Abstract
Abstract 259
Platelet adhesion, activation, and aggregation in the vasculature are necessary events in both life-saving hemostasis and pathological thrombosis. Thrombosis may occur in patients presenting with several clinical conditions including atherosclerosis, cardiovascular disease, and inflammation. Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a critical role in mediating platelet adhesion, activation, and aggregation on the exposed subendothelium in order to maintain hemostasis under arterial flow conditions. On the other hand, VWF permits the stabilization of platelets adherent to components of ruptured atherosclerotic plaques, leading to artery-occluding thrombus formation. The initial interaction of activated or hyperadhesive VWF with platelets occurs via the interaction between the A1 domain of VWF and the platelet receptor glycoprotein (GP)Ibα. This engagement is responsible for reducing the velocity of rapidly flowing platelets, allowing the rolling platelets to interact with the second binding site on VWF for the platelet receptor GPIIb/IIIa; a binding site that is located within the C domains of VWF. Therefore, the hyperadhesive property of VWF apparently relies on the synchronized interaction of the two platelet surface receptors, GPIbα and GPIIb/IIIa. Despite this concept, we and others have speculated that other binding site in VWF synergistically works with the A1 domain to quickly capture the extremely fast flowing platelets. We have obtained interesting results from studies using a monomeric A1A2A3 domain protein that lacks the binding site for GPIIb/IIIa. For example, the rolling velocity of platelets over an A1A2A3-coated surface was markedly lower than that seen with use of the single A1 domain. This observation suggests the possibility of an additional binding site in the A domains for platelets. Given the similar hyperadhesive features of the A1A2A3 protein and plasma VWF, we proposed to look for a potential receptor on platelets with a recognition site within the A domains of VWF. We suggested examining vimentin because, it was identified as a binding protein for the isolated A2 domain of VWF in our laboratory, and vimentin has been found on the surface of platelets. First, both full length VWF and recombinant A1A2A3 proteins efficiently bound to human vimentin only in the presence of the modulator ristocetin, indicating that vimentin preferably interact with the active conformation of VWF. In fact, a constitutively active A1A2A3 protein (containing a gain-of-function mutation in A1 domain) had a binding activity for vimentin higher than that of wild type (WT) A1A2A3 in the absence of ristocetin. Second, anti-vimentin monoclonal antibody blocked the interaction of that mutant A1A2A3 to activated washed platelets using flow cytometry. Third, we then examined the effect of anti-vimentin antibody on flow-dependent platelet adhesion to A1A2A3-coated surface at high shear stress. In comparison to whole blood incubated with irrelevant IgG molecule as a negative control, the anti-vimentin antibody blocked 75% platelet adhesion to the triple-A domain protein. Finally, whole blood from vimentin-deficient or WT mice was perfused over a surface coated with murine VWF at high shear rate. In comparison to platelets from WT mice, vimentin-deficient platelets had a significant reduced platelet adhesion to VWF (25% of WT). Similarly, vimentin-deficient platelets had a reduced platelet adhesion to collagen (20% of WT) under high flow conditions. This platelet-collagen interaction is initially mediated by VWF. These interesting results indicate that vimentin on platelets serves as a receptor for VWF, and this binding may participate in the initial interaction of circulating platelets with VWF under flow conditions.
Disclosures:
No relevant conflicts of interest to declare.
Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF
