Previous studies have shown that thrombin binds to the extracellular endothelial matrix and remains biologically active. In the present study, the role of matrix-bound alpha-thrombin in thrombus formation was investigated by utilizing a model system of thrombogenesis. Plastic cover-slips coated with either matrix-bound alpha-thrombin or matrix-bound active site inactivated thrombin (DIP-alpha-thrombin) were positioned in parallel-plate perfusion chambers and subsequently exposed to non-anticoagulated human blood at a venous wall shear rate of 100/s. The blood was drawn directly from an antecubital vein by a roller pump placed distally to the perfusion chamber. The thrombotic deposits on the matrix, fibrin deposition and platelet thrombus volume, were morphologically evaluated. Matrix-bound alpha-thrombin enhanced the fibrin deposition and thrombus volume on matrices of non-stimulated endothelium with 91% (P < 0.001) and 94% (P < 0.05), respectively. In contrast, binding of DIP-alpha-thrombin to matrices of stimulated endothelium reduced the fibrin deposition by 33% (P < 0.05), but had no effect on the platelet thrombus volume. Translocation of thrombin molecules from upstream matrix areas to binding sites farther downstream on the matrix was indicated in experiments with matrices of stimulated endothelium, which showed enhanced fibrin deposition on downstream areas. Our findings are compatible with a prominent role for matrix-bound alpha-thrombin in thrombogenesis, and in particular on endothelial matrices without tissue factor. The role of matrix-bound alpha-thrombin on tissue factor containing matrices appears less prominent, although it is significant.