Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial.

Science.gov (United States)

Madurantakam, Parthasarathy; Yoganarasimha, Suyog; Hasan, Fadi K

2015-09-29

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

The effect of ibuprofen on accumulation of indium-111-labeled platelets and leukocytes in experimental myocardial infarction

International Nuclear Information System (INIS)

Romson, J.L.; Hook, B.G.; Rigot, V.H.; Schark, M.A.; Swanson, D.P.; Lucchesi, B.R.

1982-01-01

To assess the ability of ibuprofen to influence the extent of platelet aggregation and leukocyte infiltration during acute myocardial infarction, autologous indium-111 ( 111 In)-labeled platelets or leukocytes were injected before 60 minutes of left circumflex coronary artery (LCx) occlusion, followed by 24 hours of reperfusion in the canine heart. Myocardial infarct size, as a percent of the area at risk, was reduced in the ibuprofen-treated group (12.5 mg/kg i.v. every 4 hours beginning 30 minutes before LCx occulsion) by 40%, from 48 +/- 4% in control animals to 29 +/- 4% in ibuprofen-treated dogs (p=0.005). Quantification of the platelet-associated 111 In radioactivity in irreversibly injured myocardium indicated that ibuprofen did not alter the accumulation of platelets in infarcted myocardium. In contrast, leukocyte accumulation in infarcted tissue was reduced significantly. In tissue samples with 0.41-0.60 gram infarct, the infarcted/normal ratio of leukocyte radioactivity was 12 +/- 2 in control dogs and 4 +/- 1 in ibuprofen-treated dogs, which represents a 67% reduction in leukocyte accumulation in ibuprofen-treated compared with control dogs. Similar reductions were found in other gram-infarct-weight categories. Although both platelets and leukocytes acumulate in infarcted canine myocardium, ibuprofen may exert its beneficial effect on ischemic myocardium by suppressing the inflammatory response associated with myocardial ischemia and infarction

The effect of ibuprofen on accumulation of 111In-labeled platelets and leukocytes in experimental myocardial infarction

International Nuclear Information System (INIS)

Romson, J.L.; Hook, B.G.; Rigot, V.H.; Schork, M.A.; Swanson, D.P.; Lucchesi, B.R.

1982-01-01

To assess the ability of ibuprofen to influence the extent of platelet aggregation and leukocyte infiltration during acute myocardial infarction, autologous indium-111 ( 111 In)-labeled platelets or leukocytes were injected before 60 minutes of left circumflex coronary artery (LCx) occlusion, followed by 24 hours of reperfusion in the canine heart. Myocardial infarct size, as a percent of the area at risk, was reduced in the ibuprofen-treated group (12.5 mg/kg i.v. every 4 hours beginning 30 minutes before LCx occlusion) by 40%, from 48 +/- 4% in control animals to 29 +/- 4% in ibuprofen-treated dogs (p . 0.005). Quantification of the platelet-associated 111 In radioactivity in irreversibly injured myocardium indicated that ibuprofen did not alter the accumulation of platelets in infarcted myocardium. In contrast, leukocyte accumulation in infarcted tissue was reduced significantly. In tissue samples with 0.41-0.60 gram infarct, the infarcted/normal ratio of leukocyte radioactivity was 12 +/- 2 in control dogs and 4 +/- 1 in ibuprofen-treated dogs, which represents a 67% reduction in leukocyte accumulation in ibuprofen-treated compared with control dogs. Similar reductions were found in other gram-infarct-weight categories. Although both platelets and leukocytes accumulate in infarcted canine myocardium, ibuprofen may exert its beneficial effect on ischemic myocardium by suppressing the inflammatory response associated with myocardial ischemia and infarction

Microbicidal properties of Leukocyte- and Platelet-Rich Plasma/Fibrin (L-PRP/L-PRF): new perspectives.

Science.gov (United States)

Cieslik-Bielecka, A; Dohan Ehrenfest, D M; Lubkowska, A; Bielecki, T

2012-01-01

Platelets, as main actors of the first stage of the healing process, play an important role in tissue repair. Their granules contain many active substances, particularly over 30 growth factors with significant effects on the resident cells at the site of injury, such as mesenchymal stem cells, chondrocytes, fibroblasts, osteoblasts. This potential may be increased by the concentration of the platelets, using platelet-rich plasma/fibrin products. In the four families of platelet concentrates, 2 families contain also significant concentrations of leukocytes: L-PRP (Leukocyte- and Platelet-Rich Plasma) and L-PRF (Leukocyte- and Platelet-Rich Fibrin). Inductive properties of platelet concentrates were widely described. However, they present also antimicrobial effects. The antibacterial effects of L-PRP were highlighted in only a few in vitro studies. Strong activity comparable to gentamicin and oxacillin for L-PRP against methicillin susceptible Staphylococcus aureus (MSSA) was already demonstrated. L-PRP also inhibited the growth of methicillin resistant Staphylococcus aureus (MRSA) and Escherichia coli. Some authors also reported clinical observations about the reduction of infections and the induction of healing processes after the use of platelet concentrates in cardiac, orthopaedic, oral and maxillofacial surgery. However, very little is yet known about the antibacterial effects of these concentrates. In this manuscript, the current data about the antimicrobial agents and cells present in the platelet-rich plasma/fibrin are highlighted and discussed, in order to introduce this new key chapter of the platelet concentrate technology history.

Analysis of leukocyte binding to depletion filters: role of passive binding, interaction with platelets, and plasma components.

Science.gov (United States)

Henschler, R; Rüster, B; Steimle, A; Hansmann, H L; Walker, W; Montag, T; Seifried, E

2005-08-01

Since limited knowledge exists on the mechanisms which regulate cell binding to leukocyte removal filter surfaces, we investigated the binding patterns of leukocytes to individual layers of leukocyte depletion filters. After passage of 1 unit of whole blood, blotting of isolated filter layers on glass slides or elution of cells from filter layers revealed that most leukocytes were located within the first 10 of a total of 28 filter layers, peaking at layers 6 to 8, with granulocytes binding on average to earlier filter layers than lymphocytes. Leukocytes preincubated with inhibitors of actin activation showed unchanged distribution between filter layers, suggesting that cytoskeletal activation does not significantly contribute to their binding. When leukocytes were directly incubated with single filter layers, binding of up to 30% of input cells was recorded in the absence of Ca(2+). Immunohistological analyses showed colocalization of platelets and leukocytes, with co-clustering of platelets and leukocytes. Monocytes and to some degree lymphocytes but not granulocytes competed with platelets for filter binding. Precoating of filter layers with individual plasma components showed that hyaluronic acid, plasma type fibronectin, and fibrinogen all increased the binding of leukocytes compared with albumin coating. In conclusion, leukocytes can bind passively to filters in a process which does not require Ca(2+), which is independent of cytoskeletal activation and which may depend on individual plasma components. These results are of importance when new selective cell enrichment or depletion strategies through specific filters are envisaged.

Extracellular fibrinogen-binding protein (Efb) from staphylococcus aureus Inhibits the formation of platelet-leukocyte complexes

NARCIS (Netherlands)

Posner, M.G; Upadhyay, A.; Abubaker, A.A.; Fortunato, T.M.; Vara, D.; Canobbio, I.; Bagby, S.; Pula, G.

2016-01-01

Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to

Characteristics of an autologous leukocyte and platelet-rich fibrin patch intended for the treatment of recalcitrant wounds

DEFF Research Database (Denmark)

Lundquist, Rasmus; Holmstrøm, Kim; Clausen, Christian

2012-01-01

We have investigated the physical, biochemical, and cellular properties of an autologous leukocyte and platelet-rich fibrin patch. This was generated in an automated device from a sample of a patient's blood at the point of care. Using microscopy, cell counting, enzyme-linked immunosorbent assay...... of chronic wound fluid. By comparison with traditional platelet-rich plasma, differences in immune components were found. The relevance of these findings was assessed by showing a mitogenic and migratory effect on cultured human dermal fibroblasts. Further, we showed that fibrocytes, a cell type important......, antibody arrays, and cell culture assays, we show that the patch is a three-layered membrane comprising a fibrin sheet, a layer of platelets, and a layer of leukocytes. Mean recovery of platelets from the donated blood was 98% (±95%CI 0.8%). Mean levels of platelet-derived growth factor AB, human...

The use of autologous platelet-leukocyte gels to enhance the healing process in surgery, a review

NARCIS (Netherlands)

Everts, P. A.; Overdevest, E. P.; Jakimowicz, J. J.; Oosterbos, C. J.; Schonberger, J. P.; Knape, J. T.; van Zundert, A.

2007-01-01

Background: The therapeutic use of autologously prepared, platelet-leukocyte-enriched gel (PLG) is a relatively new technology for the stimulation and acceleration of soft tissue and bone healing. The effectiveness of this procedure lies in the delivery of a wide range of platelet growth factors

Functional groups grafted nonwoven fabrics for blood filtration-The effects of functional groups and wettability on the adhesion of leukocyte and platelet

Energy Technology Data Exchange (ETDEWEB)

Yang Chao [State Key Lab of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240 (China); Cao Ye [Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu 610081 (China); Sun Kang, E-mail: ksun@sjtu.edu.cn [State Key Lab of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240 (China); Liu Jiaxin; Wang Hong [Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu 610081 (China)

2011-01-15

In this work, the effects of grafted functional groups and surface wettability on the adhesion of leukocyte and platelet were investigated by the method of blood filtration. The filter materials, poly(butylene terephthalate) nonwoven fabrics bearing different functional groups including hydroxyl (OH), carboxyl (COOH), sulfonic acid group (SO{sub 3}H) and zwitterionic sulfobetaine group ({sup +}N((CH{sub 3}){sub 2})(CH{sub 2}){sub 3}SO{sub 3}{sup Circled-Minus }) with controllable wettability were prepared by UV radiation grafting vinyl monomers with these functional groups. Our results emphasized that both surface functional groups and surface wettability had significant effects on the adhesion of leukocyte and platelet. In the case of filter materials with the same wettability, leukocytes adhering to filter materials decreased in the order: the surface bearing OH only > the surface bearing both OH and COOH > the surface bearing sulfobetaine group > the surface bearing SO{sub 3}H, while platelets adhering to filter materials decreased as the following order: the surface bearing SO{sub 3}H > the surface bearing both OH and COOH > the surface bearing OH only > the surface bearing sulfobetaine group. As the wettability of filter materials increased, both leukocyte and platelet adhesion to filter materials declined, except that leukocyte adhesion to the surface bearing OH only remained unchanged.

Leukocyte Inclusion within a Platelet Rich Plasma-Derived Fibrin Scaffold Stimulates a More Pro-Inflammatory Environment and Alters Fibrin Properties

Science.gov (United States)

Anitua, Eduardo; Zalduendo, Mar; Troya, María; Padilla, Sabino; Orive, Gorka

2015-01-01

One of the main differences among platelet-rich plasma (PRP) products is the inclusion of leukocytes that may affect the biological efficacy of these autologous preparations. The purpose of this study was to evaluate whether the addition of leukocytes modified the morphological, biomechanical and biological properties of PRP under normal and inflammatory conditions. The release of pro-inflammatory cytokines from plasma rich in growth factors (PRGF) and leukocyte-platelet rich plasma (L-PRP) scaffolds was determined by enzyme-linked immunosorbent assay (ELISA) and was significantly increased under an inflammatory condition when leukocytes were included in the PRP. Fibroblasts and osteoblasts treated with L-PRP, under an inflammatory situation, underwent a greater activation of NFĸB pathway, proliferated significantly less and secreted a higher concentration of pro-inflammatory cytokines. These cellular events were assessed through Western blot and fluorimetric and ELISA methods, respectively. Therefore, the inclusion of leukocytes induced significantly higher pro-inflammatory conditions. PMID:25823008

Leukocyte inclusion within a platelet rich plasma-derived fibrin scaffold stimulates a more pro-inflammatory environment and alters fibrin properties.

Directory of Open Access Journals (Sweden)

Eduardo Anitua

Full Text Available One of the main differences among platelet-rich plasma (PRP products is the inclusion of leukocytes that may affect the biological efficacy of these autologous preparations. The purpose of this study was to evaluate whether the addition of leukocytes modified the morphological, biomechanical and biological properties of PRP under normal and inflammatory conditions. The release of pro-inflammatory cytokines from plasma rich in growth factors (PRGF and leukocyte-platelet rich plasma (L-PRP scaffolds was determined by enzyme-linked immunosorbent assay (ELISA and was significantly increased under an inflammatory condition when leukocytes were included in the PRP. Fibroblasts and osteoblasts treated with L-PRP, under an inflammatory situation, underwent a greater activation of NFĸB pathway, proliferated significantly less and secreted a higher concentration of pro-inflammatory cytokines. These cellular events were assessed through Western blot and fluorimetric and ELISA methods, respectively. Therefore, the inclusion of leukocytes induced significantly higher pro-inflammatory conditions.

[Mechanisms of leukocyte formation of endogenous pyrogen].

Science.gov (United States)

Rybakina, E G; Sorokin, A V

1982-06-01

A study was made of the kinetics of endogenous pyrogen production by rabbit blood and exudate leukocytes and possible role played by the products of activated leukocytes in autoregulation of the process. It was established that accumulation of endogenous pyrogen in the cell precedes its release by stimulated cells. Then the processes of active pyrogen formation and release gel interdependent: pyrogen formed releases from the cell; the lowering of pyrogen concentration in the cell is accompanied by the decrease of its content in the medium. No stimulating effect of the products activated during leukocyte inflammation on pyrogen formation by blood leukocytes was discovered.

Exogenous application of platelet-leukocyte gel during open subacromial decompression contributes to improved patient outcome

NARCIS (Netherlands)

Everts, P. A.; Devilee, R. J. J.; Mahoney, C. Brown; van Erp, A.; Oosterbos, C. J. M.; Stellenboom, M.; Knape, J. T. A.; van Zundert, A.

2008-01-01

Background: Platelet-leukocyte gel (PLG) is being used during various surgical procedures in an attempt to enhance the healing process. We studied the effects of PLG on postoperative recovery of patients undergoing open subacromial decompression (OSD). Methods: PLG was produced from

Matrix metalloproteinase content and activity in low-platelet, low-leukocyte and high-platelet, high-leukocyte platelet rich plasma (PRP) and the biologic response to PRP by human ligament fibroblasts.

Science.gov (United States)

Pifer, Matthew A; Maerz, Tristan; Baker, Kevin C; Anderson, Kyle

2014-05-01

Recent work has shown the presence of catabolic cytokines in platelet-rich plasma (PRP), but little is known about endogenous catabolic proteases such as matrix metalloproteinases (MMPs). Hypothesis/ To quantify MMP content in 2 commercially available PRP preparation systems: Arthrex Double Syringe System autologous conditioned plasma (ACP) and Biomet GPS (GPS). The hypothesis was that MMPs are actively secreted from PRP immediately after preparation. Controlled laboratory study. PRP was prepared using either ACP (low platelet, low leukocyte) or GPS (high platelet, high leukocyte). MMP-2, MMP-3, and MMP-9 concentrations were measured using multiplex enzyme-linked immunosorbent assays for up to 6 days in 2 donors, and MMP activity was measured in 3 donors using kinetic activity kits able to detect the enzymatic cleavage of a fluorogenic peptide. Human ligament fibroblasts were cultured and exposed to both ACP and GPS from 1 donor each. MMP-2, -3, and -9 concentrations were assayed in culture media at 24 and 48 hours after exposure. GPS exhibited higher total MMP-2, -3, and -9 concentrations for up to 144 hours of release, while ACP had higher platelet-normalized MMP-2 and MMP-3 concentrations. GPS had significantly higher total and endogenous MMP-2 activity (P = .004 and .014, respectively), MMP-3 activity (P = .020 and .015, respectively), and MMP-9 activity (P = .004 and .002, respectively) compared with ACP. Once normalized to platelet count, differences in MMP activity were not significant between ACP and GPS. Compared with controls, cells stimulated with interleukin-1 beta (IL-1β) and treated with ACP showed significantly higher fold changes of MMP-2 (P = .001) and MMP-3 (P = .003) concentrations at 24 hours than did cells treated with GPS. Total MMP-9 content was higher in the media of GPS-treated, IL-1β-stimulated cells compared with ACP-treated cells (P = .001). At 48 hours, IL-1β-stimulated cells treated with GPS exhibited higher fold changes of MMP-2

Synergistic effect of signaling from receptors of soluble platelet agonists and outside-in signaling in formation of a stable fibrinogen-integrin αIIbβ3-actin cytoskeleton complex.

Science.gov (United States)

Budnik, Ivan; Shenkman, Boris; Savion, Naphtali

2015-01-01

Thrombus formation in the injured vessel wall is a highly complex process involving various blood-born components that go through specific temporal and spatial changes as observed by intravital videomicroscopy. Platelets bind transiently to the developing thrombus and may either become stably incorporated into or disengage from the thrombus. The aim of the present study was to reveal the processes involved in the formation of a stable thrombus. Platelet-rich plasma and washed platelets were studied by the aggregometer. The aggregate stability was challenged by eptifibatide. Platelet Triton-insoluble fraction was prepared and the actin and αIIb content in the cytoskeleton was analyzed by western blot. Maximal actin polymerization is achieved 1min after platelet activation while maximal αIIbβ3-actin cytoskeleton association requires 5 to 10min of activation and fibrinogen-mediated platelet-to-platelet bridging. Thus, actin polymerization is dependent on platelet activation and requires neither αIIbβ3 integrin occupation nor platelet aggregation. Formation of a stable aggregate requires platelet activation for more than 1min, complete increase in actin cytoskeleton fraction and partial association of αIIbβ3 with the actin cytoskeleton. However, direct αIIbβ3 activation is not sufficient for cytoskeleton complex formation. Thus, stable αIIbβ3-fibrinogen interaction, representing stable aggregate, is achieved after more than 1min agonist activation, involving inside-out and outside-in signaling but not after direct integrin activation, involving only outside-in signaling. Formation of a stable fibrinogen-αIIbβ3-actin cytoskeleton complex is the result of the combined effect of platelet stimulation by soluble agonists, activation of αIIbβ3, fibrinogen binding and platelet-to-platelet bridging. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cellular basis for accumulation of 111In-labeled leukocytes and platelets in rejecting cardiac allografts: concise communication

International Nuclear Information System (INIS)

Wang, T.S.; Oluwole, S.; Fawwaz, R.A.; Wolff, M.; Kuromoto, N.; Satake, K.; Hardy, M.A.; Alderson, P.O.

1982-01-01

Biodistribution and imaging studies in rats showed that 111 In-labeled leukocytes and platelets accumulate progressively with time after transplantation in cardiac allografts undergoing rejection, but do not accumulate in normal syngeneic heart grafts. Maximum heart allograft-to-blood ratios of 9:1 were obtained, and allograft-to-native heart ratios of 17:1. Microscopic studies of the rejecting cardiac allografts showed that histologic findings paralleled the cellular changes predicted by the radionuclide studies. Intravenously administered 67 Ga citrate and /sup 99m/Tc sulfur colloid failed to show significant accumulation in rejecting grafts. The findings suggest that cellular rejection, rather than nonspecific inflammatory changes, is the primary basis for accumulation of 111 In leukocytes and platelets in rejecting cardiac allografts

Cellular basis for accumulation of In-111-labeled leukocytes and platelets in rejecting cardiac allografts: concise communication

International Nuclear Information System (INIS)

Wang, T.S.T.; Oluwole, S.; Fawwaz, R.A.; Wolff, M.; Kuromoto, N.; Satake, K.; Hardy, M.A.; Alderson, P.O.

1982-01-01

Biodistribution and imaging studies in rats showed that In-111-labeled leukocytes and platelets accumulate progressively with time after transplantation in cardiac allografts undergoing rejection, but do not accumulate in normal syngeneic heart grafts. Maximum heart allograft-to-blood ratios of 9:1 were obtained, and allograft-to-native heart ratios of 17:1. Microscopic studies of the rejecting cardiac allografts showed that histologic findings paralleled the cellular changes predicted by the radionuclide studies. Intravenously administered Ga-67 citrate and Tc-99m sulfur colloid failed to show significant accumulation in rejecting grafts. The findings suggest that cellular rejection, rather than nonspecific inflammatory changes, is the primary basis for accumulation of In-111 leukocytes and platelets in rejecting cardiac allografts

Intratendon Delivery of Leukocyte-Poor Platelet-Rich Plasma Improves Healing Compared With Leukocyte-Rich Platelet-Rich Plasma in a Rabbit Achilles Tendinopathy Model.

Science.gov (United States)

Yan, Ruijian; Gu, Yanjia; Ran, Jisheng; Hu, Yejun; Zheng, Zefeng; Zeng, Mengfeng; Heng, Boon Chin; Chen, Xiao; Yin, Zi; Chen, Weishan; Shen, Weiliang; Ouyang, Hongwei

2017-07-01

Chronic tendinopathy is a commonly occurring clinical problem that affects both athletes and inactive middle-aged patients. Although some studies have shown that different platelet-rich plasma (PRP) preparations could exert various therapeutic effects in vitro, the role of leukocytes in PRP has not yet been defined under tendinopathy conditions in vivo. This study compared the effects of the intratendon delivery of leukocyte-poor PRP (Lp-PRP) versus leukocyte-rich PRP (Lr-PRP) in a rabbit chronic tendinopathy model in vivo. Controlled laboratory study. Four weeks after a local injection of collagenase in the Achilles tendon, the following treatments were randomly administered on the lesions: injections of (1) 200 μL of Lp-PRP (n = 8), (2) 200 μL of Lr-PRP (n = 8), or (3) 200 μL of saline (n = 8). Healing outcomes were assessed at 4 weeks after therapy with magnetic resonance imaging (MRI), cytokine quantification, real-time polymerase chain reaction analysis of gene expression, histology, and transmission electron microscopy (TEM). MRI revealed that the Lr-PRP and saline groups displayed higher signal intensities compared with the Lp-PRP group with T2 mapping. Histologically, the Lp-PRP group displayed significantly better general scores compared with the Lr-PRP ( P = .001) and saline ( P tendon healing and is a preferable option for the clinical treatment of tendinopathy. PRP is widely used in the clinical management of chronic tendinopathy. However, the clinical results are ambiguous. It is imperative to understand the influence of leukocytes on PRP-mediated tissue healing in vivo, which could facilitate the better clinical management of chronic tendinopathy. Further studies are needed to translate our findings to the clinical setting.

Preparation of leukocyte-poor platelet concentrates from buffy coats. I. Special inserts for centrifuge cups

NARCIS (Netherlands)

Pietersz, R. N.; Reesink, H. W.; Dekker, W. J.; Fijen, F. J.

1987-01-01

A special insert was developed for centrifuge cups in order to prepare leukocyte-poor platelet concentrates from buffy coats by using quadruple citrate phosphate dextrose-saline adenine glucose mannitol systems from different manufacturers. Each centrifuge cup could contain up to 4 sets of double

Platelet degranulation and monocyte-platelet complex formation are increased in the acute and convalescent phases after ischaemic stroke or transient ischaemic attack.

LENUS (Irish Health Repository)

McCabe, Dominick J H

2004-06-01

Flow cytometric studies suggest that platelets are activated in ischaemic stroke or transient ischaemic attack (TIA). However, few studies have measured circulating leucocyte-platelet complexes in this patient population. Whole blood flow cytometry was used to quantify the expression of CD62P-, CD63-, and PAC1-binding, and the percentages of leucocyte-platelet complexes in acute (1-27 d, n = 79) and convalescent (79-725 d, n = 70) ischaemic cerebrovascular disease (CVD) patients compared with controls without CVD (n = 27). We performed a full blood count, and measured plasma levels of soluble P-selectin, soluble E-selectin, and von Willebrand factor antigen (VWF:Ag) as additional markers of platelet and\\/or endothelial cell activation. The median percentage CD62P expression and the median percentage monocyte-platelet complexes were higher in both acute and convalescent CVD patients than controls (P <\\/= 0.02). The mean white cell count and mean VWF:Ag levels were significantly elevated in the acute and convalescent phases after ischaemic stroke or TIA (P <\\/= 0.02). Otherwise, there was no significant increase in any other marker of platelet or endothelial activation in CVD patients. There was a positive correlation between the percentage expression of CD62P and the percentages of both neutrophil-platelet and monocyte-platelet complexes in the acute phase, and the percentages of all leucocyte-platelet complexes in the convalescent phase after ischaemic CVD. This study provides evidence for ongoing excessive platelet and\\/or endothelial activation in ischaemic CVD patients despite treatment with antithrombotic therapy.

Comparison of point-of-care methods for preparation of platelet concentrate (platelet-rich plasma).

Science.gov (United States)

Weibrich, Gernot; Kleis, Wilfried K G; Streckbein, Philipp; Moergel, Maximilian; Hitzler, Walter E; Hafner, Gerd

2012-01-01

This study analyzed the concentrations of platelets and growth factors in platelet-rich plasma (PRP), which are likely to depend on the method used for its production. The cellular composition and growth factor content of platelet concentrates (platelet-rich plasma) produced by six different procedures were quantitatively analyzed and compared. Platelet and leukocyte counts were determined on an automatic cell counter, and analysis of growth factors was performed using enzyme-linked immunosorbent assay. The principal differences between the analyzed PRP production methods (blood bank method of intermittent flow centrifuge system/platelet apheresis and by the five point-of-care methods) and the resulting platelet concentrates were evaluated with regard to resulting platelet, leukocyte, and growth factor levels. The platelet counts in both whole blood and PRP were generally higher in women than in men; no differences were observed with regard to age. Statistical analysis of platelet-derived growth factor AB (PDGF-AB) and transforming growth factor β1 (TGF-β1) showed no differences with regard to age or gender. Platelet counts and TGF-β1 concentration correlated closely, as did platelet counts and PDGF-AB levels. There were only rare correlations between leukocyte counts and PDGF-AB levels, but comparison of leukocyte counts and PDGF-AB levels demonstrated certain parallel tendencies. TGF-β1 levels derive in substantial part from platelets and emphasize the role of leukocytes, in addition to that of platelets, as a source of growth factors in PRP. All methods of producing PRP showed high variability in platelet counts and growth factor levels. The highest growth factor levels were found in the PRP prepared using the Platelet Concentrate Collection System manufactured by Biomet 3i.

Platelet activation and platelet-leukocyte interaction in dogs naturally infected with Babesia rossi

DEFF Research Database (Denmark)

Goddard, Amelia; Leisewitz, Andrew L; Kristensen, Annemarie Thuri

2015-01-01

EDTA as anticoagulant. Activated platelets and PLA formation were detected by measuring surface expression of P-selectin (CD62P) on platelets, monocytes and neutrophils. Of the Babesia-infected dogs, 29 survived and seven died. The percentage of CD62P-positive monocytes was significantly higher (P = 0.......036) in the Babesia-infected dogs (54%) than in healthy control dogs (35.3%). However, there were no significant differences between the Babesia-infected and control groups for CD62P-positive platelets (4.9% and 1.2%, respectively) and CD62P-positive neutrophils (28.3% and 17.9%, respectively). The percentage of CD62...... groups for the percentage of CD62P-positive platelets (survivors 4.8%; non-survivors 5.3%; controls 1.2%) or CD62P-positive neutrophils (survivors 31.6%; non-survivors 5.6%; controls 17.9%). In conclusion, Babesia-infected dogs, specifically dogs that survived, had a significantly increased percentage...

CONGESTIVE HEART FAILURE IN DOGS IS ASSOCIATED WITH INCREASED PLATELET LEUKOCYTE AGGREGATION MEASURED BY FLOW CYTOMETRY

DEFF Research Database (Denmark)

Tarnow, Inge; Andreasen, Susanne SH; Olsen, Lisbeth Høier

2010-01-01

Sciences, Faculty of Life Science, University of Copenhagen, Denmark. Chronic congestive heart failure (CHF) in humans is associated with abnormal hemostasis, and changes in hemostatic biomarkers carry a poor prognosis. CHF in dogs has been associated with plasma markers of hypercoagulability, however......CONGESTIVE HEART FAILURE IN DOGS IS ASSOCIATED WITH ENHANCED PLATELET-LEUKOCYTE AGGREGATES - A MARKER FOR PLATELET ACTIVATION. I Tarnow1, LH Olsen2, SHS Andreasen2, SG Moesgaard2, CE Rasmussen2, AT Kristensen1, T Falk2. 1Departments of Small Animal Clinical Sciences and 2Animal and Veterinary Basic......, platelet activation markers have not been investigated in dogs with clinical signs of heart disease. We hypothesized that platelet surface activation markers are higher in dogs with CHF compared to age-matched controls without clinical signs of heart failure. Dogs with compensated congestive heart failure...

The relationship between fractional flow reserve, platelet reactivity and platelet leukocyte complexes in stable coronary artery disease

NARCIS (Netherlands)

Sels, J.W.E.M.; Rutten, B.; Holten, van T.C.; Hillaert, M.A.K.; Waltenberger, J.; Pijls, N.H.J.; Pasterkamp, G.; Groot, de P.G.; Roest, M.

2013-01-01

Background: The presence of stenoses that significantly impair blood flow and cause myocardial ischemia negatively affects prognosis of patients with stable coronary artery disease. Altered platelet reactivity has been associated with impaired prognosis of stable coronary artery disease. Platelets

Discrimination between platelet-mediated and coagulation-mediated mechanisms in a model of complex thrombus formation in vivo

International Nuclear Information System (INIS)

Cadroy, Y.; Horbett, T.A.; Hanson, S.R.

1989-01-01

To study mechanisms of complex thrombus formation in vivo, and to compare the relative antithrombotic effects of anticoagulants and antiplatelet agents, a model was developed in baboons. Segments of collagen-coated tubing followed by two sequentially placed expansion chambers exhibiting disturbed flow patterns were exposed to native blood under laminar flow conditions. The device was incorporated for 1 hour into an exteriorized arteriovenous shunt in baboons under controlled blood flow (20 ml/min). Morphologic evaluation by scanning electron microscopy showed that thrombi associated with collagen were relatively rich in platelets but thrombi in the chambers were rich in fibrin and red cells. Deposition of indium 111-labeled platelets was continuously measured with a scintillation camera. Platelet deposition increased in a linear (collagen-coated segment) or exponential (chambers 1 and 2) fashion over time, with values after 40 minutes averaging 24.1 +/- 3.3 x 10(8) platelets (collagen segment), 16.7 +/- 3.4 x 10(8) platelets (chamber 1), and 8.4 +/- 2.4 x 10(8) platelets (chamber 2). Total fibrinogen deposition after 40 minutes was determined by using iodine 125-labeled baboon fibrinogen and averaged 0.58 +/- 0.14 mg in the collagen segment, 1.51 +/- 0.27 mg in chamber 1, and 0.95 +/- 0.25 mg in chamber 2. Plasma levels of beta-thromboglobulin (beta TG), platelet-factor 4 (PF4), and fibrinopeptide A (FPA) increased fourfold to fivefold after 60 minutes of blood exposure to the thrombotic device. Platelet deposition onto the collagen segment, chamber 1, and chamber 2 was linearly dependent on the circulating platelet count. Platelet accumulation in chamber 1 and chamber 2 was also dependent on the presence of the proximal collagen segment

Platelets and infections—complex interactions with bacteria

Directory of Open Access Journals (Sweden)

Hind eHAMZEH-COGNASSE

2015-02-01

Full Text Available Platelets can be considered sentinels of vascular system due to their high number in the circulation and to the range of functional immunoreceptors they express. Platelets express a wide range of potential bacterial receptors, including complement receptors, FcγRII, Toll-Like Receptors but also integrins conventionally described in the hemostatic response, such as GPIIb-IIIa or GPIb. Bacteria bind these receptors either directly, or indirectly via fibrinogen, fibronectin, the first complement C1q, the von Willebrand Factor, etc. The fate of platelet bound bacteria is questioned. Several studies reported the ability of activated platelets to internalize bacteria such as Staphylococcus aureus or Porphyromonas gingivalis, though there is no clue on what happens thereafter. Are they sheltered from the immune system in the cytoplasm of platelets or are they lysed? Indeed, while the presence of phagolysosome has not been demonstrated in platelets, they contain antimicrobial peptides that were shown to be efficient on S. aureus. Besides, the fact that bacteria can bind to platelets via receptors involved in hemostasis suggests that they may induce aggregation; this has indeed been described for Streptococcus sanguinis, S. epidermidis or C. pneumoniae. On the other hand, platelets are able to display an inflammatory response to an infectious triggering. We, and others, have shown that platelet release soluble immunomodulatory factors upon stimulation by bacterial components. Moreover, interactions between bacteria and platelets are not limited to only these two partners. Indeed, platelets are also essential for the formation of Neutrophil Extracellular Traps by neutrophils, resulting in bacterial clearance by trapping bacteria and concentrating antibacterial factors but in enhancing thrombosis. In conclusion, the platelet-bacteria interplay is a complex game; its fine analysis is complicated by the fact that the inflammatory component adds to the

Platelet transfusions can induce transplantation tolerance

International Nuclear Information System (INIS)

Claas, F.H.J.; Blankert, J.J.; Ruigrok, R.; Moerel, L.

1982-01-01

Recently it was shown that the induction of antibodies against the H-2 antigens after multiple platelet transfusions is due to leukocyte contamination of the platelet suspensions. Pure platelets are not able to induce a primary antibody response. The present study shows that the platelets, however, can be recognized by the immune system but they induce a suppression of the response. Mice pretreated with donor platelets will not give a primary antibody response upon a subsequent injection of donor leukocytes and the survival of donor skin grafts will be prolonged. Similar results were obtained by pretreatment of the responder mice with heat-treated donor leukocytes. Furthermore, repeated injections of heat-treated leukocytes of the recipient strain to the donor before bone marrow grafting, will graft-versus-host mortality. The recipient mice were irradiated and received spleen cell injections. These data show that cells which have only class I antigens on their surface and no activating class II antigens, induce a suppression of the response against class I antigens. (Auth.)

The interaction of thrombin with platelet protease nexin

International Nuclear Information System (INIS)

Knupp, C.L.

1989-01-01

Thrombin interacts with a platelet protein which is immunologically related to fibroblast protease nexin and has been termed platelet protease nexin I (PNI). Conflicting hypotheses about the relationship of the thrombin-PNI complex formation to platelet activation have been proposed. The studies presented here demonstrate that the platelet-associated and supernatant complexes with added 125I-thrombin are formed only under conditions which produce platelet activation in normal and chymotrypsin-modified platelets. The platelet-associated complex is formed prior to the appearance of complexes in supernatants. Appearance of the supernatant complex coincides with the appearance of thrombospondin in the reaction supernatants. Excess native thrombin, dansylarginine N-(3-ethyl-1,5-pentanediyl) amide or hirudin can prevent radiolabeled platelet-associated complex formation if added before 125I-thrombin. DAPA or hirudin can prevent or dissociate complex formation if added up to one minute after thrombin but not at later time points. The surface associated complex is accessible to trypsin although a portion remains with the cytoskeletal proteins when thrombin-activated platelets are solubilized with Triton X 100. The surface-associated complex formation parallels many aspects of the specific measurable thrombin binding, yet it does not appear to involve other identified surface glycoprotein thrombin receptors or substrates. Although the time course of appearance of the complexes in supernatants is consistent with other data which suggest that PNI may be released from platelet granules during platelet activation, other explanations for the appearance of PNI on the platelet surface and in supernatants during platelet activation are possible

Current trends in platelet transfusions practice: The role of ABO-RhD and human leukocyte antigen incompatibility

Directory of Open Access Journals (Sweden)

Serena Valsami

2015-01-01

Full Text Available Platelet transfusions have contributed to the revolutionary modern treatment of hypoproliferative thrombocytopenia. Despite the long-term application of platelet transfusion in therapeutics, all aspects of their optimal use (i.e., in cases of ABO and/or Rh (D incompatibility have not been definitively determined yet. We reviewed the available data on transfusion practices and outcome in ABO and RhD incompatibility and platelet refractoriness due to anti-human leukocyte antigen (HLA antibodies. Transfusion of platelets with major ABO-incompatibility is related to reduced posttransfusion platelet (PLT count increments, compared to ABO-identical and minor, but still are equally effective in preventing clinical bleeding. ABO-minor incompatible transfusions pose the risk of an acute hemolytic reaction of the recipient that is not always related to high anti-A, B donor titers. ABO-identical PLT transfusion seems to be the most effective and safest therapeutic strategy. Exclusive ABO-identical platelet transfusion policy could be feasible, but alternative approaches could facilitate platelet inventory management. Transfusion of platelets from RhD positive donors to RhD negative patients is considered to be effective and safe though is associated with low rate of anti-D alloimmunization due to contaminating red blood cells. The prevention of D alloimmunization is recommended only for women of childbearing age. HLA alloimmunization is a major cause of platelet refractoriness. Managing patients with refractoriness with cross-matched or HLA-matched platelets is the current practice although data are still lacking for the efficacy of this practice in terms of clinical outcome. Leukoreduction contributes to the reduction of both HLA and anti-D alloimmunization.

PKCalpha regulates platelet granule secretion and thrombus formation in mice.

Science.gov (United States)

Konopatskaya, Olga; Gilio, Karen; Harper, Matthew T; Zhao, Yan; Cosemans, Judith M E M; Karim, Zubair A; Whiteheart, Sidney W; Molkentin, Jeffery D; Verkade, Paul; Watson, Steve P; Heemskerk, Johan W M; Poole, Alastair W

2009-02-01

Platelets are central players in atherothrombosis development in coronary artery disease. The PKC family provides important intracellular mechanisms for regulating platelet activity, and platelets express several members of this family, including the classical isoforms PKCalpha and PKCbeta and novel isoforms PKCdelta and PKCtheta. Here, we used a genetic approach to definitively demonstrate the role played by PKCalpha in regulating thrombus formation and platelet function. Thrombus formation in vivo was attenuated in Prkca-/- mice, and PKCalpha was required for thrombus formation in vitro, although this PKC isoform did not regulate platelet adhesion to collagen. The ablation of in vitro thrombus formation in Prkca-/- platelets was rescued by the addition of ADP, consistent with the key mechanistic finding that dense-granule biogenesis and secretion depend upon PKCalpha expression. Furthermore, defective platelet aggregation in response to either collagen-related peptide or thrombin could be overcome by an increase in agonist concentration. Evidence of overt bleeding, including gastrointestinal and tail bleeding, was not seen in Prkca-/- mice. In summary, the effects of PKCalpha ablation on thrombus formation and granule secretion may implicate PKCalpha as a drug target for antithrombotic therapy.

[In-line leukocyte depletion ov thrombocytapheresis concentrates with the Fresenius-AS-104 cell separator].

Science.gov (United States)

Zeiler, T; Kretschmer, V

1997-01-01

This study reports on in-line filtration of 72 platelet concentrates (PC) collected by the Fresenius AS 104 cell separator, using the new C4F sets with integrated leukocyte filters (Biofil P plus). 72 volunteer donors, automatic counts of platelets, microscopical counting of residual leukocytes with the Nageotte chamber, GMP-140 by flow cytometrie, beta-thromboglobulin release, platelet aggregation (ADP, collagen). Filtration reduced leukocytes by 98.5%. Residual leukocyte contamination remained clearly below 5 x 10(6) (mean 0.5 +/- 0.6 x 10(6), maximum 2.8 x 10(6). Platelet loss by filtration was found to be between 27.4 and 0.7% (median 8.5%). Filtration caused a significant decrease of platelet aggregability (p < 0.005), but no significant increase of beta-thromboglobulin release and only a slight decrease of GMP-140 expression. From these data can be concluded that in-line filtration was highly efficient with acceptable platelet retention. No significant platelet activation could be observed in the PC. The decrease of platelet aggregability have been due to the reduction of activated platelets which are believed to show reduced in vivo survival.

[Assessment study on a set of platelet-rich plasma preparation].

Science.gov (United States)

Li, Ming; Zhang, Changqing; Yuan, Ting; Chen, Shengbao; Lü, Ruju

2011-01-01

To calculate the recovery rate and enrichment factor and to analyse the correlation by measuring the concentrations of platelets, leukocyte, and growth factors in platelet-rich plasma (PRP) so as to evaluate the feasibility and stability of a set of PRP preparation. The peripheral blood (40 mL) was collected from 30 volunteers accorded with the inclusion criteria, and then 4 mL PRP was prepared using the package produced by Shandong Weigao Group Medical Polymer Company Limited. Automatic hematology analyzer was used to count the concentrations of platelets and leukocyte in whole blood and PRP. The enrichment factor and recovery rate of platelets or leukocyte were calculated; the platelet and leukocyte concentrations of male and female volunteers were measured, respectively. The concentrations of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), and vascular endothelial growth factor (VEGF) were assayed by ELISA. The platelet concentrations of whole blood and PRP were (131.40 +/- 29.44) x 10(9)/L and (819.47 +/- 136.32) x 10(9)/L, respectively, showing significant difference (t = 27.020, P = 0.000). The recovery rate of platelets was 60.85% +/- 8.97%, and the enrichment factor was 6.40 +/- 1.06. The leukocyte concentrations of whole blood and PRP were (5.57 +/- 1.91) x 10(12)/L and (32.20 +/- 10.42) x 10(12)/L, respectively, showing significant difference (t = 13.780, P = 0.000). The recovery rate of leukocyte was 58.30% +/- 19.24%, and the enrichment factor was 6.10 +/- 1.93. The concentrations of platelets and leukocyte in PRP were positively correlated with the platelet concentration (r = 0.652, P = 0.000) and leukocyte concentration (r = 0.460, P = 0.011) in whole blood. The concentrations of platelet and leukocyte in PRP between male and female were not significantly different (P > 0.05). The concentrations of PDGF, TGF-beta, and VEGF in PRP were (698.15 +/- 64.48), (681.36 +/- 65.90), and (1071.55 +/- 106.04) ng/mL, which were

Thrombopoietin contributes to enhanced platelet activation in cigarette smokers.

Science.gov (United States)

Lupia, Enrico; Bosco, Ornella; Goffi, Alberto; Poletto, Cesare; Locatelli, Stefania; Spatola, Tiziana; Cuccurullo, Alessandra; Montrucchio, Giuseppe

2010-05-01

Thrombopoietin (TPO) is a humoral growth factor that primes platelet activation in response to several agonists. We recently showed that TPO enhances platelet activation in unstable angina and sepsis. Aim of this study was to investigate the role of TPO in platelet function abnormalities described in cigarette smokers. In a case-control study we enrolled 20 healthy cigarette smokers and 20 nonsmokers, and measured TPO and C-reactive protein (CRP), as well as platelet-leukocyte binding and P-selectin expression. In vitro we evaluated the priming activity of smoker or control plasma on platelet activation, and the role of TPO in this effect. We then studied the effects of acute smoking and smoking cessation on TPO levels and platelet activation indices. Chronic cigarette smokers had higher circulating TPO levels than nonsmoking controls, as well as increased platelet-leukocyte binding, P-selectin expression, and CRP levels. Serum cotinine concentrations correlated with TPO concentrations, platelet-monocyte aggregates and P-selectin expression. In addition, TPO levels significantly correlated with ex vivo platelet-monocyte aggregation and P-selectin expression. In vitro, the plasma from cigarette smokers, but not from nonsmoking controls, primed platelet-monocyte binding, which was reduced when an inhibitor of TPO was used. We also found that acute smoking slightly increased TPO levels, but did not affect platelet-leukocyte binding, whereas smoking cessation induced a significant decrease in both circulating TPO and platelet-leukocyte aggregation. Elevated TPO contributes to enhance platelet activation and platelet-monocyte cross-talk in cigarette smokers. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Platelet size and density affect shear-induced thrombus formation in tortuous arterioles

Science.gov (United States)

Chesnutt, Jennifer K. W.; Han, Hai-Chao

2013-10-01

Thrombosis accounts for 80% of deaths in patients with diabetes mellitus. Diabetic patients demonstrate tortuous microvessels and larger than normal platelets. Large platelets are associated with increased platelet activation and thrombosis, but the physical effects of large platelets in the microscale processes of thrombus formation are not clear. Therefore, the objective of this study was to determine the physical effects of mean platelet volume (MPV), mean platelet density (MPD) and vessel tortuosity on platelet activation and thrombus formation in tortuous arterioles. A computational model of the transport, shear-induced activation, collision, adhesion and aggregation of individual platelets was used to simulate platelet interactions and thrombus formation in tortuous arterioles. Our results showed that an increase in MPV resulted in a larger number of activated platelets, though MPD and level of tortuosity made little difference on platelet activation. Platelets with normal MPD yielded the lowest amount of mural thrombus. With platelets of normal MPD, the amount of mural thrombus decreased with increasing level of tortuosity but did not have a simple monotonic relationship with MPV. The physical mechanisms associated with MPV, MPD and arteriole tortuosity play important roles in platelet activation and thrombus formation.

Assessment of canine autologous platelet-rich plasma produced with a commercial centrifugation and platelet recovery kit.

Science.gov (United States)

Frye, Chris W; Enders, Andrew; Brooks, Marjory B; Struble, Angela M; Wakshlag, Joseph J

2016-01-01

To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and confirm reproducibility of platelet enrichment, as well as determine the platelet activation status in the final product of a commercial platelet-rich plasma kit using canine blood. Venous blood from 20 sedated client-owned dogs was used to prepare platelet-rich plasma (PRP) from a commercial kit. Complete blood counts were performed to determine erythrocyte, leukocyte, and platelet numbers in both whole blood (WB) and resultant PRP. The WB and PRP samples from jugular (fast collection) and cephalic (slow collection) venipuncture were also compared. P-selectin externalization was measured in WB and PRP samples from 15 of 20 dogs. This commercial kit produced an average percent recovery in platelets of 64.7 ± 17.4; erythrocytes of 3.7 ± 0.8, and leukocytes of 31.6 ± 10.0. Neutrophil, monocyte, and lymphocyte percent recovery was 19.6 ± 7.2, 44.89 ± 19.8, and 57.5 ± 10.6, respectively. The recovery of platelets from jugular venipuncture (59.7 ± 13.6%) was lower than from cephalic recovery (68.8 ± 19.1%). The mean percent P-Selectin externalization for WB, PRP, and PRP with thrombin was 25.5 ± 30.9, 4.5 ± 6.4, and 90.6 ± 4.4 respectively. Cellular reproducibility of this kit was confirmed and platelets were concentrated within autologous serum. Additionally, measurements of P-selectin externalization showed that platelets are inactive in PRP unless stimulated to degranulate.

Transformation of ATLA-negative leukocytes by blood components from anti-ATLA-positive donors in vitro.

Science.gov (United States)

Miyamoto, K; Tomita, N; Ishii, A; Nishizaki, T; Kitajima, K; Tanaka, T; Nakamura, T; Watanabe, S; Oda, T

1984-06-15

Anti-ATLA-positive blood components transformed healthy human leukocytes in vitro. Blood components examined were packed red cells, whole blood, platelet concentrate and fresh frozen plasma. Leukocytes present in anti-ATLA-positive blood components such as packed red cells, whole blood and platelet concentrate easily transformed anti-ATLA-negative leukocytes. Co-culture in fresh frozen plasma, however, did not transform recipient leukocytes, and leukocytes of anti-ATLA-positive recipients proved refractory to transformation. The transformed cells were morphologically lymphoid, grew in suspension, and possessed normal recipient karyotypes except in the case of three platelet concentrates. A high proportion of all the transformed populations formed E-rosettes with neuraminidase-treated sheep erythrocytes. The cytoplasm of over 90% of each recipient was stained brilliantly with antibodies against ATLV-determined antigens. Electron microscopy of these transformed cells revealed many C-type virus particles in the extracellular space. Blood components, such as packed red cells, whole blood and platelet concentrate, containing leukocytes from anti-ATLA-positive donors, should be used cautiously to prevent the transmission on ATLV to anti-ATLA-negative recipients.

Combined blockade of ADP receptors and PI3-kinase p110β fully prevents platelet and leukocyte activation during hypothermic extracorporeal circulation.

Directory of Open Access Journals (Sweden)

Stefanie Krajewski

Full Text Available Extracorporeal circulation (ECC and hypothermia are used to maintain stable circulatory parameters and improve the ischemia tolerance of patients in cardiac surgery. However, ECC and hypothermia induce activation mechanisms in platelets and leukocytes, which are mediated by the platelet agonist ADP and the phosphoinositide-3-kinase (PI3K p110β. Under clinical conditions these processes are associated with life-threatening complications including thromboembolism and inflammation. This study analyzes effects of ADP receptor P(2Y(12 and P(2Y(1 blockade and PI3K p110β inhibition on platelets and granulocytes during hypothermic ECC. Human blood was treated with the P(2Y(12 antagonist 2-MeSAMP, the P(2Y(1 antagonist MRS2179, the PI3K p110β inhibitor TGX-221, combinations thereof, or PBS and propylene glycol (controls. Under static in vitro conditions a concentration-dependent effect regarding the inhibition of ADP-induced platelet activation was found using 2-MeSAMP or TGX-221. Further inhibition of ADP-mediated effects was achieved with MRS2179. Next, blood was circulated in an ex vivo ECC model at 28°C for 30 minutes and various platelet and granulocyte markers were investigated using flow cytometry, ELISA and platelet count analysis. GPIIb/IIIa activation induced by hypothermic ECC was inhibited using TGX-221 alone or in combination with P(2Y blockers (p<0.05, while no effect of hypothermic ECC or antiplatelet agents on GPIIb/IIIa and GPIbα expression and von Willebrand factor binding was observed. Sole P(2Y and PI3K blockade or a combination thereof inhibited P-selectin expression on platelets and platelet-derived microparticles during hypothermic ECC (p<0.05. P(2Y blockade alone or combined with TGX-221 prevented ECC-induced platelet-granulocyte aggregate formation (p<0.05. Platelet adhesion to the ECC surface, platelet loss and Mac-1 expression on granulocytes were inhibited by combined P(2Y and PI3K blockade (p<0.05. Combined blockade of P

Developmental endothelial locus-1 modulates platelet-monocyte interactions and instant blood-mediated inflammatory reaction in islet transplantation.

Science.gov (United States)

Kourtzelis, Ioannis; Kotlabova, Klara; Lim, Jong-Hyung; Mitroulis, Ioannis; Ferreira, Anaisa; Chen, Lan-Sun; Gercken, Bettina; Steffen, Anja; Kemter, Elisabeth; Klotzsche-von Ameln, Anne; Waskow, Claudia; Hosur, Kavita; Chatzigeorgiou, Antonios; Ludwig, Barbara; Wolf, Eckhard; Hajishengallis, George; Chavakis, Triantafyllos

2016-04-01

Platelet-monocyte interactions are strongly implicated in thrombo-inflammatory injury by actively contributing to intravascular inflammation, leukocyte recruitment to inflamed sites, and the amplification of the procoagulant response. Instant blood-mediated inflammatory reaction (IBMIR) represents thrombo-inflammatory injury elicited upon pancreatic islet transplantation (islet-Tx), thereby dramatically affecting transplant survival and function. Developmental endothelial locus-1 (Del-1) is a functionally versatile endothelial cell-derived homeostatic factor with anti-inflammatory properties, but its potential role in IBMIR has not been previously addressed. Here, we establish Del-1 as a novel inhibitor of IBMIR using a whole blood-islet model and a syngeneic murine transplantation model. Indeed, Del-1 pre-treatment of blood before addition of islets diminished coagulation activation and islet damage as assessed by C-peptide release. Consistently, intraportal islet-Tx in transgenic mice with endothelial cell-specific overexpression of Del-1 resulted in a marked decrease of monocytes and platelet-monocyte aggregates in the transplanted tissues, relative to those in wild-type recipients. Mechanistically, Del-1 decreased platelet-monocyte aggregate formation, by specifically blocking the interaction between monocyte Mac-1-integrin and platelet GPIb. Our findings reveal a hitherto unknown role of Del-1 in the regulation of platelet-monocyte interplay and the subsequent heterotypic aggregate formation in the context of IBMIR. Therefore, Del-1 may represent a novel approach to prevent or mitigate the adverse reactions mediated through thrombo-inflammatory pathways in islet-Tx and perhaps other inflammatory disorders involving platelet-leukocyte aggregate formation.

