Plasma fibronectin has recently been demonstrated to stabilize growth of platelet-rich thrombi in injured mesenteric arterioles in vivo. We have determined the impact of two forms of insolubilized plasma fibronectin on adhesion and aggregation of platelets - fibronectin-crosslinked fibrin by activated factor XIII (fibronectin-fibrin) and fibronectin assembled by adherent platelets. In static adhesion assays, 2-fold more platelets adhered to fibronectin-fibrin than to fibrin. Antibody blocking studies demonstrated that a different mix of αIIbβ3, αvβ3, and α5β1 integrins mediate platelet adhesion to both substrates. At a shear rate of 300 or 1,250s-1 in a parallel plate flow chamber, more platelets adhered and aggregated on surfaces coated with fibronectin-fibrin than surfaces coated with fibrin. The number of adherent platelets and the volume of platelet thrombi increased by 3- to 4-fold when soluble fibronectin was co-perfused with platelets, which resulted in assembly of fibronectin by adherent platelets. Increased adhesion and aggregation of platelets were dependent on the amounts of fibronectin crosslinked to fibrin prior to perfusion or included in the perfusate. The effects of perfused fibronectin were blocked by two inhibitors of fibronectin assembly, the N-terminal 70-kD fragment of fibronectin or the functional upstream domain of protein F1 of Streptococcus pyogenes, indicating that assembly of soluble fibronectin by adherent platelets is mediated by the binding of the N-terminal region of fibronectin to the platelet surface. Thus, plasma fibronectin has the potential to enhance adhesion and aggregation of platelets in vivo as a result of being crosslinked to fibrin networks and its subsequent assembly by adherent platelets.