ALBERT

Description: Thrombosis can be initiated when activated platelets adhere to injured blood vessels via the interaction of subendothelial collagen with its platelet receptor, glycoprotein (GP) VI. Here we observed that incubation of platelets with convulxin, collagen, or collagen-related peptide (CRP) resulted in GPVI signaling-dependent loss of surface GPVI and the appearance of an approximately 55-kDa soluble fragment of GPVI as revealed by immunoblotting. Ethylenediaminetetraacetic acid (EDTA) or GM6001 (a metalloproteinase inhibitor with broad specificity) prevented this loss. In other receptor systems, calmodulin binding to membrane-proximal cytoplasmic sequences regulates metalloproteinase-mediated ectodomain shedding. In this regard, we have previously shown that calmodulin binds to a positively charged, membrane-proximal sequence within the cytoplasmic tail of GPVI. Incubation of platelets with calmodulin inhibitor W7 (150 μM) resulted in a time-dependent loss of GPVI from the platelet surface. Both EDTA and GM6001 prevented this loss. Surface plasmon resonance demonstrated that W7 specifically blocked the association of calmodulin with an immobilized synthetic peptide corresponding to the calmodulin-binding sequence of GPVI. These findings suggest that disruption of calmodulin binding to receptor cytoplasmic tails by agonist binding to the receptor triggers metalloproteinase-mediated loss of GPVI from the platelet surface. This process may represent a potential mechanism to regulate GPVI-dependent platelet adhesion.

Description: Ligand-induced ectodomain shedding of glycoprotein VI (GPVI) is a metalloproteinase-dependent event. We examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. Human-citrated platelet-rich plasma or washed platelets were subjected to increasing shear rates in a cone-plate viscometer, and levels of intact and cleaved GPVI were examined by Western blot and ELISA. Pathophysiologic shear rates (3000-10 000 seconds−1) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of αIIbβ3, GPIbα, or intracellular signaling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in platelet-rich plasma or washed platelets isolated from a von Willebrand disease type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIbα ligands. Significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease and mean intracoronary shear estimates at 2935 seconds−1 (peak shear, 19 224 seconds−1). Loss of GPVI in platelets exposed to shear has potential implications for the stability of a forming thrombus at arterial shear rates.

Description: Aim Ventricular assist devices (VADs) and extracorporeal membrane oxygenation devices (ECMO) are associated with bleeding, not fully explained by anticoagulant or anti-platelet therapy. Acquired von Willebrand syndrome (AVWS) may contribute to bleeding in patients with these devices. We investigated the relationship between AVWS and platelet dysfunction, through the loss of von Willebrand factor (VWF) receptor, GPIb (of the GPIb-IX-V complex) and the major collagen receptor, GPVI. GPIb and GPVI are crucial for platelet function at arterial shear rates and we have shown that metalloproteolytic shedding of GPVI is triggered on exposure of platelets to high shear (Al-Tamimi et al. Blood, 2012). Methods A pilot analysis was performed in 21 VADs patients (0.5-30 months post-implant) and 13 ECMO patients (1-9 days post-implant). All patients were in receipt of at least one anticoagulant and/or one anti-platelet therapy. Platelet counts, coagulation tests and VWF analyses including VWF multimers were performed. Levels of platelet surface and shed receptors were measured by flow cytometry-based assays or ELISA developed in-house. The in-house assays are unaffected by routine therapeutics used in these patients. Data were analysed by non-parametric Wilcoxon-Mann-Whitney test. The National Cancer Institute (NCI) bleeding score was used and VADs patients were grouped into major (NCI ≥3) or non-major (NCI

Description: The platelet collagen receptor, glycoprotein (GP)VI, initiates platelet aggregation at low shear stress while GPIb-IX-V, which binds von Willebrand factor, elicits platelet aggregation under high shear conditions. To investigate the possibility that GPIb-IX-V and GPVI are associated on the platelet surface, we first ascertained that aggregation induced by a GPVI-specific agonist, collagen-related peptide, like collagen, is markedly cross-blocked by a GPIbα-specific monoclonal antibody, SZ2. Immunoprecipitation of GPIb-IX with anti-GPIbα from the 1% (v/v) Triton-soluble fraction of unstimulated platelets and immunoblotting with anti-GPVI demonstrated association between GPIb-IX and GPVI. This association was maintained when platelets were activated by thrombin. Pre-treatment of platelets with methyl-β-cyclodextrin to disrupt lipid rafts did not affect association in resting or activated platelets under these conditions of detergent lysis. The association is also independent of cytoskeletal attachment, since it was unaffected by treatment with N-ethylmaleimide or DNaseI, which dissociate GPIb-IX from filamin and the actin-containing cytoskeleton, respectively. Finally, the association involves an interaction between the ectodomains of GPIbα and GPVI, since soluble fragments of GPIbα (glycocalicin) and GPVI are co-precipitated from the platelet supernatant under conditions where GPVI is shed. A contribution of GPIb-IX-V to GPVI-induced platelet responses, and vice versa, therefore warrants further investigation.