A Novel Platelet Concentrate: Titanium-Prepared Platelet-Rich Fibrin

OpenAIRE

Mustafa Tunalı; Hakan Özdemir; Zafer Küçükodacı; Serhan Akman; Emre Yaprak; Hülya Toker; Erhan Fıratlı

2014-01-01

We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF). The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun's leukocyte- and platelet-rich fibrin (L-PRF) method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn us...

Indium-111 leukocyte scintigraphic detection of myocardial abscess formation in patients with endocarditis

Energy Technology Data Exchange (ETDEWEB)

Cerqueira, M.D.; Jacobson, A.F.

1989-05-01

Myocardial abscess formation in patients with bacterial endocarditis in most clinical settings, especially in patients with prosthetic valves, is a primary indicator for surgical valve replacement. We report the detection of myocardial abscesses using /sup 111/In leukocyte scintigraphy in three patients with prosthetic or native valve endocarditis and nondiagnostic echocardiograms. Leukocyte scintigraphy may allow identification of myocardial abscess formation earlier than other imaging modalities.

Indium-111 leukocyte scintigraphic detection of myocardial abscess formation in patients with endocarditis

International Nuclear Information System (INIS)

Cerqueira, M.D.; Jacobson, A.F.

1989-01-01

Myocardial abscess formation in patients with bacterial endocarditis in most clinical settings, especially in patients with prosthetic valves, is a primary indicator for surgical valve replacement. We report the detection of myocardial abscesses using 111 In leukocyte scintigraphy in three patients with prosthetic or native valve endocarditis and nondiagnostic echocardiograms. Leukocyte scintigraphy may allow identification of myocardial abscess formation earlier than other imaging modalities

Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation.

Directory of Open Access Journals (Sweden)

Naadiya Carrim

Full Text Available We have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI.To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.Human and mouse washed platelets (from WT or Pyk2 KO mice were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P surface expression and integrin αIIbβ3 activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk, PI3-K and Bruton's tyrosine kinase (Btk and upstream of Rac1, PLCγ2, Ca2+ release, PKC, Hic-5, NOX1 and αIIbβ3 activation.Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.

A novel platelet concentrate: titanium-prepared platelet-rich fibrin.

Science.gov (United States)

Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Yaprak, Emre; Toker, Hülya; Fıratlı, Erhan

2014-01-01

We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF). The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun's leukocyte- and platelet-rich fibrin (L-PRF) method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF) was processed with a scanning electron microscope (SEM). The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

A Novel Platelet Concentrate: Titanium-Prepared Platelet-Rich Fibrin

Directory of Open Access Journals (Sweden)

Mustafa Tunalı

2014-01-01

Full Text Available We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF. The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun’s leukocyte- and platelet-rich fibrin (L-PRF method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF was processed with a scanning electron microscope (SEM. The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

Leukocyte and platelet depletion improves blood flow and function in a renal transplant model.

Science.gov (United States)

Yates, Phillip J; Hosgood, Sarah A; Nicholson, Michael L

2012-01-01

Donation after cardiac death (DCD) donors are an important source of organs for transplantation. Due to warm and cold ischemic injury, DCD kidneys undergo a significant reperfusion insult when transplanted. This is manifested clinically as a high incidence of delayed graft function (DGF) and primary non-function (PNF). The importance of leukocytes in the generation of reperfusion injury is pivotal. Using an ex vivo porcine model of kidney transplantation, the effects of reperfusion with leukocyte and platelet depleted blood (LDB) and whole blood (WB) on renal blood flow and function were compared. Hemodynamic measurements were recorded, and biochemical, hematological, and histologic samples taken at set time-points. Reperfusion with LDB improved renal blood flow significantly compared with WB reperfusion. In addition, there was a significant improvement in creatinine clearance and renal oxygen consumption, but not fractional excretion of sodium, acid-base homeostasis, urinary nitric oxide (NO), or 8-isoprostane levels. This study represents a good model for the initial reperfusion period in renal transplantation. Improvement in only some functional markers and neither urinary NO nor 8-isoprostane levels indicates that improved blood flow alone is not sufficient to reverse the severe ischemic insult endured by DCD kidneys. Copyright © 2012 Elsevier Inc. All rights reserved.

An Innovative Regenerative Endodontic Procedure Using Leukocyte and Platelet-rich Fibrin Associated with Apical Surgery: A Case Report.

Science.gov (United States)

Pinto, Nelson; Harnish, Alexandra; Cabrera, Carolina; Andrade, Catherine; Druttman, Tony; Brizuela, Claudia

2017-11-01

Regenerative endodontic procedures (REPs) associated with apical surgery could represent an alternative treatment strategy for patients whose teeth present incomplete root formation and extensive apical lesions. Leukocyte platelet-rich fibrin (L-PRF) has potential benefits in REPs; it could promote apical root formation and optimal bone healing. The aim of this case report was to describe innovative regenerative endodontic therapy using L-PRF in the root canal and an extensive apical lesion in an immature tooth with dens invaginatus and asymptomatic apical periodontitis. A healthy 20-year-old woman was referred to the dental clinic of the Universidad de Los Andes, Santiago, Chile, for endodontic treatment in tooth # 22 with incomplete root development and an extensive apical lesion. The diagnosis was asymptomatic apical periodontitis associated with dens invaginatus type II. The patient was treated with an innovative approach using L-PRF in REPs associated with apical surgery. Follow-ups were performed at 6 months and 1 year later. They included periapical radiographs, cone-beam computed tomographic imaging, sensitivity, and vitality tests. The clinical evaluations performed at 6 months and 1 year revealed an absence of symptoms. The radiographic evaluations showed that the apical lesion was resolved. The cone-beam images indicated that the root length increased and the walls had thickened. The sensitivity tests were positive, and the laser Doppler flowmetry showed positive blood flow after 1 year. The success of the results in this case report indicate that L-PRF can be used as a complement in apical surgery and REPs and could provide an innovative alternative treatment strategy for complex clinical cases like these. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation

OpenAIRE

Jang, Ja-Young; Kim, Tae-Su; Cai, Jingmei; Kim, Jihyun; Kim, Youngeun; Shin, Kyungha; Kim, Kwang Sei; Park, Sung Kyeong; Lee, Sung-Pyo; Choi, Ehn-Kyoung; Rhee, Man Hee; Kim, Yun-Bae

2013-01-01

The effects of nattokinase on the in vitro platelet aggregation and in vivo thrombosis were investigated in comparison with aspirin. Rabbit platelet-rich plasma was incubated with nattokinase and aggregation inducers collagen and thrombin, and the platelet aggregation rate was analyzed. Nattokinase significantly inhibited both the collagen- and thrombin-induced platelet aggregations. Nattokinase also reduced thromboxane B2 formation from collagen-activated platelets in a concentration-depende...

Surface modification of platelet concentrate bags to reduce biofilm formation and transfusion sepsis.

Science.gov (United States)

Wilson-Nieuwenhuis, Joels S T; Dempsey-Hibbert, Nina; Liauw, Christopher M; Whitehead, Kathryn A

2017-12-01

Bacterial contamination of blood products poses a major risk in transfusion medicine, including transfusions involving platelet products. Although testing systems are in place for routine screening of platelet units, the formation of bacterial biofilms in such units may decrease the likelihood that bacteria will be detected. This work determined the surface properties of p-PVC platelet concentrate bags and investigated how these characteristics influenced biofilm formation. Serratia marcescens and Staphylococcus epidermidis, two species commonly implicated in platelet contamination, were used to study biofilm growth. The platelet concentrate bags were physically flattened to determine if reducing the surface roughness altered biofilm formation. The results demonstrated that the flattening process of the platelet bags affected the chemistry of the surface and reduced the surface hydrophobicity. Flattening of the surfaces resulted in a reduction in biofilm formation for both species after 5 days, with S. marcescens demonstrating a greater reduction. However, there was no significant difference between the smooth and flat surfaces following 7 days' incubation for S. marcescens and no significant differences between any of the surfaces following 7 days' incubation for S. epidermidis. The results suggest that flattening the p-PVC surfaces may limit potential biofilm formation for the current duration of platelet storage time of 5 days. It is hoped that this work will enhance the understanding of how surface properties influence the development of microbial biofilms in platelet concentrate bags in order to devise a solution to discourage biofilm formation. Copyright © 2017 Elsevier B.V. All rights reserved.

Reduction of relative centrifugation force within injectable platelet-rich-fibrin (PRF) concentrates advances patients' own inflammatory cells, platelets and growth factors: the first introduction to the low speed centrifugation concept.

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Choukroun, J; Ghanaati, S

2018-02-01

The aim of this study was to analyze systematically the influence of the relative centrifugation force (RCF) on leukocytes, platelets and growth factor release within fluid platelet-rich fibrin matrices (PRF). Systematically using peripheral blood from six healthy volunteers, the RCF was reduced four times for each of the three experimental protocols (I-III) within the spectrum (710-44 g), while maintaining a constant centrifugation time. Flow cytometry was applied to determine the platelets and leukocyte number. The growth factor concentration was quantified 1 and 24 h after clotting using ELISA. Reducing RCF in accordance with protocol-II (177 g) led to a significantly higher platelets and leukocytes numbers compared to protocol-I (710 g). Protocol-III (44 g) showed a highly significant increase of leukocytes and platelets number in comparison to -I and -II. The growth factors' concentration of VEGF and TGF-β1 was significantly higher in protocol-II compared to -I, whereas protocol-III exhibited significantly higher growth factor concentration compared to protocols-I and -II. These findings were observed among 1 and 24 h after clotting, as well as the accumulated growth factor concentration over 24 h. Based on the results, it has been demonstrated that it is possible to enrich PRF-based fluid matrices with leukocytes, platelets and growth factors by means of a single alteration of the centrifugation settings within the clinical routine. We postulate that the so-called low speed centrifugation concept (LSCC) selectively enriches leukocytes, platelets and growth factors within fluid PRF-based matrices. Further studies are needed to evaluate the effect of cell and growth factor enrichment on wound healing and tissue regeneration while comparing blood concentrates gained by high and low RCF.

Platelet transfusion therapy: from 1973 to 2005.

NARCIS (Netherlands)

Brand, A.; Novotny, V.M.J.; Tomson, B.

2006-01-01

Platelet transfusions are indispensable for supportive care of patients with hematological diseases. We describe the developments in platelet products for transfusion since the 1970s, when, in particular, support for patients with allo-antibodies against human leukocyte antigens was a laborious

Morphogen and proinflammatory cytokine release kinetics from PRGF-Endoret fibrin scaffolds: evaluation of the effect of leukocyte inclusion.

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Anitua, E; Zalduendo, M M; Prado, R; Alkhraisat, M H; Orive, G

2015-03-01

The potential influence of leukocyte incorporation in the kinetic release of growth factors from platelet-rich plasma (PRP) may explain the conflicting efficiency of leukocyte platelet-rich plasma (L-PRP) scaffolds in tissue regeneration. To assess this hypothesis, leukocyte-free (PRGF-Endoret) and L-PRP fibrin scaffolds were prepared, and both morphogen and proinflammatory cytokine release kinetics were analyzed. Clots were incubated with culture medium to monitor protein release over 8 days. Furthermore, the different fibrin scaffolds were morphologically characterized. Results show that leukocyte-free fibrin matrices were homogenous while leukocyte-containing ones were heterogeneous, loose and cellular. Leukocyte incorporation produced a significant increase in the contents of proinflammatory cytokines interleukin (IL)-1β and IL-16 but not in the platelet-derived growth factors release (PRGF-Endoret, the inclusion of leukocytes induced a major increase in these cytokines, which was characterized by the presence of a latent period. The PRGF-Endoret matrices were stable during the 8 days of incubation. The inclusion of leukocytes alters the growth factors release profile and also increased the dose of proinflammatory cytokines. © 2014 Wiley Periodicals, Inc.

The Use of Platelet-Rich and Platelet-Poor Plasma to Enhance Differentiation of Skeletal Myoblasts: Implications for the Use of Autologous Blood Products for Muscle Regeneration.

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Miroshnychenko, Olga; Chang, Wen-Teh; Dragoo, Jason L

2017-03-01

Platelet-rich plasma (PRP) has been used to augment tissue repair and regeneration after musculoskeletal injury. However, there is increasing clinical evidence that PRP does not show a consistent clinical effect. Purpose/Hypothesis: This study aimed to compare the effects of the following non-neutrophil-containing (leukocyte-poor) plasma fractions on human skeletal muscle myoblast (HSMM) differentiation: (1) PRP, (2) modified PRP (Mod-PRP), in which transforming growth factor β1 (TGF-β1) and myostatin (MSTN) were depleted, and (3) platelet-poor plasma (PPP). The hypothesis was that leukocyte-poor PRP would lead to myoblast proliferation (not differentiation), whereas certain modifications of PRP preparations would increase myoblast differentiation, which is necessary for skeletal muscle regeneration. Controlled laboratory study. Blood from 7 human donors was individually processed to simultaneously create leukocyte-poor fractions: PRP, Mod-PRP, PPP, and secondarily spun PRP and Mod-PRP (PRP ss and Mod-PRP ss , respectively). Mod-PRP was produced by removing TGF-β1 and MSTN from PRP using antibodies attached to sterile beads, while a second-stage centrifugal spin of PRP was performed to remove platelets. The biologics were individually added to cell culture groups. Analysis for induction into myoblast differentiation pathways included Western blot analysis, reverse-transcription polymerase chain reaction, and immunohistochemistry, as well as confocal microscopy to assess polynucleated myotubule formation. HSMMs cultured with PRP showed an increase in proliferation but no evidence of differentiation. Western blot analysis confirmed that MSTN and TGF-β1 could be decreased in Mod-PRP using antibody-coated beads, but this modification mildly improved myoblast differentiation. However, cell culture with PPP, PRP ss , and Mod-PRP ss led to a decreased proliferation rate but a significant induction of myoblast differentiation verified by increased multinucleated

Potential Effect of Leukocyte-Platelet-Rich Fibrin in Bone Healing of Skull Base: A Pilot Study

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Felipe Fredes

2017-01-01

Full Text Available Background. Reconstruction of surgical defects following cranial base surgery is challenging. Others have demonstrated that leukocyte-platelet-rich fibrin (L-PRF stimulates tissue healing and bone regeneration. However, these studies have addressed mostly maxillofacial surgical wounds. Objective. The objective of this study was to assess the possible adjuvant role of L-PRF in inducing neoossification of the surgical bone defect in anterior skull base surgery. Methods. We identified patients who had undergone an endoscopic endonasal surgery of the anterior skull base in which L-PRF membranes were used for the reconstruction of the bone defect and who were followed up with postoperative CT scans. CT findings were then correlated with baseline scans and with the CT scans of a patient who had undergone imaging and histologic analysis after maxillofacial surgery in which L-PRF was used and in which we demonstrated bone formation. Results. Five patients fulfilled the inclusion criteria. In four patients, the CT scan demonstrated closure of the bony defect by neoosteogenesis; however, the bone appeared less dense than the surrounding normal bone. A comparison with the control patient yielded similar radiological features. Conclusion. This case series suggests that L-PRF may induce bone healing and regeneration at the surgical site defect. Multi-institutional studies with a larger series of patients are required to confirm this possibility.

The Dynamics of Platelet Activation during the Progression of Streptococcal Sepsis.

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Sinead M Hurley

Full Text Available Platelets contribute to inflammation however, the role of platelet activation during the pathophysiological response to invasive bacterial infection and sepsis is not clear. Herein, we have investigated platelet activation in a mouse model of invasive Streptococcus pyogenes infection at 5, 12, and 18 hours post infection and correlated this to parameters of infection. The platelet population in ex-vivo blood samples showed no increased integrin activation or surface presentation of CD62P, however platelet-neutrophil complex formation and plasma levels of CD62P were increased during bacterial dissemination and the progression of sepsis, indicating that platelet activation had occurred in vivo. Platelet-neutrophil complex formation was the most discriminatory marker of platelet activation. Platelet-neutrophil complexes were increased above baseline levels during early sepsis but decreased to significantly lower levels than baseline during late sepsis. The removal of these complexes from the circulation coincided with a significant increase in organ damage and the accumulation of platelets in the liver sinusoids, suggesting that platelet activation in the circulation precedes accumulation of platelets in damaged organs. The results demonstrate that monitoring platelet activation using complementary methods may provide prognostic information during the pathogenesis of invasive S. pyogenes infection.

Platelet RNA as a circulating biomarker trove for cancer diagnostics.

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Best, M G; Vancura, A; Wurdinger, T

2017-07-01

Platelets are multifunctional cell fragments, circulating in blood in high abundance. Platelets assist in thrombus formation, sensing of pathogens entering the blood stream, signaling to immune cells, releasing vascular remodeling factors, and, negatively, enhancing cancer metastasis. Platelets are 'educated' by their environment, including in patients with cancer. Cancer cells appear to initiate intraplatelet signaling, resulting in splicing of platelet pre-mRNAs, and enhance secretion of cytokines. Platelets can induce leukocyte and endothelial cell modeling factors, for example, through adenine nucleotides (ATP), thereby facilitating extravasation of cancer cells. Besides releasing factors, platelets can also sequester RNAs and proteins released by cancer cells. Thus, platelets actively respond to queues from local and systemic conditions, thereby altering their transcriptome and molecular content. Platelets contain a rich repertoire of RNA species, including mRNAs, small non-coding RNAs and circular RNAs; although studies regarding the functionality of the various platelet RNA species require more attention. Recent advances in high-throughput characterization of platelet mRNAs revealed 10 to > 1000 altered mRNAs in platelets in the presence of disease. Hence, platelet RNA appears to be dynamically affected by pathological conditions, thus possibly providing opportunities to use platelet RNA as diagnostic, prognostic, predictive, or monitoring biomarkers. In this review, we cover the literature regarding the platelet RNA families, processing of platelet RNAs, and the potential application of platelet RNA as disease biomarkers. © 2017 International Society on Thrombosis and Haemostasis.

Granulocyte-platelet interactions and platelet fibrinogen receptor exposure

International Nuclear Information System (INIS)

Kornecki, E.; Ehrlich, Y.H.; Egbring, R.; Gramse, M.; Seitz, R.; Eckardt, A.; Lukasiewicz, H.; Niewiarowski, S.

1988-01-01

The authors have examined the interaction of human granulocyte elastase with human platelets. Incubation of human platelets with human granulocyte elastase exposed active fibrinogen-binding sites as evidenced by 125 I-labeled fibrinogen binding and spontaneous fibrinogen-induced platelet aggregation. The aggregation of platelets by fibrinogen occurred at low concentrations of human granulocyte elastase. Platelets pretreated with human granulocyte elastase exposed an average of 10,500 fibrinogen-binding sites per platelet, i.e., about one-third the number of binding sites exposed by optimal concentrations of ADP. With the use of a polyclonal antiplatelet membrane antibody, the glycoproteins IIb (GPIIb), IIIa (GPIIIa), and a 60,000-Da (60 kDa) protein (66 kDa in a reduced system) derived from GPIIIa were immunoprecipitated from the surface of detergent extracts of human 125 I-radiolabeled platelets pretreated with increasing concentrations of human granulocyte elastase. They conclude that (1) the proteolytic action of human granulocyte elastase on platelet GPIIIa results in the formation of two major hydrolytic products, and (2) human granulocyte elastase exposes active fibrongen-binding sites associated with the GPIIb/GPIIIa complex, resulting in direct platelet aggregation by fibrinogen

Leukocyte- and platelet-rich fibrin (L-PRF) for long-term delivery of growth factor in rotator cuff repair: review, preliminary results and future directions.

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Zumstein, Matthias A; Berger, Simon; Schober, Martin; Boileau, Pascal; Nyffeler, Richard W; Horn, Michael; Dahinden, Clemens A

2012-06-01

Surgical repair of the rotator cuff repair is one of the most common procedures in orthopedic surgery. Despite it being the focus of much research, the physiological tendon-bone insertion is not recreated following repair and there is an anatomic non-healing rate of up to 94%. During the healing phase, several growth factors are upregulated that induce cellular proliferation and matrix deposition. Subsequently, this provisional matrix is replaced by the definitive matrix. Leukocyte- and platelet-rich fibrin (L-PRF) contain growth factors and has a stable dense fibrin matrix. Therefore, use of LPRF in rotator cuff repair is theoretically attractive. The aim of the present study was to determine 1) the optimal protocol to achieve the highest leukocyte content; 2) whether L-PRF releases growth factors in a sustained manner over 28 days; 3) whether standard/gelatinous or dry/compressed matrix preparation methods result in higher growth factor concentrations. 1) The standard L-PRF centrifugation protocol with 400 x g showed the highest concentration of platelets and leukocytes. 2) The L-PRF clots cultured in medium showed a continuous slow release with an increase in the absolute release of growth factors TGF-β1, VEGF and MPO in the first 7 days, and for IGF1, PDGF-AB and platelet activity (PF4=CXCL4) in the first 8 hours, followed by a decrease to close to zero at 28 days. Significantly higher levels of growth factor were expressed relative to the control values of normal blood at each culture time point. 3) Except for MPO and the TGFβ-1, there was always a tendency towards higher release of growth factors (i.e., CXCL4, IGF-1, PDGF-AB, and VEGF) in the standard/gelatinous- compared to the dry/compressed group. L-PRF in its optimal standard/gelatinous-type matrix can store and deliver locally specific healing growth factors for up to 28 days and may be a useful adjunct in rotator cuff repair.

Prevention of alloimmunization by ultraviolet-B irradiation. Inactivation of leukocytes and the generation of active oxygen and radicals

International Nuclear Information System (INIS)

Takahashi, Tsuneo; Mogi, Yuko; Sekiguchi, Sadayoshi; Akasaka, Junichi; Kamo, Naoki; Kuwabara, Mikinori.

1994-01-01

UV-B irradiation of platelet concentrates (PC) has been tried in several institutes to inactivate leukocytes in PC and prevent alloimmunization on platelet transfusion. However, the mechanism of inactivation of leukocytes contaminating PC has not been fully understood. It is known that UV-B light is absorbed by photosensitizers in cells and produces active oxygen and radicals, such as singlet oxygen, superioxide anions and hydroxyl radicals. These active oxygen or radicals should injure cellular components and this could cause the suppression of cellular functions. In this study, we investigated the relationships among UV-B irradiation, free radical generation and leukocyte inactivation. We found the evidence that active oxygen and radicals were produced in peripheral blood mononuclear cells by UV-B irradiation. UV-B irradiation suppressed the stimulatory function of leukocytes in a mixed lymphocyte reaction (MLR), and the suppression depended on the dosage of UV-B. Even a low dosage of UV-B, 10 J/m 2 , could inhibit the MLR if the irradiated cells were incubated at 37degC for 24 hours before co-culture with responder cells. Treatments of cells with the exogenous singlet oxygen or superoxide anions also caused suppression of the stimulatory function in the MLR, inhibition of capping formation of HLA-DR antigens, and an increase of intracellular free Ca 2+ levels as did the UV-B treatment. These results indicate that the active oxygen or radicals generated in UV-B-irradiated leukocytes could be one of the causes of leukocyte inactivation. (author0

Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation.

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Jang, Ja-Young; Kim, Tae-Su; Cai, Jingmei; Kim, Jihyun; Kim, Youngeun; Shin, Kyungha; Kim, Kwang Sei; Park, Sung Kyeong; Lee, Sung-Pyo; Choi, Ehn-Kyoung; Rhee, Man Hee; Kim, Yun-Bae

2013-12-01

The effects of nattokinase on the in vitro platelet aggregation and in vivo thrombosis were investigated in comparison with aspirin. Rabbit platelet-rich plasma was incubated with nattokinase and aggregation inducers collagen and thrombin, and the platelet aggregation rate was analyzed. Nattokinase significantly inhibited both the collagen- and thrombin-induced platelet aggregations. Nattokinase also reduced thromboxane B2 formation from collagen-activated platelets in a concentration-dependent manner. Rats were orally administered with nattokinase for 1 week, and their carotid arteries were exposed. Arterial thrombosis was induced by applying 35% FeCl3-soaked filter paper for 10 min, and the blood flow was monitored with a laser Doppler probe. Nattokinase delayed the FeCl3-induced arterial occlusion in a dose-dependent manner, doubling the occlusion time at 160 mg/kg. In addition, a high dose (500 mg/kg) of nattokinase fully prevented the occlusion, as achieved with aspirin (30 mg/kg). The results indicate that nattokinase extracted from fermented soybean inhibit platelet aggregation by blocking thromboxane formation, and thereby delay thrombosis following oxidative arterial wall injury. Therefore, it is suggested that nattokinase could be a good candidate without adverse effects for the improvement of blood flow.

Treatment of endo-periodontal lesion using leukocyte-platelet-rich fibrin. A case report.

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Betancourt, Pablo; Elgueta, Ricardo; Fuentes, Ramon

2017-12-30

The main objective of this paper was to report the clinical effectiveness of leukocyte- platelet- rich fibrin (L-PRF) in the treatment of a combined endo-periodontal lesion of an upper first premolar. The tooth had a profound abfraction on the vestibular aspect and presented no mobility but revealed a deep pocket measuring of 11 mm on the mesial vestibular aspect and 14 mm on the mesial palatine aspect. The three dimensional image analysis showed total bone loss in the mesial aspect and an extensively bone loss of the vestibular aspect of the vestibular root. Endodontic treatment was performed and periodontal access surgery (surgical periodontal therapy) was done with the application of autologous L-PRF. Three month and 6 months after surgery, the cone beam computed tomography (CBCT) exams showed no bone regeneration in any aspect of the tooth. However, periodontal examination showed a significative improvement in the deepness of surcus. The mesial vestibular aspect had a deep pocket of 3 mm and 5 mm on the mesial palatine aspect showing a reduction in deepness of 8 mm and 9 mm, respectively. The actual treatment for teeth with bad prognosis is the extraction and replacement with implants. Even though implants are capable of restore function and aesthetic, the abuse of this approach have led to the loss of teeth that could be successfully treated with a less invasive technique. The prognosis of teeth with endoperiodontal lesion is poor but could be enhanced with regenerative therapies. Until now there are no clinical trials and just four case report about the treatment of these teeth with platelet rich fibrin.

Leukoreduction of platelet concentrates using a 'polishing' filter

NARCIS (Netherlands)

van der Meer, P. F.; Pietersz, R. N.; Reesink, H. W.

2000-01-01

BACKGROUND AND OBJECTIVES: Filters for removal of leukocytes from platelet concentrates (PCs) usually have a large volume to guarantee sufficient leukoreduction. In this study, a small filter, with a volume of only 8 ml and therefore minimal platelet loss, for leukoreduction of PCs was investigated.

Preparation of leukodepleted platelet concentrates from pooled buffy coats: prestorage filtration with Autostop BC

NARCIS (Netherlands)

Pietersz, R. N.; van der Meer, P. F.; Steneker, I.; Hinloopen, B.; Dekker, W. J.; van Zanten, A. P.; Reesink, H. W.

1999-01-01

BACKGROUND AND OBJECTIVES: Our requirements for leukocyte-depleted platelet concentrates (LD-PC) for an adult patient are: platelets >240x10(9), leukocytes <5x10(6), volume of 150-400 ml; and at the end of storage a pH between 6.8 and 7.4 and presence of the swirling effect. Our aim was to develop a

Increased platelet reactivity is associated with circulating platelet-monocyte complexes and macrophages in human atherosclerotic plaques.

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Bert Rutten

Full Text Available Platelet reactivity, platelet binding to monocytes and monocyte infiltration play a detrimental role in atherosclerotic plaque progression. We investigated whether platelet reactivity was associated with levels of circulating platelet-monocyte complexes (PMCs and macrophages in human atherosclerotic carotid plaques.Platelet reactivity was determined by measuring platelet P-selectin expression after platelet stimulation with increasing concentrations of adenosine diphosphate (ADP, in two independent cohorts: the Circulating Cells cohort (n = 244 and the Athero-Express cohort (n = 91. Levels of PMCs were assessed by flow cytometry in blood samples of patients who were scheduled for percutaneous coronary intervention (Circulating Cells cohort. Monocyte infiltration was semi-quantitatively determined by histological examination of atherosclerotic carotid plaques collected during carotid endarterectomy (Athero-Express cohort.We found increased platelet reactivity in patients with high PMCs as compared to patients with low PMCs (median (interquartile range: 4153 (1585-11267 area under the curve (AUC vs. 9633 (3580-21565 AUC, P<0.001. Also, we observed increased platelet reactivity in patients with high macrophage levels in atherosclerotic plaques as compared to patients with low macrophage levels in atherosclerotic plaques (mean ± SD; 8969 ± 3485 AUC vs. 7020 ± 3442 AUC, P = 0.02. All associations remained significant after adjustment for age, sex and use of drugs against platelet activation.Platelet reactivity towards ADP is associated with levels of PMCs and macrophages in human atherosclerotic carotid plaques.

Effect of red blood cells on platelet activation and thrombus formation in tortuous arterioles

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Jennifer K. W. Chesnutt

2013-12-01

Full Text Available Thrombosis is a major contributor to cardiovascular disease, which can lead to myocardial infarction and stroke. Thrombosis may form in tortuous microvessels, which are often seen throughout the human body, but the microscale mechanisms and processes are not well understood. In straight vessels, the presence of red blood cells (RBCs is known to push platelets toward walls, which may affect platelet aggregation and thrombus formation. However in tortuous vessels, the effects of RBC interactions with platelets in thrombosis are largely unknown. Accordingly, the objective of this work was to determine the physical effects of RBCs, platelet size, and vessel tortuosity on platelet activation and thrombus formation in tortuous arterioles. A discrete element computational model was used to simulate the transport, collision, adhesion, aggregation, and shear-induced platelet activation of hundreds of individual platelets and RBCs in thrombus formation in tortuous arterioles. Results showed that high shear stress near the inner sides of curved arteriole walls activated platelets to initiate thrombosis. RBCs initially promoted platelet activation, but then collisions of RBCs with mural thrombi reduced the amount of mural thrombus and the size of emboli. In the absence of RBCs, mural thrombus mass was smaller in a highly tortuous arteriole compared to a less tortuous arteriole. In the presence of RBCs however, mural thrombus mass was larger in the highly tortuous arteriole compared to the less tortuous arteriole. As well, smaller platelet size yielded less mural thrombus mass and smaller emboli, either with or without RBCs. This study shed light on microscopic interactions of RBCs and platelets in tortuous microvessels, which have implications in various pathologies associated with thrombosis and bleeding.

Functional divergence of platelet protein kinase C (PKC) isoforms in thrombus formation on collagen.

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Gilio, Karen; Harper, Matthew T; Cosemans, Judith M E M; Konopatskaya, Olga; Munnix, Imke C A; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D; Heemskerk, Johan W M; Poole, Alastair W

2010-07-23

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent alpha-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCalpha and PKCbeta, whereas the novel isoform, PKC, negatively regulates these events. PKCdelta also negatively regulates thrombus formation but not alpha-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCalpha or PKCbeta showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKC. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen*

Science.gov (United States)

Gilio, Karen; Harper, Matthew T.; Cosemans, Judith M. E. M.; Konopatskaya, Olga; Munnix, Imke C. A.; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D.; Heemskerk, Johan W. M.; Poole, Alastair W.

2010-01-01

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen. PMID:20479008

Platelets and white blood cells in acute coronary syndromes

NARCIS (Netherlands)

Smit, Jaap Jan Johannes

2008-01-01

In this thesis, we have studied the role of leukocytes and platelets as methods to measure platelets aggregation, in the clinical management of presenting with acute coronary syndromes. We have tried to incidence and to identify predictors of adverse cardiac events with function tests or

Crosstalk between Platelets and the Immune System: Old Systems with New Discoveries

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Conglei Li

2012-01-01

Full Text Available Platelets are small anucleate cells circulating in the blood. It has been recognized for more than 100 years that platelet adhesion and aggregation at the site of vascular injury are critical events in hemostasis and thrombosis; however, recent studies demonstrated that, in addition to these classic roles, platelets also have important functions in inflammation and the immune response. Platelets contain many proinflammatory molecules and cytokines (e.g., P-selectin, CD40L, IL-1β, etc., which support leukocyte trafficking, modulate immunoglobulin class switch, and germinal center formation. Platelets express several functional Toll-like receptors (TLRs, such as TLR-2, TLR-4, and TLR-9, which may potentially link innate immunity with thrombosis. Interestingly, platelets also contain multiple anti-inflammatory molecules and cytokines (e.g., transforming growth factor-β and thrombospondin-1. Emerging evidence also suggests that platelets are involved in lymphatic vessel development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2. Besides the active contributions of platelets to the immune system, platelets are passively targeted in several immune-mediated diseases, such as autoimmune thrombocytopenia, infection-associated thrombocytopenia, and fetal and neonatal alloimmune thrombocytopenia. These data suggest that platelets are important immune cells and may contribute to innate and adaptive immunity under both physiological and pathological conditions.

Platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice

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Suidan, Georgette L.; Demers, Melanie; Herr, Nadine; Carbo, Carla; Brill, Alexander; Cifuni, Stephen M.; Mauler, Maximilian; Cicko, Sanja; Bader, Michael; Idzko, Marco; Bode, Christoph

2013-01-01

The majority of peripheral serotonin is stored in platelets, which secrete it on activation. Serotonin releases Weibel-Palade bodies (WPBs) and we asked whether absence of platelet serotonin affects neutrophil recruitment in inflammatory responses. Tryptophan hydroxylase (Tph)1–deficient mice, lacking non-neuronal serotonin, showed mild leukocytosis compared with wild-type (WT), primarily driven by an elevated neutrophil count. Despite this, 50% fewer leukocytes rolled on unstimulated mesenteric venous endothelium of Tph1−/− mice. The velocity of rolling leukocytes was higher in Tph1−/− mice, indicating fewer selectin-mediated interactions with endothelium. Stimulation of endothelium with histamine, a secretagogue of WPBs, or injection of serotonin normalized the rolling in Tph1−/− mice. Diminished rolling in Tph1−/− mice resulted in reduced firm adhesion of leukocytes after lipopolysaccharide treatment. Blocking platelet serotonin uptake with fluoxetine in WT mice reduced serum serotonin by > 80% and similarly reduced leukocyte rolling and adhesion. Four hours after inflammatory stimulation, neutrophil extravasation into lung, peritoneum, and skin wounds was reduced in Tph1−/− mice, whereas in vitro neutrophil chemotaxis was independent of serotonin. Survival of lipopolysaccharide-induced endotoxic shock was improved in Tph1−/− mice. In conclusion, platelet serotonin promotes the recruitment of neutrophils in acute inflammation, supporting an important role for platelet serotonin in innate immunity. PMID:23243271

Factor H C-Terminal Domains Are Critical for Regulation of Platelet/Granulocyte Aggregate Formation

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Adam Z. Blatt

2017-11-01

Full Text Available Platelet/granulocyte aggregates (PGAs increase thromboinflammation in the vasculature, and PGA formation is tightly controlled by the complement alternative pathway (AP negative regulator, Factor H (FH. Mutations in FH are associated with the prothrombotic disease atypical hemolytic uremic syndrome (aHUS, yet it is unknown whether increased PGA formation contributes to the thrombosis seen in patients with aHUS. Here, flow cytometry assays were used to evaluate the effects of aHUS-related mutations on FH regulation of PGA formation and characterize the mechanism. Utilizing recombinant fragments of FH spanning the entire length of the protein, we mapped the regions of FH most critical for limiting AP activity on the surface of isolated human platelets and neutrophils, as well as the regions most critical for regulating PGA formation in human whole blood stimulated with thrombin receptor-activating peptide (TRAP. FH domains 19–20 were the most critical for limiting AP activity on platelets, neutrophils, and at the platelet/granulocyte interface. The role of FH in PGA formation was attributed to its ability to regulate AP-mediated C5a generation. AHUS-related mutations in domains 19–20 caused differential effects on control of PGA formation and AP activity on platelets and neutrophils. Our data indicate FH C-terminal domains are key for regulating PGA formation, thus increased FH protection may have a beneficial impact on diseases characterized by increased PGA formation, such as cardiovascular disease. Additionally, aHUS-related mutations in domains 19–20 have varying effects on control of TRAP-mediated PGA formation, suggesting that some, but not all, aHUS-related mutations may cause increased PGA formation that contributes to excessive thrombosis in patients with aHUS.

Mechanobiology of Platelets: Techniques to Study the Role of Fluid Flow and Platelet Retraction Forces at the Micro- and Nano-Scale

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Nathan J. Sniadecki

2011-12-01

Full Text Available Coagulation involves a complex set of events that are important in maintaining hemostasis. Biochemical interactions are classically known to regulate the hemostatic process, but recent evidence has revealed that mechanical interactions between platelets and their surroundings can also play a substantial role. Investigations into platelet mechanobiology have been challenging however, due to the small dimensions of platelets and their glycoprotein receptors. Platelet researchers have recently turned to microfabricated devices to control these physical, nanometer-scale interactions with a higher degree of precision. These approaches have enabled exciting, new insights into the molecular and biomechanical factors that affect platelets in clot formation. In this review, we highlight the new tools used to understand platelet mechanobiology and the roles of adhesion, shear flow, and retraction forces in clot formation.

Delayed-onset of procoagulant signalling revealed by kinetic analysis of COAT platelet formation.

Science.gov (United States)

Alberio, Lorenzo; Ravanat, Catherine; Hechler, Béatrice; Mangin, Pierre H; Lanza, François; Gachet, Christian

2017-06-02

The combined action of collagen and thrombin induces the formation of COAT platelets, which are characterised by a coat of procoagulant and adhesive molecules on their surface. Although recent work has started to highlight their clinical relevance, the exact mechanisms regulating the formation of procoagulant COAT platelets remain unclear. Therefore, we employed flow cytometry in order to visualise in real time surface and intracellular events following simultaneous platelet activation with convulxin and thrombin. After a rapid initial response pattern characterised by the homogenous activation of the fibrinogen receptor glycoprotein IIb/IIIa in all platelets, starting with a delay of about 2 minutes an increasing fraction transforms to procoagulant COAT platelets. Their surface is characterised by progressive loss of PAC-1 binding, expression of negative phospholipids and retention of α-granule von Willebrand factor. Intracellular events in procoagulant COAT platelets are a marked increase of free calcium into the low micromolar range, concomitantly with early depolarisation of the mitochondrial membrane and activation of caspase-3, while non-COAT platelets keep the intracellular free calcium in the nanomolar range and maintain an intact mitochondrial membrane. We show for the first time that the flow-cytometrically distinct fractions of COAT and non-COAT platelets differentially phosphorylate two signalling proteins, PKCα and p38MAPK, which may be involved in the regulation of the different calcium fluxes observed in COAT versus non-COAT platelets. This study demonstrates the utility of concomitant cellular and signalling evaluation using flow cytometry in order to further dissect the mechanisms underlying the dichotomous platelet response observed after collagen/thrombin stimulation.

Uptake of apoptotic leukocytes by synovial lining macrophages inhibits immune complex-mediated arthritis.

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van Lent, P L; Licht, R; Dijkman, H; Holthuysen, A E; Berden, J H; van den Berg, W B

2001-11-01

Previously we have shown that synovial lining macrophages (SLMs) determine the onset of experimental immune complex-mediated arthritis (ICA). During joint inflammation, many leukocytes undergo apoptosis, and removal of leukocytes by SLMs may regulate resolution of inflammation. In this study we investigated binding and uptake of apoptotic leukocytes by SLMs and its impact on the onset of murine experimental arthritis. We used an in vitro model to evaluate phagocytosis of apoptotic cells on chemotaxis. Phagocytosis of apoptotic thymocytes resulted in a significant decrease (58%) of chemotactic activity for polymorphonuclear neutrophils (PMNs). If apoptotic cells were injected directly into a normal murine knee joint, SLMs resulted in a prominent uptake of cells. After ICA induction, electron micrographs showed that apoptotic leukocytes were evidently present in SLMs on days 1 and 2. Injection of apoptotic leukocytes into the knee joint 1 h before induction of ICA significantly inhibited PMN infiltration into the knee joint at 24 h (61% decrease). This study indicates that uptake of apoptotic leukocytes by SLM reduces chemotactic activity and inhibits the onset of experimental arthritis. These findings indicate an important mechanism in the resolution of joint inflammation.

Exposure to acrolein by inhalation causes platelet activation

International Nuclear Information System (INIS)

Sithu, Srinivas D.; Srivastava, Sanjay; Siddiqui, Maqsood A.; Vladykovskaya, Elena; Riggs, Daniel W.; Conklin, Daniel J.; Haberzettl, Petra; O'Toole, Timothy E.; Bhatnagar, Aruni; D'Souza, Stanley E.

2010-01-01

Acrolein is a common air pollutant that is present in high concentrations in wood, cotton, and tobacco smoke, automobile exhaust and industrial waste and emissions. Exposure to acrolein containing environmental pollutants such as tobacco smoke and automobile exhaust has been linked to the activation of the coagulation and hemostasis pathways and thereby to the predisposition of thrombotic events in human. To examine the effects of acrolein on platelets, adult male C57Bl/6 mice were subjected acute (5 ppm for 6 h) or sub-chronic (1 ppm, 6 h/day for 4 days) acrolein inhalation exposures. The acute exposure to acrolein did not cause pulmonary inflammation and oxidative stress, dyslipidemia or induce liver damage or muscle injury. Platelet GSH levels in acrolein-exposed mice were comparable to controls, but acrolein-exposure increased the abundance of protein-acrolein adducts in platelets. Platelets isolated from mice, exposed to both acute and sub-chronic acrolein levels, showed increased ADP-induced platelet aggregation. Exposure to acrolein also led to an increase in the indices of platelet activation such as the formation of platelet-leukocyte aggregates in the blood, plasma PF4 levels, and increased platelet-fibrinogen binding. The bleeding time was decreased in acrolein exposed mice. Plasma levels of PF4 were also increased in mice exposed to environmental tobacco smoke. Similar to inhalation exposure, acrolein feeding to mice also increased platelet activation and established a pro-thrombotic state in mice. Together, our data suggest that acrolein is an important contributing factor to the pro-thrombotic risk in human exposure to pollutants such as tobacco smoke or automobile exhaust, or through dietary consumption.

Exposure to acrolein by inhalation causes platelet activation

Energy Technology Data Exchange (ETDEWEB)

Sithu, Srinivas D [Department of Physiology and Biophysics, University of Louisville, Louisville, KY 40202 (United States); Diabetes and Obesity Center, University of Louisville, Louisville, KY 40202 (United States); Srivastava, Sanjay; Siddiqui, Maqsood A; Vladykovskaya, Elena; Riggs, Daniel W; Conklin, Daniel J; Haberzettl, Petra; O' Toole, Timothy E; Bhatnagar, Aruni [Diabetes and Obesity Center, University of Louisville, Louisville, KY 40202 (United States); D' Souza, Stanley E., E-mail: sedsou01@louisville.ed [Department of Physiology and Biophysics, University of Louisville, Louisville, KY 40202 (United States)

2010-10-15

Acrolein is a common air pollutant that is present in high concentrations in wood, cotton, and tobacco smoke, automobile exhaust and industrial waste and emissions. Exposure to acrolein containing environmental pollutants such as tobacco smoke and automobile exhaust has been linked to the activation of the coagulation and hemostasis pathways and thereby to the predisposition of thrombotic events in human. To examine the effects of acrolein on platelets, adult male C57Bl/6 mice were subjected acute (5 ppm for 6 h) or sub-chronic (1 ppm, 6 h/day for 4 days) acrolein inhalation exposures. The acute exposure to acrolein did not cause pulmonary inflammation and oxidative stress, dyslipidemia or induce liver damage or muscle injury. Platelet GSH levels in acrolein-exposed mice were comparable to controls, but acrolein-exposure increased the abundance of protein-acrolein adducts in platelets. Platelets isolated from mice, exposed to both acute and sub-chronic acrolein levels, showed increased ADP-induced platelet aggregation. Exposure to acrolein also led to an increase in the indices of platelet activation such as the formation of platelet-leukocyte aggregates in the blood, plasma PF4 levels, and increased platelet-fibrinogen binding. The bleeding time was decreased in acrolein exposed mice. Plasma levels of PF4 were also increased in mice exposed to environmental tobacco smoke. Similar to inhalation exposure, acrolein feeding to mice also increased platelet activation and established a pro-thrombotic state in mice. Together, our data suggest that acrolein is an important contributing factor to the pro-thrombotic risk in human exposure to pollutants such as tobacco smoke or automobile exhaust, or through dietary consumption.

RhoG protein regulates platelet granule secretion and thrombus formation in mice.

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Goggs, Robert; Harper, Matthew T; Pope, Robert J; Savage, Joshua S; Williams, Christopher M; Mundell, Stuart J; Heesom, Kate J; Bass, Mark; Mellor, Harry; Poole, Alastair W

2013-11-22

Rho GTPases such as Rac, RhoA, and Cdc42 are vital for normal platelet function, but the role of RhoG in platelets has not been studied. In other cells, RhoG orchestrates processes integral to platelet function, including actin cytoskeletal rearrangement and membrane trafficking. We therefore hypothesized that RhoG would play a critical role in platelets. Here, we show that RhoG is expressed in human and mouse platelets and is activated by both collagen-related peptide (CRP) and thrombin stimulation. We used RhoG(-/-) mice to study the function of RhoG in platelets. Integrin activation and aggregation were reduced in RhoG(-/-) platelets stimulated by CRP, but responses to thrombin were normal. The central defect in RhoG(-/-) platelets was reduced secretion from α-granules, dense granules, and lysosomes following CRP stimulation. The integrin activation and aggregation defects could be rescued by ADP co-stimulation, indicating that they are a consequence of diminished dense granule secretion. Defective dense granule secretion in RhoG(-/-) platelets limited recruitment of additional platelets to growing thrombi in flowing blood in vitro and translated into reduced thrombus formation in vivo. Interestingly, tail bleeding times were normal in RhoG(-/-) mice, suggesting that the functions of RhoG in platelets are particularly relevant to thrombotic disorders.

Importance of Endogenous Fibrinolysis in Platelet Thrombus Formation.

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Gue, Ying X; Gorog, Diana A

2017-08-25

The processes of thrombosis and coagulation are finely regulated by endogenous fibrinolysis maintaining healthy equilibrium. When the balance is altered in favour of platelet activation and/or coagulation, or if endogenous fibrinolysis becomes less efficient, pathological thrombosis can occur. Arterial thrombosis remains a major cause of morbidity and mortality in the world despite advances in medical therapies. The role endogenous fibrinolysis in the pathogenesis of arterial thrombosis has gained increasing attention in recent years as it presents novel ways to prevent and treat existing diseases. In this review article, we discuss the role of endogenous fibrinolysis in platelet thrombus formation, methods of measurement of fibrinolytic activity, its role in predicting cardiovascular diseases and clinical outcomes and future directions.

Exogenous modification of platelet membranes with the omega-3 fatty acids EPA and DHA reduces platelet procoagulant activity and thrombus formation.

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Larson, Mark K; Tormoen, Garth W; Weaver, Lucinda J; Luepke, Kristen J; Patel, Ishan A; Hjelmen, Carl E; Ensz, Nicole M; McComas, Leah S; McCarty, Owen J T

2013-02-01

Several studies have implicated the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in inhibition of normal platelet function, suggesting a role for platelets in EPA- and DHA-mediated cardioprotection. However, it is unclear whether the cardioprotective mechanisms arise from alterations to platelet-platelet, platelet-matrix, or platelet-coagulation factor interactions. Our previous results led us to hypothesize that EPA and DHA alter the ability of platelets to catalyze the generation of thrombin. We tested this hypothesis by exogenously modifying platelet membranes with EPA and DHA, which resulted in compositional changes analogous to increased dietary EPA and DHA intake. Platelets treated with EPA and DHA showed reductions in the rate of thrombin generation and exposure of platelet phosphatidylserine. In addition, treatment of platelets with EPA and DHA decreased thrombus formation and altered the processing of thrombin precursor proteins. Furthermore, treatment of whole blood with EPA and DHA resulted in increased occlusion time and a sharply reduced accumulation of fibrin under flow conditions. These results demonstrate that EPA and DHA inhibit, but do not eliminate, the ability of platelets to catalyze thrombin generation in vitro. The ability of EPA and DHA to reduce the procoagulant function of platelets provides a possible mechanism behind the cardioprotective phenotype in individuals consuming high levels of EPA and DHA.

Sirt3 deficiency does not affect venous thrombosis or NETosis despite mild elevation of intracellular ROS in platelets and neutrophils in mice.

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Hideki Hayashi

Full Text Available Inflammation is a common denominator in chronic diseases of aging. Yet, how inflammation fuels these diseases remains unknown. Neutrophils are the primary leukocytes involved in the early phase of innate immunity and inflammation. As part of their anti-microbial defense, neutrophils form extracellular traps (NETs by releasing decondensed chromatin lined with cytotoxic proteins. NETs have been shown to induce tissue injury and thrombosis. Here, we demonstrated that Sirt3, a nicotinamide adenine dinucleotide (NAD+-dependent protein deacetylase, an enzyme linked to human longevity, was expressed in mouse neutrophils and platelets. Using Sirt3-/- mice as a model of accelerated aging, we investigated the effects of Sirt3 deficiency on NETosis and platelet function, aiming to detect enhancement of thrombosis. More mitochondrial reactive oxygen species (ROS were generated in neutrophils and platelets of Sirt3-/- mice compared to WT, when stimulated with a low concentration of phorbol 12-myristate 13-acetate (PMA and a high concentration of thrombin, respectively. There were no differences in in vitro NETosis, with or without stimulation. Platelet aggregation was mildly augmented in Sirt3-/- mice compared to WT mice, when stimulated with a low concentration of collagen. The effect of Sirt3 deficiency on platelet and neutrophil activation in vivo was examined by the venous thrombosis model of inferior vena cava stenosis. Elevation of plasma DNA concentration was observed after stenosis in both genotypes, but no difference was shown between the two genotypes. The systemic response to thrombosis was enhanced in Sirt3-/- mice with significantly elevated neutrophil count and reduced platelet count. However, no differences were observed in incidence of thrombus formation, thrombus weight and thrombin-antithrombin complex generation between WT and Sirt3-/- mice. We conclude that Sirt3 does not considerably impact NET formation, platelet function, or venous

Effects of leukocyte-platelet rich fibrin on postoperative complications of direct sinus lifting.

Science.gov (United States)

Gurler, Gokhan; Delilbasi, Cagri

2016-08-01

Blood products have been widely used in soft tissue and bone regeneration in oral and maxillofacial surgery. The purpose of this study is to evaluate the effects of leukocyte-platelet rich fibrin (L-PRF) following direct sinus lifting procedure. Twenty-eight patients were included in the study. Direct sinus lifting was performed via lateral window approach under conscious sedation and local anesthesia. Bony window and sinus floor elevation were performed using piezosurgery device. Two groups were formed. In the first group an allogenous bone graft and L-PRF mixture was used as grafting material. The L-PRF membrane was used to close the lateral window. In the second group, only allogenous bone was used for grafting and resorbable collagen membrane was used to close the lateral window. Pain, swelling, sleeping, eating, phonetics, activities of daily living, missed work days and soft tissue healing were evaluated postoperatively. Data of 24 patients were evaluated. Improvements were seen in the studied parameters in the L-PRF group; however, the difference was not significant between the two groups (P>0.05). The use of L-PRF and allogenous bone graft in combination with L-PRF membrane does not significantly improve postoperative complications following direct sinus lifting.

