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Objectives: Platelet activation underpins thrombus formation in ischemic stroke. The active, dimeric form of platelet receptor glycoprotein (GP) VI plays key roles by binding platelet ligands collagen and fibrin, leading to platelet activation. We investigated whether patients presenting with stroke expressed more GPVI on their platelet surface and had more active circulating platelets as measured by platelet P-selectin exposure. Methods: 129 ischemic or hemorrhagic stroke patients were recruited within 8h of symptom onset. Whole blood was analyzed for platelet-surface expression of total GPVI, GPVI-dimer, and P-selectin by flow cytometry at admission and day-90 post-stroke. Results were compared against a healthy control population (n = 301). Results: The platelets of stroke patients expressed significantly higher total GPVI and GPVI-dimer (P<0.0001) as well as demonstrating higher resting P-selectin exposure (P<0.0001), a measure of platelet activity, compared to the control group, suggesting increased circulating platelet activation. GPVI-dimer expression was strongly correlated circulating platelet activation [r2 = 0.88, P<0.0001] in stroke patients. Furthermore, higher platelet surface GPVI expression was associated with increased stroke severity at admission. At day-90 post-stroke, GPVI-dimer expression and was further raised compared to the level at admission (P<0.0001) despite anti-thrombotic therapy. All ischemic stroke subtypes and hemorrhagic strokes expressed significantly higher GPVI-dimer compared to controls (P<0.0001). Conclusions: Stroke patients express more GPVI-dimer on their platelet surface at presentation, lasting at least until day-90 post-stroke. Small molecule GPVI-dimer inhibitors are currently in development and the results of this study validate that GPVI-dimer as an anti-thrombotic target in ischemic stroke. [ABSTRACT FROM AUTHOR]

The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (Kd) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triplehelical (GPO)10-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ∼29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ∼39 and ∼44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation. [ABSTRACT FROM AUTHOR]

Of the two physiologically important platelet collagen receptors, glycoprotein (GP) VI is the receptor responsible for platelet activation. However, its reactivities towards different types of vascular collagen have not been directly and quantitatively analysed with collagen preparations of defined composition, although the other major platelet collagen receptor integrin α2β1 was shown to react with collagen types I-VI and VIII under either static or flow conditions. We analysed the collagen type specificity of GPVI binding to identify the physiological contribution of the various vascular collagens and how platelet reactivity towards the various collagens may be affected by fibril size. We used two methods to analyse the binding of recombinant GPVI (GPVI-Fc2) to different types of bovine collagen: binding to collagen microparticles in suspension and binding to immobilized collagen. GPVI-Fc2 bound to type I-III collagens that can form large fibrils, but not to type V that only forms small fibrils. The apparent GPVI binding to types IV and V could be ascribed to type I collagen that was a contaminant in each of these preparations. Kinetic analyses of the binding data showed that type III collagen fibrils have both a higher Kd and Bmax than types I and II. Flow adhesion studies demonstrated that type III collagen supports the formation of larger platelet aggregates than type I. Our present results suggest that the physiological importance of type III collagen is to induce thrombus formation. Furthermore, these studies indicate that GPVI mainly binds to collagen types that can form large collagen fibrils. [ABSTRACT FROM AUTHOR]

Abstract: The abundant Rap1 in platelets becomes activated when these cells are stimulated by various agonists, but its function has remained unknown. In view of this, we developed an assay to quantitatively measure activated Rap1 and used it to determine relationships between Rap1 activation and several platelet functions: integrin α2β1 activation, tyrosine phosphorylation, and the release reaction. We looked at how these processes are affected by the protein kinase C inhibitor BIMI, tyrosine kinase inhibitor PP2, PI 3-kinase inhibitor wortmannin, and ADP scavenger apyrase. In CRP (collagen related peptide)-activated platelets, all the inhibitors severely inhibited Rap1 activation, but had little effect on integrin α2β1 activation, indicating that the integrin activation mechanism is different from the Rap1 activation mechanism, at least in GPVI-dependent activation. With p85α-null mouse platelets, we demonstrated that Rap1 activation involves PI 3-kinase p85α-dependent tyrosine phosphorylation. All the inhibitors similarly decreased Rap1 activation and the serotonin release reaction, and the inhibition of Rap1 activation was not due to the lack of released ADP. Our results indicate that platelet Rap1 activation is closely related to the release reaction and not to integrin α2β1 activation in GPVI-mediated platelet activation. [Copyright &y& Elsevier]

