FIBRIN-STIMULATED RELEASE OF VON WILLEBRAND FACTOR FROM ENDOTHELIAL CELLS IS LINKED TO FIBRINOPEPTIDE B CLEAVAGE

von Willebrand factor (vWf) is synthesized in endothelial cells and stored in specialized organelles, the Weibel-Palade bodies. We have examined the role of fibrin as a potential physiological secretagogue of vWf from Weibel-Palade bodies using indirect immunofluorescence staining of endothelial cells to detect release. Addition of fibrinogen to endothelial cell cultures resulted in the formation of a clot, and this was temporally associated with vWf release. Addition to endothelial cells of preformed fibrin prepared by clotting fibrinogen with thrombin also stimulated release of vWf within 10 minutes. Hirudin inhibition or heat denaturation of clot-bound thrombin abolished most of the thrombin activity but did not diminish release. The role of fibrinopeptide A and B (FPA, FPB) cleavage in stimulating release was examined using reptilase or the venom from A. contortrix to selectively remove FPA or FPB. Release was stimulated by fibrin from which FPB had been cleaved by either thrombin or A. contortrix, while desAA fibrin prepared with Reptilase was an ineffective stimulus. The formation of a stimulatory fibrin clot with the contortrix enzyme, which does not cause release by itself, demonstrates that fibrin stimulation was completely independent of thrombin activity. The capacity to stimulate release was found to be independent of factor XIIIa crosslinking with both crosslinked and noncrosslinked FPB cleaved fibrins demonstrating stimulation. We conclude that fibrin stimulates rapid release of vWf from endothelial cells independent of thrombin activity and may function as a physiologic secretagogue. Furthermore, the stimulating capacity is dependant on cleavage of FPB suggesting that release is mediated by an active site near the N-terminal of the 3 chain or is dependent on a fibrin structure resulting from FPB cleavage.