Characterization of the Unique Mechanism Mediating the Shear-dependent Binding of Soluble von Willebrand Factor to Platelets

Abstract

We have studied the mechanism of interaction between soluble von Willebrand factor (vWF), labeled with fluorescein isothiocyanate (FITC), and platelets exposed to shear in a cone-and-plate viscometer. A flow cytometer calibrated with fluorescent bead standards was used to calculate the number of molecules associated with each platelet in suspension. To validate the methods and reagents used, binding of the same labeled vWF was assessed in the presence of ristocetin or ␣-thrombin and found to be saturable, with a narrow and symmetric distribution on >90% of the platelets. As expected, essentially all bound ligand interacted exclusively with platelet membrane glycoprotein (GP) Ib␣ in the presence of ristocetin and with GP IIb-IIIa after stimulation with ␣-thrombin. In contrast, only a minor proportion (<20%) of the platelets exposed to shear were found to bind vWF, with no evidence for saturation and markedly decreased interaction when the platelet count was below 100,000 l. Moreover, shear-induced vWF binding was blocked equally effectively by selected monoclonal antibodies against either GP Ib␣ or GP IIb-IIIa or against the respective binding sites in vWF. Thus, both receptors are involved in the process, possibly through initial transient interactions mediated by GP Ib␣ that lead to platelet activation and subsequent irreversible binding supported by GP IIb-IIIa. While the levels of shear stress theoretically applied to platelets in these experiments are above those thought to occur in the normal circulation, our findings demonstrate a unique vWF binding mechanism that is not mimicked by other known modulators and correlates with platelet aggregation. Similar processes may occur in response to lower shear stress when platelets are exposed to thrombogenic surfaces and agonists generated at sites of vascular injury during thrombus formation. The role of von Willebrand factor (vWF)1 in platelet thrombus formation, particularly under conditions of high shear, is well established and supported by experimental evidence as well as clinical observations (1). It is known that platelets have two distinct binding sites for vWF, glycoprotein (GP) Ib␣ in the GP Ib-IX-V complex and the integrin ␣ IIb ␤ 3 (GP IIb-IIIa complex) (2). Binding of soluble normal vWF to GP Ib␣ in the absence of flow requires the presence of exogenous modulators, like ristocetin (3) or botrocetin (4), while interaction with GP IIb-IIIa can only occur after platelet activation (5). In addition to vWF, the GP IIb-IIIa receptor can also bind fibrinogen, fibronectin (6), and vitronectin (7), but the respective role of these proteins in thrombogenesis is still a topic for investigation.Exposure of platelets to levels of shear higher than thought to occur in the normal circulation results in aggregation if soluble vWF is present (8); no other adhesive ligand can support this process (9). Shear-induced platelet aggregation may represent an important pathophysiological function of vWF, but the underlying mechanism is still poorly understood. It has been pro...

Current issues in diagnosis and treatment of von Willebrand disease

“…The multimeric form of VWF is disrupted in types 2A and 2B of VWD (Figures 2A and B), decreasing the function of VWF. Normally, upon endothelial injury VWF's A1 and A3 domains bind to collagen, and the resultant shear stress changes the conformation of VWF to allow VWF binding to platelet GPIbα 8. In this way VWF acts as a bridge to bind platelets to exposed collagen on injured endothelium, acting as a first responder in coagulation.…”

The effects of shear flow on protein structure and function

“…109 Interestingly, it has been shown that molecular confinement enhances the deformation of entangled polymers under squeeze flow, 110 a condition which models pulsatile blood flow. Similarly, there are a considerably number of reports 27,34,35,42,59,60,[111][112][113] citing haemodynamic shear stress as the primary facilitator of the proteolytic cleavage of large vWF multimers freshly released from endothelial cells as well as vWF-mediated platelet adhesion during haemostasis. The accumulation of large vWF multimers in the plasma may lead to disease states including thrombotic thrombocytopenic purpura, microvascular occlusion and atherosclerosis, due to the development of microvascular thrombi resulting from the complexation of the hyperactive vWF multimers and platelets at regions of low physiological shear.…”

AJvW-2, an Anti-vWF Monoclonal Antibody, Inhibits Enhanced Platelet Aggregation Induced by High Shear Stress in Platelet-Rich Plasma From Patients With Acute Coronary Syndromes

“…25 In contrast, it has been shown that high shear-induced aggregation of washed platelets increases as a function of the concentration of exogenous purified vWF. 26 In addition, because wall shear rates of 3000 to 10 000 s Ϫ1 have been measured at the top of plaques, causing a 50% occlusion of the coronary arteries, 27 vWFGPIb binding may lead to arterial occlusion in a patient with atherosclerosis and precipitate a disease such as an ACS. It should therefore be possible to examine the association between the plasma vWF concentration and the vWF-GPIb interaction by blocking vWF binding to GPIb, where flow generates higher shear rates, to prevent ACSs.…”