Glaucocalyxin A inhibits platelet activation and thrombus formation preferentially via GPVI signaling pathway.

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Wei Li

Full Text Available Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of glaucocalyxin A (GLA, an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f. var. glaucocalyx (Maxim. Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01 μg/ml, 0.1 μg/ml significantly inhibited platelet aggregation induced by collagen (P<0.001 and CRP (P<0.01, a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cγ2, the signaling events in collagen receptor GPⅥ pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent.

Platelet aggregation and quality control of platelet concentrates produced in the Amazon Blood Bank

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Maria José Dantas Coêlho

2011-01-01

Full Text Available BACKGROUND: The study of platelet aggregation is essential to assess in vitro platelet function by different platelet activation pathways. OBJECTIVE: To assess aggregation and biochemical parameters of random platelet concentrates produced at the Fundação HEMOAM using the quality control tests defined by law. METHODS: Whole blood samples from 80 donors and the respective platelet concentrate units were tested. Platelet concentrates were tested (platelet count, aggregation and pH on days 1, 3 and 5 of storage. Additionally a leukocyte count was done only on day 1 and microbiological tests on day 5 of storage. Collagen and adenosine diphosphate were used as inducing agonists for platelet aggregation testing. RESULTS: Donor whole blood had normal aggregation (aggregation with adenosine diphosphate = 67% and with collagen = 78%. The median aggregation in platelet concentrates with adenosine diphosphate was low throughout storage (18% on day 1, 7% on day 3 and 6% on day 5 and the median aggregation with collagen was normal only on day 1 and low thereafter (54.4% on day 1, 20.5% on day 3 and 9% on day 5. CONCLUSION: Although the results were within the norms required by law, platelet concentrates had low aggregation rates. We suggest the inclusion of a functional assessment test for the quality control of platelet concentrates for a more effective response to platelet replacement therapy.

Platelet Vascular Endothelial Growth Factor is a Potential Mediator of Transfusion-Related Acute Lung Injury.

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Maloney, James P; Ambruso, Daniel R; Voelkel, Norbert F; Silliman, Christopher C

The occurrence of non-hemolytic transfusion reactions is highest with platelet and plasma administration. Some of these reactions are characterized by endothelial leak, especially transfusion related acute lung injury (TRALI). Elevated concentrations of inflammatory mediators secreted by contaminating leukocytes during blood product storage may contribute to such reactions, but platelet-secreted mediators may also contribute. We hypothesized that platelet storage leads to accumulation of the endothelial permeability mediator vascular endothelial growth factor (VEGF), and that intravascular administration of exogenous VEGF leads to extensive binding to its lung receptors. Single donor, leukocyte-reduced apheresis platelet units were sampled over 5 days of storage. VEGF protein content of the centrifuged supernatant was determined by ELISA, and the potential contribution of VEGF from contaminating leukocytes was quantified. Isolated-perfused rat lungs were used to study the uptake of radiolabeled VEGF administered intravascularly, and the effect of unlabeled VEGF on lung leak. There was a time-dependent release of VEGF into the plasma fraction of the platelet concentrates (62 ± 9 pg/ml on day one, 149 ± 23 pg/ml on day 5; mean ± SEM, pproducts.

Platelet-Rich Fibrin Accelerates Skin Wound Healing in Diabetic Mice.

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Ding, Yinjia; Cui, Lei; Zhao, Qiming; Zhang, Weiqiang; Sun, Huafeng; Zheng, Lijun

2017-09-01

Diabetic foot ulcers (DFUs) are associated with an increased risk of secondary infection and amputation. Platelet-rich fibrin (PRF), a platelet and leukocyte concentrate containing several cytokines and growth factors, is known to promote wound healing. However, the effect of PRF on diabetic wound healing has not been adequately investigated. The aim of the study was to investigate the effect of PRF on skin wound healing in a diabetic mouse model. Platelet-rich fibrin was prepared from whole blood of 8 healthy volunteers. Two symmetrical skin wounds per mouse were created on the back of 16 diabetic nude mice. One of the 2 wounds in each mouse was treated with routine dressings (control), whereas the other wound was treated with PRF in addition to routine dressings (test), each for a period of 14 days. Skin wound healing rate was calculated.Use of PRF was associated with significantly improved skin wound healing in diabetic mice. On hematoxylin and eosin and CD31 staining, a significant increase in the number of capillaries and CD31-positive cells was observed, suggesting that PRF may have promoted blood vessel formation in the skin wound. In this study, PRF seemed to accelerate skin wound healing in diabetic mouse models, probably via increased blood vessel formation.

Mechanisms and kinetics for platelet and neutrophil localization in immune complex nephritis

Energy Technology Data Exchange (ETDEWEB)

Johnson, R.J.; Alpers, C.E.; Pruchno, C.; Schulze, M.; Baker, P.J.; Pritzl, P.; Couser, W.G. (Univ. of Washington, Seattle (USA))

1989-11-01

We have previously reported that both neutrophils (PMNs) and platelets mediate proteinuria in a model of subendothelial immune complex (IC) nephritis (GN) in the rat. In order to understand the interaction of PMNs and platelets in this model, we quantitated the uptake of {sup 111}In-labelled platelets in glomeruli and correlated this with the number of PMNs observed histologically at 10 and 30 minutes, 1, 4 and 24 hours following induction of GN. Platelet accumulation was biphasic with a major peak at 10 minutes and a minor peak at four hours. Early platelet accumulation was complement dependent, and PMN-independent. PMN accumulation occurred after the initial platelet influx, peaking at one and four hours, was complement dependent, but was not affected by platelet depletion. Complement depletion significantly reduced proteinuria. This is the first documentation that platelet accumulation in glomeruli in IC GN is complement dependent. In addition, the enhancement of PMN-mediated injury by the platelet in this model does not involve effects of platelets on PMN localization, thus implying a functional interaction between these cells within the glomerulus.

Platelets and their chemokines in atherosclerosis – clinical applications

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Philipp evon Hundelshausen

2014-08-01

Full Text Available The concept of platelets as important players in the process of atherogenesis has become increasingly accepted due to accumulating experimental and clinical evidence. Despite the progress in understanding the molecular details of atherosclerosis, particularly by using animal models, the inflammatory and thrombotic roles of activated platelet s especially in the human system remain difficult to dissect, as often only the complications of atherosclerosis i.e. stroke and myocardial infarction are definable but not the plaque burden.Platelet indices including platelet count and mean platelet volume and soluble mediators released by activated platelets are associated with atherosclerosis. The chemokine CXCL4 has multiple atherogenic activities e.g. altering the differentiation of T cells and macrophages by inhibiting neutrophil and monocyte apoptosis and by increasing the uptake of oxLDL and synergizing with CCL5. CCL5 is released and deposited on endothelium by activated platelets thereby triggering atherogenic monocyte recruitment, which can be attenuated by blocking the corresponding chemokine receptor CCR5. Atheroprotective and plaque stabilizing properties are attributed to CXCL12, which plays an important role in regenerative processes by attracting progenitor cells. Its release from luminal attached platelets accelerates endothelial healing after injury. Platelet surface molecules GPIIb/IIIa, GP1bα, P-selectin, JAM-A and the CD40/CD40L dyade are crucially involved in the interaction with endothelial cells, leukocytes and matrix molecules affecting atherogenesis. Beyond the effects on the arterial inflammatory infiltrate, platelets affect cholesterol metabolism by binding, modifying and endocytosing LDL particles via their scavenger receptors and contribute to the formation of lipid laden macrophages. Current medical therapies for the prevention of atherosclerotic therapies enable the elucidation of mechanisms linking platelets to inflammation

An investigation on platelet transport during thrombus formation at micro-scale stenosis.

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Francisco Javier Tovar-Lopez

Full Text Available This paper reports on an investigation of mass transport of blood cells at micro-scale stenosis where local strain-rate micro-gradients trigger platelet aggregation. Using a microfluidic flow focusing platform we investigate the blood flow streams that principally contribute to platelet aggregation under shear micro-gradient conditions. We demonstrate that relatively thin surface streams located at the channel wall are the primary contributor of platelets to the developing aggregate under shear gradient conditions. Furthermore we delineate a role for red blood cell hydrodynamic lift forces in driving enhanced advection of platelets to the stenosis wall and surface of developing aggregates. We show that this novel microfluidic platform can be effectively used to study the role of mass transport phenomena driving platelet recruitment and aggregate formation and believe that this approach will lead to a greater understanding of the mechanisms underlying shear-gradient dependent discoid platelet aggregation in the context of cardiovascular diseases such as acute coronary syndromes and ischemic stroke.

Platelet Rich Plasma- mechanism of action and clinical applications

OpenAIRE

Cristina N. Cozma; Laura Raducu; Cristian R. Jecan

2016-01-01

Platelet-rich plasma (PRP) is a blood-derived fraction containing high level of platelets, a high concentration of leukocytes and growth factors. PRP therapy has been growing as a viable treatment alternative for a number of clinical applications and has a potential benefit for use in wound healing. Nowadays platelet rich plasma is used in stimulating wound healing in skin and soft tissue ulcerations, accelerating wound healing in diabetic patients and facilitating bone proliferation in ortho...

Cathepsin G-dependent modulation of platelet thrombus formation in vivo by blood neutrophils.

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Nauder Faraday

Full Text Available Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.

Genetic analysis of the role of protein kinase Ctheta in platelet function and thrombus formation.

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Kellie J Hall

2008-09-01

Full Text Available PKCtheta is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCtheta(-/- T cells exhibit reduced activation and PKCtheta(-/- mice are resistant to autoimmune disease, making PKCtheta an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCtheta positively regulates outside-in signalling through integrin alpha(IIbbeta(3 in platelets, the role of PKCtheta in GPVI-dependent signalling and functional activation of platelets has not been assessed.In the present study we assessed static adhesion, cell spreading, granule secretion, integrin alpha(IIbbeta(3 activation and platelet aggregation in washed mouse platelets lacking PKCtheta. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCtheta(-/- platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCtheta positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCtheta(-/- platelets also exhibited markedly enhanced GPVI-dependent alpha-granule secretion, although dense granule secretion was unaffected, suggesting that PKCtheta differentially regulates these two granules. Inside-out regulation of alpha(IIbbeta(3 activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s(-1 was enhanced.These data suggest that PKCtheta is an important negative regulator of thrombus formation on collagen, potentially mediated by alpha-granule secretion and alpha(IIbbeta(3 activation. PKCtheta therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCtheta inhibitors.

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

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Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

2011-07-14

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

Increased platelet count and leucocyte-platelet complex formation in acute symptomatic compared with asymptomatic severe carotid stenosis.

LENUS (Irish Health Repository)

McCabe, D J H

2005-09-01

The risk of stroke in patients with recently symptomatic carotid stenosis is considerably higher than in patients with asymptomatic stenosis. In the present study it was hypothesised that excessive platelet activation might partly contribute to this difference.

Inhibition of the mixed leukocyte reaction by alloantisera in man

International Nuclear Information System (INIS)

Jonker, M.; Leeuwen, A. van; Rood, J.J. van

1977-01-01

The incidence of MLC(mixed leukocyte culture)-inhibiting antibodies was determined in 42 pregnancy sera. MLC's were carried out between the cells from the serum donor and her husband in the presence of nonimmune AB serum and the test serum. Fifty per cent of the sera reduced the MLC response to less than 40% of the control values. Only four sera had lymphocytotoxic activity. The inhibition was strong against the specific immunizor, less strong against random unrelated cells and weak against cells which were SD(serologically defined)-identical with the serum donor. Absorptions with lymphocytes and platelets were carried out. Lymphocytes removed activity in three of the four sera tested. Platelets removed activity from one serum. It was concluded that both anti-LD(lymphocyte defined) and anti-SD antibodies were able to inhibit the MLR (mixed leukocyte reaction) at the stimulator cell level. (author)

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

International Nuclear Information System (INIS)

Nygaard, Gyrid; Herfindal, Lars; Kopperud, Reidun; Aragay, Anna M.; Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune; Selheim, Frode

2014-01-01

Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation

Efficient removal of platelets from peripheral blood progenitor cell products using a novel micro-chip based acoustophoretic platform.

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Josefina Dykes

Full Text Available BACKGROUND: Excessive collection of platelets is an unwanted side effect in current centrifugation-based peripheral blood progenitor cell (PBPC apheresis. We investigated a novel microchip-based acoustophoresis technique, utilizing ultrasonic standing wave forces for the removal of platelets from PBPC products. By applying an acoustic standing wave field onto a continuously flowing cell suspension in a micro channel, cells can be separated from the surrounding media depending on their physical properties. STUDY DESIGN AND METHODS: PBPC samples were obtained from patients (n = 15 and healthy donors (n = 6 and sorted on an acoustophoresis-chip. The acoustic force was set to separate leukocytes from platelets into a target fraction and a waste fraction, respectively. The PBPC samples, the target and the waste fractions were analysed for cell recovery, purity and functionality. RESULTS: The median separation efficiency of leukocytes to the target fraction was 98% whereas platelets were effectively depleted by 89%. PBPC samples and corresponding target fractions were similar in the percentage of CD34+ hematopoetic progenitor/stem cells as well as leukocyte/lymphocyte subset distributions. Median viability was 98%, 98% and 97% in the PBPC samples, the target and the waste fractions, respectively. Results from hematopoietic progenitor cell assays indicated a preserved colony-forming ability post-sorting. Evaluation of platelet activation by P-selectin (CD62P expression revealed a significant increase of CD62P+ platelets in the target (19% and waste fractions (20%, respectively, compared to the PBPC input samples (9%. However, activation was lower when compared to stored blood bank platelet concentrates (48%. CONCLUSION: Acoustophoresis can be utilized to efficiently deplete PBPC samples of platelets, whilst preserving the target stem/progenitor cell and leukocyte cell populations, cell viability and progenitor cell colony-forming ability

Genetic Analysis of the Role of Protein Kinase Cθ in Platelet Function and Thrombus Formation

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Hall, Kellie J.; Harper, Matthew T.; Gilio, Karen; Cosemans, Judith M.; Heemskerk, Johan W. M.; Poole, Alastair W.

2008-01-01

Background PKCθ is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCθ−/− T cells exhibit reduced activation and PKCθ−/− mice are resistant to autoimmune disease, making PKCθ an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCθ positively regulates outside-in signalling through integrin αIIbβ3 in platelets, the role of PKCθ in GPVI-dependent signalling and functional activation of platelets has not been assessed. Methodology/Principal Findings In the present study we assessed static adhesion, cell spreading, granule secretion, integrin αIIbβ3 activation and platelet aggregation in washed mouse platelets lacking PKCθ. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCθ−/− platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCθ positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCθ−/− platelets also exhibited markedly enhanced GPVI-dependent α-granule secretion, although dense granule secretion was unaffected, suggesting that PKCθ differentially regulates these two granules. Inside-out regulation of αIIbβ3 activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s−1) was enhanced. Conclusions/Significance These data suggest that PKCθ is an important negative regulator of thrombus formation on collagen, potentially mediated by α-granule secretion and αIIbβ3 activation. PKCθ therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCθ inhibitors. PMID:18815612

Modulation of protein C activation by histones, platelet factor 4, and heparinoids: new insights into activated protein C formation.

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Kowalska, M Anna; Zhao, Guohua; Zhai, Li; David, George; Marcus, Stephen; Krishnaswamy, Sriram; Poncz, Mortimer

2014-01-01

Histones are detrimental in late sepsis. Both activated protein C (aPC) and heparin can reverse their effect. Here, we investigated whether histones can modulate aPC generation in a manner similar to another positively charged molecule, platelet factor 4, and how heparinoids (unfractionated heparin or oxygen-desulfated unfractionated heparin with marked decrease anticoagulant activity) may modulate this effect. We measured in vitro and in vivo effects of histones, platelet factor 4, and heparinoids on aPC formation, activated partial thromboplastin time, and murine survival. In vitro, histones and platelet factor 4 both affect thrombin/thrombomodulin aPC generation following a bell-shaped curve, with a peak of >5-fold enhancement. Heparinoids shift these curves rightward. Murine aPC generation studies after infusions of histones, platelet factor 4, and heparinoids supported the in vitro data. Importantly, although unfractionated heparin and 2-O, 3-O desulfated heparin both reversed the lethality of high-dose histone infusions, only mice treated with 2-O, 3-O desulfated heparin demonstrated corrected activated partial thromboplastin times and had significant levels of aPC. Our data provide a new contextual model of how histones affect aPC generation, and how heparinoid therapy may be beneficial in sepsis. These studies provide new insights into the complex interactions controlling aPC formation and suggest a novel therapeutic interventional strategy.

Production and characterization of monoclonal antibodies against mink leukocytes

DEFF Research Database (Denmark)

Chen, W.S.; Pedersen, Mikael; Gram-Nielsen, S.

1997-01-01

Three monoclonal antibodies (mAbs) were generated against mink leukocytes. One antibody reacted with all T lymphocytes, one with all monocytes and one had platelet reactivity. Under reducing conditions, the T lymphocyte reactive antibody immunoprecipitated 18 kDa, 23 kDa, 25 kDa and 32-40 kDa pol...

First comparative analysis concerning the plasma platelet contamination during MNC collection.

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Pfeiffer, Hella; Achenbach, Susanne; Strobel, Julian; Zimmermann, Robert; Eckstein, Reinhold; Strasser, Erwin F

2017-08-01

Monocytes can be cultured into dendritic cells with addition of autologous plasma, which is highly prone to platelet contamination due to the apheresis process. Since platelets affect the maturation process of monocytes into dendritic cells and might even lead to a diminished harvest of dendritic cells, it is very important to reduce the platelet contamination. A new collection device (Spectra Optia) was analyzed, compared to two established devices (COM.TEC, Cobe Spectra) and evaluated regarding the potential generation of source plasma. Concurrent plasma collected during leukapheresis was analyzed for residual cell contamination in a prospective study with the new Spectra Optia apheresis device (n=24) and was compared with COM.TEC and Cobe Spectra data (retrospective analysis, n=72). Donor pre-donation counts of platelets were analyzed for their predictive value of contaminating PLTs in plasma harvests. The newest apheresis device showed the lowest residual platelet count of the collected concurrent plasma (median 3.50×10 9 /l) independent of pre-donation counts. The other two devices and sets had a higher platelet contamination. The contamination of the plasma with leukocytes was very low (only 2.0% were higher than 0.5×10 9 /l). This study showed a significant reduction of platelet contamination of the concurrent plasma collected with the new Spectra Optia device. This plasma product with low residual platelets and leukocytes might also be used as plasma for fractionation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platelets release pathogenic serotonin and return to circulation after immune complex-mediated sequestration.

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Cloutier, Nathalie; Allaeys, Isabelle; Marcoux, Genevieve; Machlus, Kellie R; Mailhot, Benoit; Zufferey, Anne; Levesque, Tania; Becker, Yann; Tessandier, Nicolas; Melki, Imene; Zhi, Huiying; Poirier, Guy; Rondina, Matthew T; Italiano, Joseph E; Flamand, Louis; McKenzie, Steven E; Cote, Francine; Nieswandt, Bernhard; Khan, Waliul I; Flick, Matthew J; Newman, Peter J; Lacroix, Steve; Fortin, Paul R; Boilard, Eric

2018-02-13

There is a growing appreciation for the contribution of platelets to immunity; however, our knowledge mostly relies on platelet functions associated with vascular injury and the prevention of bleeding. Circulating immune complexes (ICs) contribute to both chronic and acute inflammation in a multitude of clinical conditions. Herein, we scrutinized platelet responses to systemic ICs in the absence of tissue and endothelial wall injury. Platelet activation by circulating ICs through a mechanism requiring expression of platelet Fcγ receptor IIA resulted in the induction of systemic shock. IC-driven shock was dependent on release of serotonin from platelet-dense granules secondary to platelet outside-in signaling by αIIbβ3 and its ligand fibrinogen. While activated platelets sequestered in the lungs and leaky vasculature of the blood-brain barrier, platelets also sequestered in the absence of shock in mice lacking peripheral serotonin. Unexpectedly, platelets returned to the blood circulation with emptied granules and were thereby ineffective at promoting subsequent systemic shock, although they still underwent sequestration. We propose that in response to circulating ICs, platelets are a crucial mediator of the inflammatory response highly relevant to sepsis, viremia, and anaphylaxis. In addition, platelets recirculate after degranulation and sequestration, demonstrating that in adaptive immunity implicating antibody responses, activated platelets are longer lived than anticipated and may explain platelet count fluctuations in IC-driven diseases.

Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane.

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Dohan Ehrenfest, David M; Del Corso, Marco; Diss, Antoine; Mouhyi, Jaafar; Charrier, Jean-Baptiste

2010-04-01

Platelet-rich fibrin (PRF; Choukroun's technique) is a second-generation platelet concentrate for surgical use. This easy protocol allows the production of leukocyte and platelet-rich fibrin clots and membranes starting from 10-ml blood samples. The purposes of this study were to determine the cell composition and three-dimensional organization of this autologous biomaterial and to evaluate the influence of different collection tubes (dry glass or glass-coated plastic tubes) and compression procedures (forcible or soft) on the final PRF-membrane architecture. After centrifugation, blood analyses were performed on the residual waste plasmatic layers after collecting PRF clots. The PRF clots and membranes were processed for examination by light microscopy and scanning electron microscopy. Approximately 97% of the platelets and >50% of the leukocytes were concentrated in the PRF clot and showed a specific three-dimensional distribution, depending on the centrifugation forces. Platelets and fibrin formed large clusters of coagulation in the first millimeters of the membrane beyond the red blood cell base. The fibrin network was very mature and dense. Moreover, there was no significant difference in the PRF architecture between groups using the different tested collection tubes and compression techniques, even if these two parameters could have influenced the growth factor content and biologic matrix properties. The PRF protocol concentrated most platelets and leukocytes from a blood harvest into a single autologous fibrin biomaterial. This protocol offers reproducible results as long as the main production principles are respected.

Platelet abnormalities in a dog suffering from gangrenous mastitis by Staphylococcus aureus infection.

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Hasegawa, T; Fujii, M; Fukada, T; Tsuji, C; Fujita, T; Goto, Y; Shinjo, T; Ogawa, H

1993-02-01

Severe gangrenous mastitis due to Staphylococcus aureus infection was diagnosed in a 7 year-old intact female beagle which was presented with swelling of mammary glands after dystocia. Leukocytosis (25,200-48,600/microliters), decreased platelets (107,000-179,000/microliters), and abnormal platelet pattern continued during the critical condition. Consistent with platelet pattern, large platelets were observed in the blood smear. The number of leukocytes and platelets rapidly returned to normal during treatment, and the platelet pattern was also restored. The number and pattern of platelet may provide a clue for the evaluation of the clinical condition and/or severity of the lesions in the dog with mastitis.

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

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Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-09-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.

In vitro studies of leukocyte labeling with /sup 99m/Tc

International Nuclear Information System (INIS)

Uchida, T.; Vincent, P.C.

1976-01-01

A method for labeling leukocytes in vitro with /sup 99m/Tc is described. Separated leukocytes are incubated with /sup 99m/Tc, followed by reduction with stannous chloride and washing with acid citrate dextrose solution. Maximum labeling occurs after at least 5 min incubation with pertechnetate, followed by at least 10 min incubation with stannous chloride. Labeling is similar at room temperature and at 37 0 C. The labeled leukocytes are viable, and reutilization of label does not occur in vitro. In acid conditions (pH 5.2), the elution of /sup 99m/Tc from leukocytes is comparable with that of 32 P-diisopropylfluorophosphate, but /sup 99m/Tc elution is greater at pH 7.2 to 7.4. Neutrophils label more heavily with /sup 99m/Tc than do monocytes, lymphocytes, erythrocytes, or platelets

Elevated thrombopoietin in plasma of burned patients without and with sepsis enhances platelet activation.

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Lupia, E; Bosco, O; Mariano, F; Dondi, A E; Goffi, A; Spatola, T; Cuccurullo, A; Tizzani, P; Brondino, G; Stella, M; Montrucchio, G

2009-06-01

Thrombopoietin (TPO) is a humoral growth factor that does not induce platelet aggregation per se, but enhances platelet activation in response to several agonists. Circulating levels of TPO are increased in patients with sepsis and are mainly related to sepsis severity. To investigate the potential contribution of elevated TPO levels in platelet activation during burn injury complicated or not by sepsis. We studied 22 burned patients, 10 without and 12 with sepsis, and 10 healthy subjects. We measured plasma levels of TPO, as well as leukocyte-platelet binding and P-selectin expression. The priming activity of plasma from burned patients or healthy subjects on platelet aggregation and leukocyte-platelet binding, and the role of TPO in these effects were also studied in vitro. Burned patients without and with sepsis showed higher circulating TPO levels and increased monocyte-platelet binding compared with healthy subjects. Moreover, TPO levels, monocyte-platelet binding and P-selectin expression were significantly higher in burned patients with sepsis than in burned patients without sepsis. In vitro, plasma from burned patients without and with sepsis, but not from healthy subjects, primed platelet aggregation, monocyte-platelet binding and platelet P-selectin expression. The effect of plasma from burned patients with sepsis was significantly higher than that of plasma from burned patients without sepsis. An inhibitor of TPO prevented the priming effect of plasma from burned patients. Increased TPO levels may enhance platelet activation during burn injury and sepsis, potentially participating in the pathogenesis of multi-organ failure in these diseases.

The Non-Hemostatic Aspects of Transfused Platelets

Directory of Open Access Journals (Sweden)

Caroline Sut

2018-02-01

Full Text Available Platelets transfusion is a safe process, but during or after the process, the recipient may experience an adverse reaction and occasionally a serious adverse reaction (SAR. In this review, we focus on the inflammatory potential of platelet components (PCs and their involvement in SARs. Recent evidence has highlighted a central role for platelets in the host inflammatory and immune responses. Blood platelets are involved in inflammation and various other aspects of innate immunity through the release of a plethora of immunomodulatory cytokines, chemokines, and associated molecules, collectively termed biological response modifiers that behave like ligands for endothelial and leukocyte receptors and for platelets themselves. The involvement of PCs in SARs—particularly on a critically ill patient’s context—could be related, at least in part, to the inflammatory functions of platelets, acquired during storage lesions. Moreover, we focus on causal link between platelet activation and immune-mediated disorders (transfusion-associated immunomodulation, platelets, polyanions, and bacterial defense and alloimmunization. This is linked to the platelets’ propensity to be activated even in the absence of deliberate stimuli and to the occurrence of time-dependent storage lesions.

The Non-Hemostatic Aspects of Transfused Platelets

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Sut, Caroline; Tariket, Sofiane; Aubron, Cécile; Aloui, Chaker; Hamzeh-Cognasse, Hind; Berthelot, Philippe; Laradi, Sandrine; Greinacher, Andreas; Garraud, Olivier; Cognasse, Fabrice

2018-01-01

Platelets transfusion is a safe process, but during or after the process, the recipient may experience an adverse reaction and occasionally a serious adverse reaction (SAR). In this review, we focus on the inflammatory potential of platelet components (PCs) and their involvement in SARs. Recent evidence has highlighted a central role for platelets in the host inflammatory and immune responses. Blood platelets are involved in inflammation and various other aspects of innate immunity through the release of a plethora of immunomodulatory cytokines, chemokines, and associated molecules, collectively termed biological response modifiers that behave like ligands for endothelial and leukocyte receptors and for platelets themselves. The involvement of PCs in SARs—particularly on a critically ill patient’s context—could be related, at least in part, to the inflammatory functions of platelets, acquired during storage lesions. Moreover, we focus on causal link between platelet activation and immune-mediated disorders (transfusion-associated immunomodulation, platelets, polyanions, and bacterial defense and alloimmunization). This is linked to the platelets’ propensity to be activated even in the absence of deliberate stimuli and to the occurrence of time-dependent storage lesions. PMID:29536007

In vitro study of the role of thrombin in platelet rich plasma (PRP) preparation: utility for gel formation and impact in growth factors release.

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Huber, Stephany Cares; Cunha Júnior, José Luiz Rosenberis; Montalvão, Silmara; da Silva, Letícia Queiroz; Paffaro, Aline Urban; da Silva, Francesca Aparecida Ramos; Rodrigues, Bruno Lima; Lana, José Fabio Santos Duarte; Annichino-Bizzacchi, Joyce Maria

2016-01-01

The use of PRP has been studied for different fields, with promising results in regenerative medicine. Until now, there is no study in the literature evaluating thrombin levels in serum, used as autologous thrombin preparation. Therefore, in the present study we evaluated the role played by different thrombin concentrations in PRP and the impact in the release of growth factors. Also, different activators for PRP gel formation were evaluated. Thrombin levels were measured in different autologous preparations: serum, L-PRP (PRP rich in leukocytes) and T-PRP (thrombin produced through PRP added calcium gluconate). L-PRP was prepared according to the literature, with platelets and leukocytes being quantified. The effect of autologous thrombin associated or not with calcium in PRP gel was determined by measuring the time of gel formation. The relationship between thrombin concentration and release of growth factors was determined by growth factors (PDGF-AA, VEGF and EGF) multiplex analysis. A similar concentration of thrombin was observed in serum, L-PRP and T-PRP (8.13 nM, 8.63 nM and 7.56 nM, respectively) with a high variation between individuals (CV%: 35.07, 43 and 58.42, respectively). T-PRP and serum with calcium chloride showed similar results in time to promote gel formation. The increase of thrombin concentrations (2.66, 8 and 24 nM) did not promote an increase in growth factor release. The technique of using serum as a thrombin source proved to be the most efficient and reproducible for promoting PRP gel formation, with some advantages when compared to other activation methods, as this technique is easier and quicker with no need of consuming part of PRP. Noteworthy, PRP activation using different thrombin concentrations did not promote a higher release of growth factors, appearing not to be necessary when PRP is used as a suspension.

Elemental composition of platelets. Part I. Sampling and sample preparation of platelets for trace-element analysis

International Nuclear Information System (INIS)

Iyengar, G.V.; Borberg, H.; Kasperek, K.; Kiem, J.; Siegers, M.; Feinendegen, L.E.; Gross, R.

1979-01-01

Sampling of platelets for trace-element analysis poses special problems: obtaining adequate sample materials, achieving a sufficient cell purity, preserving viability (integrity), correcting for trapped plasma, and controlling contamination. We used a blood-cell separator for the primary isolation of platelets from blood, and differential centrifugation in natural plasma to further isolate them. The pyrimidopyrimidine RA233 was used as a stabilizer to maintain viability. 131 I-labeled human serum albumin was used to estimate trapped plasma. Contamination was controlled by using five-times-distilled water to simulate donor's blood in the system and by comparing three fractions: the serum, the first portion of the platelet-rich plasma, and the supernatant plasma after the final centrifugation. Neutron activation analysis was used for the elemental analysis. A single differential centrifugation of the platelet-rich plasma from the blood-cell separator at 400 x g for 8 min was optimum (mean mass fractions: erythrocytes/platelets < 5 mg/g and leukocytes/platelets < 20 mg/g). The trapped plasma in the wet platelet samples amounted to about 0.40 g/g. No appreciable contamination from the sampling system was found for the elements Ag, Cd, Co, Cr, Cs, Cu, Fe, Mo, Rb, Sb, Se, and Zn. 2 figures, 3 tables

Antibody-mediated suppression of grafted lymphoma. III. Evaluation of the role of thymic function, non-thymus-derived lymphocytes, macrophages, platelets, and polymorphonuclear leukocytes in syngeneic and allogeneic hosts

International Nuclear Information System (INIS)

Shin, H.S.; Hayden, M.; Langley, S.; Kaliss, N.; Smith, M.R.

1975-01-01

Syngeneic or allogeneic mice pretreated with sublethal whole-body irradiation were rendered incapable of suppressing the growth of grafted tumor cells sensitized with alloantibody. The growth of sensitized tumor cells was suppressed when they were mixed with donor effector cells from mice syngeneic or allogeneic to the recipients and then were inoculated in irradiated recipients. Three donor-host combinations were used to study the suppression of the murine lymphoma 6C3HED indigenous to C3H mice. These were C3H donor cells in C3H recipients, C57BL/6 donor cells in C3H recipients, or C57BL/6 donor cells in C57BL/6 recipients. In all three combinations, macrophages obtained from an inflammatory exudate, exudate lymphocytes not bearing theta antigen, and platelets were, in descending order of effectiveness, consistently active in restoring antibody-mediated suppression of tumor growth in irradiated hosts. Prior irradiation of the transferred lymphocytes somewhat diminished their effectiveness. Freeze-thawed or heat-killed macrophages (but not freeze-thawed platelets or lymphocytes) were effective in restoration. Peripheral blood mononuclear leukocytes and splenic lymphoid cells were not active in the recipients syngeneic to the donor cells but were active in recipients allogeneic to the donor cells. Polymorphonuclear leukocytes isolated from peripheral blood or an inflammatory exudate were not active. Intact thymic function seems unimportant since antibody-mediated suppression took place as effectively in thymectomized mice as in normal controls. (U.S.)

Use of 8-methoxypsoralen and ultraviolet-A pretreated platelet concentrates to prevent alloimmunization against class I major histocompatibility antigens

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Grana, N.H.; Kao, K.J.

1991-01-01

The use of 8-methoxypsoralen (8-MOP) and UV-A irradiation to inactivate contaminating donor leukocytes in platelet concentrates and to prevent primary alloimmunization against donor class I major histocompatibility (MHC) antigens in mice was investigated. CBA/CaH-T6J mice with the H2k haplotype and BALB/cByJ mice with the H2d haplotype were used as donors and recipients, respectively. The mixed leukocyte reaction between these two strains of mice showed that treatment of spleen cells with 500 ng/mL 8-MOP and 5J/cm2 UV-A inhibited 99% of responder and 92% of stimulator function. There was no measurable loss of platelet aggregating activity after the treatment. After two weekly transfusions of platelets without any treatment, 93% of control mice (n = 15) developed anti-H2k antibody. In contrast, only 33% of mice (n = 15) receiving platelets treated with 8-MOP and UV-A became alloimmunized. After six weekly platelet transfusions, all mice became alloimmunized. Nevertheless, the mean titers of anti-H2k antibody in sera of the treated groups were significantly lower than the control groups. One hour posttransfusion recoveries of 51Cr-labeled donor platelets were also higher in mice transfused with the treated platelets. Thus, the pretreatment of platelet concentrates with 8-MOP and UV-A irradiation effectively reduced the alloantigenicity of class I MHC molecules. The implication of this finding in relation to the mechanism by which donor leukocytes allosensitize recipients is discussed

The role of platelets in hemostasis and the effects of snake venom toxins on platelet function.

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de Queiroz, Mayara Ribeiro; de Sousa, Bruna Barbosa; da Cunha Pereira, Déborah Fernanda; Mamede, Carla Cristine Neves; Matias, Mariana Santos; de Morais, Nadia Cristina Gomes; de Oliveira Costa, Júnia; de Oliveira, Fábio

2017-07-01

The human body has a set of physiological processes, known as hemostasis, which keeps the blood fluid and free of clots in normal vessels; in the case of vascular injury, this process induces the local formation of a hemostatic plug, preventing hemorrhage. The hemostatic system in humans presents complex physiological interactions that involve platelets, plasma proteins, endothelial and subendothelial structures. Disequilibrium in the regulatory mechanisms that control the growth and the size of the thrombus is one of the factors that favors the development of diseases related to vascular disorders such as myocardial infarction and stroke, which are among the leading causes of death in the western world. Interfering with platelet function is a strategy for the treatment of thrombotic diseases. Antiplatelet drugs are used mainly in cases related to arterial thrombosis and interfere in the formation of the platelet plug by different mechanisms. Aspirin (acetylsalicylic acid) is the oldest and most widely used antithrombotic drug. Although highly effective in most cases, aspirin has limitations compared to other drugs used in the treatment of homeostatic disorders. For this reason, research related to molecules that interfere with platelet aggregation are of great relevance. In this regard, snake venoms are known to contain a number of molecules that interfere with hemostasis, including platelet function. The mechanisms by which snake venom components inhibit or activate platelet aggregation are varied and can be used as tools for the diagnosis and the treatment of several hemostatic disorders. The aim of this review is to present the role of platelets in hemostasis and the mechanisms by which snake venom toxins interfere with platelet function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of Titanium-prepared Platelet-rich Fibrin Treatment on the ...

African Journals Online (AJOL)

2018-02-07

Feb 7, 2018 ... Defects of Patients with Chronic Periodontitis: A Randomized. Controlled ... Faculty of Dentistry Department of Periodontology, Kirikkale. University ..... using leukocyte and platelet-rich fibrin: A report of a case. Case. Rep Dent ...

Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation

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Kirkby, Nicholas S.; Reed, Daniel M.; Edin, Matthew L.; Rauzi, Francesca; Mataragka, Stefania; Vojnovic, Ivana; Bishop-Bailey, David; Milne, Ginger L.; Longhurst, Hilary; Zeldin, Darryl C.; Mitchell, Jane A.; Warner, Timothy D.

2016-01-01

Eicosanoids are important vascular regulators, but the phospholipase A2 (PLA2) isoforms supporting their production within the cardiovascular system are not fully understood. To address this, we have studied platelets, endothelial cells, and leukocytes from 2 siblings with a homozygous loss-of-function mutation in group IVA cytosolic phospholipase A2 (cPLA2α). Chromatography/mass spectrometry was used to determine levels of a broad range of eicosanoids produced by isolated vascular cells, and in plasma and urine. Eicosanoid release data were paired with studies of cellular function. Absence of cPLA2α almost abolished eicosanoid synthesis in platelets (e.g., thromboxane A2, control 20.5 ± 1.4 ng/ml vs. patient 0.1 ng/ml) and leukocytes [e.g., prostaglandin E2 (PGE2), control 21.9 ± 7.4 ng/ml vs. patient 1.9 ng/ml], and this was associated with impaired platelet activation and enhanced inflammatory responses. cPLA2α-deficient endothelial cells showed reduced, but not absent, formation of prostaglandin I2 (prostacyclin; control 956 ± 422 pg/ml vs. patient 196 pg/ml) and were primed for inflammation. In the urine, prostaglandin metabolites were selectively influenced by cPLA2α deficiency. For example, prostacyclin metabolites were strongly reduced (18.4% of control) in patients lacking cPLA2α, whereas PGE2 metabolites (77.8% of control) were similar to healthy volunteer levels. These studies constitute a definitive account, demonstrating the fundamental role of cPLA2α to eicosanoid formation and cellular responses within the human circulation.—Kirkby, N. S., Reed, D. M., Edin, M. L., Rauzi, F., Mataragka, S., Vojnovic, I., Bishop-Bailey, D., Milne, G. L., Longhurst, H., Zeldin, D. C., Mitchell, J. A., Warner, T. D. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation. PMID:26183771

Leukocyte- and endothelial-derived microparticles: a circulating source for fibrinolysis

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Lacroix, Romaric; Plawinski, Laurent; Robert, Stéphane; Doeuvre, Loïc; Sabatier, Florence; Martinez de Lizarrondo, Sara; Mezzapesa, Anna; Anfosso, Francine; Leroyer, Aurelie S.; Poullin, Pascale; Jourde, Noémie; Njock, Makon-Sébastien; Boulanger, Chantal M.; Anglés-Cano, Eduardo; Dignat-George, Françoise

2012-01-01

Background We recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. Design and Methods Circulating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. Results Circulating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. Conclusions Endothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium. PMID:22733025

Platelet-derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis.

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Wang, Yongzhi; Zhang, Su; Luo, Lingtao; Norström, Eva; Braun, Oscar Ö; Mörgelin, Matthias; Thorlacius, Henrik

2018-02-01

Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis. © 2017 Wiley Periodicals, Inc.

Roles of platelet STIM1 and Orai1 in glycoprotein VI- and thrombin-dependent procoagulant activity and thrombus formation.

Science.gov (United States)

Gilio, Karen; van Kruchten, Roger; Braun, Attila; Berna-Erro, Alejandro; Feijge, Marion A H; Stegner, David; van der Meijden, Paola E J; Kuijpers, Marijke J E; Varga-Szabo, David; Heemskerk, Johan W M; Nieswandt, Bernhard

2010-07-30

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.

Inhibition of nitric oxide synthesis enhances leukocyte rolling and adhesion in human microvasculature

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Hossain Mokarram

2012-07-01

Full Text Available Abstract Background Nitric oxide (NO is a multifunctional signaling molecule that regulates important cellular events in inflammation including leukocyte recruitment. Previous studies have shown that pharmacological inhibition of NO synthesis induces leukocyte recruitment in various in vitro and animal models. However, it is not known whether NO modulation has similar effects on leukocyte-endothelial cell interactions within the human microvasculature. The present study explored the effect of systemic L-NAME treatment on leukocyte recruitment in the SCID-hu mouse model. Methods Human skin xenografts were transplanted in SCID mice to study human leukocyte dynamics in human vasculature. Early events of human leukocyte recruitment in human vasculature were studied using intravital microscopy. NO synthesis was pharmacologically inhibited using NG-nitro-L-arginine methyl ester (L-NAME. Immunohistochemical analysis was performed to elucidate E-selectin expression in human xenograft skin. Human neutrophil-endothelial cell interactions were also studied in an in vitro flow chamber assay system. P- and E-selectin expression on cultured human umbilical vein endothelial cells (HUVECs was measured using ELISA. Platelet-activating factor (PAF synthesis was detected using a TLC-based assay. Results L-NAME treatment significantly enhanced the rolling and adhesion of human leukocytes to the human vasculature. Functional blocking of P- and E-selectins significantly inhibited rolling but not adhesion induced by inhibition of NO synthesis. Systemic L-NAME treatment enhanced E-selectin expression in human xenograft skin. L-NAME treatment significantly enhanced P- and E-selectin expression on HUVECs. L-NAME treatment did not significantly modify neutrophil rolling or adhesion to HUVECs indicating that L-NAME−induced subtle P- and E-selectin expression was insufficient to elicit dynamic neutrophil-HUVEC interactions in vitro. Moreover, synthesis of endothelial

Effects of N-acetyl cysteine on lipid levels and on leukocyte and ...

African Journals Online (AJOL)

Introduction: Many of studies have shown that increased lipid levels play a significant role in the pathogenesis of atherosclerosis after splenectomy. We investigated the effects of N-acetyl cysteine (NAC) on lipid parameters and leukocyte and platelet (PLT) levels following splenectomy. Materials and Methods: 32 Sprague.

Gene targeting implicates Cdc42 GTPase in GPVI and non-GPVI mediated platelet filopodia formation, secretion and aggregation.

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Huzoor Akbar

Full Text Available Cdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in actin cytoskeleton regulation. We have shown previously that Rac1 is involved in regulation of platelet secretion and aggregation. However, the role of Cdc42 in platelet activation remains controversial. This study was undertaken to better understand the role of Cdc42 in platelet activation.We utilized the Mx-cre;Cdc42(lox/lox inducible mice with transient Cdc42 deletion to investigate the involvement of Cdc42 in platelet function. The Cdc42-deficient mice exhibited a significantly reduced platelet count than the matching Cdc42(+/+ mice. Platelets isolated from Cdc42(-/-, as compared to Cdc42(+/+, mice exhibited (a diminished phosphorylation of PAK1/2, an effector molecule of Cdc42, (b inhibition of filopodia formation on immobilized CRP or fibrinogen, (c inhibition of CRP- or thrombin-induced secretion of ATP and release of P-selectin, (d inhibition of CRP, collagen or thrombin induced platelet aggregation, and (e minimal phosphorylation of Akt upon stimulation with CRP or thrombin. The bleeding times were significantly prolonged in Cdc42(-/- mice compared with Cdc42(+/+ mice.Our data demonstrate that Cdc42 is required for platelet filopodia formation, secretion and aggregation and therefore plays a critical role in platelet mediated hemostasis and thrombosis.

FLow-Induced PRotrusions ( FLIPRs) A Platelet-Derived Platform for the Retrieval of Microparticles by Monocytes and Neutrophils

NARCIS (Netherlands)

Tersteeg, Claudia; Heijnen, Harry F.; Eckly, Anita; Pasterkamp, Gerard; Urbanus, Rolf T.; Maas, Coen; Hoefer, Imo E.; Nieuwland, Rienk; Farndale, Richard W.; Gachet, Christian; de Groot, Philip G.; Roest, Mark

2014-01-01

Rationale: Platelets are the most important cells in the primary prevention of blood loss after injury. In addition, platelets are at the interface between circulating leukocytes and the (sub)endothelium regulating inflammatory responses. Objective: Our aim was to study the dynamic process that

Leukocytes and transfusion related adverse events: the effects of leuko-reduction process in the prevention of adverse reactions resulted from the transfusion of blood components: review article

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Ehteramolsadat Hosseini

2017-05-01

Full Text Available Blood transfusion is commonly implemented to manage life and health-threatening conditions on a rapid and short-term basis. Over the years, ongoing technical advances have dramatically improved transfusion medicine to provide more safety and effectiveness. However, transfusion is still complicated with different adverse events that mainly induced by the presence of allogeneic leukocytes in the blood products. Several lines of evidence have shown that leukocytes in blood components are involved in the induction of febrile nonhemolytic transfusion reactions (FNHTRs, HLA alloimmunization and platelet refractoriness as well as the increased risk of the infectious diseases transmitted by leukotropic viruses including cytomegalovirus (CMV, human T-lymphotropic virus (HTLV-I/II and Epstein-Barr virus (EBV. During current decades, introducing various leuko-reduction techniques have shown to be associated with less transfusion related adverse events and improved clinical outcomes. The lower incidence and severity of febrile transfusion reactions; reduced risk of transfusion related transmission of CMV or other leukocyte-associated infections, lowered incidence of alloimmune platelet refractoriness in addition to reducing risk of mortality and morbidity in patients are considered as clinical benefits of leuko-reduced products. Currently, by the use of 3rd and 4th generation of filters, the highest levels of leukoreduction in blood components have been achieved. Filtration techniques have also the advantages of being performed shortly after preparation of components (pre-storage or post-storage even at the patient’s bedside. However, it seems that pre-storage depletion of leukocytes provides better protection than post-storage techniques due to the elimination of leukocyte-derived cytokines effects which are increasingly released during storage. Particularly in platelet products, the earlier depletion of leukocyte also favors less platelet

Catabolism of exogenously supplied thymidine to thymine and dihydrothymine by platelets in human peripheral blood

International Nuclear Information System (INIS)

Pero, R.W.; Johnson, D.; Olsson, A.

1984-01-01

The interference of platelets with the estimation of unscheduled DNA synthesis in human peripheral mononuclear leukocytes following genotoxic exposure was studied. A 96% reduction in the unscheduled DNA synthesis value was achieved by incubating [ 3 H]thymidine with platelet-rich plasma for 5 hr at 37 degrees. Using radioactive thymine-containing compounds, together with quantitative analyses based on thin-layer and ion-exchange chromatographies, we have shown that thymidine was converted to thymine which, in turn, was converted to dihydrothymine in platelet-rich plasma. The enzymes responsible were separated from platelet lysates by gel filtration and were identified as thymidine phosphorylase and dihydrothymine dehydrogenase. The phosphorylase reversibly catalyzed the formation of thymine from thymidine and converted bromodeoxyuridine to bromouracil. The dehydrogenase reversibly catalyzed the interconversion of thymine and dihydrothymine in a reaction dependent on NADP(H), and it was inhibited by diazouracil and by thymine. Nearly all the thymidine-catabolizing activity found in whole blood samples supplied exogenously with thymidine was accounted for by the platelets. Since most genetic toxicological tests that use blood samples do not involve removing platelets from the blood cell cultures, then it is concluded that precautions should be taken in the future to determine the influence of platelets on these test systems. This is particularly true for methods dependent on thymidine pulses such as unscheduled DNA synthesis, or those dependent on bromodeoxyuridine, such as sister chromatid exchanges, since this nucleoside is also a substrate for thymidine phosphorylase

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation*

Science.gov (United States)

Gilio, Karen; van Kruchten, Roger; Braun, Attila; Berna-Erro, Alejandro; Feijge, Marion A. H.; Stegner, David; van der Meijden, Paola E. J.; Kuijpers, Marijke J. E.; Varga-Szabo, David; Heemskerk, Johan W. M.; Nieswandt, Bernhard

2010-01-01

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca2+ entry (SOCE) with Orai1 as principal Ca2+ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca2+ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1−/− and Orai1−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca2+ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2−/− platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca2+ signals of Stim1−/− and Orai1−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1−/− and Orai1−/− platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca2+ entry, inhibited Ca2+ and procoagulant responses even in Stim1−/− and Orai1−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca2+ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca2+ entry and PS exposure, only one relying on STIM1-Orai1 interaction. PMID:20519511

Non-redundant roles of phosphoinositide 3-kinase isoforms alpha and beta in glycoprotein VI-induced platelet signaling and thrombus formation.

Science.gov (United States)

Gilio, Karen; Munnix, Imke C A; Mangin, Pierre; Cosemans, Judith M E M; Feijge, Marion A H; van der Meijden, Paola E J; Olieslagers, Servé; Chrzanowska-Wodnicka, Magdalena B; Lillian, Rivka; Schoenwaelder, Simone; Koyasu, Shigeo; Sage, Stewart O; Jackson, Shaun P; Heemskerk, Johan W M

2009-12-04

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cgamma2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kalpha or -beta strongly and selectively suppressed GPVI-induced Ca(2+) mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cgamma2 by PI3Kalpha/beta. That PI3Kalpha and -beta have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca(2+) increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kalpha/beta was blocked or p85alpha was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kdelta or -gamma. Furthermore, PI3Kalpha/beta, but not PI3Kgamma, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kalpha and -beta isoforms are required for full GPVI-dependent platelet Ca(2+) signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kalpha an interesting new target for anti-platelet therapy.

Comparison of the Mechanical Properties of Early Leukocyte- and Platelet-Rich Fibrin versus PRGF/Endoret Membranes

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Hooman Khorshidi

2016-01-01

Full Text Available Objectives. The mechanical properties of membranes are important factors in the success of treatment and clinical handling. The goal of this study was to compare the mechanical properties of early leukocyte- and platelet-rich fibrin (L-PRF versus PRGF/Endoret membrane. Materials and Methods. In this experimental study, membranes were obtained from 10 healthy male volunteers. After obtaining 20 cc venous blood from each volunteer, 10 cc was used to prepare early L-PRF (group 1 and the rest was used to get a membrane by PRGF-Endoret system (group 2. Tensile loads were applied to specimens using universal testing machine. Tensile strength, stiffness, and toughness of the two groups of membranes were calculated and compared by paired t-test. Results. The mean tensile strength and toughness were higher in group 1 with a significant difference (P0.05. Conclusions. The results showed that early L-PRF membranes had stronger mechanical properties than membranes produced by PRGF-Endoret system. Early L-PRF membranes might have easier clinical handling and could be a more proper scaffold in periodontal regenerative procedures. The real results of the current L-PRF should be in fact much higher than what is reported here.

Comparison of the Mechanical Properties of Early Leukocyte- and Platelet-Rich Fibrin versus PRGF/Endoret Membranes.

