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Platelet Adhesion (platelet + adhesion)
Selected AbstractsMicropatterning: Patterned Hydrogels for Controlled Platelet Adhesion from Whole Blood and Plasma (Adv. Funct.ADVANCED FUNCTIONAL MATERIALS, Issue 15 2010Mater. Poly(ethylene glycol)-based hydrogel coatings patterned with selected proteins can be utilized to control and study the adhesion of human blood platelets with excellent precision, as presented by B. Liedberg et al. on page 2396. This frontispiece shows how imaging surface plasmon resonance is used in combination with fluorescence microscopy to investigate the platelet adhesion process in undiluted blood plasma. [source] Patterned Hydrogels for Controlled Platelet Adhesion from Whole Blood and PlasmaADVANCED FUNCTIONAL MATERIALS, Issue 15 2010Tobias Ekblad Abstract This work describes the preparation and properties of hydrogel surface chemistries enabling controlled and well-defined cell adhesion. The hydrogels may be prepared directly on plastic substrates, such as polystyrene slides or dishes, using a quick and experimentally simple photopolymerization process, compatible with photolithographic and microfluidic patterning methods. The intended application for these materials is as substrates for diagnostic cell adhesion assays, particularly for the analysis of human platelet function. The non-specific adsorption of fibrinogen, a platelet adhesion promoting protein, is shown to be completely inhibited by the hydrogel, provided that the film thickness is sufficient (>5,nm). This allows the hydrogel to be used as a matrix for presenting selected bioactive ligands without risking interference from non-specifically adsorbed platelet adhesion factors, even in undiluted whole blood and blood plasma. This concept is demonstrated by preparing patterns of proteins on hydrogel surfaces, resulting in highly controlled platelet adhesion. Further insights into the protein immobilization and platelet adhesion processes are provided by studies using imaging surface plasmon resonance. The hydrogel surfaces used in this work appear to provide an ideal platform for cell adhesion studies of platelets, and potentially also for other cell types. [source] Substantial Reduction of Platelet Adhesion by Heparin-Coated StentsJOURNAL OF INTERVENTIONAL CARDIOLOGY, Issue 4 2001CHRISTOPH BICKEL M.D. Although optimized antiplatelet medication has improved the clinical outcome after coronary stenting, vessel occlusion and restenosis still remain a relevant clinical problem. Platelets play a key role in this process. Therefore, the authors compared the platelet adhesion on different stent surface modifications (electropolished without coating or coated with carbon, carbon and additional heparin, silicon carbide, or heparin alone) to investigate their role in reducing platelet adhesion. All stem and additional stainless steel plates were incubated in heparinized whole blood with radiolabeled platelets. Afrer washing the stents and plates four times, radioactivity caused by the adhesion of radiolabeled platelets was measured. The adhesion of radiolabeled platelets, compared to uncoated, electropolished stents, was reduced through silicon carbide coating to 58.6%, by carbon coating with additional heparin to 32.9%, and heparin coating alone to 7.7%. Stent coating with heparin is the most effective among the examined coatings in reducing platelet adhesion in vitro. [source] Dynamic Aspects Of Platelet Adhesion Under FlowCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2001Sacha M Dopheide SUMMARY 1. Cell,cell and cell,matrix adhesive interactions are critical for a wide range of physiological processes, including embryogenesis, inflammation, immunity and haemostasis. 2. The ability of circulating blood cells, such as platelets and leucocytes, to adhere to sites of vascular injury is complicated by the presence of blood flow, which imposes hydrodynamic forces on adhesion contacts. 3. To overcome this problem, platelets and leucocytes have evolved specific adhesion receptors with unique biomechanical properties that enable these cells to adhere to the vessel wall under flow conditions. 4. Platelet adhesion in the normal circulation appears to be a multiple-step process involving an initial reversible interaction between the platelet adhesion receptor glycoprotein Ib-IX-V and the vascular adhesion protein von Willebrand factor. Once tethered to the vessel wall, platelets form irreversible adhesion contacts through the binding of one or more platelet integrins to specific subendothelial matrix proteins. 5. There is now a wealth of evidence demonstrating that these receptors not only mediate platelet adhesion, but also transduce signals leading to platelet activation. 6. In the present review, we will briefly discuss the current understanding of the specific roles of individual platelet receptors in supporting the haemostatic function of platelets and discuss mechanisms by which these receptors induce platelet activation. [source] Quantitative Analysis of Human Platelet Adhesions Under a Small-Scale Flow DeviceARTIFICIAL ORGANS, Issue 4 2010Katsuko S. Furukawa Abstract To realize real-time evaluation of human platelet adhesions onto material surfaces with small volumes of human platelet suspensions, we developed an apparatus consisting of a modified cone and plate-type viscometer, combined with an upright epi-fluorescence microscope. The apparatus allowed real-time evaluation of platelet,material interactions and the initial event of thrombus formation, using small platelet suspension volumes (7.5 µL) under shear flow