Abstract Background and aims Platelets play a fundamental role in the increased atherothrombotic risk related to central obesity since they show hyperactivation and lower sensitivity to antiplatelet therapy in obese patients. The main goal of this study was to identify platelet biomarkers related to the risk of atherothrombosis in obese patients, confirm platelet activation levels in these patients, and identify altered activation pathways. Methods Platelets were obtained from cohorts of obese patients and age- and sex-matched lean controls. Biochemical and proteome analyses were done by two-dimensional differential in-gel electrophoresis (2D-DIGE), mass spectrometry, and immunoblotting. Functional and mechanistic studies were conducted with aggregation assays and flow cytometry. Results We confirmed an up-regulation of αIIb and fibrinogen isoforms in platelets from obese patients. A complementary platelet aggregation approach showed platelets from obese patients are hyper-reactive in response to collagen and collagen-related peptide (CRP), revealing the collagen receptor Glycoprotein VI (GPVI) signalling as one of the altered pathways. We also found the active form of Src (pTyr418) is up-regulated in platelets from obese individuals, which links proteomics to aggregation data. Moreover, we showed that CRP-activated platelets present higher levels of tyrosine phosphorylated PLCγ2 in obese patients, confirming alterations in GPVI signalling. In line with the above, flow cytometry studies show higher surface expression levels of total GPVI and GPVI-dimer in obese platelets, both correlating with BMI. Conclusions Our results suggest a higher activation state of SFKs-mediated signalling pathways in platelets from obese patients, with a primary involvement of GPVI signalling. Graphical abstract Image 1 Highlights • Higher expression of the active form of Src (pTyr418) in obese patients platelets. • Increased aggregation levels of obese platelets in response to GPVI activation. • GPVI-activated platelets show higher levels of tyr-phosphorylated PLCγ2 in obesity. • Higher surface expression levels of GPVI in obese platelets, correlating with BMI. [ABSTRACT FROM AUTHOR]

Background Glycoprotein VI (GPVI) is the essential platelet collagen receptor in atherothrombosis, but its inhibition causes only a mild bleeding tendency. Thus, targeting this receptor has selective antithrombotic potential. Objectives This study sought to compare compounds interfering with platelet GPVI–atherosclerotic plaque interaction to improve current antiatherothrombotic therapy. Methods Human atherosclerotic plaque–induced platelet aggregation was measured in anticoagulated blood under static and arterial flow conditions (550/s, 1,100/s, and 1,500/s). Inhibition by dimeric GPVI fragment crystallizable region of IgG (Fc) masking GPVI binding sites on collagen was compared with that of 3 anti-GPVI antibodies: BLO8-1, a human domain antibody; 5C4, a fragment antigen-binding (Fab fragment) of monoclonal rat immunoglobulin G; and m-Fab-F, a human recombinant sFab against GPVI dimers. Results GPVI-Fc reduced plaque-triggered platelet aggregation in static blood by 51%, BLO8-1 by 88%, and 5C4 by 93%. Under arterial flow conditions, BLO8-1 and 5C4 almost completely inhibited platelet aggregation while preserving platelet adhesion on plaque. Inhibition by GPVI-Fc, even at high concentrations, was less marked but increased with shear rate. Advanced optical imaging revealed rapid persistent GPVI-Fc binding to collagen under low and high shear flow, upstream and downstream of plaque fragments. At low shear particularly, platelets adhered in plaque flow niches to GPVI-Fc–free segments of collagen fibers and recruited other platelets onto aggregates via ADP and TxA2 release. Conclusions Anti-GPVI antibodies inhibit atherosclerotic plaque-induced platelet aggregation under static and flow conditions more effectively than GPVI-Fc. However, potent platelet inhibition by GPVI-Fc at a higher shear rate (1,500/s) suggests localized antithrombotic efficacy at denuded or fissured stenotic high-risk lesions without systemic bleeding. The compound-specific differences have relevance for clinical trials targeting GPVI-collagen interaction combined with established antiplatelet therapies in patients with spontaneous plaque rupture or intervention-associated plaque injury. [ABSTRACT FROM AUTHOR]