Soluble plasma fibronectin (Fn) with its inactive compact structure requires unfolding to assemble into active fibrils, which play a role in hemostasis and thrombosis. Fn fibril assembly involves Fn binding to cell receptors, biomechanical coupling of Fn to the cytoskeleton by integrins, exposure of self-assembly sites via contractile cell forces, and elongation of fibrils by Fn polymerization. In this report, we investigated the effect of platelet integrins and actin cytoskeleton on conformational changes of Fn induced by shear. Plasma Fn, in the presence or absence of washed platelets, was exposed to dynamic shear simulating venous or arterial flow conditions. Platelet integrins (alpha IIb beta 3, alpha v beta 3, and alpha 5 beta 1) were blocked by inhibitory antibodies to determine their contribution to shear-induced Fn fibrillogenesis. To examine the role of platelet cytoskeleton in Fn fibrillogenesis induced by shear, platelets were preincubated with cytoskeleton drugs, i. e jasplakinolide to stabilize actin or cytochalasin D to inhibit actin polymerization. Microscopic analyses demonstrated that flow and resulting shear stress over a broad range of physiological and pathological rates (50-5000 s(-1)) could induce conformational changes of plasma Fn. In addition, the formation of Fn fibrils is modulated by platelet integrins. In this respect, beta 3 integrins play a dominant role in terms of Fn fibrillogenesis induced by shear. Disruption of the actin polymerization markedly diminished Fn unfolding and assembly. These observations lead to the conclusion that Fn-integrin beta 3-cytoskeleton interaction is crucial for the assembly of plasma Fn matrix under flow conditions. (C) 2018 Elsevier Inc. All rights reserved.