conditions. To study the dynamic behavior of platelet,material interaction, we chose five representative opaque and transparent materials: acrylate resin (AC), polytetrafluoroethylene (PTFE), polyvynylchrolide (PVC), glass, and a monolayer of human normal umbilical cord vein endothelial cells (EC) on glass under shear flow conditions. The values of adhesiveness of human platelets to the test materials in descending order were as follows: AC > PTFE > PVC > glass > human EC. Under this new small-scale flow system, we could obtain highly reproducible data, which were comparable with results from a previously developed large-scale flow system. Therefore, the newly developed cone and plate-type rheometer is a useful instrument for testing and screening materials, and allows precise quantitative evaluation of human platelet adhesion. [source] Platelet adhesion to dimeric ,2 -glycoprotein I under conditions of flow is mediated by at least two receptors: glycoprotein Ib, and apolipoprotein E receptor 2,JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2007M. T. T. PENNINGS Summary.,Background: The major antigen implicated in the antiphospholipid syndrome is beta2-glycoprotein I (,2GPI). Dimerized ,2GPI binds to apolipoprotein E receptor 2, (apoER2,) on platelets and increases platelet adhesion to collagen under conditions of flow. Aim: To investigate whether the interaction between dimerized ,2GPI and platelets is sufficiently strong to resist shear stresses. Methods: We studied the interaction of platelets with immobilized dimerized ,2GPI under conditions of flow, and further analyzed the interaction using surface plasmon resonance and solid phase immunoassays. Results: We found that dimerized ,2GPI supports platelet adhesion and aggregate formation under venous flow conditions. Adhesion of platelets to dimerized ,2GPI was completely inhibited by the addition of soluble forms of both apoER2, and GPIb,, and the addition of receptor-associated protein and the removal of GPIb, from the platelet surface. GPIb, co-precipitated with apoER2,, suggesting the presence of complexes between GPIb, and apoER2, on platelet membranes. The interaction between GPIb, and dimeric ,2GPI was of intermediate affinity (Kd = 180 nm) and Zn2+, but not Ca2+ -dependent. Deletion of domain V from dimeric ,2GPI strongly reduced its binding to both GPIb, and apoER2,. Antibodies that inhibit the binding of thrombin to GPIb, inhibited platelet adhesion to dimeric ,2GPI completely, while antibodies blocking the binding of von Willebrand factor to GPIb, had no effect. Dimeric ,2GPI showed reduced binding to low-sulfated GPIb, compared to the fully sulfated form. Conclusion: We show that platelets adhere to dimeric ,2GPI under both arterial and venous shear stresses. Platelets adhere via two receptors: GPIb, and apoER2,. These receptors are present in a complex on the platelet surface. [source] The von Willebrand factor self-association is modulated by a multiple domain interactionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 3 2005H. ULRICHTS Summary.,Background:,Platelet adhesion and aggregation at sites of vascular injury exposed to rapid blood flow require von Willebrand factor (VWF). VWF becomes immobilized by binding to subendothelial components or by a self-association at the interface of soluble and surface-bound VWF. Objectives:,As this self-association has been demonstrated only under shear conditions, our first goal was to determine whether the same interaction could be observed under static conditions. Furthermore, we wanted to identify VWF domain(s) important for this self-association. Results:,Biotinylated VWF (b-VWF) interacted dose-dependently and specifically with immobilized VWF in an enzyme-linked immunosorbent assay (ELISA) assay, showing that shear is not necessary to induce the VWF self-association. Whereas anti-VWF monoclonal antibodies (mAbs) had no effect on the self-association, the proteolytic VWF-fragments SpII(1366,2050) and SpIII(1,1365) inhibited the b-VWF,VWF interaction by 70 and 80%, respectively. Moreover, a specific binding of b-VWF to immobilized Sp-fragments was demonstrated. Finally, both biotinylated SpII and SpIII were able to bind specifically to both immobilized SpII and SpIII. Similar results were observed under flow conditions, which confirmed the functional relevance of our ELISA system. Conclusion:,We have developed an ELISA binding assay in which a specific VWF self-association under static conditions can be demonstrated. Our results suggest a multiple domain interaction between immobilized and soluble VWF. [source] von Willebrand factor stimulates thrombin-induced exposure of procoagulant phospholipids on the surface of fibrin-adherent plateletsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 3 2003J. J. Briedé Summary., Studies from our laboratory have demonstrated that von Willebrand factor (VWF) stimulates thrombin generation in platelet-rich plasma. The precise role of VWF and fibrin in this reaction, however, remained to be clarified. In the present study we utilized thrombin-free planar fibrin layers and washed platelets to examine the relationship between platelet,fibrin interaction and exposure of coagulation-stimulating phosphatidylserine (PS) under conditions of low and high shear stress. Our study confirms that platelet adhesion to fibrin at a shear rate of 1000 s,1 requires fibrin-bound VWF. The cytosolic calcium concentration ([Ca2+]i) of stationary platelets was not elevated and PS exposing platelets were virtually absent (2 ± 2%). However, thrombin activation resulted in a marked increase in the number of PS exposing