Science.gov (United States)

Khorshidi, Hooman; Raoofi, Saeed; Bagheri, Rafat; Banihashemi, Hodasadat

2016-01-01

Objectives. The mechanical properties of membranes are important factors in the success of treatment and clinical handling. The goal of this study was to compare the mechanical properties of early leukocyte- and platelet-rich fibrin (L-PRF) versus PRGF/Endoret membrane. Materials and Methods. In this experimental study, membranes were obtained from 10 healthy male volunteers. After obtaining 20 cc venous blood from each volunteer, 10 cc was used to prepare early L-PRF (group 1) and the rest was used to get a membrane by PRGF-Endoret system (group 2). Tensile loads were applied to specimens using universal testing machine. Tensile strength, stiffness, and toughness of the two groups of membranes were calculated and compared by paired t-test. Results. The mean tensile strength and toughness were higher in group 1 with a significant difference (P 0.05). Conclusions. The results showed that early L-PRF membranes had stronger mechanical properties than membranes produced by PRGF-Endoret system. Early L-PRF membranes might have easier clinical handling and could be a more proper scaffold in periodontal regenerative procedures. The real results of the current L-PRF should be in fact much higher than what is reported here.

Improved Human Erythropoiesis and Platelet Formation in Humanized NSGW41 Mice

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Susann Rahmig

2016-10-01

Full Text Available Human erythro-megakaryopoiesis does not occur in humanized mouse models, preventing the in vivo analysis of human hematopoietic stem cell (HSC differentiation into these lineages in a surrogate host. Here we show that stably engrafted KIT-deficient NOD/SCID Il2rg−/− KitW41/W41 (NSGW41 mice support much improved human erythropoiesis and platelet formation compared with irradiated NSG recipients. Considerable numbers of human erythroblasts and mature thrombocytes are present in the bone marrow and blood, respectively. Morphology, composition, and enucleation capacity of de novo generated human erythroblasts in NSGW41 mice are comparable with those in human bone marrow. Overexpression of human erythropoietin showed no further improvement in human erythrocyte output, but depletion of macrophages led to the appearance of human erythrocytes in the blood. Human erythropoiesis up to normoblasts and platelet formation is fully supported in NSGW41 mice, allowing the analysis of human HSC differentiation into these lineages, the exploration of certain pathophysiologies, and the evaluation of gene therapeutic approaches.

Evolution, current status and advances in application of platelet concentrate in periodontics and implantology.

Science.gov (United States)

Agrawal, Amit Arvind

2017-05-16

Platelet concentrates (PC) [platelet-rich plasma (PRP) and platelet-rich fibrin (PRF)] are frequently used for surgical procedures in medical and dental fields, particularly in oral and maxillofacial surgery, plastic surgery and sports medicine. The objective of all these technologies is to extract all the elements from a blood sample that could be used to improve healing and promote tissue regeneration. Although leukocyte rich and leukocyte poor PRP's have their own place in literature, the importance of non-platelet components in a platelet concentrate remains a mystery. PC have come a long way since its first appearance in 1954 to the T-PRF, A-PRF and i-PRF introduced recently. These PC find varied applications successfully in periodontics and implant dentistry as well. However, the technique of preparation, standing time, transfer process, temperature of centrifuge, vibration, etc ., are the various factors for the mixed results reported in the literature. Until the introduction of a proper classification of terminologies, the PC were known by different names in different countries and by different commercial companies which also created a lot of confusion. This review intends to clarify all these confusion by briefing the exact evolution of PC, their preparation techniques, recent advances and their various clinical and technical aspects and applications.

Effects of an injectable platelet-rich fibrin on osteoblast behavior and bone tissue formation in comparison to platelet-rich plasma.

Science.gov (United States)

Wang, Xuzhu; Zhang, Yufeng; Choukroun, Joseph; Ghanaati, Shahram; Miron, Richard J

2018-01-01

Platelet-rich plasma (PRP) has been utilized for many years as a regenerative agent capable of inducing vascularization of various tissues using blood-derived growth factors. Despite this, drawbacks mostly related to the additional use of anti-coagulants found in PRP have been shown to inhibit the wound healing process. For these reasons, a novel platelet concentrate has recently been developed with no additives by utilizing lower centrifugation speeds. The purpose of this study was therefore to investigate osteoblast behavior of this novel therapy (injectable-platelet-rich fibrin; i-PRF, 100% natural with no additives) when compared to traditional PRP. Human primary osteoblasts were cultured with either i-PRF or PRP and compared to control tissue culture plastic. A live/dead assay, migration assay as well as a cell adhesion/proliferation assay were investigated. Furthermore, osteoblast differentiation was assessed by alkaline phosphatase (ALP), alizarin red and osteocalcin staining, as well as real-time PCR for genes encoding Runx2, ALP, collagen1 and osteocalcin. The results showed that all cells had high survival rates throughout the entire study period irrespective of culture-conditions. While PRP induced a significant 2-fold increase in osteoblast migration, i-PRF demonstrated a 3-fold increase in migration when compared to control tissue-culture plastic and PRP. While no differences were observed for cell attachment, i-PRF induced a significantly higher proliferation rate at three and five days when compared to PRP. Furthermore, i-PRF induced significantly greater ALP staining at 7 days and alizarin red staining at 14 days. A significant increase in mRNA levels of ALP, Runx2 and osteocalcin, as well as immunofluorescent staining of osteocalcin was also observed in the i-PRF group when compared to PRP. In conclusion, the results from the present study favored the use of the naturally-formulated i-PRF when compared to traditional PRP with anti

Storage of platelets: effects associated with high platelet content in platelet storage containers.

Science.gov (United States)

Gulliksson, Hans; Sandgren, Per; Sjödin, Agneta; Hultenby, Kjell

2012-04-01

A major problem associated with platelet storage containers is that some platelet units show a dramatic fall in pH, especially above certain platelet contents. The aim of this study was a detailed investigation of the different in vitro effects occurring when the maximum storage capacity of a platelet container is exceeded as compared to normal storage. Buffy coats were combined in large-volume containers to create primary pools to be split into two equal aliquots for the preparation of platelets (450-520×10(9) platelets/unit) in SSP+ for 7-day storage in two containers (test and reference) with different platelet storage capacity (n=8). Exceeding the maximum storage capacity of the test platelet storage container resulted in immediate negative effects on platelet metabolism and energy supply, but also delayed effects on platelet function, activation and disintegration. Our study gives a very clear indication of the effects in different phases associated with exceeding the maximum storage capacity of platelet containers but throw little additional light on the mechanism initiating those negative effects. The problem appears to be complex and further studies in different media using different storage containers will be needed to understand the mechanisms involved.

Increased platelet activation in early symptomatic versus asymptomatic carotid stenosis and relationship with microembolic status: Results from the Platelets And Carotid Stenosis (PACS) Study.

LENUS (Irish Health Repository)

Kinsella, Ja

2013-04-26

-negative patients (2.8 vs. 2.3%; p=0.0085). DISCUSSION: Recently symptomatic carotid stenosis patients have higher platelet counts (potentially reflecting increased platelet production, mobilisation or reduced clearance) and platelet activation status than asymptomatic patients. MES were more frequently detected in early symptomatic than asymptomatic patients, but the differences between late symptomatic and asymptomatic groups were not significant. Increased lymphocyte-platelet complex formation in recently symptomatic vs. asymptomatic MES-negative patients indicates enhanced platelet activation in this early symptomatic subgroup. Platelet biomarkers, in combination with TCD, have the potential to aid risk-stratification in asymptomatic and symptomatic carotid stenosis patients. This article is protected by copyright. All rights reserved.

Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

Science.gov (United States)

Metcalfe, Clive; Ramasubramoni, Anjana; Pula, Giordano; Harper, Matthew T; Mundell, Stuart J; Coxon, Carmen H

2016-01-01

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.

Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

Directory of Open Access Journals (Sweden)

Clive Metcalfe

Full Text Available Thioredoxin (Trx is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12 to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase. In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb. This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.

The influence of environmental variables on platelet concentration in horse platelet-rich plasma.

Science.gov (United States)

Rinnovati, Riccardo; Romagnoli, Noemi; Gentilini, Fabio; Lambertini, Carlotta; Spadari, Alessandro

2016-07-04

Platelet-rich plasma (PRP) commonly refers to blood products which contain a higher platelet (PLT) concentration as compared to normal plasma. Autologous PRP has been shown to be safe and effective in promoting the natural processes of soft tissue healing or reconstruction in humans and horses. Variability in PLT concentration has been observed in practice between PRP preparations from different patients or from the same individual under different conditions. A change in PLT concentration could modify PRP efficacy in routine applications. The aim of this study was to test the influence of environmental, individual and agonistic variables on the PLT concentration of PRP in horses. Six healthy Standardbred mares were exposed to six different variables with a one-week washout period between variables, and PRP was subsequently obtained from each horse. The variables were time of withdrawal during the day (morning/evening), hydration status (overhydration/dehydration) treatment with anti-inflammatory drugs and training periods on a treadmill. The platelet concentration was significantly higher in horses treated with a non-steroidal anti-inflammatory drug (P = 0.03). The leukocyte concentration increased 2-9 fold with respect to whole blood in the PRP which was obtained after exposure to all the variable considered. Environmental variation in platelet concentration should be taken into consideration during PRP preparation.

Procoagulant expression in platelets and defects leading to clinical disorders.

Science.gov (United States)

Solum, N O

1999-12-01

Hemostasis is a result of interactions between fibrillar structures in the damaged vessel wall, soluble components in plasma, and cellular elements in blood represented mainly by platelets and platelet-derived material. During formation of a platelet plug at the damaged vessel wall, factors IXa and VIIIa form the "tenase" complex, leading to activation of factor X on the surface of activated platelets. Subsequently, factors Xa and Va form the "prothrombinase" complex, which catalyzes the formation of thrombin from prothrombin, leading to fibrin formation. An enhanced expression of negatively charged phosphatidylserine in the outer membrane leaflet resulting from a breakdown of the phospholipid asymmetry is essential for the formation of the procoagulant surface. An ATP-driven and inward-acting aminophospholipid "translocase" and a "floppase" counterbalancing this have been postulated to maintain the dynamic state of phospholipid asymmetry. A phospholipid-nonspecific "scramblase," believed to be responsible for the fast breakdown of the asymmetry during cell activation, has recently been isolated from erythrocytes, cloned, and characterized. An intracellular calcium-binding segment and one or more thioesterified fatty acids are probably of importance for calcium-induced activation of this transporter protein. Cytosolic calcium ions also activate the calcium-dependent protease calpain associated with shedding of microvesicles from the transformed platelet membrane. These are shed with a procoagulant surface and with surface-exposed P-selectin from the alpha-granules. Theoretically, therefore, microvesicles can be involved in both coagulation and inflammation. Scott syndrome is probably caused by a defect in the activation of an otherwise normal scramblase, resulting in a relatively severe bleeding tendency. In Stormorken syndrome, the patients demonstrate a spontaneous surface expression of aminophospholipids. Activated platelets and the presence of procoagulant

The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation.

Science.gov (United States)

Moreira Teixeira, Liliana S; Leijten, Jeroen C H; Wennink, Jos W H; Chatterjea, Anindita G; Feijen, Jan; van Blitterswijk, Clemens A; Dijkstra, Pieter J; Karperien, Marcel

2012-05-01

In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and anti-inflammatory cytokines, but mechanically unstable. We hypothesized that the advantages of these systems may be combined in one hydrogel, which can be easily translated into clinical settings. Platelet lysate was successfully incorporated into Dex-TA polymer solution prior to gelation. After enzymatic crosslinking, rheological and morphological evaluations were performed. Subsequently, the effect of platelet lysate on cell migration, adhesion, proliferation and multi-lineage differentiation was determined. Finally, we evaluated the integration potential of this gel onto osteoarthritis-affected cartilage. The mechanical properties and covalent attachment of Dex-TA to cartilage tissue during in situ gel formation were successfully combined with the advantages of platelet lysate, revealing the potential of this enhanced hydrogel as a cell-free approach. The addition of platelet lysate did not affect the mechanical properties and porosity of Dex-TA hydrogels. Furthermore, platelet lysate derived anabolic growth factors promoted proliferation and triggered chondrogenic differentiation of mesenchymal stromal cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

[Experimental research on the effects of different activators on the formation of platelet-rich gel and the release of bioactive substances in human platelet-rich plasma].

Science.gov (United States)

Yang, Y; Zhang, W; Cheng, B

2017-01-20

Objective: To explore the effects of calcium gluconate and thrombin on the formation of platelet-rich gel (PRG) and the release of bioactive substances in human platelet-rich plasma (PRP) and the clinical significance. Methods: Six healthy blood donors who met the inclusion criteria were recruited in our unit from May to August in 2016. Platelet samples of each donor were collected for preparation of PRP. (1) PRP in the volume of 10 mL was collected from each donor and divided into thrombin activation group (TA, added with 0.5 mL thrombin solution in dose of 100 U/mL) and calcium gluconate activation group (CGA, added with 0.5 mL calcium gluconate solution in dose of 100 g/L) according to the random number table, with 5 mL PRP in each group. Then the PRP of the two groups was activated in water bath at 37 ℃ for 1 h. The formation time of PRG was recorded, and the formation situation of PRG was observed within 1 hour of activation. After being activated for 1 h, one part of PRG was collected to observe the distribution of fibrous protein with HE staining, and another part of PRG was collected to observe platelet ultrastructure under transmission electron microscope (TEM). After being activated for 1 h, the supernatant was collected to determine the content of transforming growth factor β(1, )platelet-derived growth factor BB (PDGF-BB), vascular endothelial growth factor, basic fibroblast growth factor (bFGF), epidermal growth factor, and insulin-like growth factorⅠby enzyme-linked immunosorbent assay. (2) Another 10 mL PRP from each donor was collected and grouped as above, and the platelet suspension was obtained after two times of centrifugation and resuspension with phosphate buffered saline, respectively. And then they were treated with corresponding activator for 1 h as that in experiment (1). Nanoparticle tracking analyzer was used to detect the concentrations of microvesicles with different diameters and total microvesicles derived from platelet. Data

Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus, Prevents Platelet Activation in Human Platelets

Directory of Open Access Journals (Sweden)

Ye-Ming Lee

2012-01-01

Full Text Available Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.. Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca2+]i mobilization, thromboxane A2 formation, hydroxyl radical (OH● formation, and phospholipase C (PLCγ2, protein kinase C (PKC, mitogen-activated protein kinase (MAPK, and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A2 formation, thereby leading to inhibition of [Ca2+]i and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.

Release kinetics of platelet-derived and plasma-derived growth factors from autologous plasma rich in growth factors.

Science.gov (United States)

Anitua, Eduardo; Zalduendo, Mari Mar; Alkhraisat, Mohammad Hamdan; Orive, Gorka

2013-10-01

Many studies have evaluated the biological effects of platelet rich plasma reporting the final outcomes on cell and tissues. However, few studies have dealt with the kinetics of growth factor delivery by plasma rich in growth factors. Venous blood was obtained from three healthy volunteers and processed with PRGF-Endoret technology to prepare autologous plasma rich in growth factors. The gel-like fibrin scaffolds were then incubated in triplicate, in a cell culture medium to monitor the release of PDGF-AB, VEGF, HGF and IGF-I during 8 days of incubation. A leukocyte-platelet rich plasma was prepared employing the same technology and the concentrations of growth factors and interleukin-1β were determined after 24h of incubation. After each period, the medium was collected, fibrin clot was destroyed and the supernatants were stored at -80°C until analysis. The growth factor delivery is diffusion controlled with a rapid initial release by 30% of the bioactive content after 1h of incubation and a steady state release when almost 70% of the growth factor content has been delivered. Autologous fibrin matrix retained almost 30% of the amount of the growth factors after 8 days of incubation. The addition of leukocytes to the formula of platelet rich plasma did not increase the concentration of the growth factors, while it drastically increased the presence of pro-inflammatory IL-1β. Further studies employing an in vitro inflammatory model would be interesting to study the difference in growth factors and pro-inflammatory cytokines between leukocyte-free and leukocyte-rich platelet rich plasma. Copyright © 2013 Elsevier GmbH. All rights reserved.

Anti-platelet activity of water dispersible curcuminoids in rat platelets.

Science.gov (United States)

Maheswaraiah, Anikisetty; Rao, Lingamallu Jaganmohan; Naidu, Kamatham Akhilender

2015-03-01

Curcuminoids are active principle of turmeric with plethora of health beneficial properties. In this study, we have evaluated for the first time the effect of water dispersible curcuminoids on rat platelet aggregation. Curcuminoids (10-30 µg/mL) significantly inhibited platelet aggregation induced by agonists viz., collagen, ADP and arachidonic acid. Curcuminoids were found to be two-fold more potent than curcumin in inhibiting platelet aggregation. Intracellular curcuminoid concentration was relatively higher than curcumin in rat platelets. Curcuminoids significantly attenuated thromboxane A2 , serotonin levels in rat platelets which play an important role in platelet aggregation. Curcuminoid treatment increased nitric oxide (NO) levels in platelets treated with agonists. Curcuminoids inhibited free radicals such as superoxide anion released from activated platelets, which ultimately inhibits platelet aggregation. Further, curcuminoids inhibited 12-lipoxygenase activity and formation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE) in activated rat platelets which regulates platelet aggregation. The results suggest that curcuminoids have remarkable anti-platelet activity by modulating multiple mechanisms involved in platelet aggregation. Thus curcuminoids may have a therapeutic potential to prevent platelet activation related disorders. Copyright © 2015 John Wiley & Sons, Ltd.

Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo

Science.gov (United States)

von Brühl, Marie-Luise; Stark, Konstantin; Steinhart, Alexander; Chandraratne, Sue; Konrad, Ildiko; Lorenz, Michael; Khandoga, Alexander; Tirniceriu, Anca; Coletti, Raffaele; Köllnberger, Maria; Byrne, Robert A.; Laitinen, Iina; Walch, Axel; Brill, Alexander; Pfeiler, Susanne; Manukyan, Davit; Braun, Siegmund; Lange, Philipp; Riegger, Julia; Ware, Jerry; Eckart, Annekathrin; Haidari, Selgai; Rudelius, Martina; Schulz, Christian; Echtler, Katrin; Brinkmann, Volker; Schwaiger, Markus; Preissner, Klaus T.; Wagner, Denisa D.; Mackman, Nigel; Engelmann, Bernd

2012-01-01

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features. PMID:22451716

Chin Symphysis Bone, Allograft, and Platelet-Rich Fibrin: Is the Combination Effective in Repair of Alveolar Cleft?

Science.gov (United States)

Movahedian Attar, Bijan; Naghdi, Navid; Etemadi Sh, Milad; Mehdizadeh, Mojdeh

2017-05-01

Secondary grafting of alveolar defects with iliac crest bone is a common treatment method in cleft patients. The aim of this study was to evaluate the effectiveness of the combination of symphysis bone, allograft, and platelet-rich fibrin in regeneration of alveolar defects compared with iliac bone graft. In this randomized clinical trial, patients with unilateral alveolar defects were divided randomly into two categories. Group A comprised patients in whom the combination of chin symphysis bone plus allogeneic bone material plus leukocyte- and platelet-rich fibrin was considered for treatment. Group B comprised patients in whom iliac bone graft was considered. Cone beam computed tomography before treatment and 1 year postoperatively was used for measurement of bone formation (bone volume in cubic centimeters). The data were analyzed by paired t and χ 2 tests via SPSS software (version 23; IBM, Armonk, NY). P platelet-rich fibrin is a proper combination for bone regeneration in alveolar defects with a small to moderate volume range. Copyright © 2016 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

The differential effects of leukocyte-containing and pure platelet-rich plasma (PRP) on tendon stem/progenitor cells - implications of PRP application for the clinical treatment of tendon injuries.

Science.gov (United States)

Zhou, Yiqin; Zhang, Jianying; Wu, Haishan; Hogan, MaCalus V; Wang, James H-C

2015-09-15

Platelet-rich plasma (PRP) is widely used to treat tendon injuries in clinics. These PRP preparations often contain white blood cells or leukocytes, and the precise cellular effects of leukocyte-rich PRP (L-PRP) on tendons are not well defined. Therefore, in this study, we determined the effects of L-PRP on tendon stem/progenitor cells (TSCs), which play a key role in tendon homeostasis and repair. TSCs isolated from the patellar tendons of rabbits were treated with L-PRP or P-PRP (pure PRP without leukocytes) in vitro, followed by measuring cell proliferation, stem cell marker expression, inflammatory gene expression, and anabolic and catabolic protein expression by using immunostaining, quantitative real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay, respectively. Cell proliferation was induced by both L-PRP and P-PRP in a dose-dependent manner with maximum proliferation at a 10 % PRP dose. Both PRP treatments also induced differentiation of TSCs into active tenocytes. Nevertheless, the two types of PRP largely differed in several effects exerted on TSCs. L-PRP induced predominantly catabolic and inflammatory changes in differentiated tenocytes; its treatment increased the expression of catabolic marker genes, matrix metalloproteinase-1 (MMP-1), MMP-13, interleukin-1beta (IL-1β), IL-6 and tumor necrosis factor-alpha (TNF-α), and their respective protein expression and prostaglandin E2 (PGE 2) production. In contrast, P-PRP mainly induced anabolic changes; that is, P-PRP increased the gene expression of anabolic genes, alpha-smooth muscle actin (α-SMA), collagen types I and III. These findings indicate that, while both L-PRP and P-PRP appear to be "safe" in inducing TSC differentiation into active tenocytes, L-PRP may be detrimental to the healing of injured tendons because it induces catabolic and inflammatory effects on tendon cells and may prolong the effects in healing tendons. On the other hand, when P-PRP is used to

Iliac artery mural thrombus formation. Effect of antiplatelet therapy on 111In-platelet deposition in baboons

International Nuclear Information System (INIS)

Hanson, S.R.; Paxton, L.D.; Harker, L.A.

1986-01-01

To measure the rate, extent, and time course of arterial mural thrombus formation in vivo and to assess the effects of antiplatelet therapy in that setting, we have studied autologous 111 In-platelet deposition induced by experimental iliac artery aneurysms in baboons. Scintillation camera imaging analyses were performed at 1, 24, 48, and 72 hours after implantation of the device. Correction for tissue attenuation was determined by using a small, comparably located 111 In source implanted at the time of surgery. In five animals, 111 In-platelet activity accumulated progressively after device implantation, reaching a maximum after the third day. Repeat image analysis carried out 2 weeks after the surgical procedure also showed progressive accumulation of 111 In-platelets over 3 days but at markedly reduced amounts as compared with the initial study. In five additional animals, treatment with a combination of aspirin and dipyridamole begun 1 hour after surgical implantation reduced 111 In-platelet deposition to negligible levels by the third day. Although platelet survival time was shortened and platelet turnover was reciprocally increased in all operated animals, platelet survival and turnover were not affected by antiplatelet therapy. We conclude that, in contrast to platelet survival and turnover measurements, 111 In-platelet imaging is a reliable and sensitive method for localizing and quantifying focal arterial thrombi and for assessing the effects of antiplatelet therapy

Modulation of VEGF-induced migration and network formation by lymphatic endothelial cells: Roles of platelets and podoplanin.

Science.gov (United States)

Langan, Stacey A; Navarro-Núñez, Leyre; Watson, Steve P; Nash, Gerard B

2017-07-20

Lymphatic endothelial cells (LEC) express the transmembrane receptor podoplanin whose only known endogenous ligand CLEC-2 is found on platelets. Both podoplanin and CLEC-2 are required for normal lymphangiogenesis as mice lacking either protein develop a blood-lymphatic mixing phenotype. We investigated the roles of podoplanin and its interaction with platelets in migration and tube formation by LEC. Addition of platelets or antibody-mediated crosslinking of podoplanin inhibited LEC migration induced by vascular endothelial growth factors (VEGF-A or VEGF-C), but did not modify basal migration or the response to basic fibroblast growth factor or epidermal growth factor. In addition, platelets and podoplanin crosslinking disrupted networks of LEC formed in co-culture with fibroblasts. Depletion of podoplanin in LEC using siRNA negated the pro-migratory effect of VEGF-A and VEGF-C. Inhibition of RhoA or Rho-kinase reduced LEC migration induced by VEGF-C, but had no further effect after crosslinking of podoplanin, suggesting that podoplanin is required for signaling downstream of VEGF-receptors but upstream of RhoA. Together, these data reveal for the first time that podoplanin is an intrinsic specific regulator of VEGF-mediated migration and network formation in LEC and identify crosslinking of podoplanin by platelets or antibodies as mechanisms to modulate this pathway.

Enhanced ex vivo inhibition of platelet function following addition of dipyridamole to aspirin after transient ischaemic attack or ischaemic stroke: first results from the TRinity AntiPlatelet responsiveness (TrAP) study.

LENUS (Irish Health Repository)

Tobin, William Oliver

2012-02-01

Ex vivo dipyridamole \\'non-responsiveness\\' has not been extensively studied in ischaemic cerebrovascular disease. Platelet surface marker expression, leucocyte-platelet complex formation and inhibition of platelet function at high shear stress as detected by the PFA-100(R) Collagen-Adenosine-diphosphate (C-ADP) and Collagen-Epinephrine cartridges was assessed in 52 patients within 4 weeks of transient ischaemic attack (TIA) or ischaemic stroke on aspirin, and then 14 d (14 d) and >90 d (90 d) after adding dipyridamole. A novel definition of \\'Dipyridamole non-responsiveness\\' was used. The median C-ADP closure time increased following addition of dipyridamole, remained elevated at 90 d (P <\\/= 0.03), and was unaffected by aspirin dose. 59% at 14 d and 56% at 90 d were \\'dipyridamole non-responders\\' on the PFA-100. The proportion of non-responders at 14 and 90 d was similar (P= 0.9). Compared with baseline (4.6%), median monocyte-platelet complexes increased at 14 d (5.0%, P= 0.03) and 90 d (4.9%, P= 0.04). Low C-ADP closure times were associated with increased monocyte-platelet complexes at 14 d (r= -0.32, P= 0.02) and 90 d (r= -0.33, P = 0.02). Monocyte-platelet complexes increased in the subgroup of dipyridamole non-responders on the PFA-100 (P<\\/= 0.045), but not in responders (P >\\/= 0.5), at 14 and 90 d versus baseline. Additional inhibition of platelet function has been detected with the PFA-100 when dipyridamole is added to aspirin. Elevated monocyte-platelet complexes may contribute to ex vivo dipyridamole non-responsiveness.

A bioactive molecule in a complex wound healing process: platelet-derived growth factor.

Science.gov (United States)

Kaltalioglu, Kaan; Coskun-Cevher, Sule

2015-08-01

Wound healing is considered to be particularly important after surgical procedures, and the most important wounds related to surgical procedures are incisional, excisional, and punch wounds. Research is ongoing to identify methods to heal non-closed wounds or to accelerate wound healing; however, wound healing is a complex process that includes many biological and physiological events, and it is affected by various local and systemic factors, including diabetes mellitus, infection, ischemia, and aging. Different cell types (such as platelets, macrophages, and neutrophils) release growth factors during the healing process, and platelet-derived growth factor is a particularly important mediator in most stages of wound healing. This review explores the relationship between platelet-derived growth factor and wound healing. © 2014 The International Society of Dermatology.

A novel activating chicken IgY FcR is related to leukocyte receptor complex (LRC)

NARCIS (Netherlands)

Viertlboeck, B.C.; Schmitt, R.; Hanczaruk, M.A.; Crooijmans, R.P.M.A.; Groenen, M.A.M.; Gobel, T.W.

2009-01-01

FcRs have multifaceted roles in the immune system. Chicken FcRs were demonstrated on macrophages decades ago; however, only recently the chicken Ig-like receptor AB1, encoded in the leukocyte receptor complex, was molecularly identified as a high-affinity FcR. The present study was initiated to

Hypothermia and Platelet Dysfunction

National Research Council Canada - National Science Library

Michelson, A

1994-01-01

... (the GPIIb-IIIa complex). Circulating platelets are normally in a resting state and, despite the presence of platelet surface GPIb-IX and GPIIb-IIIa complexes, they bind neither plasma von Willebrand factor nor plasma fibrinogen...

Hypothermia and Platelet Dysfunction

National Research Council Canada - National Science Library

Michelson, Alan

1993-01-01

... (the OPlib-Illa complex).3 Circulating platelets are normally in a resting state and, despite the presence of platelet surface GPTh-IX and OPlib-Illa complexes, they bind neither plasma von Willebrand factor nor plasma fibrinogen...

Comparative Effects of Platelet-Rich Plasma, Platelet Lysate, and Fetal Calf Serum on Mesenchymal Stem Cells.

Science.gov (United States)

Lykov, A P; Bondarenko, N A; Surovtseva, M A; Kim, I I; Poveshchenko, O V; Pokushalov, E A; Konenkov, V I

2017-10-01

We studied the effects of human platelet-rich plasma and platelet lysate on proliferation, migration, and colony-forming properties of rat mesenchymal stem cells. Platelet-rich plasma and platelet lysate stimulated the proliferation, migration, and colony formation of mesenchymal stem cells. A real-time study showed that platelet-rich plasma produces the most potent stimulatory effect, while both platelet-rich plasma and platelet lysate stimulated migration of cells.

DioxolaneA3-phosphatidylethanolamines are generated by human platelets and stimulate neutrophil integrin expression

Directory of Open Access Journals (Sweden)

Maceler Aldrovandi

2017-04-01

Full Text Available Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3. Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1 ng/2×108 and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX, protease-activated receptors (PAR 1 and 4, cytosolic phospholipase A2 (cPLA2, phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.

Metabolism of 5,6-epoxyeicosatrienoic acid by the human platelet. Formation of novel thromboxane analogs.

Science.gov (United States)

Balazy, M

1991-12-15

Radiolabeled cis-(+-)-5,6-epoxyeicosatrienoic acid (5(6)-EpETrE) was incubated with a suspension of isolated human platelets in order to study its metabolic fate. The epoxide slowly disappeared from the suspension and was completely metabolized within 30 min. After extraction and analysis by reverse-phase high performance liquid chromatography, seven metabolites were found. Addition of either indomethacin (0.01 mM, cyclooxygenase inhibitor) or BW755C (0.1 mM, cyclooxygenase/lipoxygenase inhibitor) to the incubations blocked the formation of four and six metabolites, respectively, 1,2-Epoxy-3,3,3-trichloropropane (inhibitor of microsomal epoxide hydrolase) failed to inhibit the formation of 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETrE), a hydrolysis product of the precursor 5(6)-EpETrE. The metabolites were characterized by UV spectroscopy, negative ion chemical ionization liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry and, in one instance, coelution with synthetic standard. Three primary platelet metabolites were structurally determined to be 5,6-epoxy-12-hydroxyeicosatrienoic acid, 5,6-epoxy-12-hydroxyheptadecadienoic acid, and a unique bicyclic metabolite, 5-hydroxy-6,9-epoxy-thromboxane B1, which originated from intramolecular hydrolysis of 5,6-epoxythromboxane-B1. This thromboxane analog was partially separated into stereoisomers and coeluted with the racemic synthetic standard in gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Three other metabolites were characterized as 5,6,12-trihydroxyeicosatrienoic acid, 5,6,12-trihydroxyheptadecadienoic acid, and 5,6-dihydroxythromboxane-B1, and resulted from the hydrolysis of the corresponding epoxides rather than from the metabolism of 5,6-DiHETrE. The latter was not metabolized by platelet cyclooxygenase or lipoxygenase. The biosynthesis of two cyclooxygenase metabolites indicated the formation of unstable 5,6-epoxythromboxane-A1 as an intermediate

Tenocytes, pro-inflammatory cytokines and leukocytes: a relationship?

OpenAIRE

Al-Sadi, Onays; Schulze-Tanzil, Gundula; Kohl, Benjamin; Lohan, Anke; Lemke, Marion; Ertel, Wolfgang; John, Thilo

2012-01-01

Leukocyte derived pro-inflammatory mediators could be involved in tendon healing and scar formation. Hence, the effect of autologous leukocytes (PBMCs, peripheral blood mononuclear cells and neutrophils) on primary rabbit Achilles tenocytes gene expression was tested in insert assisted co-cultures.

LDL oxidation by platelets propagates platelet activation via an oxidative stress-mediated mechanism.

Science.gov (United States)

Carnevale, Roberto; Bartimoccia, Simona; Nocella, Cristina; Di Santo, Serena; Loffredo, Lorenzo; Illuminati, Giulio; Lombardi, Elisabetta; Boz, Valentina; Del Ben, Maria; De Marco, Luigi; Pignatelli, Pasquale; Violi, Francesco

2014-11-01

Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation. Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency. Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately -35% and -25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α-ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47(phox) translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists. Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Characteristics of canine platelet-rich plasma prepared with five commercially available systems.

Science.gov (United States)

Franklin, Samuel P; Garner, Bridget C; Cook, James L

2015-09-01

To characterize platelet-rich plasma (PRP) products obtained from canine blood by use of a variety of commercially available devices. Blood samples from 15 dogs between 18 months and 9 years of age with no concurrent disease, except for osteoarthritis in some dogs. PRP products were produced from blood obtained from each of the 15 dogs by use of each of 5 commercially available PRP-concentrating systems. Complete blood counts were performed on each whole blood sample and PRP product. The degree of platelet, leukocyte, and erythrocyte concentration or reduction for PRP, compared with results for the whole blood sample, was quantified for each dog and summarized for each concentrating system. The various PRP-concentrating systems differed substantially in the amount of blood processed, method of PRP preparation, amount of PRP produced, and platelet, leukocyte, and erythrocyte concentrations or reductions for PRP relative to results for whole blood. The characteristics of PRP products differed considerably. Investigators evaluating the efficacy of PRPs need to specify the characteristics of the product they are assessing. Clinicians should be aware of the data (or lack of data) supporting use of a particular PRP for a specific medical condition.

Platelet 3H-serotonin releasing immune complexes induced by pseudomonas aeruginosa in cystic fibrosis

International Nuclear Information System (INIS)

Permin, H.; Stahl Skov, P.; Norn, S.; Hoeiby, N.; Schioetz, P.O.

1982-01-01

In vitro formation of immune complexes was studied by 3 H-serotonin release from human platelets by P. aeruginosa antigens in the presence of serum from 22 cyctic fibrosis patients, chronically infected with mucoid P. aeruginosa (CF+P) and with a pronounced antibody response against these bacteria, and in 24 patients without P. aeruginosa (CF-P). All CF+P patients responded with 3 H-serotonin release (16-34%), whereas CF-P patients released less than 15%. In the group of CF+P patients the number of P. aeruginosa precipitins was correlated to the serotonin titer. Time courses indicated that 3 H-serotonin release was maximal between 2 and 5 min, and that no further release was observed up to 20 min. There was a gradual increase in 3 H-serotonin release with higher platelet concentrations. The response was not changed by complement inactivation, and fractionation of serum demonstrated that the serotonin release was dependent on the presence of the immunoglobulin fraction. These experiments support the suggestion of a type III reaction being involved in the lung damage in CF+P patients and also suggest a possible involvement of serotonin in the inflammatory reaction during chronic P. aeruginosa lung infection. (author)

Responsiveness of platelets during storage studied with flow cytometry--formation of platelet subpopulations and LAMP-1 as new markers for the platelet storage lesion.

Science.gov (United States)

Södergren, A L; Tynngård, N; Berlin, G; Ramström, S

2016-02-01

Storage lesions may prevent transfused platelets to respond to agonists and arrest bleeding. The aim of this study was to evaluate and quantify the capacity of platelet activation during storage using flow cytometry and new markers of platelet activation. Activation responses of platelets prepared by apheresis were measured on days 1, 5, 7 and 12. In addition, comparisons were made for platelet concentrates stored until swirling was affected. Lysosome-associated membrane protein-1 (LAMP-1), P-selectin and phosphatidylserine (PS) exposure were assessed by flow cytometry on platelets in different subpopulations in resting state or following stimulation with platelet agonists (cross-linked collagen-related peptide (CRP-XL), PAR1- and PAR4-activating peptides). The ability to form subpopulations upon activation was significantly decreased already at day 5 for some agonist combinations. The agonist-induced exposure of PS and LAMP-1 also gradually decreased with time. Spontaneous exposure of P-selectin and PS increased with time, while spontaneous LAMP-1 exposure was unchanged. In addition, agonist-induced LAMP-1 expression clearly discriminated platelet concentrates with reduced swirling from those with retained swirling. This suggests that LAMP-1 could be a good marker to capture changes in activation capacity in stored platelets. The platelet activation potential seen as LAMP-1 exposure and fragmentation into platelet subpopulations is potential sensitive markers for the platelet storage lesion. © 2015 International Society of Blood Transfusion.

Human SolCD39 Inhibits Injury-induced Development of Neointimal Hyperplasia

Science.gov (United States)

Drosopoulos, Joan H. F.; Kraemer, Rosemary; Shen, Hao; Upmacis, Rita K.; Marcus, Aaron J.; Musi, Elgilda

2010-01-01

SUMMARY Blood platelets provide the initial response to vascular endothelial injury, becoming activated as they adhere to the injured site. Activated platelets recruit leukocytes, and initiate proliferation and migration of vascular smooth muscle cells (SMC) within the injured vessel wall, leading to development of neointimal hyperplasia. Endothelial CD39/NTPDase1 and recombinant solCD39 rapidly metabolize nucleotides, including stimulatory ADP released from activated platelets, thereby suppressing additional platelet reactivity. Using a murine model of vascular endothelial injury, we investigated whether circulating human solCD39 could reduce platelet activation and accumulation, thus abating leukocyte infiltration and neointimal formation following vascular damage. Intraperitoneally-administered solCD39 ADPase activity in plasma peaked 1 hr post-injection, with an elimination half-life of 43 hr. Accordingly, mice were administered solCD39 or saline 1 hr prior to vessel injury, then either sacrificed 24 hr post-injury or treated with solCD39 or saline (3X weekly) for an additional 18 days. 24 hr post-injury, solCD39-treated mice displayed a reduction in platelet activation and recruitment, P-selectin expression, and leukocyte accumulation in the arterial lumen. Furthermore, repeated administration of solCD39 modulated the late stage of vascular injury by suppressing leukocyte deposition, macrophage infiltration and SMC proliferation/migration, resulting in abrogation of neointimal thickening. In contrast, injured femoral arteries of saline-injected mice exhibited massive platelet thrombus formation, marked P-selectin expression, and leukocyte infiltration. Pronounced neointimal growth with macrophage and SMC accretion was also observed (intimal-to-medial area ratio 1.56±0.34 at 19 days). Thus, systemic administration of solCD39 profoundly affects injury-induced cellular responses, minimizing platelet deposition and leukocyte recruitment, and suppressing

Multiple alterations of platelet functions dominated by increased secretion in mice lacking Cdc42 in platelets

DEFF Research Database (Denmark)

Pleines, Irina; Eckly, Anita; Elvers, Margitta

2010-01-01

formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form...

Freezing of Apheresis Platelet Concentrates in 6% Dimethyl Sulfoxide: The First Preliminary Study in Turkey

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Soner Yılmaz

2016-03-01

Full Text Available Objective: Transfusion of platelet suspensions is an essential part of patient care for certain clinical indications. In this pioneering study in Turkey, we aimed to assess the in vitro hemostatic functions of platelets after cryopreservation. Materials and Methods: Seven units of platelet concentrates were obtained by apheresis. Each apheresis platelet concentrate (APC was divided into 2 equal volumes and frozen with 6% dimethyl sulfoxide (DMSO. The 14 frozen units of APCs were kept at -80 °C for 1 day. APCs were thawed at 37 °C and diluted either with autologous plasma or 0.9% NaCl. The volume and residual numbers of leukocytes and platelets were tested in both before-freezing and post-thawing periods. Aggregation and thrombin generation tests were used to analyze the in vitro hemostatic functions of platelets. Flow-cytometric analysis was used to assess the presence of frozen treated platelets and their viability. Results: The residual number of leukocytes in both dilution groups was <1x106. The mean platelet recovery rate in the plasma-diluted group (88.1±9.5% was higher than that in the 0.9% NaCl-diluted group (63±10%. These results were compatible with the European Directorate for the Quality of Medicines quality criteria. Expectedly, there was no aggregation response to platelet aggregation test. The mean thrombin generation potential of postthaw APCs was higher in the plasma-diluted group (2411 nmol/L per minute when compared to both the 0.9% NaCl-diluted group (1913 nmol/L per minute and the before-freezing period (1681 nmol/L per minute. The flowcytometric analysis results for the viability of APCs after cryopreservation were 94.9% and 96.6% in the plasma and 0.9% NaCl groups, respectively. Conclusion: Cryopreservation of platelets with 6% DMSO and storage at -80 °C increases their shelf life from 7 days to 2 years. Besides the increase in hemostatic functions of platelets, the cryopreservation process also does not affect their

Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C

NARCIS (Netherlands)

Pietersz, R. N.; Loos, J. A.; Reesink, H. W.

1987-01-01

High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in

Hypersensitivity to thrombin of platelets from hypercholesterolemic rats

International Nuclear Information System (INIS)

Winocour, P.D.; Rand, M.L.; Kinlough-Rathbone, R.L.; Mustard, J.F.

1986-01-01

Hypersensitivity of platelets to thrombin has been associated with hypercholesterolemia. The authors have examined the mechanisms involved in this hypersensitivity. Rats were given diets rich in milk fat and containing added cholesterol and taurocholate to produce hypercholesterolemia (HC) (262 +/- 25 mg%) or added sitosterol as a normocholesterolemic control (NC) (89 +/- 6 mg%). Washed platelets were prelabelled with 14 C-serotonin. In the presence of acetylsalicyclic acid (ASA) (to inhibit thromboxane A 2 (TXA 2 ) formation) and creatine phosphate/creatine phosphokinase (CP/CPK) (to remove released ADP), HC platelets aggregated more (26 +/- 1%) and released more 14 C (9.1 +/- 2.0%) than NC platelets (aggregation: 0%, p 14 C release: 1.5 +/- 0.5%, p 2 formation is involved in the hypersensitivity of HC platelets to thrombin. Total binding of 125 I-thrombin to HC platelets was less than that to NC platelets but HC platelets were smaller and had less protein than NC platelets; the thrombin binding per mg platelet protein was the same for HC and NC platelets, indicating that hypersensitivity to thrombin of HC platelets does not result from increased thrombin binding. Thus, hypersensitivity of HC platelets to thrombin is not due to TXA 2 formation, the action of released ADP or increased thrombin binding

Study of CD69 antigen expression and integrity of leukocyte cellular membrane in stored platelet concentrates following irradiation and treatment with Mirasol® PRT System.

Science.gov (United States)

Lachert, Elżbieta; Woźniak, Jolanta; Antoniewicz-Papis, Jolanta; Krzywdzińska, Agnieszka; Kubis, Jolanta; Mikołowska, Agata; Letowska, Magdalena

2017-01-01

Leukocytes in transfused blood components, particularly residual lymphocytes, have been shown to contribute to the occurrence of various adverse reactions. One of the most severe is transfusionassociated graft versus host disease (TA-GvHD) following transfusion of blood components contaminated with immunocompetent T lymphocytes. Irradiation is a routine method for protection against TA-GvHD. According to the literature, some pathogen reduction methods have also been proven effective for the inactivation of T lymphocytes, and so they may be considered as an alternative to irradiation. Comparison of CD69 antigen expression and the integrity of the leukocyte cellular membrane in stored platelet concentrates (PCs) following irradiation with the Gammacell 3000 Elan (Nordion Inc., Ottawa, Canada) and treatment with the Mirasol® Pathogen Reduction Technology (PRT) System (Terumo BCT, Lakewood, USA). The study included seven experiments. For each experiment we used 3 PCs, for Mirasol® PRT System treatment (M), for Gammacell 3000 Elan irradiation (R), and for the control (C). 7-amino-actinomycin D (7-AAD, Becton Dickinson, Franklin Lakes, USA) permeability was used to determine lymphocyte viability while CD69 antigen expression was the marker of lymphocyte activation. Analyses of 7-AAD and CD69 antigen expression were performed in a FACS Canto I flow cytometer (Becton Dickinson, USA). During 6 storage days, viable lymphocyte count decreased to 28% (p = 0.001) in the Mirasol® PRT System treated PCs and to 65% (p = 0.004) in the irradiated PCs. A statistically significant increase in CD69 expression in the irradiated PCs was observed; 1.3-fold on day 3 and 1.5-fold on day 6. In the Mirasol ® PRT System treated PCs, no statistically significant increase was observed. The in vitro results suggest that the Mirasol® PRT System is as effective as irradiation due to donor leukocyte inactivation capacity.

Complement Activation Induces Neutrophil Adhesion and Neutrophil-Platelet Aggregate Formation on Vascular Endothelial Cells

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Magdalena Riedl

2017-01-01

Discussion: Therefore, our findings of (i neutrophils adhering to complement-activated endothelial cells, (ii the formation of neutrophil-platelet aggregates on endothelial cells, and (iii the ability of aHUS serum to induce similar effects identify a possible role for neutrophils in aHUS manifestation.

Platelet count is associated with cardiovascular disease, cancer and mortality

DEFF Research Database (Denmark)

Vinholt, P J; Hvas, A M; Frederiksen, H

2016-01-01

count (100-450×10(9)/L) and mortality, development of future cardiovascular disease (myocardial infarction, ischaemic stroke, or peripheral vascular disease), venous thromboembolism, bleeding or cancer in the general population. MATERIAL AND METHODS: We conducted a register-based cohort study of 21......,252 adults (≥20years) from the Danish General Suburban Population Study (GESUS). Laboratory results from GESUS were linked to information from national registers regarding morbidity and death. Cox proportional hazard regression was conducted with adjustment for age, sex, smoking status, haemoglobin......, leukocyte count, C-reactive protein and Charlson comorbidity index. RESULTS: We found a U-shaped relationship between mortality and platelet count. Mortality was significantly increased for platelet count 300×10(9)/L. When categorizing platelet count using the interval 201-250×10(9)/L...

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

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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

Responsiveness of platelets during storage studied with flow cytometry - formation of platelet subpopulations and LAMP-1 as new markers for the platelet storage lesion

OpenAIRE

Södergren, Anna; Tynngård, Nahreen; Berlin, Gösta; Ramström, Sofia

2016-01-01

Background and ObjectivesStorage lesions may prevent transfused platelets to respond to agonists and arrest bleeding. The aim of this study was to evaluate and quantify the capacity of platelet activation during storage using flow cytometry and new markers of platelet activation. Materials and MethodsActivation responses of platelets prepared by apheresis were measured on days 1, 5, 7 and 12. In addition, comparisons were made for platelet concentrates stored until swirling was affected. Lyso...

Salivary Platelet Activating Factor Levels in Periodontal Disease

Science.gov (United States)

1991-05-01

toxins may be present (Madri, 1990). In addition, endogenous mediators, such as C5a, may attract polymorphonuclear leukocytes (PMN) to the site of...relatively harmless to many cells in vitro and in vivo with most pathology resulting from release of endogenous mediators from inflammatory cells (Beutler and...and lipids dissolved in pyrogen -free saline containing 0.25% bovine serum albumin (BSA; Miles Laboratories, Elkhart, IN). Rabbit platelets were

Platelet Glycoprotein Ib-IX and Malignancy

Science.gov (United States)

2010-09-01

provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin - rich network produced by coagulation...process and can initiate the formation of a platelet - rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib... Platelet Glycoprotein Ib-IX and Malignancy PRINCIPAL INVESTIGATOR: Jerry Ware, Ph.D

Hemocompatibility studies on a degradable polar hydrophobic ionic polyurethane (D-PHI).

Science.gov (United States)

Brockman, Kathryne S; Kizhakkedathu, Jayachandran N; Santerre, J Paul

2017-01-15

Biomaterial blood compatibility is a complex process that involves four key pathways, including the coagulation cascade, the complement system, platelets, and leukocytes. While many studies have addressed the initial contact of blood with homopolymeric (e.g. Teflon) or simple copolymeric (e.g. Dacron) biomaterials, relatively less attention has been given to investigating blood coagulation with respect to complex copolymeric systems containing well defined and diverse function. The current study sought to assess the hemocompatibility of a complex polyurethane (PU) containing a unique combination of polar, hydrophobic, and ionic domains (D-PHI). This included a whole blood (WB) study, followed by tests on the intrinsic and extrinsic coagulation pathways, complement activation, platelet activation, and an assessment of the effect of leukocytes on platelet-biomaterial interactions. A small increase in blood clot formation was observed on D-PHI in WB; however, there was no significant increase in clotting via the intrinsic coagulation cascade. No significant increase in platelet adhesion and only a very slight increase in platelet activation were observed in comparison to albumin-coated substrates (negative control). D-PHI showed mild complement activation and increased initiation of the extrinsic pathway of coagulation, along with the observation that leukocytes were important in mediating platelet-biomaterial interactions. It is proposed that complement is responsible for activating coagulation by inciting leukocytes to generate tissue factor (TF), which causes extrinsic pathway activation. This low level of blood clotting on D-PHI's surface may be necessary for the beneficial wound healing of vascular constructs that has been previously reported for this material. Understanding the hemocompatibility of devices intended for blood-contacting applications is important for predicting device failure. Hemocompatibility is a complex parameter (affected by at least four

Mean platelet volume, neutrophil to lyphocyte ratio and platelet to lymphocyte ratio in psoriasis

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Mehmet Ünal

2015-06-01

Full Text Available Background and Design: It has been demonstrated that ratio of neutrophil and platelet count systemic inflammation and is associated with prognosis of many cardiovascular diseases, malignates and chronic inflammatory diseases.As far as it is known, there are no studies investigating neutrophil/lymphocyeratio(NLR, platelet/lymphocyte ratio(PLR and mean platelet volume(MPV values together within the context of psoriasis, a chronic and systemic inflammatory disease. Materials and Methods: 320 patients followed up in our polyclinic with psoriasis vulgaris and 200 healthy persons were evaluated in the study. Results: Leukocyte, neutrophil, platelet, MPV, NLR and PLR values in patients with psoriasis were significantly higher, and lymphocyte count, on the other hand, was significantly lower than those of the control group. No significant difference was found between MPV, NLR and PLR values of patients with or without a family history, nail and joint involvement. Conclusions: These parameters may be made use of as cheap and easily applicable methods in predicting which psoriasis patients are under the risk of cardiovascular disease. PLR is a better inflammation marker than MPV and NLR in patients with psoriasis. We did not observe a significant relationship between MPV, NLR and PLR values and such disease characteristics as severity of disease, joint involvement, nail involvement and duration of disease in patients with psoriasis. So, we believe that there is little information on the extent to which MPV,NLR and PLR might be useful regarding these characteristics.

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbβ3 and activated platelets.

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Marcinkiewicz, Cezary; Gerstenhaber, Jonathan A; Sternberg, Mark; Lelkes, Peter I; Feuerstein, Giora

2017-01-01

Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the α IIb β 3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, α IIb β 3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.

Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications.

Science.gov (United States)

Fortunato, Tiago M; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A; Pula, Giordano

2016-05-04

Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs.

Autologous leukocyte-reduced platelet-rich plasma therapy for Achilles tendinopathy induced by collagenase in a rabbit model.