Description: Engagement of the adhesion receptor glycoprotein (GP) Ib-IX-V by von Willebrand factor (VWF) mediates platelet adhesion to damaged vessels and triggers platelet activation and thrombus formation in heart attack and stroke. GPIb-IX-V contains distinct 14-3-3ζ–binding sites at the GPIbα C-terminus involving phosphorylation of Ser609, an upstream site involving phosphorylated Ser587/Ser590, and a protein kinase A (PKA)–dependent site on GPIbβ involving Ser166. 14-3-3ζ regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3ζ association blocks receptor signaling, suggesting a key functional role for 14-3-3ζ. We used deletion mutants of GPIbα expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3ζ binding to another GPIb-IX-V–associated signaling protein, phosphoinositide 3-kinase (PI3-kinase). Pull-down experiments involving glutathione S-transferase (GST)–PI3-kinase/p85-subunit and GST–14-3-3ζ indicated that both proteins interacted with contiguous GPIbα sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIbα expressed in CHO cells, inhibited VWF/ristocetin-dependent Akt phosphorylation, relative to wild-type receptor, confirming this region encompassed a functional PI3-kinase–binding site. Pull-down experiments with GST-p85 truncates indicated the GPIbα-binding region involved the p85 breakpoint cluster region (BCR) domain, containing RSXSXP. However, pull-down of GPIb-IX was unaltered by mutation/deletion/phosphorylation of this potential 14-3-3ζ–binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIbα independently of 14-3-3ζ; 14-3-3ζ inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3ζ but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3ζ. Together, these data suggest the GPIbα C-terminus regulates signaling through independent association of 14-3-3ζ and PI3-kinase.

Description: Thrombus formation in hemostasis or thrombotic disease is initiated by the rapid adhesion, activation, and aggregation of circulating platelets in flowing blood. At arterial or pathological shear rates, for example due to vascular stenosis or circulatory support devices, platelets may be exposed to highly pulsatile blood flow, while even under constant flow platelets are exposed to pulsation due to thrombus growth or changes in vessel geometry. The aim of this study is to investigate platelet thrombus formation dynamics within flow conditions consisting of either constant or variable shear. Human platelets in anticoagulated whole blood were exposed ex vivo to collagen type I-coated microchannels subjected to constant shear in straight channels or variable shear gradients using different stenosis geometries (50%, 70%, and 90% by area). Base wall shears between 1800 and 6600 s−1, and peak wall shears of 3700 to 29,000 s−1 within stenoses were investigated, representing arterial-pathological shear conditions. Computational flow-field simulations and stenosis platelet thrombi total volume, average volume, and surface coverage were analysed. Interestingly, shear gradients dramatically changed platelet thrombi formation compared to constant base shear alone. Such shear gradients extended the range of shear at which thrombi were formed, that is, platelets became hyperthrombotic within shear gradients. Furthermore, individual healthy donors displayed quantifiable differences in extent/formation of thrombi within shear gradients, with implications for future development and testing of antiplatelet agents. In conclusion, here, we demonstrate a specific contribution of blood flow shear gradients to thrombus formation, and provide a novel platform for platelet functional testing under shear conditions.