platelets (up to 85 ± 14%) along with a transient elevation in [Ca2+]i from 0.05 µmol L,1 up to 1.1 ± 0.2 µmol L,1. Platelet adhesion to fibrin at a shear rate of 50 s,1 is mediated by thrombin but not by fibrin-bound VWF. The [Ca2+]i of these thrombin-activated platelets was elevated (0.2 ± 0.1 µmol L,1), but only a minority of the platelets (11 ± 8%) exposed PS. The essential role of VWF in this thrombin-induced procoagulant response became apparent from low shear rate perfusion studies over fibrin that was incubated with VWF and botrocetin. After treatment with thrombin, the majority of the adherent platelets (57 ± 23%) exposed PS and had peak values of [Ca2+]i of about 0.6 µmol L,1. Taken together, these results demonstrate that thrombin-induced exposure of PS and high calcium response on fibrin-adherent platelets depends on shear- or botrocetin-induced VWF,platelet interaction. [source] Pharmacological characteristics of solid-phase von Willebrand factor in human plateletsBRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2001Anna Radomski The pharmacological characteristics of solid-phase von Willebrand factor (svWF), a novel platelet agonist, were studied. Washed platelet suspensions were obtained from human blood and the effects of svWF on platelets were measured using aggregometry, phase-contrast microscopy, flow cytometry and zymography. Incubation of platelets with svWF (0.2 , 1.2 ,g ml,1) resulted in their adhesion to the ligand, while co-incubations of svWF with subthreshold concentrations of ADP, collagen and thrombin resulted in aggregation. 6B4 inhibitory anti-glycoprotein (GP)Ib antibodies abolished platelet adhesion stimulated by svWF, while aggregation was reduced in the presence of 6B4 and N-Acetyl-Pen-Arg-Gly-Asp-Cys, an antagonist of GPIIb/IIIa. Platelet adhesion stimulated with svWF was associated with a concentration-dependent increase in expression of GPIb, but not of GPIIb/IIIa. In contrast, collagen (0.5 , 10.0 ,g ml,1) caused down-regulation of GPIb and up-regulation of GPIIb/IIIa in platelets. Solid-phase vWF (1.2 ,g ml,1) resulted in the release of MMP-2 from platelets. Inhibition of MMP-2 with phenanthroline (10 ,M), but not with aspirin or apyrase, inhibited platelet adhesion stimulated with svWF. In contrast, human recombinant MMP-2 potentiated both the effects of svWF on adhesion and up-regulation of GPIb. Platelet adhesion and aggregation stimulated with svWF were reduced by S-nitroso-n-acetyl-penicillamine, an NO donor, and prostacyclin. Thus, stimulation of human platelets with svWF leads to adhesion and aggregation that are mediated via activation of GPIb and GPIIb/IIIa, respectively. Mechanisms of activation of GPIb by svWF involve the release of MMP-2, and are regulated by NO and prostacyclin. British Journal of Pharmacology (2001) 134, 1013,1020; doi:10.1038/sj.bjp.0704345 [source] Dynamic Aspects Of Platelet Adhesion Under FlowCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2001Sacha M Dopheide SUMMARY 1. Cell,cell and cell,matrix adhesive interactions are critical for a wide range of physiological processes, including embryogenesis, inflammation, immunity and haemostasis. 2. The ability of circulating blood cells, such as platelets and leucocytes, to adhere to sites of vascular injury is complicated by the presence of blood flow, which imposes hydrodynamic forces on adhesion contacts. 3. To overcome this problem, platelets and leucocytes have evolved specific adhesion receptors with unique biomechanical properties that enable these cells to adhere to the vessel wall under flow conditions. 4. Platelet adhesion in the normal circulation appears to be a multiple-step process involving an initial reversible interaction between the platelet adhesion receptor glycoprotein Ib-IX-V and the vascular adhesion protein von Willebrand factor. Once tethered to the vessel wall, platelets form irreversible adhesion contacts through the binding of one or more platelet integrins to specific subendothelial matrix proteins. 5. There is now a wealth of evidence demonstrating that these receptors not only mediate platelet adhesion, but also transduce signals leading to platelet activation. 6. In the present review, we will briefly discuss the current understanding of the specific roles of individual platelet receptors in supporting the haemostatic function of platelets and discuss mechanisms by which these receptors induce platelet activation. [source] Isosorbide dinitrate inhibits platelet adhesion and aggregation in nonthrombolyzed patients with acute myocardial infarctionCLINICAL CARDIOLOGY, Issue 11 2000Jadwiga Gebalska M.D., Ph.D. Abstract Background: Apart from their vasodilatator properties, nitrates have been shown to inhibit platelet aggregation. The effects of nitrates on platelet adhesion have not been studied. Nonselected patients with acute myocardial infarction (AMI) have been suggested to gain no benefit from administration of nitrates. However, the importance of nitrates may be greater in a subgroup of nonthrombolyzed patients with AMI. Hypothesis: Isosorbide dinitrate (ISDN) decreases platelet adhesion and aggregation in nonthrombolyzed patients with AMI. Methods: Consecutive 48 men with AMI, not eligible for thrombolytic therapy because of late presentation (> 12 h), were prospectively randomized 2:1 to double-blind ISDN (mean dose 2.4 ± 0.9 mg/h) (n = 33) or placebo (0.9% sodium chloride) (n = 15) infusion. All patients received aspirin. Blood samples were taken at baseline (no study medication) and 3 h into ISDN or placebo infusion. Platelet adhesion to collagen was measured in the ethylene diamine