

  20. VIII Olimpíada Brasileira de Astronomia e Astronáutica