Shear-dependent tether formation during platelet translocation on von Willebrand factor

Abstract

The adhesion and aggregation of platelets at sites of vascular injury is dependent on the initial binding of the GP Ib/V/IX receptor complex to immobilized von Willebrand factor (VWF). Under flow conditions, this interaction supports platelet translocation that is characteristically stop-start in nature. High resolution imaging of platelets during surface translocation on immobilized VWF revealed that thin membrane tethers (length: 0.91 μm-47.90 μm) were pulled from the surface of these cells. Membrane tethers were dynamic structures that extended from small, localized adhesion contacts under the influence of flow. Perfusion of platelets in the presence of blocking antibodies against integrin αIIbβ3, or over isolated A1 domains, demonstrated that the VWF–GP Ib interaction was sufficient to induce membrane tether formation. The rate and extent of tether elongation was shear-dependent (shear range: 150 s−1-10 000 s−1), with mean tether length ranging from 3.23 μm to 16.55 μm, tether frequency from 2.67% to 97.33%, and tether growth rate from 0.04 μm/sec to 8.39 μm/sec. Tether formation and retraction did not require platelet activation; however, the growth rate, lifetime, and dimensions were significantly affected by the actin polymerization inhibitor, cytochalasin D, and by chelating intracellular calcium. Single-cell analysis revealed that formation of membrane tethers regulates the stop-start phases of platelet translocation on VWF, suggesting a potentially important role for this phenomenon in regulating the dynamics of the platelet-VWF interaction under flow.