tetraacetic acid (EDTA)-platelet rich plasma by recording changes in light transmission with an optical aggregometer. Platelet aggregation was measured using the Born's method. Results: Isosorbide dinitrate significantly decreased both platelet adhesion and aggregation. No effect was seen in the placebo group. Conclusions: In patients with AMI who do not receive thrombolytic therapy, ISDN effectively inhibits platelet adhesion and aggregation. These effects of nitrates may be of therapeutic and prognostic significance in this group of patients. [source] Patterned Hydrogels for Controlled Platelet Adhesion from Whole Blood and PlasmaADVANCED FUNCTIONAL MATERIALS, Issue 15 2010Tobias Ekblad Abstract This work describes the preparation and properties of hydrogel surface chemistries enabling controlled and well-defined cell adhesion. The hydrogels may be prepared directly on plastic substrates, such as polystyrene slides or dishes, using a quick and experimentally simple photopolymerization process, compatible with photolithographic and microfluidic patterning methods. The intended application for these materials is as substrates for diagnostic cell adhesion assays, particularly for the analysis of human platelet function. The non-specific adsorption of fibrinogen, a platelet adhesion promoting protein, is shown to be completely inhibited by the hydrogel, provided that the film thickness is sufficient (>5,nm). This allows the hydrogel to be used as a matrix for presenting selected bioactive ligands without risking interference from non-specifically adsorbed platelet adhesion factors, even in undiluted whole blood and blood plasma. This concept is demonstrated by preparing patterns of proteins on hydrogel surfaces, resulting in highly controlled platelet adhesion. Further insights into the protein immobilization and platelet adhesion processes are provided by studies using imaging surface plasmon resonance. The hydrogel surfaces used in this work appear to provide an ideal platform for cell adhesion studies of platelets, and potentially also for other cell types. [source] The 80th anniversary of von Willebrand's disease: history, management and researchHAEMOPHILIA, Issue 6 2006A. B. FEDERICI Summary., The history of von Willebrand's disease (VWD) is fascinating because it demonstrates how good clinical observations, genetic studies and biochemical skills can improve basic understanding of a disease and its management. The continuous efforts of scientists and clinicians during the last 80 years have significantly improved the knowledge of von Willebrand factor (VWF) structure and function and the management of VWD. Diagnosis of phenotype and genotype is now available in many countries and treatment is becoming more specific according to the VWD type. Any therapeutic agents must correct the dual defect of haemostasis, i.e. the abnormal platelet adhesion due to reduced and/or dysfunctional and low levels of factor VIII (FVIII) associated with VWF defects. Desmopressin (DDAVP) is the treatment of choice for type 1 VWD because it induces release of VWF from cellular compartments. Plasma virally inactivated VWF concentrates containing FVIII are effective and safe in patients unresponsive to DDAVP. There are advanced plans to develop a recombinant VWF but this product will require the concomitant administration of FVIII for the control of acute bleeds. Basic research studies on cellular biology, biochemistry and immunology have confirmed the role of VWF as a crucial participant in both haemostasis and thrombosis as its main biological activity is to support platelet adhesion,aggregation in the circulation. Retrospective and prospective clinical research studies, including bleeding history and laboratory markers for diagnosis as well as the use of DDAVP and VWF concentrates to manage or prevent bleeds in patients with VWD have been essential to provide general guidelines for VWD management. The large number of publications quoting VWD and VWF emphasizes the important role of VWF in medicine. [source] Management of von Willebrand disease: a guideline from the UK Haemophilia Centre Doctors' OrganizationHAEMOPHILIA, Issue 3 2004K. J. Pasi Summary., von Willebrand disease (VWD) is the commonest inherited bleeding disorder. The aim of therapy for VWD is to correct the two defects of haemostasis in this disorder, impaired primary haemostasis because of defective platelet adhesion and aggregation and impaired coagulation as a result of low levels of factor VIII. The objective of this guideline is to inform individuals making choices about the treatment and management of VWD including the use of therapeutic products. This is the second edition of this UK Haemophilia Centre Doctors' Organization (UKHCDO) guideline and supersedes the previous edition which was published in 1994. [source] Impact of platelet adhesion on Thromboelastometry in dilutional consumption coagulopathy with either HES or GelatinACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 6 2010R. Huet No abstract is available for this article. [source] Preparation and characterization of infection-resistant antibiotics-releasing hydrogels rods of poly[hydroxyethyl methacrylate- co -(poly(ethylene glycol)-methacrylate]: Biomedical application in a novel rabbit penile prosthesis modelJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2008M. Yakup Ar Abstract In this work, preparation and characterization of novel three different antibiotic loaded penile prosthesis in the rod form were investigated by copolymerization of 2-hydroxyethylmethacrylate (HEMA) with poly(ethylene glycol)-methacrylate, (PEG-MA). To