Science.gov (United States)

González, Juan C; López, Catalina; Álvarez, María E; Pérez, Jorge E; Carmona, Jorge U

2016-01-19

Leukocyte-reduced platelet-rich plasma (LR-PRP) is a therapy for tendinopathy of the Achilles tendon (TAT); however, there is scarce information regarding LR-PRP effects in rabbit models of TAT. We compared, at 4 and 12 weeks (w), the LR-PRP and placebo (PBS) effects on ultrasonography, histology and relative gene expression of collagen types I (COL1A1) and III (COL3A1) and vascular endothelial growth factor (VEGF) in 24 rabbits with TAT induced by collagenase. The rabbits (treated with both treatments) were euthanatised after either 4 or 12 w. A healthy group (HG (n = 6)) was included. At 4 and 12 w, the LR-PRP group had a no statistically different histology score to the HG. At w 4, the COL1A1 expression was significantly higher in the LR-PRP group when compared to HG, and the expression of COL3A1 from both LR-PRP and PBS-treated tendons was significantly higher when compared to the HG. At w 12, the expression of COL3A1 remained significantly higher in the PBS group in comparison to the LR-PRP group and the HG. At w 4, the LR-PRP group presented a significantly higher expression of VEGF when compared to the PBS group and the HG. In conclusion, LR-PRP treatment showed regenerative properties in rabbits with TAT.

Demonstration of a specific C3a receptor on guinea pig platelets

International Nuclear Information System (INIS)

Fukuoka, Y.; Hugli, T.E.

1988-01-01

Guinea pig platelets reportedly contain receptors specific for the anaphylatoxin C3a based on both ligand-binding studies and functional responses. A portion of the human 125I-C3a that binds to guinea pig platelets is competitively displaced by excess unlabeled C3a; however, the majority of ligand uptake was nonspecific. Uptake of 125I-C3a by guinea pig platelets is maximal in 1 min, and stimulation of guinea pig platelets by thrombin, ADP, or the Ca2+ ionophore A23187 showed little influence on binding of the ligand. Scatchard analysis indicated that approximately 1200 binding sites for C3a exist per cell with an estimated Kd of 8 x 10(-10) M. Human C3a des Arg also binds to guinea pig platelets, but Scatchard analysis indicated that no specific binding occurred. Because the ligand-binding studies were complicated by high levels of nonspecific uptake, we attempted to chemically cross-link the C3a molecule to a specific component on the platelet surface. Cross-linkage of 125I-C3a to guinea pig platelets with bis(sulfosuccinimidyl)suberate revealed radioactive complexes at 105,000 and 115,000 m.w. on SDS-PAGE gels by autoradiographic analysis. In the presence of excess unlabeled C3a, complex formation was inhibited. No cross-linkage could be demonstrated between the inactive 125I-C3a des Arg and the putative C3a-R on guinea pig platelets. Human C3a, but not C3a des Arg induces serotonin release and aggregation of the guinea pig platelets. Human C3a was unable to induce either serotonin release or promote aggregation of human platelets. Uptake of human 125I-C3a by human platelets was not saturable, and Scatchard analysis was inconclusive. Attempts to cross-link 125I-C3a to components on the surface of human platelets also failed to reveal a ligand-receptor complex. Therefore, we conclude that guinea pig platelets have specific surface receptors to C3a and that human platelets appear devoid of receptors to the anaphylatoxin

Mice with deleted multimerin 1 and alpha-synuclein genes have impaired platelet adhesion and impaired thrombus formation that is corrected by multimerin 1.

Science.gov (United States)

Reheman, Adili; Tasneem, Subia; Ni, Heyu; Hayward, Catherine P M

2010-05-01

Multimerin 1 is a stored platelet and endothelial cell adhesive protein that shows significant conservation. In vitro, multimerin 1 supports platelet adhesion and it also binds to collagen and enhances von Willebrand factor-dependent platelet adhesion to collagen. As selective, multimerin 1 deficient mice have not been generated, we investigated multimerin 1 effects on platelet adhesion using a subpopulation of C57BL/6J mice with tandem deletion of the genes for multimerin 1 and alpha-synuclein, a protein that inhibits alpha-granule release in vitro. We postulated that multimerin 1/alpha-synuclein deficient mice might show impaired platelet adhesive function from multimerin 1 deficiency and increased alpha-granule release from alpha-synuclein deficiency. Platelet function was assessed by intravital microscopy, after ferric chloride injury, using untreated and human multimerin 1-transfused multimerin 1/alpha-synuclein deficient mice, and by in vitro assays of adhesion, aggregation and thrombin-induced P-selectin release. Multimerin 1/alpha-synuclein deficient mice showed impaired platelet adhesion and their defective thrombus formation at sites of vessel injury improved with multimerin 1 transfusion. Although multimerin 1/alpha-synuclein deficient platelets showed increased P-selectin release at low thrombin concentrations, they also showed impaired adhesion to collagen, and attenuated aggregation with thrombin, that improved with added multimerin 1. Our data suggest that multimerin 1 supports platelet adhesive functions and thrombus formation, which will be important to verify by generating and testing selective multimerin 1 deficient mice. Copyright (c) 2010. Published by Elsevier Ltd.

Platelet Rich Plasma- mechanism of action and clinical applications

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Cristina N. Cozma

2016-11-01

Full Text Available Platelet-rich plasma (PRP is a blood-derived fraction containing high level of platelets, a high concentration of leukocytes and growth factors. PRP therapy has been growing as a viable treatment alternative for a number of clinical applications and has a potential benefit for use in wound healing. Nowadays platelet rich plasma is used in stimulating wound healing in skin and soft tissue ulcerations, accelerating wound healing in diabetic patients and facilitating bone proliferation in orthopedic and trauma surgery. It has also applications in maxillofacial surgery, spinal surgery, plastic and esthetic surgery, heart surgery and burns. This review of the literature shows a limited number of studies realized on humans that sustain PRP applications in orthopedic and plastic surgery. As the use of PRP increases, more properly structured clinical studies are necessary to confirm the results and to establish clearly the techniques of preparing, the conditions and the clinical indications of applying this therapy.

Real-time digital imaging of leukocyte-endothelial interaction in ischemia-reperfusion injury (IRI) of the rat cremaster muscle.

Science.gov (United States)

Thiele, Jan R; Goerendt, Kurt; Stark, G Bjoern; Eisenhardt, Steffen U

2012-08-05

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery. Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion. Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor. While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM). Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968. Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality. We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and

In vivo characterization of neutrophil extracellular traps in various organs of a murine sepsis model.

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Koji Tanaka

Full Text Available Neutrophil extracellular traps (NETs represent extracellular microbial trapping and killing. Recently, it has been implicated in thrombogenesis, autoimmune disease, and cancer progression. The aim of this study was to characterize NETs in various organs of a murine sepsis model in vivo and to investigate their associations with platelets, leukocytes, or vascular endothelium. NETs were classified as two distinct forms; cell-free NETs that were released away from neutrophils and anchored NETs that were anchored to neutrophils. Circulating cell-free NETs were characterized as fragmented or cotton-like structures, while anchored NETs were characterized as linear, reticular, membranous, or spot-like structures. In septic mice, both anchored and cell-free NETs were significantly increased in postcapillary venules of the cecum and hepatic sinusoids with increased leukocyte-endothelial interactions. NETs were also observed in both alveolar space and pulmonary capillaries of the lung. The interactions of NETs with platelet aggregates, leukocyte-platelet aggregates or vascular endothelium of arterioles and venules were observed in the microcirculation of septic mice. Microvessel occlusions which may be caused by platelet aggregates or leukocyte-platelet aggregates and heterogeneously decreased blood flow were also observed in septic mice. NETs appeared to be associated with the formation of platelet aggregates or leukocyte-platelet aggregates. These observational findings may suggest the adverse effect of intravascular NETs on the host during a sepsis.

Influence of Leukocyte- and Platelet-Rich Fibrin (L-PRF) in the Healing of Simple Postextraction Sockets: A Split-Mouth Study.

Science.gov (United States)

Marenzi, Gaetano; Riccitiello, Francesco; Tia, Mariano; di Lauro, Alessandro; Sammartino, Gilberto

2015-01-01

The aim of this study was to evaluate the effects of leukocyte- and platelet-rich fibrin (L-PRF) on the pain and soft tissue healing after tooth extractions. Twenty-six patients (9 males and 17 females) were treated with multiple extractions (2 to 8), with a total of 108 extractions. This was an exploratory single blinded randomized clinical trial with a split-mouth design. The pain after the surgery was assessed in each patient by the VAS scale (1 to 10) at intervals of 24-48-72-96 hours. The soft tissue healing was clinically evaluated at 3, 7, 14, and 21 days after surgery by the same examiner surgeon, using the modified Healing Index (4 to 12). The mean value of postextraction pain was 3.2 ± 0.3 in the experimental sides and 4.1 ± 0.1 in the control sides. After 7 days from the extractions, the values of modified Healing Index in the experimental and control groups were, respectively, 4.8 ± 0.6 and 5.1 ± 0.9. The use of L-PRF in postextraction sockets filling can be proposed as a useful procedure in order to manage the postoperative pain and to promote the soft tissue healing process, reducing the early adverse effects of the inflammation.

Effect of Leukocyte-Rich and Platelet-Rich Plasma on Healing of a Horizontal Medial Meniscus Tear in a Rabbit Model

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Kyun Ho Shin

2015-01-01

Full Text Available There are limited reports on the effect of platelet-rich plasma (PRP on meniscus healing. The purpose of this study was to investigate the effect of leukocyte-rich PRP (L-PRP on potential healing of the horizontal medial meniscus tears in a rabbit model. A horizontal medial meniscus tear was created in both knees of nine skeletally mature adult rabbits. Left or right knees were randomly assigned to a L-PRP group, or a control group. 0.5 mL of L-PRP from 10 mL of each rabbit’s whole blood was prepared and injected into the horizontal tears in a L-PRP group. None was applied to the horizontal tears in a control group. The histological assessment of meniscus healing was performed at two, four, and six weeks after surgery. We found that there were no significant differences of quantitative histologic scoring between two groups at 2, 4, and 6 weeks after surgery (p>0.05. This study failed to show the positive effect of single injection of L-PRP on enhancing healing of the horizontal medial meniscus tears in a rabbit model. Single injection of L-PRP into horizontal meniscus tears may not effectively enhance healing of horizontal medial meniscus tears.

Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

International Nuclear Information System (INIS)

Peerschke, E.I.

1991-01-01

Previous studies indicated a correlation between the formation of EDTA-resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA-resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA-resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding

Platelets retain high levels of active plasminogen activator inhibitor 1.

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Helén Brogren

Full Text Available The vascular fibrinolytic system is crucial for spontaneous lysis of blood clots. Plasminogen activator inhibitor 1 (PAI-1, the principal inhibitor of the key fibrinolytic enzyme tissue-type plasminogen activator (tPA, is present in platelets at high concentrations. However, the majority of PAI-1 stored in platelets has been considered to be inactive. Our recent finding (Brogren H, et al. Blood 2004 that PAI-1 de novo synthesized in platelets remained active for over 24 h, suggested that PAI-1 stored in the α-granules might be active to a larger extent than previously reported. To re-evaluate this issue, we performed experiments where the fraction of active PAI-1 was estimated by analyzing the tPA-PAI-1 complex formation. In these experiments platelets were lysed with Triton X-100 in the presence of serial dilutions of tPA and subsequently the tPA-PAI-1 complex was evaluated by Western blot. Also, using a non-immunologic assay, tPA was labeled with (125I, and (125I-tPA and (125I-tPA-PAI-1 was quantified by scintigraphy. Interestingly, both methods demonstrated that the majority (>50% of platelet PAI-1 is active. Further analyses suggested that pre-analytical procedures used in previous studies (sonication or freezing/thawing may have substantially reduced the activity of platelet PAI-1, which has lead to an underestimation of the proportion of active PAI-1. Our in vitro results are more compatible with the role of PAI-1 in clot stabilization as demonstrated in physiological and pathophysiological studies.

Meal-induced platelet activation in diabetes mellitus type 1 or type 2 is related to postprandial insulin rather than glucose levels.

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Spectre, Galia; Stålesen, Ragnhild; Östenson, Claes-Göran; Hjemdahl, Paul

2016-05-01

Postprandial platelet activation was related to postprandial insulin rather than glucose levels in a previous meal insulin study in type 2 diabetes mellitus (T2DM). We therefore compared postprandial platelet activation in type 1 (T1DM) patients without insulin secretion and T2DM patients with high postprandial insulin levels. Patients with T1DM (n=11) and T2DM (n=12) were studied before and 90min after a standardized meal without premeal insulin. Five T1DM patients volunteered for a restudy with their regular premeal insulin. Platelet activation was assessed by flow cytometry, with and without the thromboxane analogue U46619 or ADP, and by whole blood aggregometry (Multiplate®). Effects of insulin (100μU/mL) in vitro were also studied. Before the meal, glucose, insulin and platelet activation markers other than platelet-leukocyte aggregates (PLAs) were similar in T1DM and T2DM; PLAs were higher in T1DM. Postprandial glucose levels increased more markedly in T1DM (to 22.1±1.4 vs. 11.2±0.6mmol/L) while insulin levels increased only in T2DM (from 24.4±4.4 to 68.8±12.3μU/mL). Platelet P-selectin expression, fibrinogen binding and PLA formation stimulated by U46619 were markedly enhanced (approximately doubled) and whole blood aggregation stimulated by U46619 was increased (pinsulin in T1DM patients showed postprandial platelet activation when postprandial insulin levels increased. In vitro insulin mildly activated platelets in both groups. Postprandial platelet activation via the thromboxane pathway is related to postprandial hyperinsulinemia and not to postprandial hyperglycaemia in patients with diabetes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Use of Leukocyte Platelet (L-PRF Rich Fibrin in Diabetic Foot Ulcer with Osteomyelitis (Three Clinical Cases Report

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Alessandro Crisci

2018-04-01

Full Text Available In this study, the use of fibrin rich in leukocytes and platelets (L-PRF was explored to heal osteomyelitis ulcers in a diabetic foot. The goal was to standardize the utilization of L-PRF in patients with osteomyelitis to direct it for healing. L-PRF was obtained autologously from the peripheral blood of the diabetic patients (n = 3 having osteomyelitis and skin lesions for at least six months. The L-PRF and supernatant serum were inserted into the skin lesion to the bone after a surgical debridement. The evolution of lesions over time was analyzed. All three patients showed positivity to the Probe-to-Bone test and Nuclear Magnetic Resonance detected cortico-periosteal thickening and/or outbreaks of spongy cortical osteolysis in adjacency of the ulcer. The infections were caused by Cocci Gram-positive bacteria, such as S. Aureus, S. β-hemolytic, S. Viridans and Bacilli; and Gram-negative such as Pseudomonas, Proteus, Enterobacter; and yeast, Candida. The blood count did not show any significant alterations. To date, all three patients have healed skin lesions (in a patient for about two years with no evidence of infection. These preliminary results showed that L-PRF membranes could be a new method of therapy in such problematic diseases. Overall, the L-PRF treatment in osteomyelitis of a diabetic foot seems to be easy and cost-effective by regenerative therapy of chronic skin lesions. In addition, it will improve our understanding of wound healing.

Transfusion of pooled buffy coat platelet components prepared with photochemical pathogen inactivation treatment: the euroSPRITE trial

NARCIS (Netherlands)

D.J. van Rhenen (Dirk Jan); S. Marblie (Stephane); M. Laforet (Michel); K. Davis (Kathryn); M. Conlan (Maureen); B. Lioure (Bruno); H. Gulliksson (Hans); J.P. Cazenave; P. Metzel (Peyton); D. Pamphilon (Derwood); L. Corash (Laurence); J. Flament (Jocelyne); P. Ljungman (Per); H. Kluter; H. Vermeij (Hans); V. Mayaudon (Veronique); L. Lin (Lily); M.C. Kappers-Klunne (Mies); D. Buchholz (Don); G.E. de Greef (Georgine)

2003-01-01

textabstractA nucleic acid-targeted photochemical treatment (PCT) using amotosalen HCl (S-59) and ultraviolet A (UVA) light was developed to inactivate viruses, bacteria, protozoa, and leukocytes in platelet components. We conducted a controlled, randomized, double-blinded trial in thrombocytopenic

Of von Willebrand factor and platelets.

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Bryckaert, Marijke; Rosa, Jean-Philippe; Denis, Cécile V; Lenting, Peter J

2015-01-01

Hemostasis and pathological thrombus formation are dynamic processes that require multiple adhesive receptor-ligand interactions, with blood platelets at the heart of such events. Many studies have contributed to shed light on the importance of von Willebrand factor (VWF) interaction with its platelet receptors, glycoprotein (GP) Ib-IX-V and αIIbβ3 integrin, in promoting primary platelet adhesion and aggregation following vessel injury. This review will recapitulate our current knowledge on the subject from the rheological aspect to the spatio-temporal development of thrombus formation. We will also discuss the signaling events generated by VWF/GPIb-IX-V interaction, leading to platelet activation. Additionally, we will review the growing body of evidence gathered from the recent development of pathological mouse models suggesting that VWF binding to GPIb-IX-V is a promising target in arterial and venous pathological thrombosis. Finally, the pathological aspects of VWF and its impact on platelets will be addressed.

Absence of micronucleus formation in CHO-K1 cells cultivated in platelet lysate enriched medium.

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Bernardi, Martina; Adami, Valentina; Albiero, Elena; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

2014-03-01

Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line. PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective. Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products. Copyright © 2013 Elsevier GmbH. All rights reserved.

Platelet thromboxane B2-formation in end-stage kidney disease and after kidney transplantation

International Nuclear Information System (INIS)

Stefanovic, V.; Lecic, N.

1986-01-01

The aim of this work was to analyse TxB 2 formation by platelets in endstage kidney disease patients and in kidney graft recipients. Four groups of patients were studied: 12 preterminal chronic renal failure patients, 42 patients on maintenance hemodialysis, 8 patients on CAPD and 11 grafted patients. TxB 2 production by platelets was determined in serum following spontaneous blood clotting for 1/2 h at 37 0 C. Hemodialysis patients generated 80.7 ± 9.6 ng/ml (mean ± S.E.M.) of TxB 2 which was significantly (p 2 formation in hemodialysis patients had no relationship with the residual kidney function. Patients on CAPD produced 65.0 ± 12.7 ng/ml of TxB 2 . Very low TxB 2 generation was obtained also in preterminal chronic renal failure patients (57.0 ± 11.8 ng/ml). Kidney graft recipients had a mean TxB 2 production of 81.6 ± 24.2 ng/ml with a range from 12.5-200 ng/ml. Very low TxB 2 was formed in grafted patients with renal failure. (orig.) [de

Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry

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Irina V. Gorudko

2013-07-01

Myeloperoxidase (MPO is a heme-containing enzyme released from activated leukocytes into the extracellular space during inflammation. Its main function is the production of hypohalous acids that are potent oxidants. MPO can also modulate cell signaling and inflammatory responses independently of its enzymatic activity. Because MPO is regarded as an important risk factor for cardiovascular diseases associated with increased platelet activity, we studied the effects of MPO on human platelet functional properties. Laser scanning confocal microscopy was used to reveal carbohydrate-independent MPO binding to human platelet membrane. Adding MPO to platelets did not activate their aggregation under basal conditions (without agonist. In contrast, MPO augmented agonist-induced platelet aggregation, which was not prevented by MPO enzymatic activity inhibitors. It was found that exposure of platelets to MPO leads to actin cytoskeleton reorganization and an increase in their elasticity. Furthermore, MPO evoked a rise in cytosolic Ca2+ through enhancement of store-operated Ca2+ entry (SOCE. Together, these findings indicate that MPO is not a direct agonist but rather a mediator that binds to human platelets, induces actin cytoskeleton reorganization and affects the mechanical stiffness of human platelets, resulting in potentiating SOCE and agonist-induced human platelet aggregation. Therefore, an increased activity of platelets in vascular disease can, at least partly, be provided by MPO elevated concentrations.

A novel thromboxane receptor antagonist, nstpbp5185, inhibits platelet aggregation and thrombus formation in animal models.

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Huang, Shiu-Wen; Kuo, Heng-Lan; Hsu, Ming-Tsung; Tseng, Yufeng Jane; Lin, Shu-Wha; Kuo, Sheng-Chu; Peng, Hui-Chin; Lien, Jin-Cherng; Huang, Tur-Fu

2016-08-01

A novel benzimidazole derivative, nstpbp5185, was discovered through in vitro and in vivo evaluations for antiplatelet activity. Thromaboxane receptor (TP) is important in vascular physiology, haemostasis and pathophysiological thrombosis. Nstpbp5185 concentration-dependently inhibited human platelet aggregation caused by collagen, arachidonic acid and U46619. Nstpbp5185 caused a right-shift of the concentration-response curve of U46619 and competitively inhibited the binding of 3H-SQ-29548 to TP receptor expressed on HEK-293 cells, with an IC50 of 0.1 µM, indicating that nstpbp5185 is a TP antagonist. In murine thrombosis models, nstpbp5185 significantly prolonged the latent period in triggering platelet plug formation in mesenteric and FeCl3-induced thrombi formation, and increased the survival rate in pulmonary embolism model with less bleeding than aspirin. This study suggests nstpbp5185, an orally selective anti-thrombotic agent, acting through blockade of TXA2 receptor, may be efficacious for prevention or treatment of pathologic thrombosis.

Flavanols and Platelet Reactivity

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Debra A. Pearson

2005-01-01

Full Text Available Platelet activity and platelet-endothelial cell interactions are important in the acute development of thrombosis, as well as in the pathogenesis of cardiovascular disease. An increasing number of foods have been reported to have platelet-inhibitory actions, and research with a number of flavanol-rich foods, including, grape juice, cocoa and chocolate, suggests that these foods may provide some protection against thrombosis. In the present report, we review a series of in vivo studies on the effects of flavanol-rich cocoa and chocolate on platelet activation and platelet-dependent primary hemostasis. Consumption of flavanol-rich cocoa inhibited several measures of platelet activity including, epinephrine- and ADP-induced glycoprotein (GP IIb/IIIa and P-Selectin expression, platelet microparticle formation, and epinephrine-collagen and ADP-collagen induced primary hemostasis. The epinephrine-induced inhibitory effects on GP IIb/IIIa and primary hemostasis were similar to, though less robust than those associated with the use of low dose (81 mg aspirin. These data, coupled with information from other studies, support the concept that flavanols present in cocoa and chocolate can modulate platelet function through a multitude of pathways.

Aluminum induces lipid peroxidation and aggregation of human blood platelets

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T.J.C. Neiva

1997-05-01

Full Text Available Aluminum (Al3+ intoxication is thought to play a major role in the development of Alzheimer's disease and in certain pathologic manifestations arising from long-term hemodialysis. Although the metal does not present redox capacity, it can stimulate tissue lipid peroxidation in animal models. Furthermore, in vitro studies have revealed that the fluoroaluminate complex induces diacylglycerol formation, 43-kDa protein phosphorylation and aggregation. Based on these observations, we postulated that Al3+-induced blood platelet aggregation was mediated by lipid peroxidation. Using chemiluminescence (CL of luminol as an index of total lipid peroxidation capacity, we established a correlation between lipid peroxidation capacity and platelet aggregation. Al3+ (20-100 µM stimulated CL production by human blood platelets as well as their aggregation. Incubation of the platelets with the antioxidants nor-dihydroguaiaretic acid (NDGA (100 µM and n-propyl gallate (NPG (100 µM, inhibitors of the lipoxygenase pathway, completely prevented CL and platelet aggregation. Acetyl salicylic acid (ASA (100 µM, an inhibitor of the cyclooxygenase pathway, was a weaker inhibitor of both events. These findings suggest that Al3+ stimulates lipid peroxidation and the lipoxygenase pathway in human blood platelets thereby causing their aggregation

Platelet thrombosis in cardiac-valve prostheses

International Nuclear Information System (INIS)

Dewanjee, M.K.

1989-01-01

The contribution of platelets and clotting factors in thrombosis on cardiovascular prostheses had been quantified with several tracers. Thrombus formation in vivo could be measured semiquantitatively in animal models and patients with indium-111, Technetium-99m labeled platelets, iodine-123, iodine-131 labeled fibrinogen, and In-111 and Tc-99m labeled antibody to the fibrinogen-receptor on the platelet- membrane, or fibrin. The early studies demonstrated that certain platelet-inhibitors, e.g. sulfinpyrazone, aspirin or aspirin- persantine increased platelet survival time with mechanical valves implanted in the baboon model and patients. Thrombus localization by imaging is possible for large thrombus on thrombogenic surface of prosthesis in the acute phase. The majority of thrombus was found in the sewing ring (Dacron) in the acute phase in both the mechanical and tissue valves. The amount of retained thrombus in both mechanical and tissue valves in our one-day study in the dog model was similar (< 1% if injected In-111 platelets = 5 billion platelets). As the fibrous ingrowth covered the sewing ring, the thrombus formation decreased significantly. Only a small amount of thrombus was found on the leaflets at one month in both the dog and calf models. 38 refs., 9 figs., 5 tabs

Platelet thrombosis in cardiac-valve prostheses

Energy Technology Data Exchange (ETDEWEB)

Dewanjee, M.K.

1989-01-01

The contribution of platelets and clotting factors in thrombosis on cardiovascular prostheses had been quantified with several tracers. Thrombus formation in vivo could be measured semiquantitatively in animal models and patients with indium-111, Technetium-99m labeled platelets, iodine-123, iodine-131 labeled fibrinogen, and In-111 and Tc-99m labeled antibody to the fibrinogen-receptor on the platelet- membrane, or fibrin. The early studies demonstrated that certain platelet-inhibitors, e.g. sulfinpyrazone, aspirin or aspirin- persantine increased platelet survival time with mechanical valves implanted in the baboon model and patients. Thrombus localization by imaging is possible for large thrombus on thrombogenic surface of prosthesis in the acute phase. The majority of thrombus was found in the sewing ring (Dacron) in the acute phase in both the mechanical and tissue valves. The amount of retained thrombus in both mechanical and tissue valves in our one-day study in the dog model was similar (< 1% if injected In-111 platelets = 5 billion platelets). As the fibrous ingrowth covered the sewing ring, the thrombus formation decreased significantly. Only a small amount of thrombus was found on the leaflets at one month in both the dog and calf models. 38 refs., 9 figs., 5 tabs.

The role of platelet and endothelial GARP in thrombosis and hemostasis.

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Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

The role of platelet and endothelial GARP in thrombosis and hemostasis.

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Elien Vermeersch

Full Text Available Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32 is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish.To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice.Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice.Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected.Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

Platelet concentrates stored in plasma for 72 hours at 22 degrees C prepared from buffycoats of citrate-phosphate-dextrose blood collected in a quadruple-bag saline-adenine-glucose-mannitol system

NARCIS (Netherlands)

Pietersz, R. N.; Loos, J. A.; Reesink, H. W.

1985-01-01

A method is described to prepare platelet concentrates from the buffycoat of citrate-phosphate-dextrose (CPD) blood in a closed four-bag system and to store the platelets in autologous plasma under sterile conditions. After separation of the blood into plasma, buffycoat and leukocyte- and

Blood platelets in the progression of Alzheimer's disease.

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Nina S Gowert

Full Text Available Alzheimer's disease (AD is characterized by neurotoxic amyloid-ß plaque formation in brain parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA. Besides CAA, AD is strongly related to vascular diseases such as stroke and atherosclerosis. Cerebrovascular dysfunction occurs in AD patients leading to alterations in blood flow that might play an important role in AD pathology with neuronal loss and memory deficits. Platelets are the major players in hemostasis and thrombosis, but are also involved in neuroinflammatory diseases like AD. For many years, platelets were accepted as peripheral model to study the pathophysiology of AD because platelets display the enzymatic activities to generate amyloid-ß (Aß peptides. In addition, platelets are considered to be a biomarker for early diagnosis of AD. Effects of Aß peptides on platelets and the impact of platelets in the progression of AD remained, however, ill-defined. The present study explored the cellular mechanisms triggered by Aß in platelets. Treatment of platelets with Aß led to platelet activation and enhanced generation of reactive oxygen species (ROS and membrane scrambling, suggesting enhanced platelet apoptosis. More important, platelets modulate soluble Aß into fibrillar structures that were absorbed by apoptotic but not vital platelets. This together with enhanced platelet adhesion under flow ex vivo and in vivo and platelet accumulation at amyloid deposits of cerebral vessels of AD transgenic mice suggested that platelets are major contributors of CAA inducing platelet thrombus formation at vascular amyloid plaques leading to vessel occlusion critical for cerebrovascular events like stroke.

Proteomics strategy for identifying candidate bioactive proteins in complex mixtures: application to the platelet releasate.

LENUS (Irish Health Repository)

O'Connor, Roisin

2010-01-01

Proteomic approaches have proven powerful at identifying large numbers of proteins, but there are fewer reports of functional characterization of proteins in biological tissues. Here, we describe an experimental approach that fractionates proteins released from human platelets, linking bioassay activity to identity. We used consecutive orthogonal separation platforms to ensure sensitive detection: (a) ion-exchange of intact proteins, (b) SDS-PAGE separation of ion-exchange fractions and (c) HPLC separation of tryptic digests coupled to electrospray tandem mass spectrometry. Migration of THP-1 monocytes in response to complete or fractionated platelet releasate was assessed and located to just one of the forty-nine ion-exchange fractions. Over 300 proteins were identified in the releasate, with a wide range of annotated biophysical and biochemical properties, in particular platelet activation, adhesion, and wound healing. The presence of PEDF and involucrin, two proteins not previously reported in platelet releasate, was confirmed by western blotting. Proteins identified within the fraction with monocyte promigratory activity and not in other inactive fractions included vimentin, PEDF, and TIMP-1. We conclude that this analytical platform is effective for the characterization of complex bioactive samples.

Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb.

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Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

2016-08-12

Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb*

Science.gov (United States)

Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

2016-01-01

Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. PMID:27342778

Complementary roles of platelets and coagulation in thrombus formation on plaques acutely ruptured by targeted ultrasound treatment: a novel intravital model.

Science.gov (United States)

Kuijpers, M J E; Gilio, K; Reitsma, S; Nergiz-Unal, R; Prinzen, L; Heeneman, S; Lutgens, E; van Zandvoort, M A M J; Nieswandt, B; Egbrink, M G A Oude; Heemskerk, J W M

2009-01-01

Atherothrombosis is a major cause of cardiovascular events. However, animal models to study this process are scarce. We describe the first murine model of acute thrombus formation upon plaque rupture to study atherothrombosis by intravital fluorescence microscopy. Localized rupture of an atherosclerotic plaque in a carotid artery from Apoe(-/-) mice was induced in vivo using ultrasound. Rupture of the plaque and formation of localized thrombi were verified by two-photon laser scanning microscopy (TPLSM) in isolated arteries, and by immunohistochemistry. The thrombotic reaction was quantified by intravital fluorescence microscopy. Inspection of the ultrasound-treated plaques by histochemistry and TPLSM demonstrated local damage, collagen exposure, luminal thrombus formation as well as intra-plaque intrusion of erythrocytes and fibrin. Ultrasound treatment of healthy carotid arteries resulted in endothelial damage and limited platelet adhesion. Real-time intravital fluorescence microscopy demonstrated rapid platelet deposition on plaques and formation of a single thrombus that remained subocclusive. The thrombotic process was antagonized by thrombin inhibition, or by blocking of collagen or adenosine diphosphate receptor pathways. Multiple thrombi were formed in 70% of mice lacking CD40L. Targeted rupture of murine plaques results in collagen exposure and non-occlusive thrombus formation. The thrombotic process relies on platelet activation as well as on thrombin generation and coagulation, and is sensitive to established and novel antithrombotic medication. This model provides new possibilities to study atherothrombosis in vivo.

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

International Nuclear Information System (INIS)

Walker, G.; Bourguignon, L.Y.

1990-01-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

Energy Technology Data Exchange (ETDEWEB)

Walker, G.; Bourguignon, L.Y. (Univ. of Miami Medical School, FL (USA))

1990-08-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

Methods for evaluation of platelet function.

Science.gov (United States)

Lindahl, Tomas L; Ramström, Sofia

2009-10-01

There are a multitude of platelet function tests available, reflecting the complex nature of the platelet in haemostasis. No simple single test will ever cover all aspects of platelet function. Some tests focus on the aggregation of platelets, for example aggregometry, other on the swelling in response to hypotonic solutions, i.e. the well-known hypotonic shock response, or adhesion or coagulation and clot retraction, for example thromboelastography. In general there is a lack of clinical studies showing a predictive value of analysis of platelet concentrates.

Alternatives to allogeneic platelet transfusion.

Science.gov (United States)

Desborough, Michael J R; Smethurst, Peter A; Estcourt, Lise J; Stanworth, Simon J

2016-11-01

Allogeneic platelet transfusions are widely used for the prevention and treatment of bleeding in thrombocytopenia. Recent evidence suggests platelet transfusions have limited efficacy and are associated with uncertain immunomodulatory risks and concerns about viral or bacterial transmission. Alternatives to transfusion are a well-recognised tenet of Patient Blood Management, but there has been less focus on different strategies to reduce bleeding risk by comparison to platelet transfusion. Direct alternatives to platelet transfusion include agents to stimulate endogenous platelet production (thrombopoietin mimetics), optimising platelet adhesion to endothelium by treating anaemia or increasing von Willebrand factor levels (desmopressin), increasing formation of cross-linked fibrinogen (activated recombinant factor VII, fibrinogen concentrate or recombinant factor XIII), decreasing fibrinolysis (tranexamic acid or epsilon aminocaproic acid) or using artificial or modified platelets (cryopreserved platelets, lyophilised platelets, haemostatic particles, liposomes, engineered nanoparticles or infusible platelet membranes). The evidence base to support the use of these alternatives is variable, but an area of active research. Much of the current randomised controlled trial focus is on evaluation of the use of thrombopoietin mimetics and anti-fibrinolytics. It is also recognised that one alternative strategy to platelet transfusion is choosing not to transfuse at all. © 2016 John Wiley & Sons Ltd.

Influence of Leukocyte- and Platelet-Rich Fibrin (L-PRF) in the Healing of Simple Postextraction Sockets: A Split-Mouth Study

Science.gov (United States)

Riccitiello, Francesco; Tia, Mariano; di Lauro, Alessandro; Sammartino, Gilberto

2015-01-01

The aim of this study was to evaluate the effects of leukocyte- and platelet-rich fibrin (L-PRF) on the pain and soft tissue healing after tooth extractions. Twenty-six patients (9 males and 17 females) were treated with multiple extractions (2 to 8), with a total of 108 extractions. This was an exploratory single blinded randomized clinical trial with a split-mouth design. The pain after the surgery was assessed in each patient by the VAS scale (1 to 10) at intervals of 24-48-72-96 hours. The soft tissue healing was clinically evaluated at 3, 7, 14, and 21 days after surgery by the same examiner surgeon, using the modified Healing Index (4 to 12). The mean value of postextraction pain was 3.2 ± 0.3 in the experimental sides and 4.1 ± 0.1 in the control sides. After 7 days from the extractions, the values of modified Healing Index in the experimental and control groups were, respectively, 4.8 ± 0.6 and 5.1 ± 0.9. The use of L-PRF in postextraction sockets filling can be proposed as a useful procedure in order to manage the postoperative pain and to promote the soft tissue healing process, reducing the early adverse effects of the inflammation. PMID:26273612

Subpopulations in purified platelets adhering on glass.

Science.gov (United States)

Donati, Alessia; Gupta, Swati; Reviakine, Ilya

2016-06-22

Understanding how platelet activation is regulated is important in the context of cardiovascular disorders and their management with antiplatelet therapy. Recent evidence points to different platelet subpopulations performing different functions. In particular, procoagulant and aggregating subpopulations have been reported in the literature in platelets treated with the GPVI agonists. How the formation of platelet subpopulations upon activation is regulated remains unclear. Here, it is shown that procoagulant and aggregating platelet subpopulations arise spontaneously upon adhesion of purified platelets on clean glass surfaces. Calcium ionophore treatment of the adhering platelets resulted in one platelet population expressing both the procoagulant and the adherent population markers phosphatidylserine and the activated form of GPIIb/IIIa, while all of the platelets expressed CD62P independently of the ionophore treatment. Therefore, all platelets have the capacity to express all three activation markers. It is concluded that platelet subpopulations observed in various studies reflect the dynamics of the platelet activation process.

The life cycle of platelet granules.

Science.gov (United States)

Sharda, Anish; Flaumenhaft, Robert

2018-01-01

Platelet granules are unique among secretory vesicles in both their content and their life cycle. Platelets contain three major granule types-dense granules, α-granules, and lysosomes-although other granule types have been reported. Dense granules and α-granules are the most well-studied and the most physiologically important. Platelet granules are formed in large, multilobulated cells, termed megakaryocytes, prior to transport into platelets. The biogenesis of dense granules and α-granules involves common but also distinct pathways. Both are formed from the trans -Golgi network and early endosomes and mature in multivesicular bodies, but the formation of dense granules requires trafficking machinery different from that of α-granules. Following formation in the megakaryocyte body, both granule types are transported through and mature in long proplatelet extensions prior to the release of nascent platelets into the bloodstream. Granules remain stored in circulating platelets until platelet activation triggers the exocytosis of their contents. Soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, located on both the granules and target membranes, provide the mechanical energy that enables membrane fusion during both granulogenesis and exocytosis. The function of these core fusion engines is controlled by SNARE regulators, which direct the site, timing, and extent to which these SNAREs interact and consequently the resulting membrane fusion. In this review, we assess new developments in the study of platelet granules, from their generation to their exocytosis.

Radiolabeled leukocytes

International Nuclear Information System (INIS)

Datz, F.L.; Taylor, A.T.

1986-01-01

Leukocytes are a heterogeneous group of nucleated cells that follow similar patterns of differentiation in the bone marrow. Although the various leukocyte cell types perform somewhat different functions, they act as a group to protect the host from hazards of the internal and external environment, such as infection and neoplasia, and they assist in the repair of damaged tissue. Leukocytes spend a small fraction of their life in the peripheral blood, using it only for transportation to sites where they are needed to perform their defensive functions. In adults, the mature types of leukocytes are neutrophils (59 percent of the leukocyte population), lymphocytes (34 percent), monocytes (four percent), eosinophils (three percent), and basophils (0.5 percent). Neutrophils, eosinophils, and basophils all contain nuclei with finitely granular, evenly distributed chromatin and are collectively called granulocytes. In addition to the main categories of leukocytes listed above, there are subsets of many of these classes of cells; for example, natural killer cells are a subset of lymphocytes

Platelets are required for enhanced activation of the endothelium and fibrinogen in a mouse thrombosis model of APS.

Science.gov (United States)

Proulle, Valerie; Furie, Richard A; Merrill-Skoloff, Glenn; Furie, Barbara C; Furie, Bruce

2014-07-24

Antiphospholipid syndrome (APS) is defined by thrombosis, fetal loss, and the presence of antiphospholipid antibodies, including anti-β2-glycoprotein-1 autoantibodies (anti-β2GP1) that have a direct role in the pathogenesis of thrombosis in vivo. The cellular targets of the anti-β2GP1 autoantibody/β2GP1 complex in vivo were studied using a laser-induced thrombosis model of APS in a live mouse and human anti-β2GP1 autoantibodies affinity-purified from APS patients. Cell binding of fluorescently labeled β2GP1 and anti-β2GP1 autoantibodies revealed their colocalization on the platelet thrombus but not the endothelium. Anti-β2GP1 autoantibodies enhanced platelet activation, monitored by calcium mobilization, and endothelial activation, monitored by intercellular adhesion molecule-1 expression. When eptifibatide was infused to block platelet thrombus formation, enhanced fibrin generation and endothelial cell activation were eliminated. Thus, the anti-β2GP1 autoantibody/β2GP1 complex binds to the thrombus, enhancing platelet activation, and platelet secretion leads to enhanced endothelium activation and fibrin generation. These results lead to a paradigm shift away from the concept that binding of the anti-β2GP1 autoantibody/β2GP1 complex activates both endothelial cells and platelets toward one in which activation of platelets in response to anti-β2GP1 autoantibody/β2GP1 complex binding leads to subsequent enhanced endothelium activation and fibrin generation. © 2014 by The American Society of Hematology.

TARM1 Is a Novel Leukocyte Receptor Complex-Encoded ITAM Receptor That Costimulates Proinflammatory Cytokine Secretion by Macrophages and Neutrophils

DEFF Research Database (Denmark)

Radjabova, Valeria; Mastroeni, Piero; Skjødt, Karsten

2015-01-01

We identified a novel, evolutionarily conserved receptor encoded within the human leukocyte receptor complex and syntenic region of mouse chromosome 7, named T cell-interacting, activating receptor on myeloid cells-1 (TARM1). The transmembrane region of TARM1 contained a conserved arginine residu...

Erythrocyte sedimentation rate and fibrinogen concentration of whole blood influences the cellular composition of platelet-rich plasma obtained from centrifugation methods.

Science.gov (United States)

Yin, Wenjing; Xu, Zhengliang; Sheng, Jiagen; Xie, Xuetao; Zhang, Changqing

2017-09-01

Erythrocyte sedimentation rate (ESR), which reflects the sedimentation rate of platelets, leukocytes and erythrocytes in response to centrifugal force, may influence the cellular composition of platelet-rich plasma (PRP) obtained via centrifugation methods. However, no relevant studies have substantiated this. In the present study, blood was collected from 40 healthy volunteers and used to prepare PRP with two plasma-based preparation systems [YinPRP and Plasma Rich in Growth Factor (PRGF) systems] and two buffy coat-based systems (RegenPRP and WEGOPRP systems) in a single-donor model. Volumes of PRP and platelet-poor plasma (PPP) that were removed in the preparation process were recorded. Analyses of ESR, haematocrit, C-reaction protein, coagulation, serum glucose and serum lipid of the whole blood used for PRP preparation were performed to evaluate the levels of ESR and the factors known to influence it. Whole blood analysis was performed to evaluate the cellular composition of PRP. Results demonstrated that there were marked positive correlations between the ESR of the whole blood used for PRP preparation and PPP removal efficiencies, platelet concentrations, platelet capture efficiencies and platelet enrichment factors of PRP formulations obtained from plasma-based systems, and PRP yield efficiency of RegenPRP and PPP removal efficiency of WEGOPRP. Furthermore, there were marked negative correlations between ESR and concentrations and enrichment factors of platelets, leukocytes and erythrocytes of RegenPRP. Fibrinogen concentration of the whole blood, which had a marked positive correlation with ESR, also influenced the cellular composition of PRP. These findings may increase the understanding of PRP preparation and provide substantial evidence for the individualised optimisation of PRP preparation systems used in clinical practice.

Vessel wall and indium-111-labelled platelet response to carotid endarterectomy

International Nuclear Information System (INIS)

Lusby, R.J.; Ferrell, L.D.; Englestad, B.L.; Price, D.C.; Lipton, M.J.; Stoney, R.J.

1983-01-01

Postendarterectomy platelet deposition and thrombus formation may play an important role not only in vessel wall healing but also in the small incidence of postoperative cerebral ischemia and postoperative stenosis. A study has been performed using a canine model to investigate the healing response to carotid endarterectomy and the validity of an in vivo indium-111 (In-111) radiotracer technique in the assessment of postendarterectomy deposition of autologous labelled platelets. Sixteen endarterectomized carotid arteries showed uptake of autologous In-111 platelets immediately after infusion, reaching a maximum by 1 hour with little increase at 24 or 48 hours. No uptake was seen in ten control vessels following platelet infusion (P less than 0.05). At autopsy, seven vessels were demonstrated to have In-111 platelet deposition immediately prior to sacrifice of the animals. Postmortem scanning confirmed the localization to the vessel lumens, and microscopy revealed thrombus formation with or without partial endothelialization. Complete reendothelialization had occurred in the vessels that failed to show platelet deposition. Delayed healing was associated with continuing platelet deposition, excessive thrombus formation, and luminal stenosis. Arteriotomy closure with a vein patch altered the healing characteristics of the vessel with segmental thrombus formation over the vein patch. A preliminary study of the postendarterectomy in vivo In-111 platelet response in humans demonstrated platelet deposition that was not influenced by the administration of antiplatelet drugs at currently prescribed levels

The content of bone morphogenetic proteins in platelets varies greatly between different platelet donors

International Nuclear Information System (INIS)

Kalen, Anders; Wahlstroem, Ola; Linder, Cecilia Halling; Magnusson, Per

2008-01-01

Platelet derivates and platelet rich plasma have been used to stimulate bone formation and wound healing because of the rich content of potent growth factors. However, not all reports have been conclusive since some have not been able to demonstrate a positive effect. We investigated the interindividual variation of bone morphogenetic proteins (BMPs) in platelets from healthy donors, and the pH-dependent effect on the release of BMPs in preparations of lysed platelets in buffer (LPB). Platelet concentrates from 31 healthy donors were prepared in pH 4.3 and pH 7.4 buffers and investigated with respect to BMP-2, -4, -6, and -7. BMP-2 and BMP-4 were significantly more common in acidic LPBs in comparison with neutral preparations. We also observed a considerable variation among platelet donors with respect to the release of BMPs at pH 4.3 and 7.4. In conclusion, a considerable variation was found among platelet donors, which may be of importance considering the ambiguous results previously reported on osteoblast proliferation and differentiation

The role of platelets during reproduction.

Science.gov (United States)

Isermann, Berend; Nawroth, Peter P

2006-01-01

The availability of mice with defined defects within the hemostatic system enabled researchers to identify a role the coagulation system for embryonic and placental development. However, the role of platelets during development has only recently been experimentally addressed, giving some insight into potential functions of platelets during development. Thus, a quantitative embryonic platelet defect (severe thrombopenia secondary to NF-E2 deficiency) is associated with an embryonic growth retardation and reduced vascularisation of the placenta. Maternal platelet deficiency is associated with placental hemorrhage, which, however, does not impair embryonic or maternal survival. In vitro studies established that platelets or platelet conditioned medium regulate the invasive properties of human extravillous trophoblast cells and induce a phenotypical switch of trophoblast cells. These data imply that platelets are of relevance during placentation. Conversely, platelets and the formation of platelet-fibrin aggregates are dispensable for the development of the embryo proper, establishing that the lethal phenotypes observed in some embryo slacking coagulation regulators does not result from an inability to form platelet-fibrin aggregates, but likely reflects altered protease dependent signaling during vascular development.

Sports medicine applications of platelet rich plasma.

Science.gov (United States)

Mishra, Allan; Harmon, Kimberly; Woodall, James; Vieira, Amy

2012-06-01

Platelet rich plasma (PRP) is a powerful new biologic tool in sports medicine. PRP is a fraction of autologous whole blood containing and increased number of platelets and a wide variety of cytokines such as platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transforming growth factor beta-1 (TGF-B1), fibroblast growth factor (FGF), Insulin-like growth factor-1 (IGF-1) among many others. Worldwide interest in this biologic technology has recently risen sharply. Basic science and preclinical data support the use of PRP for a variety of sports related injuries and disorders. The published, peer reviewed, human data on PRP is limited. Although the scientific evaluation of clinical efficacy is in the early stages, elite and recreational athletes already use PRP in the treatment of sports related injuries. Many questions remain to be answered regarding the use of PRP including optimal formulation, including of leukocytes, dosage and rehabilitation protocols. In this review, a classification for platelet rich plasma is proposed and the in-vitro, preclinical and human investigations of PRP applications in sports medicine will be reviewed as well as a discussion of rehabilitation after a PRP procedure. The regulation of PRP by the World Anti-Doping Agency will also be discussed. PRP is a promising technology in sports medicine; however, it will require more vigorous study in order to better understand how to apply it most effectively.

[3H]52770 RP, a platelet-activating factor receptor antagonist, and tritiated platelet-activating factor label a common specific binding site in human polymorphonuclear leukocytes

International Nuclear Information System (INIS)

Marquis, O.; Robaut, C.; Cavero, I.

1988-01-01

In human polymorphonuclear leukocytes (PMNs), the tritiated platelet activating factor ([ 3 H]PAF) labels in a saturable manner a single class of binding sites with a Kd of 3.5 +/- 0.5 nM (n = 7) and a maximum binding capacity (Bmax) of 206 +/- 13 fmol/2.5 X 10(6) PMNs (n = 7). 52770 RP, a nonphospholipid antagonist of PAF receptors, fully and competitively displaced the [ 3 H]PAF from its binding sites with a Ki of 7.0 +/- 0.7 nM (n = 4). The high potency and the low solubility in cellular membranes of this compound led us to prepare [ 3 H]52770 RP. This ligand was characterized by a binding which was rapid, reversible, confined to a single site, saturable, specific and stereoselective. Its Kd and Bmax were 4.2 +/- 0.3 nM and 181 +/- 11 fmol/2.5 X 10(6) PMNs, respectively. The stereoselectivity of the binding was suggested by the 600- and 1050-fold higher potency of the d-enantiomer with respect to l-52770 RP in displacing [ 3 H]52770 RP or [ 3 H]PAF, respectively. Several PAF analogs (e.g., lyso-PAF, 2-O-methyl-lyso-PAF), which are poorly active as PAF receptor agonists in functional tests, were weak displacers of [ 3 H]PAF and [ 3 H]52770 RP. Furthermore, for a series of 14 known PAF receptor agonists or antagonists belonging to different chemical families, there was an excellent correlation (r = 0.98) between their ability to displace [ 3 H]PAF and [ 3 H]52770 RP. Thus, [ 3 H]52770 RP and [ 3 H]PAF appear to interact with the same binding site on human PMNs which is proposed to be the PAF receptor mediating functional responses

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbβ3 and activated platelets

Directory of Open Access Journals (Sweden)

Marcinkiewicz C

2017-05-01

Full Text Available Cezary Marcinkiewicz,1,2 Jonathan A Gerstenhaber,1 Mark Sternberg,2 Peter I Lelkes,1 Giora Feuerstein1,2 1Department of Bioengineering, College of Engineering, Temple University, Philadelphia, 2Debina Diagnostic, Inc., Newton Square, PA, USA Abstract: Thromboembolic events (TEE underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs functionalized with bitistatin (Bit, a disintegrin that specifically binds to the αIIbβ3 integrin, platelet fibrinogen receptor (PFR on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP–Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, αIIbβ3 receptor antagonist, specifically blocked F-NDP–Bit–PFR complex formation. Moreover, F-NDP–Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP. Our results suggest that engineered F-NDP–Bit particles could serve as noninvasive, “real-time” optical diagnostics for clots present in blood vessels. Keywords: carbon nanoparticles, blood clots, imaging, platelet fibrinogen receptor, fluorescence, disintegrin, thromboembolic complications, thrombosis

86Rb(K) influx and [3H]ouabain binding by human platelets: Evidence for beta-adrenergic stimulation of Na-K ATPase activity

International Nuclear Information System (INIS)

Turaihi, K.; Khokher, M.A.; Barradas, M.A.; Mikhailidis, D.P.; Dandona, P.

1989-01-01

Although active transport of potassium into human platelets has been demonstrated previously, there is hitherto no evidence that human platelets have an ouabain-inhibitable Na-K ATPase in their membrane. The present study demonstrates active rubidium (used as an index of potassium influx), 86 Rb(K), influx into platelets, inhibitable by ouabain, and also demonstrates the presence of specific [ 3 H]ouabain binding by the human platelet. This 86 Rb(K) influx was stimulated by adrenaline, isoprenaline, and salbutamol, but noradrenaline caused a mild inhibition. Active 86 Rb(K) influx by platelets was inhibited markedly by timolol, mildly by atenolol, but not by phentolamine. Therefore, active 86 Rb(K) influx in human platelets is enhanced by stimulation of beta adrenoceptors of the beta 2 subtype. The platelet may therefore replace the leukocyte in future studies of Na-K ATPase activity. This would be a considerable advantage in view of the ease and rapidity of preparation of platelets

Platelet-Derived Microvesicles in Cardiovascular Diseases

Directory of Open Access Journals (Sweden)

Maria T. K. Zaldivia

2017-11-01

Full Text Available Microvesicles (MVs circulating in the blood are small vesicles (100–1,000 nm in diameter derived from membrane blebs of cells such as activated platelets, endothelial cells, and leukocytes. A growing body of evidence now supports the concept that platelet-derived microvesicles (PMVs, the most abundant MVs in the circulation, are important regulators of hemostasis, inflammation, and angiogenesis. Compared with healthy individuals, a large increase of circulating PMVs has been observed, particularly in patients with cardiovascular diseases. As observed in MVs from other parent cells, PMVs exert their biological effects in multiple ways, such as triggering various intercellular signaling cascades and by participating in transcellular communication by the transfer of their “cargo” of cytoplasmic components and surface receptors to other cell types. This review describes our current understanding of the potential role of PMVs in mediating hemostasis, inflammation, and angiogenesis and their consequences on the pathogenesis of cardiovascular diseases, such as atherosclerosis, myocardial infarction, and venous thrombosis. Furthermore, new developments of the therapeutic potential of PMVs for the treatment of cardiovascular diseases will be discussed.

