Subsequent Platelet Aggregation Research Articles

Comprehensive In Vitro and In Vivo Characterization of Loss and Gain-of-Function Von Willebrand Factor Collagen Binding Variants Using a Mouse Model System,

Abstract 3266Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of injury. Rare mutations in the VWF A3 domain, that disrupt collagen binding, have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been relatively limited. Thus, in this study, we have developed mouse models of collagen binding mutants and analyzed the function of the A3 and A1 domains using comprehensive in vitro and in vivo approaches. All of the collagen binding variant AAs are conserved in mice. 6 loss-of-function (S1731T, W1745C, S1783A, H1786D, A1 deletion, A3 deletion) and 1 gain-of-function (L1757A) variant was generated in the context of the mouse VWF cDNA. The 4 loss-of-function missense mutants have all been described in patients with mild bleeding phenotypes. The recombinant mouse VWFs (rmVWF) were synthesized in HEK293T cells and analyzed for type I and III collagen binding in both a static assay (CBA) and a flow-based assay at 2,500s−1 in which VWF is bound to collagen on a surface, and labeled platelet adhesion is quantified. The multimer profile of all the rmVWFs was normal. The expression level of the rmVWF derived from HEK293T cells was quantified. W1745C and the A3 deletion showed significantly lower levels of expression and the A1 deletion mutant showed strong intracellular retention. In the static collagen binding assay, S1731T showed almost normal binding to collagen type I and a 50% reduction in binding to collagen type III. The other 3 missense variants, W1745C, S1783A and H1786D, showed reduced binding to both collagens I and III, and the A3 deletion mutant showed absent binding. In the in vitro flow assay, the sensitivity to detect defects in collagen binding was superior to the static assay, although the patterns of binding defects were similar. W1745C showed similar low levels of platelet adhesion to both types of collagen, while S1783A and H1786D showed a lack of platelet binding on the collagen III surface similar to the A3 deletion mutant, and a reduced binding to collagen type I similar to W1745C. The gain-of-function mutant showed consistent enhanced collagen binding and platelet adhesion in the static and flow assays, respectively. In vivo studies delivered the mVWF cDNAs with a strong liver specific promoter by hydrodynamic injection. At 7 days post-delivery, the VWF:Ag levels in the WT and collagen binding variant mice were similar, apart from the W1745C mutant, that showed 14.6% levels compared to WT. Platelet counts and multimer patterns were normal with the collagen binding variants. In vivo intravital microscopy studies were performed using the cremaster arteriolar model when VWF levels were in a physiological range. Thrombosis was induced by 10%FeCl3 applied for 3 mins. Platelets were labeled in vivo by Rhodamine 6G and the thrombus development was analyzed by spinning disc confocal microscopy. Loss-of-function mutants showed transient platelet adhesion at the site of injury, however the adhesion was unstable and vessel occlusion was not observed. Using three complementary experimental systems we have been able to confirm the collagen binding defects in this group of variant VWFs. There is a differential sensitivity to the two forms of collagen and of the three experimental systems. The A3 deletion mutant consistently resulted in the most severe phenotype while the missense mutants showed variable degrees of functional deficit. Disclosures:No relevant conflicts of interest to declare.

Prasugrel

Prasugrel

The Many Faces of Platelet Glycoprotein Ibα - Thrombin Interaction

The platelet glycoprotein receptor regulates the adhesion of blood platelets to damaged blood vessel walls and the subsequent platelet aggregation. One of the subunits, platelet glycoprotein Ibalpha (GpIbalpha), binds thrombin, a serine protease with both procoagulant and anticoagulant activities. Two groups reported the crystal structures of the complex between thrombin and the N-terminal extracellular domain (leucine-rich repeat [LRR] domain) of GpIbalpha. In both these structures, GpIbalpha was reported to bind two thrombin molecules, but both the primary and secondary thrombin binding sites differed between them. We performed a detailed comparison of the two structures to look for insights that may explain the differences. Our results show that the 1:1 GpIbalpha-thrombin complex detected in solution between the crystallized proteins is likely the only strong interaction. The anionic sequence (residues 268-284) of GpIbalpha is likely responsible for the initial interaction with thrombin and the interaction with the rest of LRR domain of GpIbalpha occurs subsequently and may alternate between two or more different binding modes. Our modelling suggests the interaction between GpIbalpha and thrombin is highly pH-dependent and a small change in pH is likely to contribute to the formation of alternate binding modes. The differences in the crystal structures reported for the GpIbalpha-thrombin complex suggest a fascinating plasticity in this protein-protein interaction that may be biologically significant.