achieve this goal, a series of novel copolymer hydrogels were prepared in rod form using HEMA and PEG-MA monomers via UV initiated photopolymerization. The thermal stability of the copolymer was found to be lowered by increase in the ratio of PEG-MA in the rod structure. Contact angle measurements on the surface of copolymer hydrogel demonstrated that the copolymer gave rise to a significant hydrophilic surface compared with pure poly(HEMA). The blood protein adsorption and platelet adhesion were significantly reduced on the surface of the copolymer hydrogels compared with control pure poly(HEMA). Poly(HEMA:PEG-MA;1:1)-1 formulation containing different antibiotics (20 mg antibiotic/g polymer) released about 90, 91, and 55% of the total loaded cephtriaxon, vancomycin, and gentamicin in 48 h at pH 7.4, respectively. Finally, antibiotics loaded biocompatible poly(HEMA:PEG-MA;1:1)-1 hydrogel compositions was used as a penile prosthesis in preventing cavernous tissue infections in a rabbit prosthesis model. The efficacy of the three different antibiotics loaded hydrogel system was evaluated in four different groups of rabbits, in which various infectious agents were inoculated. The animals were sacrificed after predetermined time periods, and clinical, histological and microbiological assessment on the implant side were carried out to detect infections. Eventually, we concluded that three different antibiotic loaded penile prostheses (i.e. poly(HEMA:PEG-MA;1:1)-1 hydrogel systems) were as effective as parenteral antibiotics applications. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2008 [source] Biocompatibility investigation and urea removal from blood by urease-immobilized HEMA incorporated poly(ethyleneglycol dimethacrylate) microbeadsJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2003F. Ayhan Abstract The biocompatibility of modified and urease-immobilized poly(ethyleneglycol dimethacrylate/2-hydroxyethylmetacrylate) [poly(EGDMA/HEMA)] microbeads was tested through blood compatibility tests. Twelve percent HEMA incorporated nonporous particles of 105,125 ,m were used in the research. Hydroxyl groups on microbeads were chemically modified by following a three-step procedure that is composed of activation, spacer-arm incorporation (hexamethylene diamine) and, finally, glutaraldehyde bounding. Enzyme urease was immobilized on microbead surfaces, and adsorption of blood proteins in serum and plasma, blood coagulation time, and leukocyte and platelet adhesion were tested. Incubation of 1.5 cc of biological fluid with 100 mg of urease-immobilized poly(EGDMA/HEMA) microbeads at room temperature shows that protein adsorption on surfaces occurs, but protein content after treatment was in the range of healthy people. Adsorbed albumin and total globulin amounts per gram of microbeads is much greater than fibrinogen. Immobilization of urease reduced the protein adsorption and blood coagulation times compared with those of modified microbeads. Prothrombin time (PT) was not altered much, whereas poly(EGDMA/HEMA) microbeads induced a significant increase of activated partial thromboplastin time (APTT). The platelet and leukocyte adhesion slightly increased with the modification of poly(EGDMA/HEMA) and decreased with the introduction of urease. When blood samples were treated with urease-immobilized microbeads, BUN values of patients were lowered to almost acceptable amounts. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 64B: 13,18, 2003 [source] Substantial Reduction of Platelet Adhesion by Heparin-Coated StentsJOURNAL OF INTERVENTIONAL CARDIOLOGY, Issue 4 2001CHRISTOPH BICKEL M.D. Although optimized antiplatelet medication has improved the clinical outcome after coronary stenting, vessel occlusion and restenosis still remain a relevant clinical problem. Platelets play a key role in this process. Therefore, the authors compared the platelet adhesion on different stent surface modifications (electropolished without coating or coated with carbon, carbon and additional heparin, silicon carbide, or heparin alone) to investigate their role in reducing platelet adhesion. All stem and additional stainless steel plates were incubated in heparinized whole blood with radiolabeled platelets. Afrer washing the stents and plates four times, radioactivity caused by the adhesion of radiolabeled platelets was measured. The adhesion of radiolabeled platelets, compared to uncoated, electropolished stents, was reduced through silicon carbide coating to 58.6%, by carbon coating with additional heparin to 32.9%, and heparin coating alone to 7.7%. Stent coating with heparin is the most effective among the examined coatings in reducing platelet adhesion in vitro. [source] The glycoprotein Ib,,von Willebrand factor interaction induces platelet apoptosisJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2010S. LI Summary.,Background: The interaction of glycoprotein (GP) Ib, with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIb,,VWF interaction. Objectives: To investigate whether the GPIb,,VWF interaction induces platelet apoptosis and the role of 14-3-3, in apoptotic signaling. Methods: Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild-type (1b9) or mutant GPIb,IX interacting with VWF by flow cytometry or western blotting. Results: Ristocetin-induced GPIb,,VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear-induced apoptosis was eliminated by the anti-GPIb, antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb,IX with GPIb, truncated at residue 551 or a serine-to-alanine mutation at the 14-3-3,-binding site in GPIb,. Conclusions: This study demonstrates