Platelet fibrinogen binding in Basset Hound Hereditary Thrombopathy

International Nuclear Information System (INIS)

Patterson, W.; Estry, D.; Schwartz, K.; Bell, T.

1986-01-01

Platelets from dogs with Basset Hound Hereditary Thrombopathy (BHT) display a thrombasthenia-like aggregation defect but have been shown to have normal amounts of platelet membrane glycoproteins IIb and IIIa (GP IIb-IIIa). In order to investigate the possibility of a functionally abnormal GPIIb-IIIa complex, which might be unable to bind fibrinogen after stimulation, fibrinogen binding in BHT was evaluated. Two canine fibrinogen preparations were used, one from BHT dogs and one from normal control dogs, as well as a human fibrinogen preparation. Platelets from BHT and normal dogs were activated with 1 x 10 -5 M ADP in the presence of 125 I-labeled fibrinogen and the surface bound radioactivity quantitated. For all fibrinogen preparations, the amount of fibrinogen bound by BHT platelets was not significantly different than that bound by normal dog platelets. BHT platelets bound 23,972 +/- 3612 and normal dog platelets bound 23,033 +/- 3971 molecules of fibrinogen per platelet. The BHT platelet aggregation defect does not seem to be caused by a functionally abnormal GP IIb-IIIa complex, since BHT platelets bind normal amounts of fibrinogen. The results suggest that fibrinogen binding is not sufficient for platelet aggregation, and other factors, perhaps receptor mobility and membrane phospholipid content should be investigated in BHT

Molecular cloning and characterization of rhesus monkey platelet glycoprotein Ibα, a major ligand-binding subunit of GPIb-IX-V complex.

Science.gov (United States)

Qiao, Jianlin; Shen, Yang; Shi, Meimei; Lu, Yanrong; Cheng, Jingqiu; Chen, Younan

2014-05-01

Through binding to von Willebrand factor (VWF), platelet glycoprotein (GP) Ibα, the major ligand-binding subunit of the GPIb-IX-V complex, initiates platelet adhesion and aggregation in response to exposed VWF or elevated fluid-shear stress. There is little data regarding non-human primate platelet GPIbα. This study cloned and characterized rhesus monkey (Macaca Mullatta) platelet GPIbα. DNAMAN software was used for sequence analysis and alignment. N/O-glycosylation sites and 3-D structure modelling were predicted by online OGPET v1.0, NetOGlyc 1.0 Server and SWISS-MODEL, respectively. Platelet function was evaluated by ADP- or ristocetin-induced platelet aggregation. Rhesus monkey GPIbα contains 2,268 nucleotides with an open reading frame encoding 755 amino acids. Rhesus monkey GPIbα nucleotide and protein sequences share 93.27% and 89.20% homology respectively, with human. Sequences encoding the leucine-rich repeats of rhesus monkey GPIbα share strong similarity with human, whereas PEST sequences and N/O-glycosylated residues vary. The GPIbα-binding residues for thrombin, filamin A and 14-3-3ζ are highly conserved between rhesus monkey and human. Platelet function analysis revealed monkey and human platelets respond similarly to ADP, but rhesus monkey platelets failed to respond to low doses of ristocetin where human platelets achieved 76% aggregation. However, monkey platelets aggregated in response to higher ristocetin doses. Monkey GPIbα shares strong homology with human GPIbα, however there are some differences in rhesus monkey platelet activation through GPIbα engagement, which need to be considered when using rhesus monkey platelet to investigate platelet GPIbα function. Copyright © 2014 Elsevier Ltd. All rights reserved.

Advances in RNAi therapeutic delivery to leukocytes using lipid nanoparticles.

Science.gov (United States)

Ramishetti, Srinivas; Landesman-Milo, Dalit; Peer, Dan

2016-11-01

Small interfering RNAs (siRNAs) therapeutics has advanced into clinical trials for liver diseases and solid tumors, but remain a challenge for manipulating leukocytes fate due to lack of specificity and safety issues. Leukocytes ingest pathogens and defend the body through a complex network. They are also involved in the pathogeneses of inflammation, viral infection, autoimmunity and cancers. Modulating gene expression in leukocytes using siRNAs holds great promise to treat leukocyte-mediated diseases. Leukocytes are notoriously hard to transduce with siRNAs and are spread throughout the body often located deep in tissues, therefore developing an efficient systemic delivery strategy is still a challenge. Here, we discuss recent advances in siRNA delivery to leukocyte subsets such as macrophages, monocytes, dendritic cells and lymphocytes. We focus mainly on lipid-based nanoparticles (LNPs) comprised of new generation of ionizable lipids and their ability to deliver siRNA to primary or malignant leukocytes in a targeted manner. Special emphasis is made on LNPs targeted to subsets of leukocytes and we detail a novel microfluidic mixing technology that could aid in changing the landscape of process development of LNPs from a lab tool to a potential novel therapeutic modality.

In vitro phagocytosis of several Candida berkhout species by murine leukocytes.

Science.gov (United States)

Fontenla de Petrino, S E; Bibas Bonet de Jorrat, M E; Sirena, A

1985-03-01

In vitro phagocytosis of thirteen Candida berkhout species by rat leukocytes was studied to assess a possible correlation between pathogenicity and phagocytosis Yeast-leukocyte suspensions were mixed up for 3 h and phagocytic index, germ-tube formation and leukocyte candidacidal activity were evaluated. Highest values for phagocytosis were reached in all cases at the end of the first hour. Leukocyte candidacidal activity was absent. Only C. albicans produced germ-tubes. The various phagocytosis indices were determined depending on the Candida species assayed. Under these conditions, the more pathogenic species presented the lower indices of phagocytosis. It is determined that the in vitro phagocytic index may bear a close relationship with the pathogenicity of the Candida berkhout.

Flow cytometric analysis reveals the high levels of platelet activation parameters in circulation of multiple sclerosis patients.

Science.gov (United States)

Morel, Agnieszka; Rywaniak, Joanna; Bijak, Michał; Miller, Elżbieta; Niwald, Marta; Saluk, Joanna

2017-06-01

The epidemiological studies confirm an increased risk of cardiovascular disease in multiple sclerosis, especially prothrombotic events directly associated with abnormal platelet activity. The aim of our study was to investigate the level of blood platelet activation in the circulation of patients with chronic phase of multiple sclerosis (SP MS) and their reactivity in response to typical platelets' physiological agonists. We examined 85 SP MS patients diagnosed according to the revised McDonald's criteria and 50 healthy volunteers as a control group. The platelet activation and reactivity were assessed using flow cytometry analysis of the following: P-selectin expression (CD62P), activation of GP IIb/IIIa complex (PAC-1 binding), and formation of platelet microparticles (PMPs) and platelet aggregates (PA) in agonist-stimulated (ADP, collagen) and unstimulated whole blood samples. Furthermore, we measured the level of soluble P-selectin (sP-selectin) in plasma using ELISA method, to evaluate the in vivo level of platelet activation, both in healthy and SP MS subjects. We found a statistically significant increase in P-selectin expression, GP IIb/IIIa activation, and formation of PMPs and PA, as well as in unstimulated and agonist-stimulated (ADP, collagen) platelets in whole blood samples from patients with SP MS in comparison to the control group. We also determined the higher sP-selectin level in plasma of SP MS subjects than in the control group. Based on the obtained results, we might conclude that during the course of SP MS platelets are chronically activated and display hyperreactivity to physiological agonists, such as ADP or collagen.

Platelet Preparations for Use in Facial Rejuvenation and Wound Healing: A Critical Review of Current Literature.

Science.gov (United States)

Sclafani, Anthony P; Azzi, James

2015-08-01

In facial plastic surgery, the potential for direct delivery of growth factors from platelet preparations has been of particular interest for use in facial rejuvenation, recovery after facial surgery, and wound healing. A literature search was conducted through PubMed for the terms PRP, PRFM, platelet-rich plasma, platelet-rich fibrin matrix, platelet preparations, platelet therapy, growth factors, platelet facial, platelet facial rejuvenation, platelet wound healing, platelet plastic surgery. Articles pertaining to the use of platelet preparations in facial surgery and wound healing in plastic surgery after 2001 were included. Thirteen in vitro studies showed use of platelet-rich plasma (PRP) and platelet-rich fibrin matrix (PRFM) had a significant effect on cellular activity. Twenty-four out of 28 animal studies exhibited favorable results with use of a platelet preparation, including five of six studies that showed enhanced fat graft survival with addition of a platelet preparation. Twenty-three case series and clinical trials were identified, only two of which showed no differences. Twenty-one reported favorable results with use of various platelet preparations. A total of 47 studies used PRP, four studies evaluated Leukocyte-rich PRP, and fourteen studies used PRFM. The vast majority of studies examined show a significant and measurable effect on cellular changes, wound healing, and facial esthetic outcomes with use of platelet preparations, both topical and injectable. One must also consider possible publication bias against null results that may have had an influence on the data that were available for review. However, the preponderance of studies suggests that platelet preparations might represent an as-of-yet untapped adjunct in facial plastic surgery.

Soluble CD40 ligand stimulates CD40-dependent activation of the β2 integrin Mac-1 and protein kinase C zeda (PKCζ in neutrophils: implications for neutrophil-platelet interactions and neutrophil oxidative burst.

Directory of Open Access Journals (Sweden)

Rong Jin

Full Text Available Recent work has revealed an essential involvement of soluble CD40L (sCD40L in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40, i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better

Glycoprotein Ibα clustering in platelet storage and function

NARCIS (Netherlands)

Gitz, E.

2013-01-01

Platelets are anucleated, discoid-shaped cells that play an essential role in the formation of a hemostatic plug to prevent blood loss from injured vessels. Initial platelet arrest at the damaged arterial vessel wall is mediated through the interaction between the platelet receptor glycoprotein (GP)

Modeling leukocyte-leukocyte non-contact interactions in a lymph node.

Directory of Open Access Journals (Sweden)

Nicola Gritti

Full Text Available The interaction among leukocytes is at the basis of the innate and adaptive immune-response and it is largely ascribed to direct cell-cell contacts. However, the exchange of a number of chemical stimuli (chemokines allows also non-contact interaction during the immunological response. We want here to evaluate the extent of the effect of the non-contact interactions on the observed leukocyte-leukocyte kinematics and their interaction duration. To this aim we adopt a simplified mean field description inspired by the Keller-Segel chemotaxis model, of which we report an analytical solution suited for slowly varying sources of chemokines. Since our focus is on the non-contact interactions, leukocyte-leukocyte contact interactions are simulated only by means of a space dependent friction coefficient of the cells. The analytical solution of the Keller-Segel model is then taken as the basis of numerical simulations of interactions between leukocytes and their duration. The mean field interaction force that we derive has a time-space separable form and depends on the chemotaxis sensitivity parameter as well as on the chemokines diffusion coefficient and their degradation rate. All these parameters affect the distribution of the interaction durations. We draw a successful qualitative comparison between simulated data and sets of experimental data for DC-NK cells interaction duration and other kinematic parameters. Remarkably, the predicted percentage of the leukocyte-leukocyte interactions falls in the experimental range and depends (~25% increase upon the chemotactic parameter indicating a non-negligible direct effect of the non-contact interaction on the leukocyte interactions.

Modeling leukocyte-leukocyte non-contact interactions in a lymph node.

Science.gov (United States)

Gritti, Nicola; Caccia, Michele; Sironi, Laura; Collini, Maddalena; D'Alfonso, Laura; Granucci, Francesca; Zanoni, Ivan; Chirico, Giuseppe

2013-01-01

The interaction among leukocytes is at the basis of the innate and adaptive immune-response and it is largely ascribed to direct cell-cell contacts. However, the exchange of a number of chemical stimuli (chemokines) allows also non-contact interaction during the immunological response. We want here to evaluate the extent of the effect of the non-contact interactions on the observed leukocyte-leukocyte kinematics and their interaction duration. To this aim we adopt a simplified mean field description inspired by the Keller-Segel chemotaxis model, of which we report an analytical solution suited for slowly varying sources of chemokines. Since our focus is on the non-contact interactions, leukocyte-leukocyte contact interactions are simulated only by means of a space dependent friction coefficient of the cells. The analytical solution of the Keller-Segel model is then taken as the basis of numerical simulations of interactions between leukocytes and their duration. The mean field interaction force that we derive has a time-space separable form and depends on the chemotaxis sensitivity parameter as well as on the chemokines diffusion coefficient and their degradation rate. All these parameters affect the distribution of the interaction durations. We draw a successful qualitative comparison between simulated data and sets of experimental data for DC-NK cells interaction duration and other kinematic parameters. Remarkably, the predicted percentage of the leukocyte-leukocyte interactions falls in the experimental range and depends (~25% increase) upon the chemotactic parameter indicating a non-negligible direct effect of the non-contact interaction on the leukocyte interactions.

Enhancement by platelets of oxygen radical responses of human neutrophils

International Nuclear Information System (INIS)

McCulloch, K.K.; Powell, J.; Johnson, K.J.; Ward, P.A.

1986-01-01

When human blood neutrophils were incubated with immune complexes (consisting of IgG antibody) in the presence of platelets, there was a 2 to 10 fold enhancement in the generation of O- 2 and H 2 O 2 . This enhancement phenomenon was proportional to the dose of immune complex added and the number of platelets present. The response was not agonist specific since similar enhancement also occurred with the following agonists: phorbol myristate acetate, opsonized zymosan particles and the chemotactic peptide N-formyl-met-leu-phe. The platelet related phenomenon of enhanced O- 2 generation could not be reproduced by the addition of serotonin, histamine or platelet-derived growth factor and was not affected by prior treatment of platelets with cyclooxygenase inhibitors (indomethacin, piroxicam) or lipoxygenase inhibitors (nafazatrom, BW755C or nordihydroguaiaretic acid). However, activation of platelets by thrombin caused release into the platelet supernatant fluid of a factor that, only in the presence of immune complexes, caused enhanced O- 2 responses to neutrophils. These data indicate that platelets potentiate oxygen radical responses of human neutrophils and suggest a mechanisms by which platelets may participate in tissue injury which is mediated by oxygen radical products from activated neutrophils

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

NARCIS (Netherlands)

Barendrecht, Arjan D; Verhoef, Johan J F; Pignatelli, Silvia; Pasterkamp, Gerard; Heijnen, Harry F G; Maas, Coen

2017-01-01

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet

PLATELET ADHESION TO POLYURETHANE UREA UNDER PULSATILE FLOW CONDITIONS

Science.gov (United States)

Navitsky, Michael A.; Taylor, Joshua O.; Smith, Alexander B.; Slattery, Margaret J.; Deutsch, Steven; Siedlecki, Christopher A.; Manning, Keefe B.

2014-01-01

Platelet adhesion to a polyurethane urea surface is a precursor to thrombus formation within blood-contacting cardiovascular devices, and platelets have been found to adhere strongly to polyurethane surfaces below a shear rate of approximately 500 s−1. The aim of the current work is to determine platelet adhesion properties to the polyurethane urea surface as a function of time varying shear exposure. A rotating disk system is used to study the influence of steady and pulsatile flow conditions (e.g. cardiac inflow and sawtooth waveforms) for platelet adhesion to the biomaterial surface. All experiments retain the same root mean square angular rotation velocity (29.63 rad/s) and waveform period. The disk is rotated in platelet rich bovine plasma for two hours with adhesion quantified by confocal microscopy measurements of immunofluorescently labeled bovine platelets. Platelet adhesion under pulsating flow is found to exponentially decay with increasing shear rate. Adhesion levels are found to depend upon peak platelet flux and shear rate regardless of rotational waveform. In combination with flow measurements, these results may be useful for predicting regions susceptible to thrombus formation within ventricular assist devices. PMID:24721222

Platelet kinetics with indium-111 platelets: comparison with chromium-51 platelets

International Nuclear Information System (INIS)

Peters, A.M.; Lavender, J.P.

1983-01-01

The application of 111In-oxine to platelet labeling has contributed to the understanding of platelet kinetics along three lines: 1. It allows the measurement of new parameters of splenic function, such as the intrasplenic platelet transit time, which has shed new light on the physiology of splenic blood cell handling. 2. It facilitates the measurement of platelet life span in conditions, such as ITP, in which 51Cr may undergo undesirable elution from the platelet as a result of platelet-antibody interaction. 3. It allows the determination of the fate of platelets, that is, the site of platelet destruction in conditions in which reduced platelet life span is associated with abnormal platelet consumption, as a result of either premature destruction of ''abnormal'' platelets by the RE system, or the consumption (or destruction) of normal platelets after their interaction with an abnormal vasculature. Future research using 111In platelets may yield further valuable information on the control as well as the significance of intrasplenic platelet pooling, on the role of platelets in the development of chronic vascular lesions, and on the sites of platelet destruction in ITP. With regard to the latter, methods will have to be developed for harvesting sufficient platelets representative of the total circulating platelet population from severely thrombocytopenic patients for autologous platelet labeling. This would avoid the use of homologous platelets, which is likely to be responsible for some of the contradictory data relating to the use of radiolabeled platelet studies for the prediction of the response of patients with ITP to splenectomy

Diminished ability of erythrocytes from patients with systemic lupus erythematosus to limit opsonized immune complex deposition on leukocytes and activation of granulocytes

DEFF Research Database (Denmark)

Nielsen, C H; Rasmussen, J M; Voss, A

1998-01-01

OBJECTIVE: To compare the ability of normal erythrocytes and erythrocytes from systemic lupus erythematosus (SLE) patients to bind immune complexes (IC), thereby inhibiting IC deposition on polymorphonuclear leukocytes (PMN) and the consequent induction of a PMN respiratory burst (RB). METHODS...

The combined use of kartogenin and platelet-rich plasma promotes fibrocartilage formation in the wounded rat Achilles tendon entheses.

Science.gov (United States)

Zhang, J; Yuan, T; Zheng, N; Zhou, Y; Hogan, M V; Wang, J H-C

2017-04-01

After an injury, the biological reattachment of tendon to bone is a challenge because healing takes place between a soft (tendon) and a hard (bone) tissue. Even after healing, the transition zone in the enthesis is not completely regenerated, making it susceptible to re-injury. In this study, we aimed to regenerate Achilles tendon entheses (ATEs) in wounded rats using a combination of kartogenin (KGN) and platelet-rich plasma (PRP). Wounds created in rat ATEs were given three different treatments: kartogenin platelet-rich plasma (KGN-PRP); PRP; or saline (control), followed by histological and immunochemical analyses, and mechanical testing of the rat ATEs after three months of healing. Histological analysis showed well organised arrangement of collagen fibres and proteoglycan formation in the wounded ATEs in the KGN-PRP group. Furthermore, immunohistochemical analysis revealed fibrocartilage formation in the KGN-PRP-treated ATEs, evidenced by the presence of both collagen I and II in the healed ATE. Larger positively stained collagen III areas were found in both PRP and saline groups than those in the KGN-PRP group. Chondrocyte-related genes, SOX9 and collagen II, and tenocyte-related genes, collagen I and scleraxis (SCX), were also upregulated by KGN-PRP. Moreover, mechanical testing results showed higher ultimate tensile strength in the KGN-PRP group than in the saline control group. In contrast, PRP treatment appeared to have healed the injured ATE but induced no apparent formation of fibrocartilage. The saline-treated group showed poor healing without fibrocartilage tissue formation in the ATEs. Our results show that injection of KGN-PRP induces fibrocartilage formation in the wounded rat ATEs. Hence, KGN-PRP may be a clinically relevant, biological approach to regenerate injured enthesis effectively. Cite this article: J. Zhang, T. Yuan, N. Zheng, Y. Zhou, M. V. Hogan, J. H-C. Wang. The combined use of kartogenin and platelet-rich plasma promotes

sup 86 Rb(K) influx and ( sup 3 H)ouabain binding by human platelets: Evidence for beta-adrenergic stimulation of Na-K ATPase activity

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Turaihi, K.; Khokher, M.A.; Barradas, M.A.; Mikhailidis, D.P.; Dandona, P. (Royal Free Hospital and School of Medicine, London (England))

1989-08-01

Although active transport of potassium into human platelets has been demonstrated previously, there is hitherto no evidence that human platelets have an ouabain-inhibitable Na-K ATPase in their membrane. The present study demonstrates active rubidium (used as an index of potassium influx), {sup 86}Rb(K), influx into platelets, inhibitable by ouabain, and also demonstrates the presence of specific ({sup 3}H)ouabain binding by the human platelet. This {sup 86}Rb(K) influx was stimulated by adrenaline, isoprenaline, and salbutamol, but noradrenaline caused a mild inhibition. Active {sup 86}Rb(K) influx by platelets was inhibited markedly by timolol, mildly by atenolol, but not by phentolamine. Therefore, active {sup 86}Rb(K) influx in human platelets is enhanced by stimulation of beta adrenoceptors of the beta 2 subtype. The platelet may therefore replace the leukocyte in future studies of Na-K ATPase activity. This would be a considerable advantage in view of the ease and rapidity of preparation of platelets.

Effects of platelet-poor plasma, platelet-rich plasma, and platelet-rich fibrin on healing of extraction sockets with buccal dehiscence in dogs.

Science.gov (United States)

Hatakeyama, Ichiro; Marukawa, Eriko; Takahashi, Yukinobu; Omura, Ken

2014-02-01

Alveolar bone resorption generally occurs during healing after tooth extraction. This study aimed to evaluate the effects of platelet-poor plasma (PPP), platelet-rich plasma (PRP), and platelet-rich fibrin (PRF) on healing in a ridge-augmentation model of the canine socket with dehiscence of the buccal wall. The third mandibular premolars of 12 beagle dogs were extracted and a 3 mm buccal dehiscence from the alveolar crest to the buccal wall of the extraction socket was created. These sockets were then divided into four groups on the basis of the material used to fill the sockets: PPP, PRP, PRF, and control (no graft material) groups. Results were evaluated at 4 and 8 weeks after surgery. The ultrastructural morphology and constructs of each blood product were studied by a scanning electron microscope (SEM) or calculating concentrations of platelets, fibrinogen, platelet-derived growth factor, and transforming growth factor-β. A total of five microcomputed tomography images of specimens were selected for measurement, and the area occupied by the newly formed bone as well as the horizontal bone width were measured. Moreover, decalcified tissue specimens from each defect were analyzed histologically. The median area of new bone at 4 and 8 weeks and median horizontal bone width at 8 weeks were the highest in the PPP group. However, bone maturation in the PRF and the PRP groups was more progressed than that in the PPP and control groups. By SEM findings, the PRF group showed a more highly condensed fibrin fiber network that was regularly arranged when compared with the PPP and PRP groups. The growth factors released from platelets in PRP indicated higher concentrations than that in PRF. Under more severe conditions for bone formation, as in this experiment, the growth factors released from platelets had a negative effect on bone formation. This study showed that PPP is an effective material for the preservation of sockets with buccal dehiscence.

Leukocyte adhesion deficiencies

NARCIS (Netherlands)

van de Vijver, Edith; van den Berg, Timo K.; Kuijpers, Taco W.

2013-01-01

During inflammation, leukocytes play a key role in maintaining tissue homeostasis through elimination of pathogens and removal of damaged tissue. Leukocytes migrate to the site of inflammation by crawling over and through the blood vessel wall, into the tissue. Leukocyte adhesion deficiencies (ie,

Enhancement by platelets of oxygen radical responses of human neutrophils

Energy Technology Data Exchange (ETDEWEB)

McCulloch, K.K.; Powell, J.; Johnson, K.J.; Ward, P.A.

1986-03-01

When human blood neutrophils were incubated with immune complexes (consisting of IgG antibody) in the presence of platelets, there was a 2 to 10 fold enhancement in the generation of O-/sub 2/ and H/sub 2/O/sub 2/. This enhancement phenomenon was proportional to the dose of immune complex added and the number of platelets present. The response was not agonist specific since similar enhancement also occurred with the following agonists: phorbol myristate acetate, opsonized zymosan particles and the chemotactic peptide N-formyl-met-leu-phe. The platelet related phenomenon of enhanced O-/sub 2/ generation could not be reproduced by the addition of serotonin, histamine or platelet-derived growth factor and was not affected by prior treatment of platelets with cyclooxygenase inhibitors (indomethacin, piroxicam) or lipoxygenase inhibitors (nafazatrom, BW755C or nordihydroguaiaretic acid). However, activation of platelets by thrombin caused release into the platelet supernatant fluid of a factor that, only in the presence of immune complexes, caused enhanced O-/sub 2/ responses to neutrophils. These data indicate that platelets potentiate oxygen radical responses of human neutrophils and suggest a mechanisms by which platelets may participate in tissue injury which is mediated by oxygen radical products from activated neutrophils.

Salivary Thromboxane A2-Binding Proteins from Triatomine Vectors of Chagas Disease Inhibit Platelet-Mediated Neutrophil Extracellular Traps (NETs Formation and Arterial Thrombosis.

Directory of Open Access Journals (Sweden)

Daniella M Mizurini

Full Text Available The saliva of blood-feeding arthropods contains a notable diversity of molecules that target the hemostatic and immune systems of the host. Dipetalodipin and triplatin are triatomine salivary proteins that exhibit high affinity binding to prostanoids, such as TXA2, thus resulting in potent inhibitory effect on platelet aggregation in vitro. It was recently demonstrated that platelet-derived TXA2 mediates the formation of neutrophil extracellular traps (NETs, a newly recognized link between inflammation and thrombosis that promote thrombus growth and stability.This study evaluated the ability of dipetalodipin and triplatin to block NETs formation in vitro. We also investigated the in vivo antithrombotic activity of TXA2 binding proteins by employing two murine models of experimental thrombosis. Remarkably, we observed that both inhibitors abolished the platelet-mediated formation of NETs in vitro. Dipetalodipin and triplatin significantly increased carotid artery occlusion time in a FeCl3-induced injury model. Treatment with TXA2-binding proteins also protected mice from lethal pulmonary thromboembolism evoked by the intravenous injection of collagen and epinephrine. Effective antithrombotic doses of dipetalodipin and triplatin did not increase blood loss, which was estimated using the tail transection method.Salivary TXA2-binding proteins, dipetalodipin and triplatin, are capable to prevent platelet-mediated NETs formation in vitro. This ability may contribute to the antithrombotic effects in vivo. Notably, both molecules inhibit arterial thrombosis without promoting excessive bleeding. Our results provide new insight into the antihemostatic effects of TXA2-binding proteins and may have important significance in elucidating the mechanisms of saliva to avoid host's hemostatic responses and innate immune system.

Platelet Glycoprotein lb-1X and Malignancy

Science.gov (United States)

2011-09-01

initiate coagulation, resulting in the formation of a fibrin - rich tumor cell- platelet emboli (Figure 1). Many of these coagulation factor-tumor cell...the tumor cell in a fibrin - rich web. (13;23;24) During this process, the platelet integrin receptor, aIIb 3, serves as a receptor linking fibrin ... platelets , and tumor cells into a fibrin rich clot normally associated with a thrombus. (25;25) Indeed, it can be speculated the fibrin - rich clot

Calcium-binding proteins from human platelets

International Nuclear Information System (INIS)

Gogstad, G.O.; Krutnes, M.B.; Solum, N.O.

1983-01-01

Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45 Ca 2 + and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45 Ca 2 + . These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4 Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45 Ca 2 + prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was wekly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelt-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIB-IIIa precipitate also became apparent. No increased incorporation of calcium occured in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45 Ca 2 + . (orig.)

Niacin and biosynthesis of PGD₂ by platelet COX-1 in mice and humans

DEFF Research Database (Denmark)

Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela

2012-01-01

during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis....... Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD₂ biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased...... thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD₂ was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD₂, like PGI₂, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular...

Abnormal megakaryocyte development and platelet function in Nbeal2(-/-) mice.

Science.gov (United States)

Kahr, Walter H A; Lo, Richard W; Li, Ling; Pluthero, Fred G; Christensen, Hilary; Ni, Ran; Vaezzadeh, Nima; Hawkins, Cynthia E; Weyrich, Andrew S; Di Paola, Jorge; Landolt-Marticorena, Carolina; Gross, Peter L

2013-11-07

Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with macrothrombocytopenia and α-granule-deficient platelets. GPS has been linked to loss of function mutations in NEABL2 (neurobeachin-like 2), and we describe here a murine GPS model, the Nbeal2(-/-) mouse. As in GPS, Nbeal2(-/-) mice exhibit splenomegaly, macrothrombocytopenia, and a deficiency of platelet α-granules and their cargo, including von Willebrand factor (VWF), thrombospondin-1, and platelet factor 4. The platelet α-granule membrane protein P-selectin is expressed at 48% of wild-type levels and externalized upon platelet activation. The presence of P-selectin and normal levels of VPS33B and VPS16B in Nbeal2(-/-) platelets suggests that NBEAL2 acts independently of VPS33B/VPS16B at a later stage of α-granule biogenesis. Impaired Nbeal2(-/-) platelet function was shown by flow cytometry, platelet aggregometry, bleeding assays, and intravital imaging of laser-induced arterial thrombus formation. Microscopic analysis detected marked abnormalities in Nbeal2(-/-) bone marrow megakaryocytes, which when cultured showed delayed maturation, decreased survival, decreased ploidy, and developmental abnormalities, including abnormal extracellular distribution of VWF. Our results confirm that α-granule secretion plays a significant role in platelet function, and they also indicate that abnormal α-granule formation in Nbeal2(-/-) mice has deleterious effects on megakaryocyte survival, development, and platelet production.

Radioimmune assay of human platelet prostaglandin synthetase

International Nuclear Information System (INIS)

Roth, G.J.; Machuga, E.T.

1982-01-01

Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH 2 from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and [ 125 I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [ 125 I]antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10 9 platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency

Use of platelet lysate for bone regeneration - are we ready for clinical translation?

Science.gov (United States)

Altaie, Ala; Owston, Heather; Jones, Elena

2016-02-26

Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells (MSCs). Although MSCs are most commonly grown in media containing fetal calf serum, human platelet lysate (PL) offers an effective alternative. Bone marrow - derived MSCs grown in PL-containing media display faster proliferation whilst maintaining good osteogenic differentiation capacity. Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo. In an alternative approach, nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices. Even though methods to coat scaffolds with PL vary, in vitro studies suggest that PL allows for MSC adhesion, migration and differentiation inside these scaffolds. Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo. This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration. To minimise inconsistency between the studies, further work is required towards standardisation of PL preparation in terms of the starting material, platelet concentration, leukocyte depletion, and the method of platelet lysis. PL quality control procedures and its "potency" assessment are urgently needed, which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation. Furthermore, different PL formulations could be tailor-made for specific bone repair indications. Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration.

Dual mechanism of integrin αIIbβ3 closure in procoagulant platelets.

Science.gov (United States)

Mattheij, Nadine J A; Gilio, Karen; van Kruchten, Roger; Jobe, Shawn M; Wieschhaus, Adam J; Chishti, Athar H; Collins, Peter; Heemskerk, Johan W M; Cosemans, Judith M E M

2013-05-10

Inactivation of integrin αIIbβ3 reverses platelet aggregate formation upon coagulation. Platelets from patient (Scott) and mouse (Capn1(-/-) and Ppif(-/-)) blood reveal a dual mechanism of αIIbβ3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling. These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets. Aggregation of platelets via activated integrin αIIbβ3 is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that αIIbβ3 inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the β3 chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed αIIbβ3 inactivation. Integrin αIIbβ3 inactivation was unchanged in platelets from Capn1(-/-) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced αIIbβ3 inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(-/-) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and αIIbβ3 inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of αIIbβ3 inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation.

Effect of the order of He{sup +} and H{sup +} ion co-implantation on damage generation and thermal evolution of complexes, platelets, and blisters in silicon

Energy Technology Data Exchange (ETDEWEB)

Daghbouj, N. [CEMES-CNRS and Université de Toulouse, 29 rue J. Marvig, 31055 Toulouse (France); Faculté des Sciences de Monastir, Université de Monastir, Monastir (Tunisia); Cherkashin, N., E-mail: nikolay.cherkashin@cemes.fr; Darras, F.-X.; Paillard, V.; Claverie, A. [CEMES-CNRS and Université de Toulouse, 29 rue J. Marvig, 31055 Toulouse (France); Fnaiech, M. [Faculté des Sciences de Monastir, Université de Monastir, Monastir (Tunisia)

2016-04-07

Hydrogen and helium co-implantation is nowadays used to efficiently transfer thin Si layers and fabricate silicon on insulator wafers for the microelectronic industry. The synergy between the two implants which is reflected through the dramatic reduction of the total fluence needed to fracture silicon has been reported to be strongly influenced by the implantation order. Contradictory conclusions on the mechanisms involved in the formation and thermal evolution of defects and complexes have been drawn. In this work, we have experimentally studied in detail the characteristics of Si samples co-implanted with He and H, comparing the defects which are formed following each implantation and after annealing. We show that the second implant always ballistically destroys the stable defects and complexes formed after the first implant and that the redistribution of these point defects among new complexes drives the final difference observed in the samples after annealing. When H is implanted first, He precipitates in the form of nano-bubbles and agglomerates within H-related platelets and nano-cracks. When He is implanted first, the whole He fluence is ultimately used to pressurize H-related platelets which quickly evolve into micro-cracks and surface blisters. We provide detailed scenarios describing the atomic mechanisms involved during and after co-implantation and annealing which well-explain our results and the reasons for the apparent contradictions reported at the state of the art.

Allogeneic hematopoietic stem-cell transplantation for leukocyte adhesion deficiency

DEFF Research Database (Denmark)

Qasim, Waseem; Cavazzana-Calvo, Marina; Davies, E Graham

2009-01-01

OBJECTIVES: Leukocyte adhesion deficiency is a rare primary immune disorder caused by defects of the CD18 beta-integrin molecule on immune cells. The condition usually presents in early infancy and is characterized by deep tissue infections, leukocytosis with impaired formation of pus, and delayed...... of leukocyte adhesion deficiency who underwent hematopoietic stem-cell transplantation between 1993 and 2007 was retrospectively analyzed. Data were collected by the registries of the European Society for Immunodeficiencies/European Group for Blood and Marrow Transplantation, and the Center for International......, with full donor engraftment in 17 cases, mixed multilineage chimerism in 7 patients, and mononuclear cell-restricted chimerism in an additional 3 cases. CONCLUSIONS: Hematopoietic stem-cell transplantation offers long-term benefit in leukocyte adhesion deficiency and should be considered as an early...

Impaired leukocyte influx in cervix of postterm women not responding to prostaglandin priming

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Masironi Britt

2008-09-01

Full Text Available Abstract Background Prolonged pregnancies are associated with increased rate of maternal and fetal complications. Post term women could be divided into at least two subgroups, one where parturition is possible to induce by prostaglandins and one where it is not. Our aim was to study parameters in cervical biopsies in women with spontaneous delivery at term (controls and compare to those that are successfully induced post term (responders, and those that are not induced (non-responders, by local prostaglandin treatment. Methods Stromal parameters examined in this study were the accumulation of leukocytes (CD45, CD68, mRNAs and/or proteins for the extracellular matrix degrading enzymes (matrix metalloproteinase (MMP-2, MMP-8 and MMP-9, their inhibitors (tissue inhibitor of MMP (TIMP-1 and TIMP-2, interleukin-8 (IL-8, the platelet activating factor-receptor (PAF-R, syndecan-1 and estrogen binding receptors (estrogen receptor (ERα, ERβ and G-coupled protein receptor (GPR 30 as well as the proliferation marker Ki-67. Results The influx of leukocytes as assessed by CD45 was strongest in the responders, thereafter in the controls and significantly lower in the non-responders. IL-8, PAF-R and MMP-9, all predominantly expressed in leukocytes, showed significantly reduced immunostaining in the group of non-responders, while ERα and GPR30 were more abundant in the non-responders, as compared to the controls. Conclusion The impaired leukocyte influx, as reflected by the reduced number of CD45 positive cells as well as decreased immunostaining of IL-8, PAF-R and MMP-9 in the non-responders, could be one explanation of the failed ripening of the cervix in post term women. If the decreased leukocyte influx is a primary explanation to absent ripening or secondary, as a result of other factors, is yet to be established.

Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets

International Nuclear Information System (INIS)

Laduca, F.M.; Bell, W.R.; Bettigole, R.E.

1987-01-01

Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of [ 3 H]serotonin, or alter the dose-responsive binding of 125 I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF

The origin and function of platelet glycosyltransferases

DEFF Research Database (Denmark)

Wandall, Hans H; Rumjantseva, Viktoria; Sørensen, Anne Louise Tølbøll

2012-01-01

Platelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and anti-angiogenic factors throughout the vascular system. Platelets are anucleated cells and lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartme...

Altered mitochondrial function and oxidative stress in leukocytes of anorexia nervosa patients.

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Victor M Victor

Full Text Available CONTEXT: Anorexia nervosa is a common illness among adolescents and is characterised by oxidative stress. OBJECTIVE: The effects of anorexia on mitochondrial function and redox state in leukocytes from anorexic subjects were evaluated. DESIGN AND SETTING: A multi-centre, cross-sectional case-control study was performed. PATIENTS: Our study population consisted of 20 anorexic patients and 20 age-matched controls, all of which were Caucasian women. MAIN OUTCOME MEASURES: Anthropometric and metabolic parameters were evaluated in the study population. To assess whether anorexia nervosa affects mitochondrial function and redox state in leukocytes of anorexic patients, we measured mitochondrial oxygen consumption, membrane potential, reactive oxygen species production, glutathione levels, mitochondrial mass, and complex I and III activity in polymorphonuclear cells. RESULTS: Mitochondrial function was impaired in the leukocytes of the anorexic patients. This was evident in a decrease in mitochondrial O2 consumption (P<0.05, mitochondrial membrane potential (P<0.01 and GSH levels (P<0.05, and an increase in ROS production (P<0.05 with respect to control subjects. Furthermore, a reduction of mitochondrial mass was detected in leukocytes of the anorexic patients (P<0.05, while the activity of mitochondrial complex I (P<0.001, but not that of complex III, was found to be inhibited in the same population. CONCLUSIONS: Oxidative stress is produced in the leukocytes of anorexic patients and is closely related to mitochondrial dysfunction. Our results lead us to propose that the oxidative stress that occurs in anorexia takes place at mitochondrial complex I. Future research concerning mitochondrial dysfunction and oxidative stress should aim to determine the physiological mechanism involved in this effect and the physiological impact of anorexia.

Full blood count and haemozoin-containing leukocytes in children with malaria: diagnostic value and association with disease severity

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Lell Bertrand

2008-06-01

Full Text Available Abstract Background Diligent and correct laboratory diagnosis and up-front identification of risk factors for progression to severe disease are the basis for optimal management of malaria. Methods Febrile children presenting to the Medical Research Unit at the Albert Schweitzer Hospital (HAS in Lambaréné, Gabon, were assessed for malaria. Giemsa-stained thick films for qualitative and quantitative diagnosis and enumeration of malaria pigment, or haemozoin (Hz-containing leukocytes (PCL were performed, and full blood counts (FBC were generated with a Cell Dyn 3000® instrument. Results Compared to standard light microscopy of Giemsa-stained thick films, diagnosis by platelet count only, by malaria pigment-containing monocytes (PCM only, or by pigment-containing granulocytes (PCN only yielded sensitivities/specificities of 92%/93%; 96%/96%; and 85%/96%, respectively. The platelet count was significantly lower in children with malaria compared to those without (p ® instrument detected significantly more patients with PCL (p Conclusion In the age group examined in the Lambaréné area, platelets are an excellent adjuvant tool to diagnose malaria. Pigment-containing leukocytes (PCL are more readily detected by automated scatter flow cytometry than by microscopy. Automated Hz detection by an instrument as used here is a reliable diagnostic tool and correlates with disease severity. However, clinical usefulness as a prognostic tool is limited due to an overlap of PCL numbers recorded in severe versus non-severe malaria. However, this is possibly because of the instrument detection algorithm was not geared towards this task, and data lost during processing; and thus adjusting the instrument's algorithm may allow to establish a meaningful cut-off value.

111In,113In-labelled platelets. I

International Nuclear Information System (INIS)

Komarek, P.; Poledne, R.; Charvat, J.; Konopkova, M.; Komarkova, I.

1983-01-01

111 In-8-hydroxyquinoline (oxine) is used for labelling blood platelets which serves diagnostic purposes in medicine. For the preparation of the complex of radioactive indium with oxine, 111 In and sup(113m)In were used under optimal conditions. Two different preparation methods for the labelled complex are described. Both methods studied the effect of pH, the amount of chelate agents and the mode of filtration on the yields of the labelled oxine. The blood platelets labelling is influenced not only by the quality of labelled oxine but also by their number. The organ distribution in laboratory animals proved that platelets were not impaired during separation and labelling and that they were suitable for diagnostic use. (author)

Dose- and time-related platelet response with apheresis platelet concentrates and pooled platelets

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Mohammad Mizanur Rahman

2017-02-01

Full Text Available This study was carried out to compare the post-transfusion platelet increment between the apheresis platelet concentrate (n=74 and pooled platelets (n=54. Pre- and post-transfusion platelet count of the recipient were carried out by automated hematology analyzer. In apheresis platelet concentrate group, the mean 24 hours post-transfusion platelet increment was 47 x 109/L which was statistically significant (p<0.001. On the other hand, in pooled platelets group, the mean 24 hours post–transfusions platelet count increment was 11.0 x 109/L which was also statistically significant (p<0.001. This study concluded that the transfusion of apheresis platelet concentrate was more useful than the transfusion of pooled platelets in terms of platelet count increment and requirement of donor.

Osteomyelitis in leukocyte adhesion deficiency type 1 syndrome

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Jabbari Azad, Farahzad; Ardalan, Maryam; H.Rafati, Ali

2010-01-01

Leukocyte adhesion deficiency type 1 (LAD-1) is a rare, inherited immunodeficiency that affects one per million people yearly and usually presents with recurrent, indolent bacterial infections of the skin, mouth, and respiratory tract and impaired pus formation and wound healing. A 13-year-old girl...

Platelet-activating factor podoplanin: from discovery to drug development.

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Takemoto, Ai; Miyata, Kenichi; Fujita, Naoya

2017-06-01

Tumor cell-induced platelet aggregation facilitates hematogenous metastasis by promoting tumor embolization, preventing immunological assaults and shear stress, and the platelet-releasing growth factors support tumor growth and invasion. Podoplanin, also known as Aggrus, is a type I transmembrane mucin-like glycoprotein and is expressed on wide range of tumor cells. Podoplanin has a role in platelet aggregation and metastasis formation through the binding to its platelet receptor, C-type lectin-like receptor 2 (CLEC-2). The podoplanin research was originally started from the cloning of highly metastatic NL-17 subclone from mouse colon 26 cancer cell line and from the establishment of 8F11 monoclonal antibody (mAb) that could neutralize NL-17-induced platelet aggregation and hematogenous metastasis. Later on, podoplanin was identified as the antigen of 8F11 mAb, and its ectopic expression brought to cells the platelet-aggregating abilities and hematogenous metastasis phenotypes. From the 8F11 mAb recognition epitopes, podoplanin is found to contain tandemly repeated, highly conserved motifs, designated platelet aggregation-stimulating (PLAG) domains. Series of analyses using the cells expressing the mutants and the established neutralizing anti-podoplanin mAbs uncovered that both PLAG3 and PLAG4 domains are associated with the CLEC-2 binding. The neutralizing mAbs targeting PLAG3 or PLAG4 could suppress podoplanin-induced platelet aggregation and hematogenous metastasis through inhibiting the podoplanin-CLEC-2 binding. Therefore, these domains are certainly functional in podoplanin-mediated metastasis through its platelet-aggregating activity. This review summarizes the platelet functions in metastasis formation, the role of platelet aggregation-inducing factor podoplanin in pathological and physiological situations, and the possibility to develop podoplanin-targeting drugs in the future.

Effects of Platelets on Platelet Concentrate Product on the Activation of Human Peripheral Blood Monocyte Cells

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N Sadat Razavi Hoseini

2016-02-01

Full Text Available Introduction: Monocytes can interact with platelets due to their surface molecules such as P-selectin glycoprotein ligand-1 (PSGL-1, and form monocyte-platelet complex. In the present study, the effects of platelets interaction of platelet concentrates (PCs and peripheral blood monocytes were investigated in vitro as a model to predict the probable interactions of these cells and consequently activation of monocytes. Methods: In this experimental study, units of whole blood and PCs were prepared from Tehran Blood Transfusion Center. After isolation of monocytes from the whole blood, these cells were treated with PC- derived platelets. The activation of monocytes was assessed before and after treatment by the analysis of the respiratory burst of monocytes using dihydrorhodamine 123 (DHR-123. The study data were analyzed using the non-parametric test of Wilcoxon. Results: The purity of monocytes was determined as 86.1±2 using NycoPrep method. The respiratory burst of monocytes was increased after exposure with platelets. In fact, the difference was significant when platelets were used on the 5th day of storage (P=0.001. Conclusions: The study findings revealed that platelets have an efficient capacity to stimulate and activate monocytes. The possible involvement of molecules in the interaction of platelet-monocyte demand to be further studied in future.

Adhesion of leukocytes under oscillating stagnation point conditions: a numerical study.

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Walker, P G; Alshorman, A A; Westwood, S; David, T

2002-01-01

Leukocyte recruitment from blood to the endothelium plays an important role in atherosclerotic plaque formation. Cells show a primary and secondary adhesive process with primary bonds responsible for capture and rolling and secondary bonds for arrest. Our objective was to investigate the role played by this process on the adhesion of leukocytes in complex flow. Cells were modelled as rigid spheres with spring like adhesion molecules which formed bonds with endothelial receptors. Models of bond kinetics and Newton's laws of motion were solved numerically to determine cell motion. Fluid force was obtained from the local shear rate obtained from a CFD simulation of the flow over a backward facing step.In stagnation point flow the shear rate near the stagnation point has a large gradient such that adherent cells in this region roll to a high shear region preventing permanent adhesion. This is enhanced if a small time dependent perturbation is imposed upon the stagnation point. For lower shear rates the cell rolling velocity may be such that secondary bonds have time to form. These bonds resist the lower fluid forces and consequently there is a relatively large permanent adhesion region.

Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets.

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Mangin, P H; Receveur, N; Wurtz, V; David, T; Gachet, C; Lanza, F

2009-09-01

Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib-IX complex initiates a signaling cascade leading to integrin alpha(IIb)beta(3) activation, a key process in hemostasis and thrombosis. Interaction of 14-3-3zeta with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. The aim of our study was to determine whether other members of the 14-3-3 family bind to GPIb-IX. In this study, western blot analyses showed that platelets also contain the 14-3-3beta, 14-3-3gamma, 14-3-3epsilon, 14-3-3eta and 14-3-3theta isoforms, but lack 14-3-3sigma. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14-3-3 isoforms expressed in platelets, including, as previously reported, 14-3-3zeta, bind to GPIb-IX. In addition, their interaction was found to critically require the same GPIbalpha domains (580-590 and 605-610) already identified as essential for 14-3-3zeta binding, in agreement with the conservation of the sequence of the I-helix among these different isoforms. Pull-down experiments indicated that all six 14-3-3 isoforms present in platelets bind to GPIbbeta. In contrast, deletion or mutation of the GPIbbeta intracytoplasmic tail did not affect the interaction of GPIb-IX with the 14-3-3 isoforms, questioning the importance of this domain. Our study suggests that, to inhibit GPIb-induced integrin alpha(IIb)beta(3) activation, a more appropriate strategy than inhibiting individual 14-3-3 isoforms would be to target the 14-3-3-binding motif on GPIb or, alternatively, the conserved 14-3-3 I-helix.

Blood platelet kinetics and platelet transfusion.

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Aster, Richard H

2013-11-01

The discovery of citrate anticoagulant in the 1920s and the development of plastic packs for blood collection in the 1960s laid the groundwork for platelet transfusion therapy on a scale not previously possible. A major limitation, however, was the finding that platelet concentrates prepared from blood anticoagulated with citrate were unsuitable for transfusion because of platelet clumping. We found that this could be prevented by simply reducing the pH of platelet-rich plasma to about 6.5 prior to centrifugation. We used this approach to characterize platelet kinetics and sites of platelet sequestration in normal and pathologic states and to define the influence of variables such as anticoagulant and ABO incompatibility on post-transfusion platelet recovery. The "acidification" approach enabled much wider use of platelet transfusion therapy until alternative means of producing concentrates suitable for transfusion became available.

New gene functions in megakaryopoiesis and platelet formation

NARCIS (Netherlands)

Gieger, Christian; Radhakrishnan, Aparna; Cvejic, Ana; Tang, Weihong; Porcu, Eleonora; Pistis, Giorgio; Serbanovic-Canic, Jovana; Elling, Ulrich; Goodall, Alison H.; Labrune, Yann; Lopez, Lorna M.; Mägi, Reedik; Meacham, Stuart; Okada, Yukinori; Pirastu, Nicola; Sorice, Rossella; Teumer, Alexander; Voss, Katrin; Zhang, Weihua; Ramirez-Solis, Ramiro; Bis, Joshua C.; Ellinghaus, David; Gögele, Martin; Hottenga, Jouke-Jan; Langenberg, Claudia; Kovacs, Peter; O'Reilly, Paul F.; Shin, So-Youn; Esko, Tõnu; Hartiala, Jaana; Kanoni, Stavroula; Murgia, Federico; Parsa, Afshin; Stephens, Jonathan; van der Harst, Pim; van der Schoot, C. Ellen; Allayee, Hooman; Attwood, Antony; Balkau, Beverley; Bastardot, François; Basu, Saonli; Baumeister, Sebastian E.; Biino, Ginevra; Bomba, Lorenzo; Bonnefond, Amélie; Cambien, François; Chambers, John C.; Cucca, Francesco; D'Adamo, Pio; Davies, Gail; de Boer, Rudolf A.; de Geus, Eco J. C.; Döring, Angela; Elliott, Paul; Erdmann, Jeanette; Evans, David M.; Falchi, Mario; Feng, Wei; Folsom, Aaron R.; Frazer, Ian H.; Gibson, Quince D.; Glazer, Nicole L.; Hammond, Chris; Hartikainen, Anna-Liisa; Heckbert, Susan R.; Hengstenberg, Christian; Hersch, Micha; Illig, Thomas; Loos, Ruth J. F.; Jolley, Jennifer; Khaw, Kay Tee; Kühnel, Brigitte; Kyrtsonis, Marie-Christine; Lagou, Vasiliki; Lloyd-Jones, Heather; Lumley, Thomas; Mangino, Massimo; Maschio, Andrea; Mateo Leach, Irene; McKnight, Barbara; Memari, Yasin; Mitchell, Braxton D.; Montgomery, Grant W.; Nakamura, Yusuke; Nauck, Matthias; Navis, Gerjan; Nöthlings, Ute; Nolte, Ilja M.; Porteous, David J.; Pouta, Anneli; Pramstaller, Peter P.; Pullat, Janne; Ring, Susan M.; Rotter, Jerome I.; Ruggiero, Daniela; Ruokonen, Aimo; Sala, Cinzia; Samani, Nilesh J.; Sambrook, Jennifer; Schlessinger, David; Schreiber, Stefan; Schunkert, Heribert; Scott, James; Smith, Nicholas L.; Snieder, Harold; Starr, John M.; Stumvoll, Michael; Takahashi, Atsushi; Tang, W. H. Wilson; Taylor, Kent; Tenesa, Albert; Lay Thein, Swee; Tönjes, Anke; Uda, Manuela; Ulivi, Sheila; van Veldhuisen, Dirk J.; Visscher, Peter M.; Völker, Uwe; Wichmann, H.-Erich; Wiggins, Kerri L.; Willemsen, Gonneke; Yang, Tsun-Po; Hua Zhao, Jing; Zitting, Paavo; Bradley, John R.; Dedoussis, George V.; Gasparini, Paolo; Hazen, Stanley L.; Metspalu, Andres; Pirastu, Mario; Shuldiner, Alan R.; Joost van Pelt, L.; Zwaginga, Jaap-Jan; Boomsma, Dorret I.; Deary, Ian J.; Franke, Andre; Froguel, Philippe; Ganesh, Santhi K.; Jarvelin, Marjo-Riitta; Martin, Nicholas G.; Meisinger, Christa; Psaty, Bruce M.; Spector, Timothy D.; Wareham, Nicholas J.; Akkerman, Jan-Willem N.; Ciullo, Marina; Deloukas, Panos; Greinacher, Andreas; Jupe, Steve; Kamatani, Naoyuki; Khadake, Jyoti; Kooner, Jaspal S.; Penninger, Josef; Prokopenko, Inga; Stemple, Derek; Toniolo, Daniela; Wernisch, Lorenz; Sanna, Serena; Hicks, Andrew A.; Rendon, Augusto; Ferreira, Manuel A.; Ouwehand, Willem H.; Soranzo, Nicole

2011-01-01

Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a

A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice.