Description: Platelets respond rapidly to injury, infection and changes in blood shear stress where they engage subendothelial collagen and von Willebrand Factor via platelet-specific receptors glycoprotein (GP)VI and the GPIb-IX-V complex, respectively. Metalloproteolytic shedding of GPVI is one important consequence of platelet activation and occurs via activation of the receptor sheddase A Disintegrin and Metalloproteinase (ADAM)10. We demonstrated that exposure of platelets to brief, elevated shear was sufficient to activate ADAM10-mediated shedding of GPVI and that this activation did not require platelet receptor engagement, intracellular signalling or release of soluble mediators (Al Tamimi et al., Blood, 2012). A critical global question, however, is how ADAM10 activity towards vascular substrates can be so rapidly upregulated in a high shear environment? To directly examine shear-induced activation of a vascular metalloproteinase, we developed the first sensor capable of visualising ADAM10 activity on a forming thrombus, consisting of a GPVI sequence-based ADAM10-sensitive fluorescent peptide with an ADAM10-releasable quencher (GPVI-Cy3). Rapid recombinant (r) ADAM10 (Km = 24.3 μM, kcat = 0.27 s-1) but not rADAM17 cleavage of GPVI-Cy3 permitted direct ADAM10 monitoring on platelets. First, suspensions of human washed platelets were exposed to variable rates of uniform shear in a cone-plate viscometer, or were treated with 10 μg/ml collagen-related peptide (CRP; a GPVI ligand), or 5 mM NEM (a potent generic activator of ADAMs) then mixed with 5-10 μM GPVI-Cy3. Real-time fluorescence was monitored in a fluorescence plate reader. Untreated platelets displayed basal (37% of maximal) levels of ADAM10 activity (2.3 pmol/min/106 platelets; 100% denoting NEM-induced levels) consistent with active ADAM10, lacking an inhibitory pro-domain by western blot and flow cytometry, being present on the non-activated platelet surface. ADAM10 activity increased to 59% of maximal activity following CRP treatment and 86% of maximal activity after exposure to 10,000 s-1 shear stress for 5 min (5.4 pmol/min/106 platelets). Cleavage of GPVI-Cy3 was completely blocked by inclusion of 100 μM GM6001 or 2 μM GI254023 (specific ADAM10 inhibitor). Second, ADAM10 activity was visualised using fluorescence multi-channel confocal imaging (Nikon A1R Plus si, Piezo z-stage and perfect focus system) of thrombi formed by perfusion of collagen-coated capillaries with hirudinated whole blood (input wall shear rate = 1,800 s-1). The channel profile was reconstructed digitally, and a Computational Fluid Dynamics package (OPEN-Foam) accurately simulated the shear forces acting on the flow throughout the domain as a function of time. Importantly, this technique permits shear rates to be precisely determined at the thrombi surface, and provides time-varying data on shear rates experienced by platelets traversing the domain in vitro. Highest ADAM10 activity was observed 5-10 minutes post thrombus formation and colocalized with areas of high (〉6000 s-1) shear on the surface of the thrombus. When GPVI-Cy3 was included for the duration of thrombus formation, serial z-stack thrombus cross section images revealed areas of high and low ADAM10 activity within the thrombus core, consistent with elevated ADAM10 activity on platelets involved in the initial stages of thrombus formation. Together, our findings (i) define a novel ADAM10-selective substrate GPVI-Cy3 which reports on ADAM10 activity in vitro and under hydrodynamic flow ex vivo, (ii) enable simultaneous quantitation of ADAM10 activity and local shear rates, and (iii) demonstrate correlation between ADAM10 activity and regions of elevated shear stress for the first time. Future studies will investigate how shear stress under pulsatile or continuous flow activates ADAM10 on vascular cell membranes, ultimately enabling the design of therapeutic agents that discretely target shear-mediated up-regulation of ADAMs activity. Disclosures: No relevant conflicts of interest to declare.

Description: Collagen binding to glycoprotein VI (GPVI) induces signals critical for platelet activation in thrombosis. Both ligand-induced GPVI signaling through its coassociated Fc-receptor γ-chain (FcRγ) immunoreceptor tyrosine-activation motif (ITAM) and the calmodulin inhibitor, W7, dissociate calmodulin from GPVI and induce metalloproteinase-mediated GPVI ectodomain shedding. We investigated whether signaling by another ITAM-bearing receptor on platelets, FcγRIIa, also down-regulates GPVI expression. Agonists that signal through FcγRIIa, the mAbs VM58 or 14A2, potently induced GPVI shedding, inhibitable by the metalloproteinase inhibitor, GM6001. Unexpectedly, FcγRIIa also underwent rapid proteolysis in platelets treated with agonists for FcγRIIa (VM58/14A2) or GPVI/FcRγ (the snake toxin, convulxin), generating an approximate 30-kDa fragment. Immunoprecipitation/pull-down experiments showed that FcγRIIa also bound calmodulin and W7 induced FcγRIIa cleavage. However, unlike GPVI, the approximate 30-kDa FcγRIIa fragment remained platelet associated, and proteolysis was unaffected by GM6001 but was inhibited by a membrane-permeable calpain inhibitor, E64d; consistent with this, μ-calpain cleaved an FcγRIIa tail-fusion protein at 222Lys/223Ala and 230Gly/231Arg, upstream of the ITAM domain. These findings suggest simultaneous activation of distinct extracellular (metalloproteinase-mediated) and intracellular (calpain-mediated) proteolytic pathways irreversibly inactivating platelet GPVI/FcRγ and FcγRIIa, respectively. Activation of both pathways was observed with immunoglobulin from patients with heparin-induced thrombocytopenia (HIT), suggesting novel mechanisms for platelet dysfunction by FcγRIIa after immunologic insult.