Differential effects of quercetin, apigenin and genistein on signalling pathways of protease‐activated receptors PAR1 and PAR4 in platelets

The modulation by flavonoids of platelet responses induced by thrombin has been little investigated, and the antiplatelet activity, as well as possible inhibitory mechanisms of these compounds on thrombin signalling, has not yet been elucidated. We explored whether flavonoids affect platelet signalling pathways triggered by thrombin and by the selective activation of its protease-activated receptors (PARs) 1 and 4, and analysed the antagonism of these polyphenols at thrombin receptors. We investigated the effect of a range of polyphenolic compounds on platelet aggregation, 5-HT secretion, intracellular calcium mobilization, protein kinase activity and tyrosine phosphorylation, triggered by thrombin and PAR agonist peptides (PAR-APs). The ability of these flavonoids to bind to thrombin receptors was investigated by competitive radioligand binding assays using (125)I-thrombin. Quercetin, apigenin and genistein impaired platelet aggregation, as well as 5-HT release and calcium mobilization, induced by thrombin and PAR-APs. Quercetin and apigenin were inhibitors of protein kinases, but genistein exhibited a minimal ability to suppress platelet phosphorylation. Binding assays did not establish any kind of interaction between thrombin receptors and any of the flavonoids tested. Quercetin, apigenin and genistein did not inhibit thrombin responses by interacting with thrombin receptors, but by interfering with intracellular signalling. While inhibition by genistein may be a consequence of affecting calcium mobilization, subsequent platelet secretion and aggregation, for quercetin and apigenin, inhibition of kinase activation may also be involved in the impairment of platelet responses.

Blood group O and vWf expression may be involved in development of peptic ulcer disease secondary to Helicobacter pylori infection

Helicobacter pylori is a gram negative organism involved in peptic ulcer disease and has been linked to a number of extra-intestinal diseases. There is a large body of evidence describing the link between blood group O/non-secretor phenotypes with H. pylori infection and the risk of peptic ulcer disease. Blood group O individuals also have a higher risk of bleeding disorders due to low levels of the circulating plasma protein von Willebrand factor (vWf). vWf is one of the main proteins that binds platelets during platelet activation and aggregation. The mechanisms of how ulcers develop during H. pylori infection are not fully understood. There is however recent evidence of vWf involvement in platelet aggregation in H. pylori infection. Our new hypothesis states that H. pylori bacteria present in blood group O/non-secretor individuals are binding the available vWf to promote adhesion and subsequent platelet aggregation within the microvasculature. This in turn may deplete any available vWf for wound repair to take place leading to an increased risk of peptic ulceration and bleeding and eventually leading to an ulcer.

Development of an animal model of microvascular thrombosis

We have developed a model of thrombosis to test in vivo the efficacy of antithrombotic agents in a microvascular network. The model involves the induction of a platelet thrombus within venules of the spinotrapezius muscle in anesthetized Sprague Dawley rats using a compound normally employed for microvascular oxygenation measurements (Pd‐tetra‐4‐carboxyphenylporphyrin). The photoactive chemical is injected intravascularly and is excited with a 532 nm pulsed, Er:YAG laser ( 8‐10 ?W, 0.4‐0.6 kHz). As a result, free radicals are formed causing injury to the endothelial wall, subsequent platelet aggregation and thrombus formation without microvascular constriction. Red blood cell (RBC) velocity and intravascular diameter at thrombus site are monitored periodically. After testing venules of different sizes and RBC velocities, the most repeatable data were gathered from venules with diameters ranging from 40 to 60 µm and with RBC velocities ranging from 2 to 4 mm/sec. Upon establishing these control parameters, the model is being used to induce local ischemia and to test the effects of injected anti‐thrombotic agents on platelet thrombus formation and local blood flow.Support: AD Willliams Trust Funds (VCU).

Recombinant Protein Infusion and Hydrodynamic Plasmid Delivery Strategies Provide Distinct and Complementary Approaches to the Characterization of Variant Forms of Von Willeband Factor (VWF) in VWF Knockout Mice.

Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of vascular injury. The size of VWF multimers is regulated by the metalloprotease, ADAMTS13. Alterations in VWF sequence can lead to an increase or decrease in ADAMTS13-mediated cleavage, resulting in either a loss or increase in high molecular weight VWF multimers, respectively. With the availability of a VWF knockout mouse, variant forms of VWF can be evaluated in vivo in terms of their contribution to hemostasis and thrombosis. In these studies, we have taken into account the significant differences in VWF-GPIb binding and ADAMTS13 cleavage efficiency seen between mice and humans and have also assumed that functionally important residues are likely to be conserved between species. With these considerations in mind, the protocols described in this report utilize mouse-exclusive reagents. Previous reports of correction of the VWF KO phenotype using hydrodynamic gene delivery have shown contradictory results for correction of the bleeding time and blood volume loss as well as a grossly abnormal multimer structure of the rescued VWF protein, due initially to an inadvertent C799R mutation and latterly to factors possibly related to the site of VWF synthesis. In this study, we compare wild type VWF clearance to a cleavage site knockout, Y1605A/M1606A, a type 2A Von Willebrand Disease (VWD) mutation, R1597W, and the common type 1 VWD mutation, Y1584C. The murine R1597W variant exhibited increased ADAMTS13-mediated cleavage (0.36- fold ADAMTS13 concentration), and the Y1605A/M1606A variant greatly decreased cleavage in vitro (>100-fold ADAMTS13 concentration). VWF KO mice, 7-10 weeks old, were injected with recombinant murine VWF (200U/kg) produced in HEK293 cells. VWF antigen levels (VWF:Ag), multimers, and complete blood counts (CBCs) were performed. Compared to the wild type infused protein (T1/2=33.2 minutes), the Y1605A/M1606A and R1597W mutant proteins show faster clearance (T1/2=17.7 minutes, p<0.001, and 27.5 minutes, p=0.025, respectively). Although, surprisingly, the Y1605A/M1606A variant showed a preferential loss of high molecular weight material compared to wild type recombinant protein, the R1597W type 2A mutant had a much more rapid loss of the high molecular multimers compared to the other proteins analyzed. No statistical differences were observed for platelet counts or other CBC parameters post protein infusion regardless of mutation compared to resting VWF KO mice. Hydrodynamic injections of a plasmid containing the ubiquitous synthetic CAG promoter and wild type VWF were performed on eight week old mice, with delivery of 100μg plasmid DNA in 10% body weight Ringer's solution over 5-7 seconds. Maximum VWF:Ag levels of 10.67 U/ml were observed two days post infusion, with a significant proportion of observable high molecular weight VWF, indistinguishable from normal C57BL6 mouse plasma pool. Mouse FVIII:C levels follow a similar trend over the time course with maximum levels observed on day 3 at 206.7% activity. Mouse platelet counts were affected, with lower platelet counts rebounding to normal levels by day 7. No other changes in CBCs were observed. Intriguingly, none of the recombinant forms of VWF nor the hydrodynamically produced VWF protein show the typical triplet structure observed in normal mouse and human plasma, regardless of circulation time. The recombinant proteins migrate with the central band of the multimer triplet, while that of the hydrodynamic protein migrates with the lower triplet band, showing a lower molecular weight. These differences could be due to changes in protein structure or glycosylation from normally produced platelet and endothelial VWF.CAG-mVWF DNA Hydrodynamic Injection ResultsDays post infusionVWF:Ag (U/ml)FVIII:C (%)Platelets (103/μl)14.82±0.1862±16.7440±101210.67±1.06128.5±53.6547±3039.92±0.53206.7±29.4558±3971.03±0.4778.3±3.3699±53100.51±0.5330.4±16502±189140.13±0.0826.8±0470±276Resting0.0±0.0(-)569±167All data presented as value ± SD from 2-3 mice. Resting values are from at least 20 VWF KO mice.

Prasugrel versus clopidogrel in acute coronary syndrome patients undergoing percutaneous coronary intervention

Introduction: The thienopyridine clopidogrel is part of the standard of care for patients presenting with acute coronary syndromes (ACS) [ 1 ]. Clopidogrel inhibits the adenosine diphosphate receptor on platelets, thereby decreasing platelet activation and subsequent platelet aggregation. The Clopidogrel in Unstable Angina to Prevent Recurrent Events (CURE) study supports up to 12 months of therapy with clopidogrel in addition to aspirin in patients presenting with ACS [ 2 , 3 ]. The results from CURE apply to patients managed invasively or conservatively. A potential limitation of clopidogrel has been that not all patients achieve a high level of platelet inhibition.

Human von Willebrand Disease Mutations in Mouse von Willebrand Factor Alter Its Cleavage by ADAMTS13.

Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of vascular injury. The size of VWF multimers in plasma is regulated by the specific VWF cleaving protease, ADAMTS13, that cleaves VWF at the Y1605-M1606 bond in the VWF A2 domain. The adhesive properties of VWF is directly related to the multimer size, with loss of high molecular weight VWF leading to the bleeding phenotype in Type 2A von Willebrand disease (VWD) and ultra high molecular weight VWF multimers observed in the prothrombotic state of thrombotic thrombocytopenic purpura (TTP). VWF mutations leading to multimer changes have not been examined in an animal model. Although the human and mouse VWF genes are highly conserved, mutations in the two genes have not been compared experimentally. To that end, we have made mutations in the mouse VWF (mVWF) cDNA to compare the ADAMTS13 cleavage patterns seen with human VWF mutations in two in vitro assays. Recombinant multimerized full-length mVWF was digested with different concentrations of recombinant mouse ADAMTS13 (mADAMTS13) and 1.5M urea, and analyzed via multimer migration distance. The GST and histidine-tagged G1554-T1668 A2 domain region of mVWF (mVWF115) was assayed via ELISA as follows: the mVWF115 was bound to anti-GST coated plates, digested with mADAMTS13, and intact mVWF115 detected via HRP-labeled anti-histidine tag antibody. We examined R1597W (VWD Type 2A), R1306W, (VWD Type 2B), Y1584C (VWD Type 1), as well as two previously described changes that drastically lower VWF cleavage, Y1605A/M1606A, and D1614A/E1615A/K1617A. Y1584C showed a statistically insignificant 41% decrease in cleavage in the full-length assay, but a 20% increase in the mVWF115 assay. Conversely, R1597W had a 64% decrease in the full-length assay, but a 28% increase in the mVWF115 assay. Y1605A/M1606A showed a dramatic loss of cleavage, with no observable loss of high molecular weight multimers at 32 units/ml of mADAMTS13, and a 126-fold higher mADAMTS13 concentration than wild type in the mVWF115 assay. This contrasted with the 8-fold increase observed with D1614A/E1615A/K1617A. The R1306W A1 domain mutation showed a significant increase in cleavage, with 63% less ADAMTS13 necessary in the full-length assay. The triple mutant R1306W/Y1605A/M1606A did not cleave at the highest concentration, 32 U/ml, similar to that of Y1605A/M1606A, showing that the R1306W mutation was not able to increase cleavage of the Y1605A/M1606A change. These results show a strong correlation between the mouse mutations described here and previously reported human VWF mutations in their susceptibility to ADAMTS13 cleavage. These results provide a rationale for the development of mouse models of type 1, 2A, and 2B VWD, as well as mVWF mutations that might lead to a prothrombotic state similar to TTP in humans.mADAMTS13 Concentration for 50% mVWF CleavageADAMTS13 Concentration (Units/ml)Relative mADAMTS13 Concentration (to Wild Type)mVWF Multimer (n=1)mVWF115 (n=4)mVWF Multimer (n=1)mVWF115 (n=4)Wild Type0.3010.19711Y1584C0.4230.157*1.410.80*R1597W0.109**0.253*0.36**1.28*Y1605A/M1606A>32**24.8**>100**126**D1614A/E1615A/K1617A(−)1.58**(−)8.0**R1306W0.112**(−)0.37**(−)R1306W/Y1605A/M1606A>32**(−)>100**(−)*: P value <0.05, **: P Value < 0.001 in 2 tailed T test.