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Trouillas, Marina; Prat, Marie; Doucet, Christelle; Ernou, Isabelle; Laplace-Builhé, Corinne; Blancard, Patrick Saint; Holy, Xavier; Lataillade, Jean-Jacques

2013-01-04

This study investigated the promising effect of a new Platelet Glue obtained from Cryoprecipitation of Apheresis Platelet products (PGCAP) used in combination with Mesenchymal Stromal Cells (MSC) loaded on ceramic biomaterials to provide novel strategies enhancing bone repair. PGCAP growth factor content was analyzed by ELISA and compared to other platelet and plasma-derived products. MSC loaded on biomaterials (65% hydroxyapatite/35% beta-TCP or 100% beta-TCP) were embedded in PGCAP and grown in presence or not of osteogenic induction medium for 21 days. Biomaterials were then implanted subcutaneously in immunodeficient mice for 28 days. Effect of PGCAP on MSC was evaluated in vitro by proliferation and osteoblastic gene expression analysis and in vivo by histology and immunohistochemistry. We showed that PGCAP, compared to other platelet-derived products, allowed concentrating large amount of growth factors and cytokines which promoted MSC and osteoprogenitor proliferation. Next, we found that PGCAP improves the proliferation of MSC and osteogenic-induced MSC. Furthermore, we demonstrated that PGCAP up-regulates the mRNA expression of osteogenic markers (Collagen type I, Osteonectin, Osteopontin and Runx2). In vivo, type I collagen expressed in ectopic bone-like tissue was highly enhanced in biomaterials embedded in PGCAP in the absence of osteogenic pre-induction. Better results were obtained with 65% hydroxyapatite/35% beta-TCP biomaterials as compared to 100% beta-TCP. We have demonstrated that PGCAP is able to enhance in vitro MSC proliferation, osteoblastic differentiation and in vivo bone formation in the absence of osteogenic pre-induction. This clinically adaptable platelet glue could be of interest for improving bone repair.

Platelet antiheparin activity. The isolation and characterisation of platelet factor 4 released from thrombin-aggregated washed human platelets and its dissociation into subunits and the isolation of membrane-bound antiheparin activity.

Science.gov (United States)

Moore, S; Pepper, D S; Cash, J D

1975-02-27

Platelet factor 4 was isolated by gel filtration from the soluble release products of thrombin-aggregated washed human platelets as a proteoglycan-platelet factor 4 complex of molecular weight 358 000, Stokes radius (r-s) of 14.0 nm, sedimentation coefficient (s) of 7.1 S and frictional ratio (f/f-o) of 3.04. The complex was dissociated at high ionic strength (I equals 0.75) and the proteoglycan separated from platelet factor 4 by gel filtration. Platelet factor 4 had a molecular weight of 27 100, r-s of 2.52 nm, s of 2.4 S and f/f-o of 1.26, was insoluble under physiological conditions but readily soluble at pH 3. Under these conditions platelet factor 4 dissociated into four subunits with a molecular weight of 6900, r-s of 1.92 nm, s of 0.8 S, and f/f-o of 1.52. Qualitative N-terminal amino acid analysis showed the presence of glutamic acid or glutamine as the major end group. Platelet factor 4 was compared with protamine sulphate, which has similar biological properties, by electrophoresis at pH 2.2, in which both migrated as single bands but with differing mobility, and by amino acid analysis which showed a more normal distribution of residues than occurred in protamine sulphate. Of the basic amino acids platelet factor 4 (molecular weight 27 100) contained 5.97% arginine, 3.18% histidine, and 12.31% lysine compared to protamine sulphate with 64.2% arginine, 0.6% lysine and no histidine. A partial specific volume (v) of 0.747 was calculated for platelet factor 4 from its amino acid analysis. A membrane fraction with antiheparin activity, an isopycnic density of 1.090-1.110 and r-s of 15-35 nm, was also isolated by sucrose density gradient centrifugation from the ultrasonicated insoluble platelet residue remaining after thrombin-induced aggregation of washed human platelets. Trypsin treatment of the membrane fraction neither solubilised nor destroyed the activity.

Platelet size and age determine platelet function independently

International Nuclear Information System (INIS)

Thompson, C.B.; Jakubowski, J.A.; Quinn, P.G.; Deykin, D.; Valeri, C.R.

1984-01-01

A study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se-methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms

Global Proteome Analysis Identifies Active Immunoproteasome Subunits in Human Platelets*

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Klockenbusch, Cordula; Walsh, Geraldine M.; Brown, Lyda M.; Hoffman, Michael D.; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-01-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. PMID:25146974

Examining platelet-fibrin interactions during traumatic shock in a swine model using platelet contractile force and clot elastic modulus.

Science.gov (United States)

White, Nathan J; Martin, Erika J; Brophy, Donald F; Ward, Kevin R

2011-07-01

A significant proportion of severely injured patients develop early coagulopathy, characterized by abnormal clot formation, which impairs resuscitation and increases mortality. We have previously demonstrated an isolated decrease in clot strength by thrombelastography in a swine model of nonresuscitated traumatic shock. In order to more closely examine platelet-fibrin interactions in this setting, we define the observed decrease in clot strength in terms of platelet-induced clot contraction and clot elastic modulus using the Hemostasis Analysis System (HAS) (Hemodyne Inc., Richmond, Virginia, USA). Whole blood was sampled for HAS measurements, metabolic measurements, cell counts, and fibrinogen concentration at baseline prior to injury and again at a predetermined level of traumatic shock defined by oxygen debt. Male swine (N=17) received femur fracture and controlled arterial hemorrhage to achieve an oxygen debt of 80 ml/kg. Platelet counts were unchanged, but fibrinogen concentration was reduced significantly during shock (167.6 vs. 66.7 mg/dl, P=0.0007). Platelet contractile force generated during clot formation did not change during shock (11.7 vs. 10.4 kdynes, P=0.41), but clot elastic modulus was dynamically altered, resulting in a lower final value (22.9 vs. 17.3 kdynes/cm, Pshock, platelet function was preserved, whereas terminal clot elastic modulus was reduced during shock in a manner most consistent with early changes in the mechanical properties of the developing fibrin fiber network.

Niacin and biosynthesis of PGD₂by platelet COX-1 in mice and humans.

Science.gov (United States)

Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela; Alamuddin, Naji; Ibrahim, Salam; Crichton, Irene; Prempeh, Maxwell; Lawson, John A; Wilensky, Robert L; Rasmussen, Lars Melholt; Puré, Ellen; FitzGerald, Garret A

2012-04-01

The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1-dependent formation of PGD₂ and PGE₂ followed by COX-2-dependent production of PGE₂. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD₂ receptor DP1. NSAID-mediated suppression of COX-2-derived PGI₂ has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD₂. Here, we show that PGD₂ biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD₂ biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD₂ was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD₂, like PGI₂, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy.

Evaluation of thrombelastographic platelet-mapping in healthy cats.

Science.gov (United States)

Blois, Shauna L; Banerjee, Amrita; Wood, R Darren

2012-06-01

Thrombelastography (TEG) permits analysis of clot formation but it is not specific for platelet activity. TEG PlateletMapping (TEG-PM) is a modification of TEG that uses adenosine diphosphate (ADP) and arachidonic acid (AA) as platelet agonists to define the contribution of platelets to clot formation. The objectives of this study were to determine values for TEG-PM in healthy cats and the interassay variation of TEG-PM. TEG-PM analysis was performed on blood specimens collected from 12 healthy cats and was repeated using a second blood specimen collected 2 hours later. Maximum amplitudes generated by thrombin (MA(thrombin)), fibrin (MA(fibrin)), ADP-stimulated platelet activity (MA(ADP)), and AA-stimulated platelet activity (MA(AA)) were recorded. Mean ± SD for MA(thrombin) was 51.1 ± 8.5 mm, for MA(fibrin) was 32.3 ± 17.7 mm, for MA(ADP) was 32.3 ± 15.0 mm, and for MA(AA) was 24.5 ± 12.2 mm. Mean MA(ADP) and MA(fibrin) were not significantly different, whereas mean MA(AA) was significantly lower than mean MA(fibrin). Results from the first and second specimens were not significantly different. Correlation between the first and second specimens was moderate for MA(thrombin), MA(fibrin), and MA(ADP), but was poor for MA(AA). A high degree of variability (coefficient of variation 47.7-60.0%) was observed for MA(fibrin), MA(ADP), and MA(AA). As MA(ADP) and MA(AA) (AA) were the same as or lower than MA(fibrin), a valid baseline to determine platelet-stimulated clot formation could not be established. Considerable interassay variation and wide intervals for MA(fibrin), MA(ADP), and MA(AA) values in this study indicate that TEG-PM should be used cautiously in feline patients. Several preanalytical factors should be examined in further detail. © 2012 American Society for Veterinary Clinical Pathology.

Neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios: are they useful for predicting gestational diabetes mellitus during pregnancy?

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Sargın MA

2016-04-01

Full Text Available Mehmet Akif Sargın, Murat Yassa, Bilge Dogan Taymur, Ayhan Celik, Emrah Ergun, Niyazi Tug Department of Obstetrics and Gynecology, Fatih Sultan Mehmet Research and Training Hospital, Istanbul, Turkey Objective: We aimed to investigate whether the neutrophil-to-lymphocyte ratio (NLR and platelet-to-lymphocyte ratio (PLR could be utilized to screen for gestational diabetes mellitus (GDM.Subjects and methods: NLR and PLR were assessed by retrospective analysis of 762 healthy and pregnant women with GDM. The patients were stratified into four groups, as follows: GDM (n=144, impaired glucose tolerance (n=76, only screen positive (n=238, and control (n=304.Results: The leukocyte, neutrophil, and lymphocyte counts were significantly higher in the study groups compared with the control group (P=0.001; P<0.01. There were no statistically significant differences between the groups with respect to the NLR and PLR (P>0.05.Conclusion: We do not recommend that blood NLR and PLR can be used to screen for GDM. However, increase in the leukocyte count is an important marker for GDM as it provides evidence of subclinical inflammation. Keywords: inflammation, lymphocytes, neutrophils, platelets, pregnancy

Platelet granule exocytosis: A comparison with chromaffin cells

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Jennifer eFitch-Tewfik

2013-06-01

Full Text Available The rapid secretion of bioactive amines from chromaffin cells constitutes an important component of the fight or flight response of mammals to stress. Platelets respond to stresses within the vasculature by rapidly secreting cargo at sites of injury, inflammation, or infection. Although chromaffin cells derive from the neural crest and platelets from bone marrow megakaryocytes, both have evolved a heterogeneous assemblage of granule types and a mechanism for efficient release. This article will provide an overview of granule formation and exocytosis in platelets with an emphasis on areas in which the study of chromaffin cells has influenced that of platelets and on similarities between the two secretory systems. Commonalities include the use of transporters to concentrate bioactive amines and other cargos into granules, the role of cytoskeletal remodeling in granule exocytosis, and the use of granules to provide membrane for cytoplasmic projections. The SNAREs and SNARE accessory proteins used by each cell type will also be considered. Finally, we will discuss the newly appreciated role of dynamin family proteins in regulated fusion pore formation. This evaluation of the comparative cell biology of regulated exocytosis in platelets and chromaffin cells demonstrates a convergence of mechanisms between two disparate cell types both tasked with responding rapidly to physiological stimuli.

Polyphosphate nanoparticles on the platelet surface trigger contact system activation

NARCIS (Netherlands)

Verhoef, Johan J F; Barendrecht, Arjan D; Nickel, Katrin F; Dijkxhoorn, Kim; Kenne, Ellinor; Labberton, Linda; McCarty, Owen J T; Schiffelers, Raymond; Heijnen, Harry F G; Hendrickx, Antoni P A; Schellekens, Huub; Fens, Marcel H; de Maat, Steven; Renné, Thomas; Maas, Coen

2017-01-01

Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII.

Standardization of a Protocol for Obtaining Platelet Rich Plasma from blood Donors; a Tool for Tissue Regeneration Procedures.

Science.gov (United States)

Gómez, Lina Andrea; Escobar, Magally; Peñuela, Oscar

2015-01-01

To develop a protocol for obtaining autologous platelet rich plasma in healthy individuals and to determine the concentration of five major growth factors before platelet activation. This protocol could be integrated into the guidelines of good clinical practice and research in regenerative medicine. Platelet rich plasma was isolated by centrifugation from 38 healthy men and 42 women ranging from 18 to 59 years old. The platelet count and quantification of growth factors were analyzed in eighty samples, stratified for age and gender of the donor. Analyses were performed using parametric the t-test or Pearson's analysis for non-parametric distribution. P platelet counts from 1.6 to 4.9 times (mean = 2.8). There was no correlation between platelet concentration and the level of the following growth factors: VEGF-D (r = 0.009, p = 0.4105), VEGF-A (r = 0.0068, p = 0.953), PDGF subunit AA (p = 0.3618; r = 0.1047), PDGF-BB (p = 0.5936; r = 0.6095). In the same way, there was no correlation between donor gender and growth factor concentrations. Only TGF-β concentration was correlated to platelet concentration (r = 0.3163, p = 0.0175). The procedure used allowed us to make preparations rich in platelets, low in leukocytes and red blood cells, and sterile. Our results showed biological variations in content of growth factors in PRP. The factors influencing these results should be further studied.

Complexity and formative experiences

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Roque Strieder

2017-12-01

Full Text Available The contemporaneity is characterized by instability and diversity calling into question certainties and truths proposed in modernity. We recognize that the reality of things and phenomena become effective as a set of events, interactions, retroactions and chances. This different frame extends the need for revision of the epistemological foundations that sustain educational practices and give them sense. The complex thinking is an alternative option for acting as a counterpoint to classical science and its reductionist logic and knowledge compartmentalization, as well as to answer to contemporary epistemological and educational challenges. It aims to associate different areas and forms of knowledge, without, however merge them, distinguishing without separating the several disciplines and instances of the realities. This study, in theoretical references, highlights the relevance of complex approaches to support formative experiences because also able to produce complexities in reflections about educational issues. We conclude that formative possibilities from complexity potentialize the resignification of human’s conception and the understanding of its singularity in interdependence; The understanding that pedagogical and educational activities is a constant interrogation about the possibilities of knowing the knowledge and reframe learning, far beyond knowing its functions and utilitarian purposes; and, as a formative possibility, places us on the trail of responsibility, not as something eventual, but present and indicative of freedom to choose to stay or go beyond.

MEAN PLATELET VOLUME AND RISK OF THROMBOTIC STROKE

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Prasantha Kumar Thankappan

2017-07-01

Full Text Available BACKGROUND Stroke is a major cause of long term morbidity and mortality. Several factors are known to increase the liability to stroke. Platelets play a crucial role in the pathogenesis of atherosclerotic complications, contributing to thrombus formation. Platelet size (mean platelet volume, MPV is a marker and possible determinant of platelet function, large platelets being potentially more reactive. Hence an attempt has-been made to study the association if any between mean platelet volume and thrombotic stroke. The aim of this study was to determine whether an association exists between Mean Platelet Volume (MPV and thrombotic stroke. MATERIALS AND METHODS The study is a case control study and data was collected at Government Medical College Hospital, Kottayam, Kerala a tertiary care referral centre. The study was carried out among fifty patients diagnosed with thrombotic stroke and presenting to the hospital within forty eight hours of onset of symptoms. Fifty age group and sex matched controls were also recruited. Mean platelet volume was obtained using a SYSMEX automated analyser. RESULTS This study has shown a statistically significant relation between mean platelet volume and risk of thrombotic stroke but no statistically significant correlation between clinical severity of stroke and mean platelet volume. CONCLUSION This study has shown an elevation of MPV in acute phase of thrombotic stroke. Platelet mass was found to be more or less a constant. This study did not find a statistically significant correlation between clinical severity of stroke and mean platelet volume.

Abnormal megakaryocyte development and platelet function in Nbeal2−/− mice

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Lo, Richard W.; Li, Ling; Pluthero, Fred G.; Christensen, Hilary; Ni, Ran; Vaezzadeh, Nima; Hawkins, Cynthia E.; Weyrich, Andrew S.; Di Paola, Jorge; Landolt-Marticorena, Carolina; Gross, Peter L.

2013-01-01

Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with macrothrombocytopenia and α-granule-deficient platelets. GPS has been linked to loss of function mutations in NEABL2 (neurobeachin-like 2), and we describe here a murine GPS model, the Nbeal2−/− mouse. As in GPS, Nbeal2−/− mice exhibit splenomegaly, macrothrombocytopenia, and a deficiency of platelet α-granules and their cargo, including von Willebrand factor (VWF), thrombospondin-1, and platelet factor 4. The platelet α-granule membrane protein P-selectin is expressed at 48% of wild-type levels and externalized upon platelet activation. The presence of P-selectin and normal levels of VPS33B and VPS16B in Nbeal2−/− platelets suggests that NBEAL2 acts independently of VPS33B/VPS16B at a later stage of α-granule biogenesis. Impaired Nbeal2−/− platelet function was shown by flow cytometry, platelet aggregometry, bleeding assays, and intravital imaging of laser-induced arterial thrombus formation. Microscopic analysis detected marked abnormalities in Nbeal2−/− bone marrow megakaryocytes, which when cultured showed delayed maturation, decreased survival, decreased ploidy, and developmental abnormalities, including abnormal extracellular distribution of VWF. Our results confirm that α-granule secretion plays a significant role in platelet function, and they also indicate that abnormal α-granule formation in Nbeal2−/− mice has deleterious effects on megakaryocyte survival, development, and platelet production. PMID:23861251

Effects of sodium citrate and acid citrate dextrose solutions on cell counts and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel.

Science.gov (United States)

Giraldo, Carlos E; Álvarez, María E; Carmona, Jorge U

2015-03-14

There is a lack information on the effects of the most commonly used anticoagulants for equine platelet rich plasmas (PRPs) elaboration on cell counts and growth factor release from platelet rich gels (PRGs). The aims of this study were 1) to compare the effects of the anticoagulants sodium citrate (SC), acid citrate dextrose solution A (ACD-A) and ACD-B on platelet (PLT), leukocyte (WBC) and on some parameters associated to platelet activation including mean platelet volume (MPV) and platelet distribution width (PDW) between whole blood, pure PRP (P-PRP) and platelet-poor plasma (PPP); 2) to compare transforming growth factor beta 1 (TGF-β(1)) and platelet-derived growth factor isoform BB (PDGF-BB) concentrations in supernatants from pure PRG (P-PRG), platelet-poor gel (PPG), P-PRP lysate (positive control) and plasma (negative control); 3) to establish the possible correlations between all the studied cellular and molecular parameters. In all cases the three anticoagulants produced P-PRPs with significantly higher PLT counts compared with whole blood and PPP. The concentrations of WBCs were similar between P-PRP and whole blood, but significantly lower in PPP. The type of anticoagulant did not significantly affect the cell counts for each blood component. The anticoagulants also did not affect the MPV and PDW parameters. Independently of the anticoagulant used, all blood components presented significantly different concentrations of PDGF-BB and TGF-β(1). The highest growth factor (GF) concentrations were observed from P-PRP lysates, followed by PRG supernatants, PPP lysates, PPG supernatants and plasma. Significant correlations were observed between PLT and WBC counts (ρ = 0.80), PLT count and TGF-β(1) concentration (ρ = 0.85), PLT count and PDGF-BB concentration (ρ = 0.80) and PDGF-BB and TGF-β(1) concentrations (ρ = 0.75). The type of anticoagulant was not correlated with any of the variables evaluated. The anticoagulants did not

Potential fluid mechanic pathways of platelet activation.

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Shadden, Shawn C; Hendabadi, Sahar

2013-06-01

Platelet activation is a precursor for blood clotting, which plays leading roles in many vascular complications and causes of death. Platelets can be activated by chemical or mechanical stimuli. Mechanically, platelet activation has been shown to be a function of elevated shear stress and exposure time. These contributions can be combined by considering the cumulative stress or strain on a platelet as it is transported. Here, we develop a framework for computing a hemodynamic-based activation potential that is derived from a Lagrangian integral of strain rate magnitude. We demonstrate that such a measure is generally maximized along, and near to, distinguished material surfaces in the flow. The connections between activation potential and these structures are illustrated through stenotic flow computations. We uncover two distinct structures that may explain observed thrombus formation at the apex and downstream of stenoses. More broadly, these findings suggest fundamental relationships may exist between potential fluid mechanic pathways for mechanical platelet activation and the mechanisms governing their transport.

Global proteome analysis identifies active immunoproteasome subunits in human platelets.

Science.gov (United States)

Klockenbusch, Cordula; Walsh, Geraldine M; Brown, Lyda M; Hoffman, Michael D; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-12-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The binding of fibrinogen to platelets in diabetes mellitus

International Nuclear Information System (INIS)

Minno, G. di; Cerbone, A.M.; Iride, C.; Mancini, M.

1986-01-01

Platelets from diabetics are known to be more sensitive in vitro to a variety of aggregating agents, to produce more prostaglandin endoperoxides and thromboxane and to bind more 125 I-fibrinogen than platelets from normal controls. Fibrinogen binding to platelets is a pre-requisite for platelet aggregation. Previous studies suggested a role for prostaglandins and/or thrombaxane A 2 in the exposure of fibrinogen receptors on platelets. The present study compares fibrinogen binding to hyperaggregable platelets from diabetic patients and to normal platelets when prostaglandin/thromboxane formation is suppressed by aspirin. It was found that pre-treatment with aspirin reduced collagen or thrombin-induced binding to platelets from none-retinopathic diabetics to the values seen in controls. By contrast, aspirin did not normalize binding to platelets obtained from retinopathic diabetics. The combination of aspirin with apyrase (an ADP scavenger) almost completely inhibited binding and aggregation of platelets from normal controls or non-retinophatic diabetics exposed to collagen or thrombin, whereas it only partially affected binding and aggregation of platelets from retinopathics. By using a monoclonal antibody (B59.2) to the platelet receptor for fibrinogen, we determined that this receptor was quantitatively and qualitatively the same on platelets from normal controls and diabetics. We conclude that increased fibrinogen binding and hyperaggregability of platelets from none-retinopathic diabetics is related to their capacity to form more prostaglandin endoperoxides/thromboxane than normal platelets. In contrast, hyperaggregability and increased binding of platelets from retinopathics appear at least partly related to a mechanism independent of ADP release and thromboxane synthesis. (Author)

Advances and controversies in neonatal ICU platelet transfusion practice.

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Christensen, Robert D

2008-01-01

Some of the platelet transfusions currently given to NICU patients are unnecessary and convey no benefits. Although ordered with good intentions, unnecessary platelet transfusions carry known and unknown risks. Identifying and eliminating any unnecessary platelet transfusions in NICUs would be a step toward better care, lower costs, and more careful preservation of blood component resources. A renewed interest in platelet transfusion studies is needed, if essential data is to be gathered to improve NICU platelet transfusion practice. Retrospective studies can be of value: for instance, seeking associations between bleeding events and platelet counts can suggest the possibility of cause and effect relationships. Such studies might identify approximate platelet count levels that convey high hemorrhagic risk and might help focus future prospective trials. Prospective indirect studies also can be of value, for instance, measuring the template bleeding time and the PFA-100 closure time as a function of platelet count and perhaps as a function of circulating platelet mass, and would provide new information with relevance to platelet transfusion benefits. Such studies might give a better awareness of how low the platelet count can fall before platelet plug formation is impaired. It seems inescapable, however, that new, multicentered, randomized, prospective studies are needed, where NICU patients are assigned different platelet transfusion triggers and then carefully tracked for bleeding events and long-term neurodevelopmental outcomes. Only that type of study is likely to generate the evidence base needed for widespread implementation of improvements in NICU platelet transfusion practice.

Failure to demonstrate a major role for Kupffer cells and radiosensitive leukocytes in immunoglobulin-mediated elimination of Trypanosoma musculi

International Nuclear Information System (INIS)

Kongshavn, P.A.; Shaw, K.; Ghadirian, E.; Ulczak, O.

1990-01-01

Previous studies have indicated that elimination of parasitemia in Trypanosoma musculi infection is brought about by immunoglobulin G2a antibodies, C3, and an effector cell. Experiments were designed to identify the putative effector cell by using several approaches. Infected C5-deficient or C5-sufficient mice treated with silica particles or given 900 rads of radiation 3 days earlier effectively eliminated trypanosomes following administration of immune plasma (IP). Silica-treated, noninfected mice given T. musculi preincubated with IP also cleared the parasites. Radiolabeling studies revealed that uptake of the cleared trypanosomes by the liver in normal mice was relatively low and fell only slightly (19%) in silica-treated mice. In contrast, uptake of radiolabeled sheep erythrocytes by the liver was normally much higher and fell drastically (7%) in silica-treated mice. Mice were then immunocompromised by 900 rads of radiation, silica particles, and anti-platelet serum combined before IP-sensitized trypanosomes were given. Leukocyte and platelet counts were both reduced by 95% and sheep erythrocyte uptake by the liver fell from 77 to 5%; however, greater than 99% of the injected trypanosomes were cleared in these mice and uptake of radiolabeled trypanosomes by the liver was similar to that of normal mice. Lastly, in anesthetized mice in which Kupffer cells were excluded surgically from the circulation, greater than 99% of the IP-sensitized trypanosomes disappeared rapidly from the blood. Only 7% of the radiolabel was found in the liver versus 60% in sham-operated mice. The results are interpreted as showing that hepatic Kupffer cells play a minor role in the immune elimination of T. musculi. Likewise, radiosensitive leukocytes and platelets are unlikely to be sole candidates for the putative effector cell that mediates a cure of murine trypanosomiasis

Low level of procoagulant platelet microparticles is associated with impaired coagulation and transfusion requirements in trauma patients

DEFF Research Database (Denmark)

Windeløv, Nis Agerlin; Johansson, Pär Ingemar; Sørensen, Anne Marie

2014-01-01

BACKGROUND: Following activation, platelets release small vesicles called platelet-derived microparticles (PMPs). PMPs accelerate thrombin generation and thus clot formation at sites of injury by exposing the procoagulant membrane phospholipid phosphatidylserine (PS). The role of PMPs in coagulop......BACKGROUND: Following activation, platelets release small vesicles called platelet-derived microparticles (PMPs). PMPs accelerate thrombin generation and thus clot formation at sites of injury by exposing the procoagulant membrane phospholipid phosphatidylserine (PS). The role of PMPs...

In vitro model of platelet aggregation in stenotic arteries

International Nuclear Information System (INIS)

Morley, D.; Santamore, W.P.

1988-01-01

Clinical and experimental evidence suggest a strong relationship between arterial stenosis, platelet aggregation, and subsequent thrombus formation. To facilitate the study of platelet accumulation in stenotic arteries, we developed an in vitro preparation. Arterial segments were perfused with whole citrated blood. A stenosis was created by applying an external plastic constrictor to the artery. Platelet accumulation within the stenosis was assessed by scanning electron microscopy and by radioactive counts from Indium-111 labeled platelets. Utilizing this preparation, 30 carotid arterial segments from 10 mongrel dogs were perfused at 100 mmHg for 15 min. In 10 arteries without a stenosis, scanning electron microscopy and radioactive counts demonstrated little platelet accumulation. In contrast, extensive platelet aggregation was observed in 10 arteries with stenoses. Moreover, in 10 stenotic arteries exposed to the thromboxane mimetic, U46619 (Upjohn Diagnostic Group), scanning electron microscopy and radioactive counts demonstrated a significant increase in platelet deposition. Conversely, we demonstrated a dimunition of platelet accumulation in stenosed arterial segments exposed to the prostacyclin analogue platelet inhibitor, Iloprost. The in vitro preparation allows precise control of hemodynamic variables and makes it possible to perform multiple tests on segments of the same vessel from the same animal

Imaging thrombus with radiolabelled monoclonal antibody to platelets

Energy Technology Data Exchange (ETDEWEB)

Peters, A.M.; Lavender, J.P.; Needham, S.G.; Loutfi, I.; Snook, D.; Epenetos, A.A.; Lumley, P.; Keery, R.J.; Hogg, N.

1986-12-13

A study was conducted evaluating a method of imaging thrombus with platelets radiolabelled with a /sup 111/In labelled monoclonal antibody, P256, directed to the platelet surface glycoprotein complex IIb/IIIa. when the number of receptors occupied by P256 was less than 3% of the total available on the platelet surface, platelet function was undisturbed. P256 was radiolabelled with /sup 111/In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The /sup 111/In kinetics recorded after intravenous P256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P256, three had documented thrombus, two of whom gave positive results on P256 platelet scintigraphy. The third had chronic deep venous thrombosis and was scintigraphically negative.

DREAM plays an important role in platelet activation and thrombogenesis

Science.gov (United States)

Kim, Kyungho; Tseng, Alan; Barazia, Andrew; Italiano, Joseph E.

2017-01-01

Downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, is known to modulate pain responses. However, it is unknown whether DREAM is expressed in anucleate platelets and plays a role in thrombogenesis. By using intravital microscopy with DREAM-null mice and their bone marrow chimeras, we demonstrated that both hematopoietic and nonhematopoietic cell DREAMs are required for platelet thrombus formation following laser-induced arteriolar injury. In a FeCl3-induced thrombosis model, we found that compared with wild-type (WT) control and nonhematopoietic DREAM knockout (KO) mice, DREAM KO control and hematopoietic DREAM KO mice showed a significant delay in time to occlusion. Tail bleeding time was prolonged in DREAM KO control mice, but not in WT or DREAM bone marrow chimeric mice. In vivo adoptive transfer experiments further indicated the importance of platelet DREAM in thrombogenesis. We found that DREAM deletion does not alter the ultrastructural features of platelets but significantly impairs platelet aggregation and adenosine triphosphate secretion induced by numerous agonists (collagen-related peptide, adenosine 5′-diphosphate, A23187, thrombin, or U46619). Biochemical studies revealed that platelet DREAM positively regulates phosphoinositide 3-kinase (PI3K) activity during platelet activation. Using DREAM-null platelets and PI3K isoform-specific inhibitors, we observed that platelet DREAM is important for α-granule secretion, Ca2+ mobilization, and aggregation through PI3K class Iβ (PI3K-Iβ). Genetic and pharmacological studies in human megakaryoblastic MEG-01 cells showed that DREAM is important for A23187-induced Ca2+ mobilization and its regulatory function requires Ca2+ binding and PI3K-Iβ activation. These results suggest that platelet DREAM regulates PI3K-Iβ activity and plays an important role during thrombus formation. PMID:27903531

Measurement of platelet aggregation, independently of patient platelet count

DEFF Research Database (Denmark)

Vinholt, P J; Frederiksen, H; Hvas, A-M

2017-01-01

with collagen-related peptide). Platelet aggregation had a negative predictive value of 100% for a bleeding tendency among patients. Conclusion The established platelet aggregation assay was applicable for thrombocytopenic patients, and improved the identification of bleeding risk.......Essentials •Platelet function may influence bleeding risk in thrombocytopenia, but useful tests are needed. •A flow cytometric platelet aggregation test independent of the patient platelet count was made. •Platelet aggregation was reduced in thrombocytopenic patients with hematological cancer....... •High platelet aggregation ruled out bleeding tendency in thrombocytopenic patients. Summary Background Methods for testing platelet aggregation in thrombocytopenia are lacking. Objective To establish a flow-cytometric test of in vitro platelet aggregation independently of the patient's platelet count...

Radiolabeled platelets

International Nuclear Information System (INIS)

Datz, F.L.; Taylor, A.T.

1986-01-01

Initial interest in developing techniques to radiolabel platelets was spurred by the lack of an accurate method for measuring platelet life span in both normals and in thrombocytopenic patients. Early investigators could obtain only rough estimates of platelet life spans by monitoring the platelet counts of thrombocytopenic patients undergoing platelet transfusions. Labels were also sought that would allow imaging of platelets in vivo in order to better understand the pathophysiology of atherosclerosis, thrombophlebitis, and clotting disorders, and to improve the clinical diagnosis of these diseases. Two types of platelet labels were investigated: cohort (pulse) labels and random labels. Cohort labels are taken up by megakaryocytes in the bone marrow and incorporated in the DNA and other components of the forming platelet. In theory, only freshly released platelets of a uniform age are labeled. Random labels, on the other hand, tag platelets in the peripheral blood, labeling platelets of all ages

Generation of Platelet Microparticles after Cryopreservation of Apheresis Platelet Concentrates Contributes to Hemostatic Activity

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İbrahim Eker

2017-03-01

Full Text Available Objective: In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs, platelet degranulation, and release of platelet-derived growth factors (PDGFs. We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation. Materials and Methods: Apheresis platelet concentrates (APCs from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing. Results: The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/μL vs. 319.9±80.5/μL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively, but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<0.001 than those of fresh APCs. The mean endogenous thrombin potential (ETP of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001. Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014. Conclusion: Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused

Altered mitochondrial function and oxidative stress in leukocytes of anorexia nervosa patients.

Science.gov (United States)

Victor, Victor M; Rovira-Llopis, Susana; Saiz-Alarcon, Vanessa; Sangüesa, Maria C; Rojo-Bofill, Luis; Bañuls, Celia; Falcón, Rosa; Castelló, Raquel; Rojo, Luis; Rocha, Milagros; Hernández-Mijares, Antonio

2014-01-01

Anorexia nervosa is a common illness among adolescents and is characterised by oxidative stress. The effects of anorexia on mitochondrial function and redox state in leukocytes from anorexic subjects were evaluated. A multi-centre, cross-sectional case-control study was performed. Our study population consisted of 20 anorexic patients and 20 age-matched controls, all of which were Caucasian women. Anthropometric and metabolic parameters were evaluated in the study population. To assess whether anorexia nervosa affects mitochondrial function and redox state in leukocytes of anorexic patients, we measured mitochondrial oxygen consumption, membrane potential, reactive oxygen species production, glutathione levels, mitochondrial mass, and complex I and III activity in polymorphonuclear cells. Mitochondrial function was impaired in the leukocytes of the anorexic patients. This was evident in a decrease in mitochondrial O2 consumption (Panorexia takes place at mitochondrial complex I. Future research concerning mitochondrial dysfunction and oxidative stress should aim to determine the physiological mechanism involved in this effect and the physiological impact of anorexia.

Platelets and hemophilia: A review of the literature.

Science.gov (United States)

Riedl, Julia; Ay, Cihan; Pabinger, Ingrid

2017-07-01

Hemophilia A and B are inherited bleeding disorders due to deficiencies of the clotting factors VIII and IX, respectively. The severity of the disease correlates with remaining factor levels, although individual differences in bleeding tendency are seen despite similar factor levels. While thrombin generation is severely impaired in persons with hemophilia, primary hemostasis, i.e. platelet function, has been generally considered to be normal. However, some studies reported prolonged bleeding times in hemophilia, suggesting that also primary hemostasis is affected. In several other studies different aspects of platelet function in hemophilia have been investigated in more detail and various alterations were discovered, such as increased platelet P-selectin expression, a lower number of procoagulant, so-called 'coated' platelets, lower aggregation upon co-incubation with tissue factor, or reduced platelet contractile forces during clot formation in comparison to healthy individuals. An influence of platelet function on clinical phenotype was suggested, which might contribute in part to variations in bleeding tendency in hemophilic patients with similar factor levels. However, the available evidence is currently limited and no clear correlations between platelet function parameters and clinical phenotypes have been demonstrated. The impact of alterations of platelet function in hemophilia remains to be better defined. Another interesting role of platelets in hemophilia has been reported recently by establishing a novel gene-therapeutic strategy using platelets as a delivery system for FVIII, showing promising results in animal models. This review gives an overview on the currently published literature on platelet function and the potential roles of platelets in hemophilia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of newly formed platelets in thrombus formation in rat after clopidogrel treatment

DEFF Research Database (Denmark)

Kuijpers, Marijke J E; Megens, Remco T A; Nikookhesal, Elham

2011-01-01

Platelet P2Y₁₂ receptors play an important role in arterial thrombosis by stimulating thrombus growth. Both irreversibly (clopidogrel) and reversibly binding (ticagrelor, AZD6140) P2Y₁₂ antagonists are clinically used for restricted periods, but possible differences in platelet function recovery ...

[Thrombopenia and radial aplasia: 2 cases with platelet function and ultrastructural studies of megakaryocytes and platelets (author's transl)].

Science.gov (United States)

Juhan, I; Bayle, J; Mattei, J F; Thevenieau, D; Perrimond, H; Muratore, R

1979-10-01

The authors report on two cases of congenital thrombopenia with radial aplasia. Both children display several formative abnormalities and a mild thrombopenia; hemorragic manifestations occurred in the first case only. Megacryoblastic to platelets series, as studied with electronic microscopy, show small-sized, "microcytic" and hypogranular megacaryocytes, displaying a maturative disorder (dysmegacaryocytopoiesis). In functional studies, platelets of the first patient show an imperfect nucleotidic release and do not agregate normally with ristocetin. The second case exhibits mostly a PF3 reduction. The variety of expression of the megacaryocytic-platelets disorders appears likewise in the squelettal and visceral malformations. The whole disorder could be ascribed to a pleiotropic abnormal gene with a variable expressivity.

Platelet size does not correlate with platelet age.

Science.gov (United States)

Thompson, C B; Love, D G; Quinn, P G; Valeri, C R

1983-08-01

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense-body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75Se-methionine, (2) in vitro labeling with 51Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51Cr and 14C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation.

Platelet size does not correlate with platelet age

International Nuclear Information System (INIS)

Thompson, C.B.; Love, D.G.; Quinn, P.G.; Valeri, C.R.

1983-01-01

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense-body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75 Se-methionine, (2) in vitro labeling with 51 Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75 Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51 Cr and 14 C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

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Monique Ramos de Oliveira Trugilho

2017-05-01

Full Text Available Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet

Complex formation of p-carboxybenzeneboronic acid with fructose

International Nuclear Information System (INIS)

Bulbul Islam, T.M.; Yoshino, K.

2000-01-01

To increase the solubility of p-caboxybenzeneboronic acid (PCBA) in physiological pH 7.4, the complex formation of PCBA with fructose has been studied by 11 B-NMR. PCBA formed complex with fructose and the complex increased the solubility of PCBA. The complex formation constant (log K) was obtained in pH 7.4 as 2.75 from the 11 B-NMR spectra. Based on this result the complex formation ability of PCBA with fructose has been discussed. (author)

The life cycle of platelet granules [version 1; referees: 2 approved

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Anish Sharda

2018-02-01

Full Text Available Platelet granules are unique among secretory vesicles in both their content and their life cycle. Platelets contain three major granule types—dense granules, α-granules, and lysosomes—although other granule types have been reported. Dense granules and α-granules are the most well-studied and the most physiologically important. Platelet granules are formed in large, multilobulated cells, termed megakaryocytes, prior to transport into platelets. The biogenesis of dense granules and α-granules involves common but also distinct pathways. Both are formed from the trans-Golgi network and early endosomes and mature in multivesicular bodies, but the formation of dense granules requires trafficking machinery different from that of α-granules. Following formation in the megakaryocyte body, both granule types are transported through and mature in long proplatelet extensions prior to the release of nascent platelets into the bloodstream. Granules remain stored in circulating platelets until platelet activation triggers the exocytosis of their contents. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE proteins, located on both the granules and target membranes, provide the mechanical energy that enables membrane fusion during both granulogenesis and exocytosis. The function of these core fusion engines is controlled by SNARE regulators, which direct the site, timing, and extent to which these SNAREs interact and consequently the resulting membrane fusion. In this review, we assess new developments in the study of platelet granules, from their generation to their exocytosis.

Glu- and Lys-forms of plasminogen differentially affect phosphatidylserine exposure on the platelet surface

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D. D. Zhernossekov

2017-04-01

Full Text Available Plasminogen/plasmin system is known for its ability to support hemostatic balance of blood. However, plasminogen may be considered as an adhesive ligand and in this way could affect the functioning of blood cells. We showed that exogenous Lys-plasminogen, but not its Glu-form, inhibited platelet aggregation and suppressed platelet α-granule secretion. The aim of this work was to investigate the influence of Glu- and Lys-form of plasminogen on the formation of platelet procoagulant surface using phosphatidylserine exposure as a marker. Human platelets were obtained from human platelet-rich plasma (donors were healthy volunteers, men aged 30-40 years by gel-filtration on Sepharose 2B. Phosphatidylserine exposure on the platelet surface was evaluated by flow cytometry with FITC-conjugated annexin A5. Glu- and Lys-plasminogen have different impact on the platelet functioning. Exogenous Lys-plasminogen has no significant effect on phosphatidylserine exposure, while Glu-plasminogen increases phosphatidylserine exposure on the surface of thrombin- and collagen-activated human platelets. Glu-plasminogen can be considered as a co-stimulator of agonist-induced platelet secretion and procoagulant surface formation. Meanwhile effects of Lys-plasminogen are probably directed at platelet-platelet interactions and not related to agonist-stimulated pro-apoptotic changes. The observed different effects of Glu- and Lys-plasminogen on phosphatidylserine exposure can be explained by their structural peculiarities.

Niacin and biosynthesis of PGD2 by platelet COX-1 in mice and humans

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Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela; Alamuddin, Naji; Ibrahim, Salam; Crichton, Irene; Prempeh, Maxwell; Lawson, John A.; Wilensky, Robert L.; Rasmussen, Lars Melholt; Puré, Ellen; FitzGerald, Garret A.

2012-01-01

The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1–dependent formation of PGD2 and PGE2 followed by COX-2–dependent production of PGE2. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD2 receptor DP1. NSAID-mediated suppression of COX-2–derived PGI2 has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD2. Here, we show that PGD2 biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1–derived PGD2 biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD2 was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD2, like PGI2, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy. PMID:22406532

Blood platelet kinetics and platelet transfusion

OpenAIRE

Aster, Richard H.

2013-01-01

The discovery of citrate anticoagulant in the 1920s and the development of plastic packs for blood collection in the 1960s laid the groundwork for platelet transfusion therapy on a scale not previously possible. A major limitation, however, was the finding that platelet concentrates prepared from blood anticoagulated with citrate were unsuitable for transfusion because of platelet clumping. We found that this could be prevented by simply reducing the pH of platelet-rich plasma to about 6.5 pr...

Evaluation of the TEG® platelet mappingTM assay in blood donors

DEFF Research Database (Denmark)

Bochsen, Louise; Wiinberg, Bo; Kjelgaard-Hansen, Mads Jens

2007-01-01

for quantification of platelet function, including the contribution of the adenosine diphosphate (ADP) and thromboxane A2 (TxA2) receptors to clot formation. Methods In 43 healthy blood donors, the analytical (CVa) and inter-individual variability (CVg) of the TEG® Platelet MappingTM assay were determined together......Background Monitoring of antiplatelet therapy in patients at cardiovascular risk is difficult because existing platelet function tests are too sophisticated for clinical routine. The whole blood TEG® Platelet MappingTM assay measures clot strength as maximal amplitude (MA) and enables...

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

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Lynch, D M; Howe, S E

1985-11-01

Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

Technetium-99m HMPAO labeled leukocytes in inflammation imaging

International Nuclear Information System (INIS)

Uno, Kimiichi; Yoshikawa, Kyousan; Imazeki, Keiko; Minoshima, Satoshi; Arimizu, Noboru

1991-01-01

Technetium-99m-HMPAO (Tc-99m-HMPAO) labeled leukocyte imaging was carried out in 19 patients at 3-5 hr after reinjection. There were no side effects noted. Tc-99m leukocyte images showed gall bladder, colon, kidney, and urinary bladder activity in normal distribution as a result of excretion of the eluted Tc-99m complex. They yielded a sensitivity of 93%, a specificity of 100% and an accuracy of 95%. They were correctly positive in 14 out of 19 cases. But one false negative case was seen in a patient with pyonephrosis showing a lack of renal function with decreased renal blood flow. It was concluded that they have some advantages over In-111 leukocyte images, but we have to consider the fact that the ureteral obstruction or the lack of renal function with decreased renal blood flow may result in a false positive or a false negative case. (author)

Cryopreservation of Human Mucosal Leukocytes.

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Sean M Hughes

Full Text Available Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible.To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension.Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

Comparison of platelet formation in hydrogen and helium-implanted silicon

International Nuclear Information System (INIS)

Hebras, X.; Nguyen, P.; Bourdelle, K.K.; Letertre, F.; Cherkashin, N.; Claverie, A.

2007-01-01

A comparative transmission electron microscopy study of the extended defects formed in (0 0 1) Si after hydrogen or helium implantation was performed. Quantitative data on the size and density of the defects with different crystallographic variants have been obtained. Common defects observed after implants with a dose of 1 x 10 16 cm -2 and isothermal anneals at 350 o C in the presence of a stiffener were platelet-like structures lying on {1 0 0} habit planes parallel and perpendicular to the wafer surface. The differences in the defect morphology and in the variant platelet population are correspondingly related to the different chemical reactivity of H and He and the different compressive biaxial stresses generated by the H and He implants

Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors

Science.gov (United States)

2013-01-01

Introduction Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed. Methods PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure. Results Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 106 to 1.9 × 106 platelets/μl). Platelets were highly purified, because only blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of

Preliminary investigations on the effects of a Strongylus vulgaris larval extract, mononuclear factors and platelet factors on equine smooth muscle cells in vitro.

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Morgan, S J; Storts, R W; Stromberg, P C; Sowa, B A; Lay, J C

1989-01-01

Factors involved in the proliferation of equine vascular smooth muscle cells were studied in vitro. The most prominent proliferative responses in cultured vascular smooth muscle cells were induced by Strongylus vulgaris larval antigen extract (LAE) and platelet-derived factors. Less significant proliferative responses were obtained with conditioned media from S. vulgaris LAE stimulated and from unstimulated equine mononuclear leukocytes. Additionally, vascular smooth muscle cells exposed to S. vulgaris LAE developed numerous perinuclear vacuoles and were more spindle-shaped than control or smooth muscle cells exposed to other factors. Equine mononuclear leukocytes exposed to LAE developed prominent morphological changes, including enlargement, clumping and increased numbers of mitotic figures.

Indium-111 tropolone, a new tracer for platelet labeling

International Nuclear Information System (INIS)

Dewanjee, M.K.; Rao, S.A.; Rosemark, J.A.; Chowdhury, S.; Didisheim, P.

1982-01-01

Platelets have been labeled with a new neutral, lipid-soluble metal complex of indium 111 ( 111 In) and tropolone. Unlike oxine, which is soluble in ethyl alcohol, tropolone is soluble in isotonic saline. Platelet labeling with 111 In tropolone can be performed in both acid-citrate-dextrose (ACD) plasma and ACD saline within two hours. Labeling efficiency has been 80% to 90%. 111 In tropolone in ACD saline and ACD plasma at tropolone concentrations of 5 and 10 micrograms/ml, respectively, and incubation of the platelets with the tracer at room temperature for 20 minutes were optimal conditions for labeling. The authors have developed an ACD-saline kit for convenient preparation of 111 In-labeled platelets. No adverse effect of 111 In tropolone on platelets has been observed in studies of biodistribution, recovery, and survival of platelets in rabbits and dogs

Indium-111 tropolone, a new tracer for platelet labeling

International Nuclear Information System (INIS)

Dewanjee, M.K.; Rao, S.A.; Rosemark, J.A.; Chowdhury, S.; Didisheim, P.

1982-01-01

Platelets have been labeled with a new neutral, lipid-soluble metal complex of indium 111 ( 111 In) and tropolone. Unlike oxine, which is soluble in ethyl alcohol, tropolone is soluble in isotonic saline. Platelet labeling with 111 In tropolone can be performed in both acid-citrate-dextrose (ACD) plasma and ACD saline within two hours. Labeling efficiency has been 80% to 90%. 111 In tropolone in ACD saline and ACD plasma at tropolone concentrations of 5 to 10 μg/ml, respectively, and incubation of the platelets with the tracer at room temperature for 20 minutes were optimal conditions for labeling. The authors have developed an ACD-saline kit for convenient preparation of 111 In-labeled platelets. No adverse effect of 111 In tropolone on platelets has been observed in studies of biodistribution, recovery, and survival of platelets in rabbits and dogs

Deletion of GLUT1 and GLUT3 Reveals Multiple Roles for Glucose Metabolism in Platelet and Megakaryocyte Function

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Trevor P. Fidler

2017-07-01

Full Text Available Anucleate platelets circulate in the blood to facilitate thrombosis and diverse immune functions. Platelet activation leading to clot formation correlates with increased glycogenolysis, glucose uptake, glucose oxidation, and lactic acid production. Simultaneous deletion of glucose transporter (GLUT 1 and GLUT3 (double knockout [DKO] specifically in platelets completely abolished glucose uptake. In DKO platelets, mitochondrial oxidative metabolism of non-glycolytic substrates, such as glutamate, increased. Thrombosis and platelet activation were decreased through impairment at multiple activation nodes, including Ca2+ signaling, degranulation, and integrin activation. DKO mice developed thrombocytopenia, secondary to impaired pro-platelet formation from megakaryocytes, and increased platelet clearance resulting from cytosolic calcium overload and calpain activation. Systemic treatment with oligomycin, inhibiting mitochondrial metabolism, induced rapid clearance of platelets, with circulating counts dropping to zero in DKO mice, but not wild-type mice, demonstrating an essential role for energy metabolism in platelet viability. Thus, substrate metabolism is essential for platelet production, activation, and survival.