that the GPIb,,VWF interaction induces apoptotic events in platelets, and that the association of 14-3-3, with the cytoplasmic domain of GPIb, is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases. [source] Staphylococcal superantigen-like 5 activates platelets and supports platelet adhesion under flow conditions, which involves glycoprotein Ib, and ,IIb,3JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2009C. J. C. DE HAAS Summary.,Objectives: Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus aureus that has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). Methods and Results: When purified recombinant SSL5 was added to washed platelets in an aggregometry set-up, complete and irreversible aggregation was observed. Proteolysis of the extracellular part of GPIb, or the addition of dRGDW abrogated platelet aggregation. When a mixture of isolated platelets and red cells was perfused over immobilized SSL5 at a shear rate of 300 s,1, stable platelet aggregates were observed, and platelet deposition was substantially reduced after proteolysis of GPIb or after addition of dRGDW. SSL5 was shown to interact with glycocalicin, a soluble GPIb, fragment, and binding of SSL5 to platelets resulted in GPIb-mediated signal transduction as evidenced by translocation of 14-3-3,. In addition, SSL5 was shown to interact with endothelial cell matrix (ECM) and this interaction enhanced aggregation of platelets from whole blood to this ECM. Conclusions: SSL5 activates and aggregates platelets in a GPIb,-dependent manner, which could be important in colonization of the vascular bed and evasion of the immune system by S. aureus. [source] Complementary roles of platelets and coagulation in thrombus formation on plaques acutely ruptured by targeted ultrasound treatment: a novel intravital modelJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2009M. J. E. KUIJPERS Summary.,Background:,Atherothrombosis is a major cause of cardiovascular events. However, animal models to study this process are scarce. Objectives:,We describe the first murine model of acute thrombus formation upon plaque rupture to study atherothrombosis by intravital fluorescence microscopy. Methods:,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. Results:,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. Conclusions:,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. [source] Nitric oxide specifically inhibits integrin-mediated platelet adhesion and spreading on collagenJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2008W. ROBERTS Summary.,Background:,Nitric oxide (NO) inhibits platelet adhesion to collagen, although the precise molecular mechanisms underlying this process are unclear. Objectives:,Collagen-mediated adhesion is a multifaceted event requiring multiple receptors and platelet-derived soluble agonists. We investigated the influence of NO on these processes. Results:,S-nitrosoglutathione (GSNO) induced a concentration-dependent inhibition of platelet adhesion to immobilized collagen. Maximal adhesion to collagen required platelet-derived ADP and TxA2. GSNO-mediated inhibition was lost in the presence of apyrase and indomethacin, suggesting that NO reduced the availability of, or signaling by, ADP and TxA2. Exogenous ADP, but not the TxA2 analogue U46619, reversed the inhibitory actions of GSNO on adhesion. Under adhesive conditions NO inhibited dense granule secretion but did not influence TxA2 generation. These data indicated that NO may block signaling by TxA2 required for dense granule secretion, thereby reducing the availability of ADP. Indeed, we found TxA2 -mediated activation of PKC was required to drive dense granule secretion, a pathway that was inhibited by NO. Because our data demonstrated that NO only inhibited the activation-dependent component of adhesion, we investigated the effects of NO on individual collagen receptors. GSNO inhibited platelet adhesion and spreading on ,2,1 specific peptide ligand GFOGER. In contrast, GSNO did not inhibit GPVI-mediated adhesion to collagen, or adhesion to the GPVI specific ligand, collagen related peptide (CRP). Conclusions:,NO targets activation-dependent adhesion mediated by ,2,1, possibly by reducing bioavailability of platelet-derived ADP, but has no effect on activation-independent adhesion mediated by GPVI. Thus, NO regulates platelet spreading and stable adhesion to collagen. [source] A snake venom metalloproteinase, kistomin, cleaves platelet glycoprotein VI and impairs platelet functionsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 9 2008C. C. HSU Summary.,Background and objectives:,Injuries to the vessel wall and subsequent exposure of the matrix of the subendothelial layer resulted in thrombus formation. Platelet glycoprotein (GP) Ib and VI play a crucial role in matrix-induced activation and aggregation of platelets. Methods and results:,In the present study, we reported that the GPIb-cleaving snake venom metalloproteinase (SVMP), kistomin, inhibited collagen-induced platelet aggregation. Moreover, kistomin inhibited platelet aggregation induced by convulxin (CVX, a GPVI agonist) and a GPVI-specific antibody in a concentration and time-dependent manner. Kistomin treatment decreased platelet GPVI but not integrin ,2,1 and ,IIb,3, accompanied with the formation of GPVI cleavage fragments, as determined by flow cytometric and Western blot analyses. In addition, intact platelet GPVI and recombinant GPVI were digested by kistomin to release 25- and 35-kDa fragments, suggesting that kistomin cleaved GPVI near the mucin-like region. We designed four synthetic peptides ranging from Leu180 to Asn249 as the substrates for kistomin and found that kistomin cleaved these synthetic peptides at FSE205/A206TA and NKV218/F219TT, as analyzed by MALDI-TOF-MS. In addition, GPVI-specific antibody-induced tyrosine kinase phosphorylation in platelets was reduced after kistomin pretreatment, and platelet adhesion to collagen but not to fibrinogen was attenuated by kistomin. Conclusions:,We provided here the first evidence that a P-I snake venom metalloproteinase, kistomin, inhibits the interaction between collagen and platelet GPVI through its proteolytic activity on GPVI, thus providing an alternative strategy for developing new anti-thrombotic agents. [source] Role of the transmembrane domain of glycoprotein IX in assembly of the glycoprotein Ib,IX complexJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2007S.