Invited commentary

Aspirin is widely used to reduce graft thrombosis after coronary artery bypass grafting. However, aspirin only prevents approximately 25% of all ischemic vascular events and there are many causes, including all the causes of aspirin resistance for the other events. Aspirin resistance is the inability of aspirin to block platelet production of thromboxane A2 and subsequent platelet activation and aggregation. Increasing degrees of aspirin resistance may correlate independently with increasing risk of cardiovascular events. The study by Lim and colleagues [1Lim E. Carballo S. Cornelissen J. et al.Dose-related efficacy of aspirin after coronary surgery in patients with PlA2 polymorphism (NCT00262275).Ann Thorac Surg. 2007; 83: 134-139Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar] analyzed a genetic polymorphism within a subgroup of a prospective randomized trial. They attempted to evaluate the role of genetic polymorphism on the aspirin response in patients after coronary artery bypass grafting. The authors postulated that several genetic polymorphisms are common and some may affect responses to aspirin. In particular, PIA2 polymorphism seems to cause aspirin resistance. As with most genetic studies, the results were not clear-cut and statistical significance was not achieved, although trends were similar to published results. The design of this study is a major weakness for determining correlation between clinical findings and genetic polymorphisms. Basically most studies on genetic polymorphism need several populations of subjects. Several different subject populations or a highly selected group can establish a correlation between clinical findings and polymorphisms. Analytic studies that investigate the association of biochemical aspirin resistance and clinical events must quantitatively account for potential confounders (eg, age, sex, ethnicity, and clinical conditions), hematologic and biochemical factors (eg, hyperlipidemia, platelet count, and hemoglobin level), and nonadherence. The analysis in this study did not consider aspirin resistance that can be diagnosed in the laboratory by measurement of platelet thromboxane A2 production or thromboxane-dependent platelet function. Potential causes of aspirin resistance include inadequate dose, drug interactions, genetic polymorphisms involved in thromboxane biosynthesis, upregulation of nonplatelet sources of thromboxane biosynthesis, and increased platelet turnover. To prove the association between the polymorphism and clinical risk, statistical exclusion of confounders is required to reach a conclusion. Differential sensitivity to aspirin may have potential clinical implications, whereby specific antiplatelet therapy is best tailored to a patient’s PIA2 genotype. A well-designed study in a larger population is necessary to confirm the association between genetic polymorphisms and aspirin resistance. Dose-Related Efficacy of Aspirin After Coronary Surgery in Patients With PlA2 Polymorphism (NCT00262275)The Annals of Thoracic SurgeryVol. 83Issue 1PreviewTo evaluate the impact of the genetic polymorphisms affecting aspirin response using platelet aggregation and the response to different aspirin doses after cardiopulmonary bypass, we performed a subanalysis of the results from a randomized trial evaluating low- and medium-dose aspirin and clopidogrel. Full-Text PDF

Aspirin Resistance: Detection, Mechanisms and Clinical Implications

Aspirin, the most widely used antiplatelet agent, irreversibly acetylates the enzyme cyclooxygenase 1 (COX-1), thereby inhibiting platelet thromboxane synthesis and subsequent platelet aggregation. Although aspirin has been demonstrated to reduce the odds of serious atherothrombotic events and death in high-risk patients by 25%, subsets of patients fail to respond to therapy and continue to suffer atherothrombotic events. This aspirin treatment failure may be due to sub-optimal bioavailability (e.g. because of non-compliance or under-dosing) or may be a consequence of the as yet poorly understood phenomenon of aspirin resistance. In this review, we summarize the current laboratory methods used to identify aspirin-resistant patients, outline the cellular mechanisms that may contribute to aspirin resistance, and discuss the clinical implications of this important issue.

The Impact of Mutations in the A2 Domain of Von Willebrand Factor on its Cleavage by ADAMTS13.

Von Willebrand factor (VWF) plays an important role in primary hemostasis as it mediates platelet adhesion to the vessel wall and subsequent platelet aggregation at the site of vascular injury. Since the adhesive function of VWF depends on its multimer size, the loss of high molecular weight multimers (HMWM) results in hemorrhagic diathesis as seen in classical von Willebrand disease (VWD) type 2A, which represents the most common qualitative defect of VWD. The size distribution of VWF multimers in plasma is strongly influenced - if not regulated - by the specific VWF cleaving protease ADAMTS13 that cleaves VWF at the Y1605-M1606 bond in the A2 domain. Mutations in classical VWD type 2A cluster in the A2 domain and earlier studies suggest that some of the mutations make VWF more susceptible to ADAMTS13 dependent proteolysis (group 2) while others decrease the secretion of VWF HMWM (group 1). Our aim was to investigate the impact of VWF A2 domain mutations on ADAMTS13 dependent proteolysis of VWF. We used recombinant human ADAMTS13 (rhuADAMTS13) to digest recombinant full-length VWF and a fragment spanning the VWF A1-A2-A3 domains, harboring 13 different mutations that we found in patients with VWD type 2A. Proteolysis was monitored by VWF multimer analysis and by SDS-PAGE of the VWF A1-A2-A3 fragment. Cleavage of full-length VWF resulted in multimer patterns similar to that seen in plasma of patients with VWD type 2A, confirming the specifity of the reaction. Eleven VWF mutants (C1272S, G1505R, S1506L, M1528V, delR1569, R1597W, V1607D, G1609R, G1629E, G1631D, E1638K) showed less HMWM and more pronounced proteolytic fragments than wildtype (wt) VWF digested under the same conditions. Co-expression of the wt allele attenuated the proteolysis-permissive phenotype. The G1629E mutation resulted in highly increased proteolysis, suggesting an important role of this residue in the interaction between VWF and ADAMTS13. Surprisingly, G1505E and I1628T mutations failed to increase cleavage of the full-length VWF by rhuADAMTS13. However, when these mutations were introduced in the monomeric VWFA1-A2-A3 fragments they (like others) allowed cleavage of the Y1605-M1606 bond even under non-denaturing conditions, suggestive of an increased proteolytic susceptibility since wt VWF is only cleaved under denaturing conditions. The differences between the assays of full-length VWF and A1-A2-A3 domain fragment might be due to the lack of shear in our assay. This study provides direct evidence that in VWD type 2A, VWF with mutations in the A2 domain is subject to increased cleavage by ADAMTS13. This includes mutations previously designated as group 1 (G1505R, S1506L and V1607D) suggesting that increased susceptibility to ADAMTS13 is a more general property of VWF with A2 domain mutations. Therefore future therapies for patients with VWD type 2A might target VWF cleavage by ADAMTS13. RhuADAMTS13 and VWF constructs with mutations in the A2 domain are valuable tools to investigate VWF cleavage under varying conditions. Further work should address the question how shear influences ADAMTS13 dependent cleavage of VWF mutants.

