We have used confocal videomicroscopy in real time to delineate the adhesive interactions supporting platelet thrombus formation on biologically relevant surfaces. Type I collagen fibrils exposed to flowing blood adsorb von Willebrand factor (vWF), to which platelets become initially tethered with continuous surface translocation mediated by the membrane glycoprotein Ib alpha. This step is essential at high wall shear rates to allow subsequent irreversible adhesion and thrombus growth mediated by the integrins alpha2beta1 and alpha(IIb)beta3. On subendothelial matrix, endogenous vWF and adsorbed plasma vWF synergistically initiate platelet recruitment, and alpha2beta1 remains key along with alpha(IIb)beta3 for normal thrombus development at all but low shear rates. Thus, hemodynamic forces and substrate characteristics define the platelet adhesion pathways leading to thrombogenesis.

Using flow channel, we report that the application of a laminar shear stress induced a transient increase of tissue factor (TF) procoagulant activity in human umbilical vein endothelial cells (HUVEC), which was accompanied by a rapid and transient induction of the TF mRNA in the HUVEC. Functional analysis of the 2.2 kb TF 5 Ј promoter indicated that a GC-rich region containing three copies each of the EGR-1 and Sp1 sites was required for induction. Mutation of the Sp1 sites, but not the EGR-1 sites, attenuated the response of TF promoter to shear stress. Thus, Sp1 is a newly defined shear stress responsive element. Electrophoretic mobility shift assays showed there was no increase in binding of nuclear extracts from sheared cells to an Sp1 consensus site. In contrast, immunoblotting of these nuclear extracts with antibody against transcription factor Sp1 demonstrated that shear stress increased the phosphorylation of Sp1. We also showed that shear stress, like the phosphatase inhibitor okadaic acid, increased the transcriptional activity of Sp1. These findings suggest that the shear stress induction of TF gene expression is mediated through an increased Sp1 transcriptional activity with a concomitant hyperphosphorylation of Sp1. ( J. Clin. Invest. 1997. 99:737-744.)

Summary. Background: Collagen and von Willebrand factor (VWF) are considered essential to initiate platelet deposition at sites of vascular injury, but their respective roles remain to be elucidated. Methods: We used a model of carotid artery thrombosis induced by a ferric chloride injury to compare the time to first occlusion and occlusion rate at 25 min postinjury in mice lacking the collagen receptor, glycoprotein (GP) VI, or the ligand-binding domain of the VWF receptor, GP Iba. Results: In normal mice used as controls (n ¼ 12), a complete obstruction of blood flow developed within 8.05 ± 0.47 min (mean ± SEM), and the occlusion rate was 100%. The results were variable in 26 GP VI )/) mice. The artery never occluded in eight mice, but the time to first occlusion in the remaining 18 (8.36 ± 0.27 min) was not different from normal (P ¼ 0.556). Nonetheless, the occlusion rate was 42%, because in seven mice the occluded artery reopened and stayed patent at 25 min. In contrast, the artery never occluded in 12 mice lacking GP Iba. In ex vivo perfusion experiments, GP VI )/) platelets failed to form thrombi onto collagen type I fibrils, but formed thrombi of normal size when exposed to endothelial or fibroblast extracellular matrix. Conclusions: Absence of GP Iba function has a more profound antithrombotic effect in vivo than absence of the GP VI-dependent pathway of collagen-induced adhesion/ activation. Components of the extracellular matrix may elicit a thrombogenic response in the absence of GP VI but not GP Iba.