-Z. LUO Summary.,Background:,The glycoprotein (GP) Ib,IX complex is critically involved in platelet adhesion to von Willebrand factor and in the initial step of platelet activation. How this complex is assembled is not clear. We previously showed that the transmembrane (TM) domains of the GPIb, and GPIb, subunits interact and participate in complex assembly. Objectives and methods:,Here, we have investigated the role of the TM and cytoplasmic domains of GPIX in assembly of the GPIb,IX complex, by analyzing the mutational effects on complex expression and assembly in transiently transfected Chinese hamster ovary cells. Results:,Replacing the cytoplasmic domain of GPIX with a poly-alanine sequence had little effect on surface expression and structural integrity of the GPIb,IX complex. In contrast, replacing the GPIX TM domain (residues 132,153) with a poly-leucine-alanine sequence markedly disrupted complex formation of GPIX with GPIb,, interfered with GPIb formation, and decreased surface expression of the host complex. We further analyzed the contributions of a number of GPIX TM residues to complex formation by mutagenesis and found significant roles for Asp135 and several Leu residues. Conclusions:,The TM domain, rather than the cytoplasmic domain, of GPIX plays an important role in expression and assembly of the GPIb,IX complex by interacting with its counterparts of GPIb. These TM domains may form a parallel four-helical bundle structure in the complex. [source] Two novel monoclonal antibodies to VWFA3 inhibit VWF-collagen and VWF-platelet interactionsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 9 2007Y. ZHAO Summary.,Background:,The interaction of collagen-von Willebrand factor (VWF)-GPIb is essential for platelet adhesion, especially under high shear conditions. VWF, which acts as a bridge between platelets and exposed subendothelium, interacts with collagen through its A3 domain, which is a new target for the antithrombotic agent. Objective:,To develop functional blockers that specifically inhibit VWF-dependent adhesion of platelets to collagen under high shear stress. Methods:,To develop murine antihuman VWF A3 monoclonal antibodies (mAbs) by standard hybridoma technology, and characterize their abilities to block interactions between VWF A3 and collagen as well as platelet function. Results:,Thirty anti-VWF-A3 mAbs were obtained. Among them, two mAbs, designated as SZ-123 and SZ-125, were found to inhibit VWF-collagen type III interaction. SZ-123 and SZ-125 inhibited the binding of purified human VWF (1.5 or 3 ,g mL,1) to human placenta collagen type III (IC50 = 0.07 ± 0.02 and 0.15 ± 0.03 ,g mL,1, respectively) or to calf skin collagen type III (IC50 = 0.48 ± 0.06 and 0.51 ± 0.07 ,g mL,1, respectively) coated on plates. Under flow shear condition (1000 s,1), SZ-123 and SZ-125 inhibited platelet adhesion on human placenta collagen- or calf skin collagen-coated surfaces. Both mAbs also inhibited platelet aggregation induced by ristocetin, botrocetin or bovine plasma. Conclusions:,SZ-123 and SZ-125 inhibited VWF-collagen and VWF-platelet interactions. [source] Purified A2 domain of von Willebrand factor binds to the active conformation of von Willebrand factor and blocks the interaction with platelet glycoprotein Ib,JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2007C. MARTIN Summary.,Background:,von Willebrand factor (VWF) does not interact with circulating platelets unless it is induced to expose the binding site for platelet glycoprotein (GP)Ib, in the A1 domain by high shear stress, immobilization, and/or a modulator. Previous studies have implied indirectly that the A2 domain may be involved in regulating A1,GPIb, binding. Objective and methods:,Because the relationship between the A1 and A2 domains has not been defined, we have investigated the effect of the A2 domain on the binding activity of the A1 domain using recombinant A domain polypeptides, multimeric VWF, and monoclonal antibodies (mAb). Results:,The A2 domain polypeptide bound specifically to the immobilized A1 domain polypeptide or full-length VWF, with half-maximal binding being obtained at 60 or 168 nm, respectively. This A1,A2 interaction was inhibited by mAb against the A2 or A1 domain and by the A1 domain polypeptide. The A2 domain polypeptide effectively blocked GPIb,-mediated platelet adhesion under high flow conditions. The A2 domain polypeptide specifically recognizes the GPIb,-binding conformation in the A1 domain, as it only interacted with VWF activated by the modulator ristocetin or immobilized VWF. Furthermore, in contrast to plasma VWF, the ultra-large (UL)VWF multimers or a recombinant VWF,A1A2A3 polypeptide containing a gain-of-function mutation (R1308 L) of type 2B von Willebrand disease bound to the A2 domain polypeptide without the