Rac2 GTPase Plays a Critical Role in Platelet Adhesion as Well as in Sustenance and Perpetuation of Platelet Aggregation.

The Rac GTPases, members of the Ras related family of Rho GTPases, have been shown to be involved in the regulation of cell spreading, adhesion and actin cytoskeleton reorganization in hematopoietic cells (Roberts, A.W., et al. Immunity 10: 183–196, 1999; Gu Y., et al. Science 302: 445–449, 2003). Adhesion of platelets to vascular matrix containing fibrinogen or collagen is the first and a critical step that leads to platelet aggregation. The possibility that individual Rac GTPases may play a role in platelet activation was examined in this study. The Rac2 gene targeted (Rac2−/−) mice were used to investigate the contribution of Rac2 GTPase in platelet adhesion and aggregation. All experiments were conducted using an equal number (1–2 x 108/ml) of washed platelets from WT and Rac2−/− mice. Firstly, the role of Rac2 in platelet adhesion was examined by quantifying adhesion of platelets to fibrinogen coated wells. Platelets from Rac2−/−mice exhibited a 35% less adhesion to fibrinogen than platelets from WT mice. Secondly, binding of platelets to soluble fibrinogen was assessed by stimulating the platelets with ADP, collagen or phospholipase C (PLC) in the presence of Oregon Green conjugated fibrinogen followed by flow cytometry analysis. ADP-, PLC- and collagen-induced fibrinogen binding to platelets from Rac2−/−mice, as compared to platelets from WT mice, was decreased by 65%, 40% and 35%, respectively. Thirdly, platelet aggregation was monitored by an optical density method using a Chronolog Aggregometer. Agonists that induce aggregation responses via specific receptors for example thrombin, collagen and U46619 (a stable analog of thromboxane A2) as well as agonists that bypass receptors and directly activate protein kinase C by generating diacylglycerol (e.g. PLC from Clostridium perfringens) or by mimicking diacylglycerol (e.g. phorbol 12-myristate 13-acetate, PMA) were used in this study. At threshold concentrations, thrombin (0.04 U/ml), collagen (4 μg/ml), U46619 (0.5 μM) and PLC (0.04 U/ml) all elicited a slower onset of aggregation, as depicted by a prolonged shape change phase, in platelets from Rac2−/−than platelets from WT mice. Aggregation responses induced by thrombin, collagen and U46619 were all diminished in platelets from Rac2−/−mice as compared to platelets from WT mice. Moreover, platelets from Rac2−/−, but not from WT, mice failed to exhibit irreversible aggregation even when challenged with higher concentrations of U46619 (10 μM) or collagen (8 μg/ml). Platelet aggregation responses induced by PLC and PMA, agents that bypass receptors, were also decreased in platelets from Rac2−/−mice than in platelets from WT mice. These data lead us to suggest that Rac2 plays a critical role not only in platelet adhesion to fibrinogen, a step necessary for subsequent platelet aggregation, but also in sustenance and perpetuation of platelet aggregation. Furthermore, Rac2 appears to be involved in regulation of agonist-receptor mediated as well as direct, receptor-independent, activation of platelets.

