Shear-induced platelet aggregation (SIPA) involves the sequential interaction of von Willebrand factor (VWF) with both glycoprotein Ib (GPIb) and ␣IIb3 receptors. Type 2B recombinant VWF (2B-rVWF), characterized by an increased affinity for GPIb, induces strong SIPA at a high shear rate (4000 s ؊1 ). Despite the increased affinity of 2B-rVWF for GPIb, patients with type 2B von Willebrand disease have a paradoxical bleeding disorder, which is not well understood. The purpose of this study was to determine if SIPA induced by 2B-rVWF was associated with ␣IIb3-dependent platelet activation. To this end, we have addressed the influence of 2B-rVWF (Val553Met substitution) on SIPAdependent variations of tyrosine protein phosphorylation (P-Tyr) and the effect of ␣IIb3 blockers. At a high shear rate, 2B-rVWF induced a strong SIPA, as shown by a 92.7% ؎ 0.4% disappearance of single platelets (DSP) after 4.5 minutes. In these conditions, increased P-Tyr of proteins migrating at positions 64 kd, 72 kd, and 125 kd were observed. The band at 125 kd was identified as pp125FAK using anti-phospho-FAK antibody. This effect, which required a high level of SIPA (> 70% DSP), was observed at 4000 s ؊1 but not at 200 s ؊1 . Monoclonal antibodies (MoAbs) 6D1 (anti-GPIb) and 328 (anti-VWF A1 domain), completely abolished SIPA and p125FAK phosphorylation mediated by 2B-rVWF. In contrast, neither RGDS peptide nor MoAb 7E3, both known to block ␣IIb3 engagement, had any effect on SIPA and pp125FAK. The size of aggregates formed at a high shear rate in the presence of 2B-rVWF was decreased by genistein, demonstrating the biologic relevance of pp125FAK. These findings provide a unique mechanism whereby the enhanced interaction of 2B-rVWF with GPIb, without engagement of ␣IIb3, is sufficient to induce SIPA but does not lead to stable thrombus formation.
IntroductionPlatelets play a critical role in the arrest of bleeding by adhering to vascular matrix proteins and to other activated platelets at sites of vessel wall injury. High shear rates generate forces between platelets and the damaged vessel wall that induce specific ligandreceptor interactions required for platelet adhesion, activation, and aggregation. A key adhesive protein initiating platelet-vessel wall interactions is the large multimeric protein, von Willebrand factor (VWF). When immobilized at the site of injury, VWF exhibits its A1 domain, allowing the capture of platelets from rapidly flowing blood by an interaction with the glycoprotein Ib/V/IX (GPIb/V/IX) complex. This binding generates signals leading to ␣IIb3 integrin activation which becomes competent to interact with VWF and to promote irreversible platelet adhesion. [1][2][3][4] In vitro, the change in VWF can be mimicked by the binding of VWF to its modulators ristocetin or botrocetin. 5,6 High shear rates are important not only in the initial contact of platelets with the vessel wall, but also in the process of platelet aggregation, where fluid-phase VWF is required to stabilize platelet-platelet interactions and...