Effect of small doses of ionizing radiation on motility, rosette formation, and antioxidant state of leukocytes under modification of G-proteins by cholera and pertussis toxins

International Nuclear Information System (INIS)

Zhirnov, V.V.; Luik, A.I.; Metelitsa, L.A.; Mogilevich, S.E.; Charochkina, L.L.

2000-01-01

The responses of motility and rosette formation of leukocytes to small radioactive doses (from 6 centre dot 10 -10 to 6 centre dot 10 -4 Gy) are studied. The influence of these doses on cell functions and oxidative homeostasis are investigated under the modification of transducing components of membrane signal pathways (adenylate cyclase and polyphosphoinositide cascades) with pertussis and cholera toxins

Platelet-derived growth factor inhibits platelet activation in heparinized whole blood.

Science.gov (United States)

Selheim, F; Holmsen, H; Vassbotn, F S

1999-08-15

We previously have demonstrated that human platelets have functionally active platelet-derived growth factor alpha-receptors. Studies with gel-filtered platelets showed that an autocrine inhibition pathway is transduced through this tyrosine kinase receptor during platelet activation. The physiological significance of this inhibitory effect of platelet-derived growth factor on gel-filtered platelets activation is, however, not known. In the present study, we investigated whether platelet-derived growth factor inhibits platelet activation under more physiological conditions in heparinized whole blood, which represents a more physiological condition than gel-filtered platelets. Using flow cytometric assays, we demonstrate here that platelet-derived growth factor inhibits thrombin-, thrombin receptor agonist peptide SFLLRN-, and collagen-induced platelet aggregation and shedding of platelet-derived microparticles from the platelet plasma membrane during platelet aggregation in stirred heparinized whole blood. The inhibitory effect of platelet-derived growth factor was dose dependent. However, under nonaggregating conditions (no stirring), we could not demonstrate any significant effect of platelet-derived growth factor on thrombin- and thrombin receptor agonist peptide-induced platelet surface expression of P-selectin. Our results demonstrate that platelet-derived growth factor appears to be a true antithrombotic agent only under aggregating conditions in heparinized whole blood.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

Science.gov (United States)

O'Connor, Marie N; Salles, Isabelle I; Cvejic, Ana; Watkins, Nicholas A; Walker, Adam; Garner, Stephen F; Jones, Chris I; Macaulay, Iain C; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H; Deckmyn, Hans; Stemple, Derek L; Ouwehand, Willem H

2009-05-07

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

A critical role of platelet TGF-β release in podoplanin-mediated tumour invasion and metastasis.

Science.gov (United States)

Takemoto, Ai; Okitaka, Mina; Takagi, Satoshi; Takami, Miho; Sato, Shigeo; Nishio, Makoto; Okumura, Sakae; Fujita, Naoya

2017-02-08

The tumour microenvironment is critical for various characteristics of tumour malignancies. Platelets, as part of the tumour microenvironment, are associated with metastasis formation via increasing the rate of tumour embolus formation in microvasculature. However, the mechanisms underlying the ability of tumour cells to acquire invasiveness and extravasate into target organs at the site of embolization remain unclear. In this study, we reported that platelet aggregation-inducing factor podoplanin expressed on tumour cell surfaces were found to not only promote the formation of tumour-platelet aggregates via interaction with platelets, but also induced the epithelial-mesenchymal transition (EMT) of tumour cells by enhancing transforming growth factor-β (TGF-β) release from platelets. In vitro and in vivo analyses revealed that podoplanin-mediated EMT resulted in increased invasiveness and extravasation of tumour cells. Treatment of mice with a TGF-β-neutralizing antibody statistically suppressed podoplanin-mediated distant metastasis in vivo, suggesting that podoplanin promoted haematogenous metastasis in part by releasing TGF-β from platelets that was essential for EMT of tumour cells. Therefore, our findings suggested that blocking the TGF-β signalling pathway might be a promising strategy for suppressing podoplanin-mediated haematogenous metastasis in vivo.

The Phosphatase Inhibitor Calyculin-A Impairs Clot Retraction, Platelet Activation, and Thrombin Generation

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Renáta Hudák

2017-01-01

Full Text Available The aim of this study was to investigate the effect of the serine/threonine protein phosphatase inhibitor, calyculin-A (CLA, on clot formation and on the procoagulant activity of human platelets. Platelet-rich plasma (PRP samples were preincubated with buffer or CLA and subsequently platelets were activated by the protease-activated receptor 1 (PAR-1 activator, thrombin receptor activating peptide (TRAP. Clot retraction was detected by observing clot morphology up to 1 hour, phosphatidylserine- (PS- expression was studied by flow cytometry, and thrombin generation was measured by a fluorimetric assay. For the intracellular Ca2+ assay, platelets were loaded with calcium-indicator dyes and the measurements were carried out using a ratiometric method with real-time confocal microscopy. CLA preincubation inhibited clot retraction, PS-expression, and thrombin formation. TRAP activation elicited Ca2+ response and PS-expression in a subset of platelets. The activated PRP displayed significantly faster and enhanced thrombin generation compared to nonactivated samples. CLA pretreatment abrogated PS-exposure and clot retraction also in TRAP-activated samples. As a consequence of the inhibitory effect on calcium elevation and PS-expression, CLA significantly downregulated thrombin generation in PRP. Our results show that CLA pretreatment may be a useful tool to investigate platelet activation mechanisms that contribute to clot formation and thrombin generation.

Defining Platelet Function During Polytrauma

Science.gov (United States)

2014-04-01

scaffold of the clot after its thrombin-induced polymerization to fibrin fibers. Formation of a stiff fibrin fiber network and its contraction by platelet...In another study (PROTEC T) Refused Participat ion (Yes=Y, No=N, W=Unqua lified , Refused further draws (agreed to keep previous data

Pharmacological modulation of human platelet leukotriene C4-synthase.

Science.gov (United States)

Sala, A; Folco, G; Henson, P M; Murphy, R C

1997-03-21

The aim of this study was to test if human platelet leukotriene C4-synthase (LTC4-S) is pharmacologically different from cloned and expressed LTC4-S and, in light of the significant homologies between 5-lipoxygenase activating protein (FLAP) and LTC4-S, if different potencies of leukotriene synthesis inhibitors acting through binding with FLAP (FLAP inhibitors) reflect in different potencies as LTC4-S inhibitors. Leukotriene C4 (LTC4) synthesis by washed human platelets supplemented with synthetic leukotriene A4 (LTA4) was studied in the absence and presence of two different, structurally unrelated FLAP inhibitors (MK-886 and BAY-X1005) as well as a direct 5-lipoxygenase inhibitor (zileuton). LTC4 production was analyzed by RP-HPLC coupled to diode array detection. We report that human platelet LTC4-S was inhibited by MK-886 and BAY-X1005 (IC50 of 4.7 microM and 91.2 microM, respectively), but not by zileuton (inactive up to 300 microM); all 3 compounds were able to inhibit 5-lipoxygenase metabolite biosynthesis in intact human polymorphonuclear leukocytes (IC50 of 0.044 microM, 0.85 microM, and 1.5 microM, respectively). Platelet LTC4-S does not appear pharmacologically different from expression cloned LTC4-S. LTC4-S inhibition by FLAP inhibitors is in agreement with the significant homology reported for expression-cloned LTC4-S with FLAP, Furthermore, functional homology of the binding sites for inhibitors on LTC4-S and FLAP is suggested by the conservation of the relative potencies of MK-886 and BAY-X1005 vs FLAP-dependent 5-lipoxygenase activity and LTC4-S inhibition: MK-886 was 19.3-fold more potent than BAY-X1005 as FLAP inhibitor and 19.6-fold more potent than BAY-X1005 as LTC4-S inhibitor.

The accumulation of a platelet protein, thrombospondin, at the site of arterial thrombus formation: Preliminary report

International Nuclear Information System (INIS)

Perlman, S.B.; Hammes, R.J.; Besozzi, M.C.; Folts, J.D.; Mosher, D.F.

1987-01-01

There are many methods available for the detection of thrombosis, none of which are noninvasive, rapid and accurate. Thrombosponding is a platelet protein that is present in the developing thrombus and may be an effective substance to use for imaging thrombosis. Vascular stenosis and thrombosis were produced in coronary, carotid and femoral arteries in eleven adult mongrel dogs. 131 I labeled thrombosponding was administered to each animal to determine whether the radiotracer accumulated at the site of thrombus formation. The radioactivity per gram of vessels with thrombi was significantly different from the control vessels or whole blood (p=0.0037 and p=0.015, respectively, paired t-test). This preliminary work suggests that iodinated thrombosponding accumulates at the site of thrombus formation. Labeled thrombosponding may be a rapid, safe and accurate method of detecting arterial thrombosis. (orig.)

Effect of Cold Storage on Shear-induced Platelet Aggregation and Clot Strength

Science.gov (United States)

2014-09-01

hemostasis and are lifesavingwhen transfused to treat thrombocytopenia. In response to vascular injury, platelets adhere, aggregate, and along with fibrin ...Handling Platelet - rich plasma (PRP) was obtained from phlebot- omized blood or AP collection. Blood was drawn in acid cit- rate dextroseYcontaining...aging and senes- cence during storage.35,38 Platelet activation and aggregation lead to clot formation, beginning with the linear polymerization of fibrin

Competitions between fibrinogen with its degradation products for interactions with the platelet-fibrinogen receptor

International Nuclear Information System (INIS)

Thorsen, L.I.; Brosstad, F.; Gogstad, G.; Sletten, K.; Solum, N.O.

1986-01-01

Direct binding of 125 -I-labelled plasmic and CNBr-derived fibrin (ogen) fragments (pre-X, X, Y, D, Degta, Efg, E1, N-DSK, N-dsk) to gel-filtered platelets was compared to their ability to support or inhibit ADP-induced aggregation, and to compete with fibrinogen for binding to ADP-stimulated platelets. Pre-X was the only fragment that supported aggregation. All fragments tested except for E derived from fibrinogen (Efg) and Degta bound specifically to the platelets and inhibited ADP-induced aggregation in the presence of fibrinogen. Competitive binding studies with fibrinogen and fragments labelled with different isotopes of iodine, or inhibition of binding of labelled fibrinogen with unlabelled fragments showed that all of the fragments except Efg and Degta were able to compete with fibrinogen for binding. When simultaneous binding of N-dsk and fibrinogen was studied, an increased binding of both ligands was observed probably due to complex formation. The results fully agree with previous findings of binding to immunoprecipitated glycoprotein IIb-IIIa after crossed immunoelectrophoresis. We conclude that the fibrinogen molecule contains at least six sequences responsible for platelet interaction, two in the E domain and two in each of the C-terminal parts of the fibrinogen molecule

Collagen can selectively trigger a platelet secretory phenotype via glycoprotein VI.

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Véronique Ollivier

Full Text Available Platelets are not only central actors of hemostasis and thrombosis but also of other processes including inflammation, angiogenesis, and tissue regeneration. Accumulating evidence indicates that these "non classical" functions of platelets do not necessarily rely on their well-known ability to form thrombi upon activation. This suggests the existence of non-thrombotic alternative states of platelets activation. We investigated this possibility through dose-response analysis of thrombin- and collagen-induced changes in platelet phenotype, with regards to morphological and functional markers of platelet activation including shape change, aggregation, P-selectin and phosphatidylserine surface expression, integrin activation, and release of soluble factors. We show that collagen at low dose (0.25 µg/mL selectively triggers a platelet secretory phenotype characterized by the release of dense- and alpha granule-derived soluble factors without causing any of the other major platelet changes that usually accompany thrombus formation. Using a blocking antibody to glycoprotein VI (GPVI, we further show that this response is mediated by GPVI. Taken together, our results show that platelet activation goes beyond the mechanisms leading to platelet aggregation and also includes alternative platelet phenotypes that might contribute to their thrombus-independent functions.

Effect of radiographic contrast agents on leukocyte metabolic response

International Nuclear Information System (INIS)

Hernanz-Schulman, M.; Vanholder, R.; Waterloos, M.A.; Hakim, R.; Schulman, G.

2000-01-01

Barium, at clinical dilutions, causes a significant increase of baseline ''resting state'' phagocytic activity, which in turn leads to significant blunting of subsequent response to phagocytic challenge and adversely affects the response to all bacteria tested. There is no baseline activation of leukocytes by the water-soluble media, although there was some inhibition (rather than activation) of leukocyte metabolic activity. The effect of the water-soluble media in bacteria was more complex (although inhibition is minor compared to barium). Our data demonstrate that barium is a significant activator of phagocytic cells, which results in deactivation of phagocytic response when challenged; these data serve to explain the enhanced adverse effect of barium in cased of fecal peritonitis. (orig.)

Using autologous platelet-rich plasma for the treatment of complex fistulas

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Almudena Moreno-Serrano

Full Text Available Objective: This study aims to demonstrate the effectiveness and safety of autologous fibrin gel rich in platelet growth factors for the treatment of complex perianal fistulas. Material and methods: Prospective epidemiological study. Patients with complex perianal fistula or perianal fistula mere alteration of continence are included. identification of both holes and the journey, curettage of it and instillation of Vivostat PRF® in the way it is done to observe excess material by OFE. The variables analyzed were: age, sex, use of prior Seton clinic prevalent type of fistula, postoperative complications, fistula closure and impaired quality of life using the SF-36 test (v2. Results: From January 2011 to May 2013 have involved 23 patients, 12 men and 11 women, with an average age of 49 years and a minimum follow-up of 12 months. Two dropped out. 17 patients had low transsphincteric fistulas, 2 and 2 high transsphincteric intersphincteric with impaired continence. The most common symptom is the discharge. Twelve patients had a loose seton (62%, of which nine cured. Of all the patients we have operated the success rate is 62%. No patient developed incontinence after treatment. Only two reported a worse quality of life after surgery. Conclusion: This study demonstrates that there is a clear benefit to the use of Vivostat PRF® as a treatment for complex perianal fistulas. It is a highly reproducible technique with acceptable results and does not produce impairment of continence.

Imaging thrombus with radiolabelled monoclonal antibody to platelets

International Nuclear Information System (INIS)

Loutfi, I.; Peters, A.M.; Lavender, J.P.; Epenetos, A.A.

1988-01-01

Indium-111-hydroxyquinoline labelled platelets, though useful in the detection of thrombus, have not gained widespread use owing to the time and technical skill required for their preparation. A study was therefore conducted evaluating a new method of imaging thrombus with platelets radiolabelled with a 111 In labelled monoclonal antibody, P 256 , directed to the platelet surface glycoprotein complex IIb/IIIa. When the number of receptors occupied by P 256 was less than 3% of the total available on the platelet surface platelet function, as assessed by platelet aggregometry, was undisturbed. P 256 was radiolabelled with 111 In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo - that is, by direct intravenous injection of P 256 - in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The 111 In kinetics recorded after intravenous P 256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P 256 , three had documented thrombus, tow of whom gave positive results on P 256 platelet scintigraphy. The third subject had chromic deep venous thrombosis and was scintigraphically negative. Imaging thrombus using a radiolabelled monoclonal antibody directed to platelets appears to offer great potential as a simple, non-invasive approach to the diagnosis of thrombosis. 3 refs. (Author)

Equid herpesvirus type 1 activates platelets.

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Tracy Stokol

Full Text Available Equid herpesvirus type 1 (EHV-1 causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression and platelet microvesiculation (increased small events double positive for CD41 and Annexin V. Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM. A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis

Heterogeneity of rabbit platelets

International Nuclear Information System (INIS)

Karpatkin, S.

1978-01-01

Rabbits were injected intravenously with a cohort platelet label, 75 Se-selenomethionine. Platelet-rich plasma was separated into five different platelet density fractions on each of seven days by repetitively centrifuging the same sample of platelet-rich plasma at increasing gravitational force. The heaviest platelet sediment fraction was enriched with larger platelets. The lightest platelet sediment fraction was enriched with smaller platelets. Incorporation of isotope into the heaviest platelet fraction was considerably greater than incorporation into the lightest platelet fraction. The mean platelet survival of the lightest two fractions was significantly shorter than that of the heaviest three fractions. SDS-polyacrylamide gel electrophoresis of the platelet cell sap generally revealed 10 prominent protein bands for the heaviest platelet fractions. The lightest platelet fraction had six absent to markedly diminished platelet proteins. The data are compatible with two models, (1) heavy-large platelets are, on average, young platelets which become lighter-smaller platelets while losing platelet membranes and cell sap components with time. (2) Heavy-large platelets and light-small platelets are produced independently by specific megakarocytes. The heavy-large platelets incorporate more isotope that lighter-smaller platelets (possibly because of their megakarocyte precursor). However, they are released earlier into the circulation than lighter-smaller platelets and are therefore younger platelets. The light-smaller platelets which are released later into the circulation have a shorter survival. (author)

Emerging Evidence for Platelets as Immune and Inflammatory Effector Cells

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Matthew Thomas Rondina

2014-12-01

Full Text Available While traditionally recognized for their roles in hemostatic pathways, emerging evidence demonstrates that platelets have previously unrecognized, dynamic roles that span the immune continuum. These newly-recognized platelet functions, including the secretion of immune mediators, interactions with endothelial cells, monocytes, and neutrophils, toll-like receptor (TLR mediated responses, and induction of neutrophil extracellular trap (NET formation, bridge thrombotic and inflammatory pathways and contribute to host defense mechanisms against invading pathogens. In this focused review, we highlight several of these emerging aspects of platelet biology and their implications in clinical infectious syndromes.

Clot formation and lysis in platelet rich plasma of healthy donors and patients with resistant hypertension

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I. I. Patalakh

2018-04-01

Full Text Available Hemostatic balance in blood is affected by numerous factors, including coagulation and fibrinolytic proteins, the wide spectrum of their inhibitors, and blood cells. Since platelets can participate in contradictory processes, they significantly complicate the whole picture. Therefore, nowadays the development of global assays of hemostasis, which can reflect the physiological process of hemostasis and can be used for point-of-care diagnosis of thrombosis, is crucial. This paper outlines a new approach we used to analyze the capabilities of clot waveform analysis tools to distinguish the response of platelet-rich plasma from healthy donors and patients with arterial hypertension caused by stimulation of coagulation and lysis (with exogenous thrombin and recombinant tissue-type plasminogen activator, respectively. In donor plasma, when the clot degradation was accompanied by 40 IU/ml of recombinant tissue-type plasminogen activator, platelets potentiated fibrinolysis more than coagulation, which ultimately shifts the overall balance to a profibrinolytic state. At the same time, for patients with hypertension, platelets, embedded in clot obtained from platelet-rich plasma, showed a weaker ability to stimulate fibrinolysis. The obtained data gives the evidence that platelets can act not only as procoagulants but also as profibrinolytics. By simultaneously amplifying coagulation and fibrinolysis, making their rates comparable, platelets would control plasma procoagulant activity, thereby regulating local hemostatic balance, the size and lifetime of the clot. Moreover, clot waveform analysis may be used to distinguish the effects of platelet-rich plasma on clotting or lysis of fibrin clots in healthy donors and patients with essential hypertension.

EMMPRIN (CD147) is a novel receptor for platelet GPVI and mediates platelet rolling via GPVI-EMMPRIN interaction.

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Seizer, Peter; Borst, Oliver; Langer, Harald F; Bültmann, Andreas; Münch, Götz; Herouy, Yared; Stellos, Konstantinos; Krämer, Björn; Bigalke, Boris; Büchele, Berthold; Bachem, Max G; Vestweber, Dietmar; Simmet, Thomas; Gawaz, Meinrad; May, Andreas E

2009-04-01

The Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147, basigin) is an immunoglobulin-like receptor expressed in various cell types. During cellular interactions homotypic EMMPRIN-EMMPRIN interactions are known to induce the synthesis of matrix metalloproteinases. Recently, we have identified EMMPRIN as a novel receptor on platelets. To our knowledge EMMPRIN has not been shown to serve as adhesion receptor, yet. Here we characterise platelet glycoprotein VI (GPVI) as a novel adhesion receptor for EMMPRIN. Human platelets were prestimulated with ADP and perfused over immobilised recombinant EMMPRIN-Fc or Fc-fragments under arterial shear conditions. ADP-stimulated platelets showed significantly enhanced rolling (but not enhanced firm adhesion) on immobilised EMMPRIN-Fc compared to Fc. Pretreatment of platelets with blocking mAbs anti-EMMPRIN or anti-GPVI leads to a significant reduction of rolling platelets on immobilised EMMPRIN-Fc, whereas pretreatment with blocking mAbs anti-p-selectin, anti-alpha4-integrin or anti-GPIIb/IIIa complex (20 microg/ml each) had no effect. Consistently, chinese hamster ovary (CHO) cells stably transfected with GPVI showed enhanced rolling (but not adhesion) on immobilised EMMPRIN-Fc in comparison to non-transfected CHO cells. Similarly, CHO cells stably transfected with EMMPRIN showed enhanced rolling on immobilised GPVI-Fc (or EMMPRIN-Fc) compared to non transfected CHO-cells. Finally, specific binding of EMMPRIN to GPVI was demonstrated by a modified ELISA and surface plasmon resonance technology with a dissociation constant of 88 nM. Platelet GPVI is a novel receptor for EMMPRIN and can mediate platelet rolling via GPVI-EMMPRIN interaction.

Dissociation of SERPINE1 mRNA from the translational repressor proteins Ago2 and TIA-1 upon platelet activation.

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Corduan, Aurélie; Plé, Hélène; Laffont, Benoit; Wallon, Thérèse; Plante, Isabelle; Landry, Patricia; Provost, Patrick

2015-05-01

Platelets play an important role in haemostasis, as well as in thrombosis and coagulation processes. They harbour a wide variety of messenger RNAs (mRNAs), that can template de novo protein synthesis, and an abundant array of microRNAs, which are known to mediate mRNA translational repression through proteins of the Argonaute (Ago) family. The relationship between platelet microRNAs and proteins capable of mediating translational repression, however, remains unclear. Here, we report that half of platelet microRNAs is associated to mRNA-regulatory Ago2 protein complexes, in various proportions. Associated to these Ago2 complexes are platelet mRNAs known to support de novo protein synthesis. Reporter gene activity assays confirmed the capacity of the platelet microRNAs, found to be associated to Ago2 complexes, to regulate translation of these platelet mRNAs through their 3'UTR. Neither the microRNA repertoire nor the microRNA composition of Ago2 complexes of human platelets changed upon activation with thrombin. However, under conditions favoring de novo synthesis of Plasminogen Activator Inhibitor-1 (PAI-1) protein, we documented a rapid dissociation of the encoding platelet SERPINE1 mRNA from Ago2 protein complexes as well as from the translational repressor protein T-cell-restricted intracellular antigen-1 (TIA-1). These findings are consistent with a scenario by which lifting of the repressive effects of Ago2 and TIA-1 protein complexes, involving a rearrangement of proteinmRNA complexes rather than disassembly of Ago2microRNA complexes, would allow translation of SERPINE1 mRNA into PAI-1 in response to platelet activation.

Defining an appropriate leucoreduction strategy by serial assessment of cytokine levels in platelet concentrates prepared by different methods

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Daljit Kaur

2015-01-01

Full Text Available Background and Objectives: Different methods of platelet concentrate preparations leave behind certain number of residual leukocytes, accounting for most of the febrile nonhemolytic transfusion reactions, especially in multitransfused patients. Various inflammatory cytokines, such as tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β, and IL-6 are generated during storage and have been implicated for these adverse effects. We have studied the levels of these cytokines and their correlation with leucocyte contents in platelet concentrates prepared by three different methods. Study Design and Methods: Five pools of platelet rich plasma platelet concentrates (PRP-PC and buffy-coat platelet concentrates (BC-PC each were prepared and divided into two halves. One half of the pool was leucofiltered (LF, whereas the other half was stored as such. Ten apheresis units were also included in the study. All the platelet concentrates were assessed for leucocyte load and cytokine content (IL-1β, IL-6, and TNF-α on different days of storage (0, 3, and 5 using Nageotte chamber and commercially available immunoassays respectively. Results: There was a statistically significant rise in cytokine levels (IL-1β, IL-6, and TNF-α in nonleucofiltered (NLF random donor platelet concentrates (RDPs (PRP-PC and BC-PC during storage (day 3 and 5 whereas LF RDP concentrates (PRP-PC and BC-PC and apheresis platelet concentrates (AP-PC did not show any significant rise in cytokine levels (on day 3 and 5 over the baseline values at day 0. Conclusion: This data suggests that although AP-PCs are superior to PRP-PC (NLF and BC-PC (NLF in terms of in vitro quality control parameters and cytokine generation during storage, BC-PC mode of platelet preparation followed by leucofiltration is the best method to store platelets and minimise the cytokine accumulation. This strategy is best suited for transfusion in multitransfused hematooncologic patients, who cannot afford

Alkali treatment of microrough titanium surfaces affects macrophage/monocyte adhesion, platelet activation and architecture of blood clot formation

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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.

Use of platelets labelled with 111In-Oxine for detection of aortic endocarditis

International Nuclear Information System (INIS)

Rodriguez, V.; Pulpon, L.A.; Rodriguez, E.; Marin, D.; Chamorro, J.L.; Ortiz-Berrocal, J.

1982-01-01

The vegetation is the most characteristic lesion of infective endocarditis. It is constituted mainly of platelets immersed in a fibrin magma, wherein microorganism nest. The damaged vascular endothelium constitutes the first stimulus for the formation of an aseptic platelet thrombus. For this reason, the use of labelled platelets permits first the external detection of formed vegetations and second, by means of serial studies, the study of the evolution of the pathological lesion under treatment with various pharmacological agent. 111 In forms a chelate with the 8-hydroxyquinolein molecule (oxine), and this chelate passes through the platelets membranes and forms a stable bond with cytoplasmic structures. The procedure used in rabbits was useful for the external detection and study of the formation of vegetation induced by infective endocarditis. The technique could be highly useful in the experimental evaluation of antiaggregant agents

HK2 Recruitment to Phospho-BAD Prevents Its Degradation, Promoting Warburg Glycolysis by Theileria-Transformed Leukocytes.

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Haidar, Malak; Lombès, Anne; Bouillaud, Frédéric; Kennedy, Eileen J; Langsley, Gordon

2017-03-10

Theileria annulata infects bovine leukocytes, transforming them into invasive, cancer-like cells that cause the widespread disease called tropical theileriosis. We report that in Theileria-transformed leukocytes hexokinase-2 (HK2) binds to B cell lymphoma-2-associated death promoter (BAD) only when serine (S) 155 in BAD is phosphorylated. We show that HK2 recruitment to BAD is abolished by a cell-penetrating peptide that acts as a nonphosphorylatable BAD substrate that inhibits endogenous S155 phosphorylation, leading to complex dissociation and ubiquitination and degradation of HK2 by the proteasome. As HK2 is a critical enzyme involved in Warburg glycolysis, its loss forces Theileria-transformed macrophages to switch back to HK1-dependent oxidative glycolysis that down-regulates macrophage proliferation only when they are growing on glucose. When growing on galactose, degradation of HK2 has no effect on Theileria-infected leukocyte proliferation, because metabolism of this sugar is independent of hexokinases. Thus, targeted disruption of the phosphorylation-dependent HK2/BAD complex may represent a novel approach to control Theileria-transformed leukocyte proliferation.

Covalent modification of platelet proteins by palmitate

International Nuclear Information System (INIS)

Muszbek, L.; Laposata, M.

1989-01-01

Covalent attachment of fatty acid to proteins plays an important role in association of certain proteins with hydrophobic membrane structures. In platelets, the structure of many membrane glycoproteins (GPs) has been examined in detail, but the question of fatty acid acylation of platelet proteins has not been addressed. In this study, we wished to determine (a) whether platelet proteins could be fatty acid acylated; and, if so, (b) whether these modified proteins were present in isolated platelet membranes and cytoskeletal fractions; and (c) if the pattern of fatty acid acylated proteins changed on stimulation of the platelets with the agonist thrombin. We observed that in platelets allowed to incorporate 3H-palmitate, a small percentage (1.37%) of radioactivity incorporated into the cells became covalently bound to protein. Selective cleavage of thioester, thioester plus O-ester, and amide-linked 3H-fatty acids from proteins, and their subsequent analysis by high-performance liquid chromatography (HPLC) indicated that the greatest part of 3H-fatty acid covalently bound to protein was thioester-linked 3H-palmitate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, at least ten major radiolabeled proteins were detected. Activation of platelets by thrombin greatly increased the quantity of 3H-palmitoylated proteins associated with the cytoskeleton. Nearly all radiolabeled proteins were recovered in the membrane fraction, indicating that these proteins are either integral or peripheral membrane proteins or proteins tightly associated to membrane constituents. Components of the GPIIb-IIIa complex were not palmitoylated. Thus, platelet proteins are significantly modified posttranslationally by 3H-palmitate, and incorporation of palmitoylated proteins into the cytoskeleton is a prominent component of the platelet response to thrombin stimulation

Reproducibility of Manual Platelet Estimation Following Automated Low Platelet Counts

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Zainab S Al-Hosni

2016-11-01

Full Text Available Objectives: Manual platelet estimation is one of the methods used when automated platelet estimates are very low. However, the reproducibility of manual platelet estimation has not been adequately studied. We sought to assess the reproducibility of manual platelet estimation following automated low platelet counts and to evaluate the impact of the level of experience of the person counting on the reproducibility of manual platelet estimates. Methods: In this cross-sectional study, peripheral blood films of patients with platelet counts less than 100 × 109/L were retrieved and given to four raters to perform manual platelet estimation independently using a predefined method (average of platelet counts in 10 fields using 100× objective multiplied by 20. Data were analyzed using intraclass correlation coefficient (ICC as a method of reproducibility assessment. Results: The ICC across the four raters was 0.840, indicating excellent agreement. The median difference of the two most experienced raters was 0 (range: -64 to 78. The level of platelet estimate by the least-experienced rater predicted the disagreement (p = 0.037. When assessing the difference between pairs of raters, there was no significant difference in the ICC (p = 0.420. Conclusions: The agreement between different raters using manual platelet estimation was excellent. Further confirmation is necessary, with a prospective study using a gold standard method of platelet counts.

Mean platelet volume and mean platelet volume/platelet count ratio

African Journals Online (AJOL)

Amira M. Elsayed

2016-03-30

Mar 30, 2016 ... The aim of this study was to compare the MPV and mean platelet volume/platelet count ... brain stroke, both in the acute phase and long after disease.17 ... males, while the healthy controls comprised 12 females and 8.

Quality assessment of platelet concentrates prepared by platelet rich plasma-platelet concentrate, buffy coat poor-platelet concentrate (BC-PC and apheresis-PC methods

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Singh Ravindra

2009-01-01

Full Text Available Background: Platelet rich plasma-platelet concentrate (PRP-PC, buffy coat poor-platelet concentrate (BC-PC, and apheresis-PC were prepared and their quality parameters were assessed. Study Design: In this study, the following platelet products were prepared: from random donor platelets (i platelet rich plasma - platelet concentrate (PRP-PC, and (ii buffy coat poor- platelet concentrate (BC-PC and (iii single donor platelets (apheresis-PC by different methods. Their quality was assessed using the following parameters: swirling, volume of the platelet concentrate, platelet count, WBC count and pH. Results: A total of 146 platelet concentrates (64 of PRP-PC, 62 of BC-PC and 20 of apheresis-PC were enrolled in this study. The mean volume of PRP-PC, BC-PC and apheresis-PC was 62.30±22.68 ml, 68.81±22.95 ml and 214.05±9.91 ml and ranged from 22-135 ml, 32-133 ml and 200-251 ml respectively. The mean platelet count of PRP-PC, BC-PC and apheresis-PC was 7.6±2.97 x 1010/unit, 7.3±2.98 x 1010/unit and 4.13±1.32 x 1011/unit and ranged from 3.2-16.2 x 1010/unit, 0.6-16.4 x 1010/unit and 1.22-8.9 x 1011/unit respectively. The mean WBC count in PRP-PC (n = 10, BC-PC (n = 10 and apheresis-PC (n = 6 units was 4.05±0.48 x 107/unit, 2.08±0.39 x 107/unit and 4.8±0.8 x 106/unit and ranged from 3.4 -4.77 x 107/unit, 1.6-2.7 x 107/unit and 3.2 - 5.2 x 106/unit respectively. A total of 26 units were analyzed for pH changes. Out of these units, 10 each were PRP-PC and BC-PC and 6 units were apheresis-PC. Their mean pH was 6.7±0.26 (mean±SD and ranged from 6.5 - 7.0 and no difference was observed among all three types of platelet concentrate. Conclusion: PRP-PC and BC-PC units were comparable in terms of swirling, platelet count per unit and pH. As expected, we found WBC contamination to be less in BC-PC than PRP-PC units. Variation in volume was more in BC-PC than PRP-PC units and this suggests that further standardization is required for preparation of BC

STAR FORMATION ACROSS THE W3 COMPLEX

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Román-Zúñiga, Carlos G.; Ybarra, Jason E.; Tapia, Mauricio [Instituto de Astronomía, Universidad Nacional Autónoma de México, Unidad Académica en Ensenada, Km 103 Carr. Tijuana–Ensenada, Ensenada 22860 (Mexico); Megías, Guillermo D. [Facultad de Física. Universidad de Sevilla. Dpto. Física Atómica, Molecular y Nuclear, Sevilla, E-41080 (Spain); Lada, Elizabeth A. [Astronomy Department, University of Florida, 211 Bryant Space Sciences Center, FL 32611 (United States); Alves, Joáo F. [Institute of Astronomy, University of Vienna, Türkenschanzstr. 17, A-1180 Vienna (Austria)

2015-09-15

We present a multi-wavelength analysis of the history of star formation in the W3 complex. Using deep, near-infrared ground-based images combined with images obtained with Spitzer and Chandra observatories, we identified and classified young embedded sources. We identified the principal clusters in the complex and determined their structure and extension. We constructed extinction-limited samples for five principal clusters and constructed K-band luminosity functions that we compare with those of artificial clusters with varying ages. This analysis provided mean ages and possible age spreads for the clusters. We found that IC 1795, the centermost cluster of the complex, still hosts a large fraction of young sources with circumstellar disks. This indicates that star formation was active in IC 1795 as recently as 2 Myr ago, simultaneous to the star-forming activity in the flanking embedded clusters, W3-Main and W3(OH). A comparison with carbon monoxide emission maps indicates strong velocity gradients in the gas clumps hosting W3-Main and W3(OH) and shows small receding clumps of gas at IC 1795, suggestive of rapid gas removal (faster than the T Tauri timescale) in the cluster-forming regions. We discuss one possible scenario for the progression of cluster formation in the W3 complex. We propose that early processes of gas collapse in the main structure of the complex could have defined the progression of cluster formation across the complex with relatively small age differences from one group to another. However, triggering effects could act as catalysts for enhanced efficiency of formation at a local level, in agreement with previous studies.

Effect of radiographic contrast agents on leukocyte metabolic response

Energy Technology Data Exchange (ETDEWEB)

Hernanz-Schulman, M. [Dept. of Pediatric Radiology, Vanderbilt Children' s Hospital, Nashville, TN (United States); Vanholder, R.; Waterloos, M.A. [Dept. of Internal Medicine, Nephrology Section, University Hospital, Gent (Belgium); Hakim, R.; Schulman, G. [Department of Nephrology, Vanderbilt University Medical Center, Nashville, TN (United States)

2000-06-01

Barium, at clinical dilutions, causes a significant increase of baseline ''resting state'' phagocytic activity, which in turn leads to significant blunting of subsequent response to phagocytic challenge and adversely affects the response to all bacteria tested. There is no baseline activation of leukocytes by the water-soluble media, although there was some inhibition (rather than activation) of leukocyte metabolic activity. The effect of the water-soluble media in bacteria was more complex (although inhibition is minor compared to barium). Our data demonstrate that barium is a significat activator of phagocytic cells, which results in deactivation of phagocytic response when challenged; these dsata serve to explain the enhanced adverse effect of barium in cased of fecal peritonitis. (orig.)

In vivo evaluation of titanium-prepared platelet-rich fibrin (T-PRF): a new platelet concentrate.

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Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Fıratlı, Erhan

2013-07-01

We have developed a new, titanium-prepared, platelet-rich fibrin (T-PRF) together with the protocol for forming it, which is based on the hypothesis that titanium tubes may be more effective at activating platelets than the glass tubes used by Chouckroun in his platelet-rich fibrin (PRF) method. The aim of this study was to find a suitable animal model in which to evaluate the method and to investigate the efficacy of T-PRF for wound healing. Blood samples from 6 rabbits were used to confirm the protocol for formation of T-PRF. We evaluated T-PRF or T-PRF-like clots morphologically using scanning electron microscopy (EM). Blood samples from 5 rabbits were used to develop an experiment in which to evaluate the effects of T-PRF on wound healing. The mucoperiosteal flaps were filled with autologous T-PRF membranes from the vestibule in the anterior mandibular regions. Samples collected from the surgical sites were stained with haematoxylin and eosin. We found a mature fibrin network in T-PRF clots that had been centrifuged for 15 min at 3500 rpm and, 15 days after placement of the membrane, we found newly-forming connective tissue and islets of bony tissue in the T-PRF membrane. These results show that T-PRF could induce the formation of new bone with new connective tissue in a rabbit model of wound healing within 30 days of treatment. Published by Elsevier Ltd.

Prevalence of risk factors for platelet transfusion refractoriness in multitransfused hemato-oncological patients at tertiary care center in North India

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Vijay Kumawat

2015-01-01

Full Text Available Background: This study was designed to determine the prevalence and assess the risk factors responsible for platelet transfusion refractoriness in hemato-oncological patients. Materials and Methods: The study included 30 patients. Twelve were clinically diagnosed as aplastic anemia and the 18 were of acute myeloid leukemia. A prospective 3 months follow-up was planned to monitor the response of platelet transfusion therapy, based on their posttransfusion corrected count increment at 1 st and 24 th h. Based on the observations, patients were categorized into refractory and nonrefractory groups. Common nonimmunological causes such as fever, sepsis, bleeding, disseminated intravascular coagulation, chemotherapy, splenomegaly, ABO mismatch, and antithymocyte globulin therapy were monitored. Among the immunological causes, presence of antihuman leukocyte antigen (HLA class I antibodies and platelet glycoprotein antibodies in patient′s serum were monitored. Results: During the study period, 17 (56.66% patients did not show desired platelet count increment. Transfusion requirements of refractory group for both red cell and platelet product were significantly higher (P < 0.05 in comparison to nonrefractory group. Among immunological causes, anti HLA class I antibodies (P < 0.013, antihuman platelet antigen-5b antibodies (P < 0.033 were significantly associated with refractoriness. Among nonimmunological causes, bleeding (P < 0.019, odd ratio 8.7, fever (P < 0.08, odd ratio 5.2, and infection (P < 0.07, odd ratio 5.4 were found to associated with refractoriness. Conclusion: Platelet refractoriness should be suspected in multitransfused patients not showing expected increment in platelet counts and thoroughly investigated to frame further guidelines in order to ensure proper management of these kind of patients.

Kaempferol inhibits thrombosis and platelet activation.

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Choi, Jun-Hui; Park, Se-Eun; Kim, Sung-Jun; Kim, Seung

2015-08-01

The objectives of the present study were to investigate whether kaempferol affects pro-coagulant proteinase activity, fibrin clot formation, blood clot and thrombin (or collagen/epinephrine)-stimulated platelet activation, thrombosis, and coagulation in ICR (Imprinting Control Region) mice and SD (Sprague-Dawley) rats. Kaempferol significantly inhibited the enzymatic activities of thrombin and FXa by 68 ± 1.6% and 52 ± 2.4%, respectively. Kaempferol also inhibited fibrin polymer formation in turbidity. Microscopic analysis was performed using a fluorescent conjugate. Kaempferol completely attenuated phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) 1/2, and phosphoinositide 3-kinase (PI3K)/PKB (AKT) in thrombin-stimulated platelets and delayed aggregation time (clotting) by 34.6% in an assay of collagen/epinephrine-stimulated platelet activation. Moreover, kaempferol protected against thrombosis development in 3 animal models, including collagen/epinephrine- and thrombin-induced acute thromboembolism models and an FeCl3-induced carotid arterial thrombus model. The ex vivo anticoagulant effect of kaempferol was further confirmed in ICR mice. This study demonstrated that kaempferol may be clinically useful due to its ability to reduce or prevent thrombotic challenge. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

[27- Hydroxycholesterol reverses estradiol induced inhibition of platelet aggregation in postmenopausal women].

Science.gov (United States)

Rocha, Gladys; Sierralta, Walter; Valladares, Luis

2016-11-01

The decline of estrogen levels increases cardiovascular risk in women. Platelets express estrogen receptors and 17β-estradiol- (E2) can produce a protective effect on thrombus formation. The hydroxylation of cholesterol generates several sterols and 27-hydroxycholesterol (27HC) predominates in circulation. To evaluate the effect of 27HC as an endogenous antagonist of the anti-aggregating properties of E2 in platelets of postmenopausal women. Platelet function of postmenopausal women was evaluated ex-vivo. Platelets pre-incubated with 27HC in the presence or absence of E2, were stimulated with collagen. Aggregation was evaluated using turbidimetry using a Chrono-log aggregometer. Collagen-stimulated platelet aggregation was significantly inhibited by E2. The inhibitory effect of E2 on collagen-stimulated platelet aggregation was significantly reversed in the presence of 27HC. The suppressive effect of E2 on platelet aggregation is inhibited by 27HC, which could contribute to increase cardiovascular risk in postmenopausal women.

Platelet "first responders" in wound response, cancer, and metastasis.

Science.gov (United States)

Menter, David G; Kopetz, Scott; Hawk, Ernest; Sood, Anil K; Loree, Jonathan M; Gresele, Paolo; Honn, Kenneth V

2017-06-01

Platelets serve as "first responders" during normal wounding and homeostasis. Arising from bone marrow stem cell lineage megakaryocytes, anucleate platelets can influence inflammation and immune regulation. Biophysically, platelets are optimized due to size and discoid morphology to distribute near vessel walls, monitor vascular integrity, and initiate quick responses to vascular lesions. Adhesion receptors linked to a highly reactive filopodia-generating cytoskeleton maximizes their vascular surface contact allowing rapid response capabilities. Functionally, platelets normally initiate rapid clotting, vasoconstriction, inflammation, and wound biology that leads to sterilization, tissue repair, and resolution. Platelets also are among the first to sense, phagocytize, decorate, or react to pathogens in the circulation. These platelet first responder properties are commandeered during chronic inflammation, cancer progression, and metastasis. Leaky or inflammatory reaction blood vessel genesis during carcinogenesis provides opportunities for platelet invasion into tumors. Cancer is thought of as a non-healing or chronic wound that can be actively aided by platelet mitogenic properties to stimulate tumor growth. This growth ultimately outstrips circulatory support leads to angiogenesis and intravasation of tumor cells into the blood stream. Circulating tumor cells reengage additional platelets, which facilitates tumor cell adhesion, arrest and extravasation, and metastasis. This process, along with the hypercoagulable states associated with malignancy, is amplified by IL6 production in tumors that stimulate liver thrombopoietin production and elevates circulating platelet numbers by thrombopoiesis in the bone marrow. These complex interactions and the "first responder" role of platelets during diverse physiologic stresses provide a useful therapeutic target that deserves further exploration.

Osteoblastic mesenchymal stem cell sheet combined with Choukroun platelet-rich fibrin induces bone formation at an ectopic site.

Science.gov (United States)

Wang, Zhifa; Weng, Yanming; Lu, Shengjun; Zong, Chunlin; Qiu, Jianyong; Liu, Yanpu; Liu, Bin

2015-08-01

To analyze the effects of platelet-rich fibrin (PRF) on mesenchymal stem cells (MSCs) in vitro and investigate in vivo bone formation by MSC sheets with PRF. Cell proliferation and expression of osteogenesis-related genes within MSC sheets were assessed upon exposure to PRF from the same donors. We then injected MSC sheet fragments with or without PRF subcutaneously in nude mice and assessed bone formation by micro-computed tomography and histological analyses. PRF significantly stimulated MSC proliferation and osteogenesis in vitro. MSC sheets injected with or without PRF formed new bone, but those with PRF produced significantly more and denser bone. MSC sheets can be used to generate tissue engineered bone upon injection, and PRF increases the osteogenic capacity of MSC sheets in vitro and in vivo. © 2014 Wiley Periodicals, Inc.

Techniques for measuring red cell, platelet, and WBC survival

International Nuclear Information System (INIS)

Mayer, K.; Freeman, J.E.

1986-01-01

Blood cell survival studies yield valuable information concerning production and destruction of cells circulating in the bloodstream. Methodologies for the measurement of red cell survival include nonisotopic methods such as differential agglutination and hemolysis. The isotopic label may be radioactive or, if not, will require availability of a mass spectrograph. These methods fall into two categories, one where red cells of all ages are labeled ( 51 Cr, DFP32, etc.) and those employing a cohort label of newly formed cells ( 14 C glycine, 75 Se methionine, etc.). Interpretation of results for methodology employed and mechanism of destruction, random or by senescence, are discussed. A similar approach is presented for platelet and leukocyte survival studies. The inherent difficulties and complications of sequestration, storage, and margination of these cells are emphasized and discussed. 38 references

Further studies on the relationship between platelet buoyant density and platelet age

International Nuclear Information System (INIS)

Boneu, B.; Vigoni, F.; Boneu, A.; Caranobe, C.; Sie, P.

1982-01-01

The relationship between platelet buoyant density and platelet age was investigated in eight human subjects submitted to an autologous chromium labeled platelet survival study. Platelets were isolated after isopycnic centrifugation using eight discontinuous isoosmotic stractan gradients (five subjects), or various continuous and linear isoosmolar gradients (three subjects). A paradoxical radioactivity enrichment of the dense platelets and a premature loss of radioactivity in the light platelets were observed. These results are explained by a shift of the radioactivity distribution curve toward higher densities during the 3-4 days after platelet injection, while the standard deviation of the distribution was conserved throughout the platelet life span. These results suggest that young platelets are heterogeneous and slightly less dense than the total platelet population

Insomnia, platelet serotonin and platelet monoamine oxidase in chronic alcoholism.

Science.gov (United States)

Nenadic Sviglin, Korona; Nedic, Gordana; Nikolac, Matea; Mustapic, Maja; Muck-Seler, Dorotea; Borovecki, Fran; Pivac, Nela

2011-08-18

Insomnia is a common sleep disorder frequently occurring in chronic alcoholic patients. Neurobiological basis of insomnia, as well as of alcoholism, is associated with disrupted functions of the main neurotransmitter systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Blood platelets are considered a limited peripheral model for the central 5-HT neurons, since both platelets and central 5-HT synaptosomes have similar dynamics of 5-HT. Platelet 5-HT concentration and platelet monoamine oxidase type B (MAO-B) are assumed to represent biomarkers for particular symptoms and behaviors in psychiatric disorders. The hypothesis of this study was that platelet 5-HT concentration and platelet MAO-B activity will be altered in chronic alcoholic patients with insomnia compared to comparable values in patients without insomnia. The study included 498 subjects: 395 male and 103 female medication-free patients with alcohol dependence and 502 healthy control subjects: 325 men and 177 women. The effects of early, middle and late insomnia (evaluated using the Hamilton Depression Rating Scale), as well as sex, age and smoking on platelet 5-HT concentration and platelet MAO-B activity were evaluated using one-way ANOVA and multiple regression analysis by the stepwise method. Platelet 5-HT concentration, but not platelet MAO-B activity, was significantly reduced in alcoholic patients with insomnia compared to patients without insomnia. Multiple regression analysis revealed that platelet 5-HT concentration was affected by middle insomnia, smoking and sex, while platelet MAO activity was affected only by sex and age. The present and previous data suggest that platelet 5-HT concentration might be used, after controlling for sex and smoking, as a biomarker for insomnia in alcoholism, PTSD and in rotating shift workers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Does autologous leukocyte-platelet-rich plasma improve tendon healing in arthroscopic repair of large or massive rotator cuff tears?

Science.gov (United States)

Charousset, Christophe; Zaoui, Amine; Bellaïche, Laurence; Piterman, Michel

2014-04-01

To evaluate the clinical and magnetic resonance imaging (MRI) outcome of arthroscopic rotator cuff repair with the use of leukocyte-platelet-rich plasma (L-PRP) in patients with large or massive rotator cuff tears. A comparative cohort of patients with large or massive rotator cuff tears undergoing arthroscopic repair was studied. Two consecutive groups of patients were included: rotator cuff repairs with L-PRP injection (group 1, n = 35) and rotator cuff repairs without L-PRP injection (group 2, n = 35). A double-row cross-suture cuff repair was performed by a single surgeon with the same rehabilitation protocol. Patients were clinically evaluated with the Constant score; Simple Shoulder Test score; University of California, Los Angeles (UCLA) score; and strength measurements by use of a handheld dynamometer. Rotator cuff healing was evaluated by postoperative MRI using the Sugaya classification (type 1 to type 5). We prospectively evaluated the 2 groups at a minimum 2-year follow-up. The results did not show differences in cuff healing between the 2 groups (P = .16). The size of recurrent tears (type 4 v type 5), however, was significantly smaller in group 1 (P = .008). There was no statistically significant difference in the recurrent tear rate (types 4 and 5) between the 2 groups (P = .65). There was no significant difference between group 1 and group 2 in terms of University of California, Los Angeles score (29.1 and 30.3, respectively; P = .90); Simple Shoulder Test score (9.9 and 10.2, respectively; P = .94); Constant score (77.3 and 78.1, respectively; P = .82); and strength (7.5 and 7.0, respectively; P = .51). In our study the use of autologous L-PRP did not improve the quality of tendon healing in patients undergoing arthroscopic repair of large or massive rotator cuff tears based on postoperative MRI evaluation. The only significant advantage was that the L-PRP patients had smaller iterative tears. However, the functional outcome was similar in

Platelet biomechanics, platelet bioenergetics, and applications to clinical practice and translational research.

Science.gov (United States)

George, Mitchell J; Bynum, James; Nair, Prajeeda; Cap, Andrew P; Wade, Charles E; Cox, Charles S; Gill, Brijesh S

2018-07-01

The purpose of this review is to explore the relationship between platelet bioenergetics and biomechanics and how this relationship affects the clinical interpretation of platelet function devices. Recent experimental and technological advances highlight platelet bioenergetics and biomechanics as alternative avenues for collecting clinically relevant data. Platelet bioenergetics drive energy production for key biomechanical processes like adhesion, spreading, aggregation, and contraction. Platelet function devices like thromboelastography, thromboelastometry, and aggregometry measure these biomechanical processes. Platelet storage, stroke, sepsis, trauma, or the activity of antiplatelet drugs alters measures of platelet function. However, the specific mechanisms governing these alterations in platelet function and how they relate to platelet bioenergetics are still under investigation.

Dual-specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis.

Science.gov (United States)

Musumeci, Lucia; Kuijpers, Marijke J; Gilio, Karen; Hego, Alexandre; Théâtre, Emilie; Maurissen, Lisbeth; Vandereyken, Maud; Diogo, Catia V; Lecut, Christelle; Guilmain, William; Bobkova, Ekaterina V; Eble, Johannes A; Dahl, Russell; Drion, Pierre; Rascon, Justin; Mostofi, Yalda; Yuan, Hongbin; Sergienko, Eduard; Chung, Thomas D Y; Thiry, Marc; Senis, Yotis; Moutschen, Michel; Mustelin, Tomas; Lancellotti, Patrizio; Heemskerk, Johan W M; Tautz, Lutz; Oury, Cécile; Rahmouni, Souad

2015-02-17

A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug. © 2014 American Heart Association, Inc.

Platelet Function Tests