Monensin augments capacitative Ca 2+ entry and subsequent aggregation of platelets via an intracellular alkalosis-mediated mechanism

Effects of monensin, an ionophore that facilitates the transmembrane exchange of Na + for H +, on capacitative Ca 2+ entry (CCE) of platelets were investigated. CCE of human platelets was induced by addition of Ca 2+ to a nominally Ca 2+-free medium after release of intracellular stored Ca 2+ caused by thapsigargin. CCE was strongly inhibited by SKF-96365 (1-[β-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1 H-imidazole hydrochloride). Monensin significantly increased SKF-96365-sensitive CCE and subsequent platelet aggregation. Monensin also induced a sustained increase in intracellular pH. The augmenting effect of monensin on CCE and subsequent platelet aggregation was not observed in the presence of sodium propionate, which canceled intracellular alkalinization induced by monensin. These results suggest that monensin augments CCE of platelets by a mechanism mediated by intracellular alkalosis.

Evidence for a role for Gαi1 in mediating weak agonist-induced platelet aggregation in human platelets: reduced Gαi1 expression and defective Gi signaling in the platelets of a patient with a chronic bleeding disorder

AbstractWe have examined platelet functional responses and characterized a novel signaling defect in the platelets of a patient suffering from a chronic bleeding disorder. Platelet aggregation responses stimulated by weak agonists such as adenosine diphosphate (ADP) and adrenaline were severely impaired. In comparison, both aggregation and dense granule secretion were normal following activation with high doses of collagen, thrombin, or phorbol-12 myristate-13 acetate (PMA). ADP, thrombin, or thromboxane A2 (TxA2) signaling through their respective Gq-coupled receptors was normal as assessed by measuring either mobilization of intracellular calcium, diacylglycerol (DAG) generation, or pleckstrin phosphorylation. In comparison, Gi-mediated signaling induced by either thrombin, ADP, or adrenaline, examined by suppression of forskolin-stimulated rise in cyclic AMP (cAMP) was impaired, indicating dysfunctional Gαi signaling. Immunoblot analysis of platelet membranes with specific antiserum against different Gα subunits indicated normal levels of Gαi2,Gαi3,Gαz, and Gαq in patient platelets. However, the Gαi1level was reduced to 25% of that found in normal platelets. Analysis of platelet cDNA and gDNA revealed no abnormality in either the Gαi1 or Gαi2 gene sequences. Our studies implicate the minor expressed Gαi subtype Gαi1 as having an important role in regulating signaling pathways associated with the activation of αIIbβ3 and subsequent platelet aggregation by weak agonists.

Aspects of hydrodynamic shear regulating shear-induced platelet activation and self-association of von Willebrand factor in suspension

The binding of plasma von Willebrand factor (VWF) to platelet receptor GpIb under high hydrodynamic shear leads to platelet activation and subsequent shear-induced platelet aggregation (SIPA). We quantitatively examined the aspects of fluid flow that regulate platelet activation by subjecting human blood and isolated platelets to well-defined shear conditions in a cone-plate viscometer. We made the following observations. First, Annexin V binding to phosphatidyl serine expressed on activated cells was detectable within 10 seconds of shear application. Second, fluid shear stress rather than shear rate controls platelet activation, and a threshold shear stress of approximately 80 dyn/cm(2) is necessary to induce significant activation. Under these conditions, individual domains of soluble VWF and platelet GpIb are subjected to similar magnitudes of fluid forces on the order of 0.1 pN, whereas GpIb with bound VWF is subjected to 1 pN. Third, cell-cell collisions and time-varying stresses are not essential for platelet activation. Fourth, the mechanism of platelet activation can be resolved in 2 steps based on the contribution of VWF and fluid forces. Fluid shear and VWF are required during the first step, when GpIb-VWF binding likely occurs. Subsequently, high shear forces alone in the absence of VWF in suspension can induce platelet activation. In other experiments, purified VWF was subjected to shear in the viscometer, and VWF morphology was assessed using light scattering. These studies demonstrate, for the first time, the ability of hydrodynamic forces to induce VWF aggregation in suspension. This VWF self-association may be an additional feature involved in controlling cell adhesion rates in circulation.

Clopidogrel in the management of ischemic heart disease.

Ischemic heart disease (IHD) represents a pathophysiologic continuum consisting of stable angina, unstable angina, non-ST-segment elevation myocardial infarction, and ST-segment elevation myocardial infarction. Patients who develop a change in their usual stable pattern of ischemia are classified as having an acute coronary syndrome, which includes patients with unstable angina, non-ST-segment elevation myocardial infarction, and ST-segment elevation myocardial infarction. Such progression from a stable to an unstable state is believed to result from disruption of an atherosclerotic plaque with subsequent platelet aggregation and thrombus formation. This, in turn, leads to the clinical manifestations of unstable angina, MI or death. Because platelets play a central role in the thrombotic complications of atherosclerosis, antiplatelet agents have been the cornerstone of the therapy for IHD. Aspirin has been the traditional antiplatelet agent, and remains the mainstay of treatment for all forms of IHD. However, aspirin is a weak antiplatelet agent and is often poorly tolerated by many patients. Clopidogrel is a new antiplatelet agent of the thienopyridine class. Clopidogrel, when used alone, and especially in combination with aspirin, has been shown to improve outcomes in patients with IHD across a variety of syndromes. However, combination therapy with aspirin and clopidogrel has been associated with an increased risk of bleeding. Therefore, despite improved outcomes, further studies are required to determine the optimal duration and dosage regimen of such combination therapy to maximize its risk-benefit ratio.

Platelet aggregation by membrane-expressed A1 domains of von Willebrand Factor is dependent on residues Asp 560 and Gly 561

We have previously demonstrated that the isolated von Willebrand Factor (vWF)-A1-domain can activate platelets in a GPIb-dependent manner. Here we evaluated the functional impact of targeted point mutations Gly561His (G1324(561)H), an analog of a previously described 2M von Willebrand disease variant, and Asp560Ser (D1323(560)S) in the model of the membrane expressed A1-domain. Platelet aggregation in response to COS-7-cells stably transfected with wild type A1-domain was abrogated by both substitutions. Ristocetin did not increase the aggregatory potential of mutant vWF-A1, in contrast to native forms. Botrocetin boosted the aggregatory responses of all A1-domains tested. These data suggest that G1324(561) and D1323(560) comprise part of the GPIb binding motif essential for subsequent platelet aggregation. Botrocetin seems to alter the potential of vWF for GPIb interaction independently of that motif. The experimental system tested here provides a rapid and reproducible approach for the functional analysis of isolated A1-domain interactions with platelet-GPIb.

Intracellular alkalinization augments capacitative Ca2+ entry in platelets.

In order to elucidate the significance of intracellular alkalinization in signal transduction of platelets, we investigated the effects on capacitative Ca(2+) entry (CCE) of intracellular alkalinization that was induced by NH(4)Cl. Addition of NH(4)Cl (10 mM) to the medium resulted in an elevation of intracellular pH by about 0.35, which was eliminated by simultaneous addition of propionate (20 mM), an inducer of intracellular acidification, to the medium. CCE was induced by an extracellular addition of Ca(2+) to platelets in which Ca(2+) stores had been depleted by stimulation with thapsigargin in nominally Ca(2+)-free medium. NH(4)Cl markedly augmented CCE and subsequent platelet aggregation, both of which were abolished in the presence of SKF-96365, an inhibitor of capacitative Ca(2+) entry in non-excitable cells such as platelets. The augmentation of CCE and subsequent aggregation by NH(4)Cl was not observed in the presence of propionate or SKF-96365. Extracellular alkalosis induced by Tris also markedly augmented CCE and subsequent aggregation. These augmenting effects of extracellular alkalosis by Tris were significantly but incompletely inhibited by simultaneous addition of propionate (20 mM), which completely eliminated elevation of intracellular pH elicited by Tris. Thus, the augmenting effect of extracellular alkalosis on CCE was in part mediated by intracellular alkalosis. These findings suggest that intracellular alkalinization is a potent signal that augments CCE in platelets.

Activation of Caspase-3/Caspase-3-Like Activity in Rat Cardiomyocytes by an RGD Peptide, but not the GPIIb/IIIa Antagonist Lotrafiban

Platelet glycoprotein GPIIb/IIIa antagonists are antithrombotic compounds that interfere with fibrinogen binding to this integrin receptor and prevent subsequent platelet aggregation [1]. Several intravenous GPIIb/IIIa antagonists (including abciximab, eptifibatide, and tirofiban) are currently employed during acute coronary syndrome and/or percutaneous coronary intervention in order to reduce the incidence of associated thrombotic events. These acute treatments are well tolerated and have been shown to decrease the rates of subsequent myocardial infarction (MI) and death (for review, see Refs. [2–4]). In contrast, clinical trials examining the potential benefit of chronic treatment with orally active GPIIb/IIIa antagonists for the prevention of thrombotic events (i.e., stroke, MI) have all failed due to lack of efficacy, often with an increase in the incidence of mortality and MI [5–8].