need for ristocetin. Conclusions:,The recombinant A2 domain polypeptide specifically binds to the active conformation of the A1 domain in VWF and effectively blocks the interaction with platelet GPIb, under high-flow conditions. [source] Programmed autologous cleavage of platelet receptorsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2007M. C. BERNDT Summary., Platelet adhesion receptors play a critical role in vascular pathophysiology, and control platelet adhesion, activation and aggregation in hemostasis, thrombotic disease and atherogenesis. One of the key emerging mechanisms for regulating platelet function is the programmed autologous cleavage of platelet receptors. Induced by ligand binding or platelet activation, proteolysis at extracellular (ectodomain shedding) or intracellular (cytoplasmic domain deactivation) sites down-regulates the adheso-signaling function of receptors, thereby controlling not only platelet responsiveness, but in the case of ectodomain shedding, liberating soluble ectodomain fragments into plasma where they constitute potential modulators or markers. This review discusses the underlying mechanisms for dual proteolytic pathways of receptor regulation, and the impact of these pathways on thrombus formation and stability in vivo. [source] A mechanism to safeguard platelet adhesion under high-shear flow: von Willebrand factor,glycoprotein Ib and integrin ,2,1,collagen interactions make complementary, collagen-type-specific contributions to adhesion: a rebuttalJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2007T. LISMAN [source] Formation of platelet strings and microthrombi in the presence of ADAMTS-13 inhibitor does not require P-selectin or ,3 integrinJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 3 2007A. K. CHAUHAN Summary. Background:,Ultra-large von Willebrand factor (ULVWF) and the receptor P-selectin are released from endothelial Weibel,Palade bodies during injury or inflammation. VWF mediates platelet adhesion and P-selectin promotes leukocyte rolling. ADAMTS-13 limits the duration of platelet adhesion by cleaving the ULVWF. In the absence of ADAMTS-13, long VWF filaments decorated with platelets form. Recent in vitro studies suggested that P-selectin might anchor these platelet strings to endothelium, but whether the same mechanism exists in vivo remains to be elucidated. Methods:,We address the role of P-selectin and ,3 integrin in platelet string formation in vivo using intravital microscopy by infusing inhibitory ADAMTS-13 antibody in P-selectin-/- and ,3 -deficient mice and activating the endothelium by injecting histamine. Results:,We show that inhibition of ADAMTS-13 combined with endothelial activation leads to similar extents of platelet string formation in wild-type, P-selectin- and integrin ,3 -deficient mice. Further, in venules the platelet strings can coalesce into VWF-platelet aggregates. This process utilizes neither the platelet ,3 integrin nor P-selectin. We also show in vitro that platelets can act as a bridge between the VWF fibers and that VWF can self-associate even in areas devoid of platelets. Conclusions:,The formation or retention of the platelet strings does not require P-selectin or the endothelial VWF receptor ,v,3. Furthermore, in the presence of low ADAMTS-13 activity, VWF-dependent and ,IIb,3 -independent platelet clustering occurs in veins, as has been shown at high arterial shear rates. Our study further supports the importance of regulation of VWF multimer size upon secretion from Weibel,Palade bodies. [source] Platelet adhesion to dimeric ,2 -glycoprotein I under conditions of flow is mediated by at least two receptors: glycoprotein Ib, and apolipoprotein E receptor 2,JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2007M. T. T. PENNINGS Summary.,Background: The major antigen implicated in the antiphospholipid syndrome is beta2-glycoprotein I (,2GPI). Dimerized ,2GPI binds to apolipoprotein E receptor 2, (apoER2,) on platelets and increases platelet adhesion to collagen under conditions of flow. Aim: To investigate whether the interaction between dimerized ,2GPI and platelets is sufficiently strong to resist shear stresses. Methods: We studied the interaction of platelets with immobilized dimerized ,2GPI under conditions of flow, and further analyzed the interaction using surface plasmon resonance and solid phase immunoassays. Results: We found that dimerized ,2GPI supports platelet adhesion and aggregate formation under venous flow conditions. Adhesion of platelets to dimerized ,2GPI was completely inhibited by the addition of soluble forms of both apoER2, and GPIb,, and the addition of receptor-associated protein and the removal of GPIb, from the platelet surface. GPIb, co-precipitated with apoER2,, suggesting the presence of complexes between GPIb, and apoER2, on platelet membranes. The interaction between GPIb, and dimeric ,2GPI was of intermediate affinity (Kd = 180 nm) and Zn2+, but not Ca2+ -dependent. Deletion of domain V from dimeric ,2GPI strongly reduced its binding to both GPIb, and apoER2,. Antibodies that inhibit the binding of thrombin to GPIb, inhibited platelet adhesion to dimeric ,2GPI completely, while antibodies blocking the binding of von Willebrand factor to GPIb, had no effect. Dimeric ,2GPI showed reduced binding to low-sulfated GPIb, compared to the fully sulfated form. Conclusion: We show that platelets adhere to dimeric ,2GPI under both arterial and venous shear stresses. Platelets adhere via two receptors: GPIb, and apoER2,. These receptors are present in a complex on the platelet surface. [source] | ||||||||||||||||||||||||||||