Transient interactions of platelet-receptor glycoprotein Ibalpha (GpIbalpha) and the plasma protein von Willebrand factor (VWF) reduce platelet velocity at sites of vascular damage and play a role in haemostasis and thrombosis. Here we present structures of the GpIbalpha amino-terminal domain and its complex with the VWF domain A1. In the complex, GpIbalpha wraps around one side of A1, providing two contact areas bridged by an area of solvated charge interaction. The structures explain the effects of gain-of-function mutations related to bleeding disorders and provide a model for shear-induced activation. These detailed insights into the initial interactions in platelet adhesion are relevant to the development of antithrombotic drugs.

Rhamnogalacturonan (RG), known as the hairy portion of the pectin network of the primary plant cell wall, is composed of repeating dimeric units of-(1-2)

Recent studies have revealed that the platelet adhesive process under flow is tightly regulated by multiple ligand-receptor interactions. However, platelet morphological changes during this process, particularly its physiological relevance, remain unknown under blood flow conditions. Using epifluorescence and scanning electron microscopy, we evaluated the real-time changes in platelet morphology during a platelet adhesive process on a von Willebrand factor-coated surface under physiological high shear flow in a perfusion chamber. Here, we show that dynamic platelet shape changes occurring during distinct phases of the adhesive process are precisely regulated by "inside-out" and "outside-in" integrin signals and are also a key regulatory element in successful platelet thrombogenesis opposing rapid blood flow in vivo.

We evaluated real-time processes of platelet thrombus formation on a collagen surface in a flow chamber with whole blood from patients with various platelet aggregation disorders, such as Bernard-Soulier syndrome (BSS), Glanzmann’s thrombasthenia (GTA), type 3 von Willebrand disease (vWD), and congenital afibrinogenemia (Af), who lack platelet glycoprotein (GP) Ib-IX complex, GP IIb-IIIa, von Willebrand factor (vWF), and fibrinogen, respectively. Blood from GTA patients showed impaired thrombus growth but significant initial platelet-surface interaction under all shear conditions tested (50 to 1,500 s−1). By contrast, blood from patients with BSS or type 3 vWD showed no platelet-surface interaction under high shear (≥1,210 s−1) but normal thrombus formation under low shear (≤340 s−1). When shear rate was increased stepwise to 1,500 s−1 during perfusion, the thrombus growth observed in type 3 vWD or BSS under low shear was arrested, whereas that in control blood was sharply accelerated as a function of shear rate. Overall thrombus formation in Af appeared indistinguishable from that of a control under shear rates between 50 and 1,500 s−1. However, Af thrombi formed under such conditions collapsed immediately when shear rate was further increased to 4,500 s−1, whereas thrombi of type 3 vWD or BSS formed under low shear were stable even when shear rate was elevated to 9,000 s−1 during perfusion. These findings suggest that distinct molecular mechanisms underlie the pathologic bleeding in these diseases and point to the distinct roles of two major adhesive proteins, vWF and fibrinogen. In mural thrombus formation under flow conditions, vWF, perhaps mainly through its interaction with GP Ib-IX, acts as an “initiator and promoter,” whereas fibrinogen, via its binding to GP IIb-IIIa, acts as a “stabilizer” against heightened shear forces that could lead to peeling off of platelets from the surface.

Using a perfusion chamber and confocal laser scanning microscopy, we analyzed the interplay of von Willebrand factor (VWF) and fibrinogen during thrombus growth on a collagen surface under physiologic high shear rate conditions. During initial thrombogenesis, platelet thrombi were constructed totally by VWF, not by fibrinogen. Fibrinogen accumulated predominantly inside the growing thrombi as a function of time, whereas the thrombus surfaces directly exposed to flow were occupied constantly by VWF throughout the observation period. In perfusion of afibrinogenemia (AF) blood lacking both plasma and platelet fibrinogen, the final height and volume of thrombi were significantly reduced compared with controls, albeit the area of surface coverage was normal. The impaired thrombus growth in AF was only partially corrected by the addition of purified fibrinogen to AF blood, whereas the addition of purified VWF to blood of severe von Willebrand disease (VWD) completely normalized the defective thrombus growth in this disease.Thus, the initial 2-dimensional thrombus expansion involves only VWF, whereas the time-dependent accumulation of fibrinogen, released from activated platelets, acts as a core adhesive ligand, increasing thrombus strength and height and resulting in 3-dimensional thrombus development against rapid blood flow. (Blood. 2002;100:3604-3610)

To explore the mechanisms that underlie the bleeding tendency in type 2A and 2B von Willebrand disease (VWD), we analyzed the mural thrombus generation process on a collagen surface under physiologic blood flow in a perfusion chamber using whole blood from these VWD patients. At a low shear rate (50 s ؊1 ), thrombus generation in all type 2A and 2B VWD patients was comparable to that of healthy controls. At a high shear rate (1500 s ؊1 ), thrombus generation was impaired in all type 2A patients, whereas that in type 2B VWD patients varied from normal to significantly defective, as judged by epifluorescence microscopy of thrombus surface coverage. However, in type 2B patients who showed normal thrombus generation at 1500 s ؊1 , the height and volume of thrombi was significantly reduced, albeit with the normal surface coverage, compared with control thrombi, and von Willebrand factor (VWF) was poorly distributed within the type 2B thrombus mass when analyzed in detail by confocal laser scanning microscopy. Addition of purified VWF to patient blood completely reversed the defective spatial thrombus growth in type 2B VWD. Thus, our results confirm the impaired thrombus generation in type 2B VWD, which has never been demonstrable in previous in vitro soluble-phase platelet aggregation assays, and point to the critical function of larger VWF multimers in the proper spatial growth of mural thrombi under high shear rate conditions. (Blood. 2003; 101:915-920)

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Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca2+]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca2+ indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca2+]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca2+]i) to green (high [Ca2+]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s−1, no change in [Ca2+]i was observed during translocation of platelets, but [Ca2+]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca2+]i elevation. The intracellular Ca2+chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca2+]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca2+]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.