PROTHROMBIN CONVERSION TO THROMBIN BY PLASMINOGEN-ACTIVATOR RESIDING IN THE SUBENDOTHELIAL EXTRACELLULAR-MATRIX

Prothrombin, a blood glycoprotein with two kringle domains, is the zymogen for the blood-clotting enzyme thrombin. Incubation of prothrombin with sulfate-labeled ECM synthesized by cultured bovine endothelial cells, resulted in generation of enzyme activity as evident from the release of nearly intact labeled ECM proteoglycans. Generation of proteolytic activity by the ECM was time- and dose-dependent and was completely abolished by preheating the ECM. Both t-PA and u-PA, as well as PAI-1, were previously shown to reside in the subendothelial ECM. When exogenously added t-PA was incubated with prothrombin followed by incubation with labeled preheated ECM, a marked release of high molecular mass labeled degradation products was obtained, compared with heated ECM treated with t-PA or prothrombin alone. Moreover, addition of anti-t-PA and anti-u-PA antibodies to native ECM, inhibited its ability to convert prothrombin to thrombin. When the prothrombin-ECM cleavage products were analyzed by immunoblotting using anti-prothrombin/thrombin antibodies, a 35 kDa protein representing thrombin B-chain was detected. These results indicate that the circulating zymogen prothrombin may be converted to thrombin through interaction with the subendothelial ECM. Furthermore, this conversion is most likely mediated via t-PA and u-PA, deposited by endothelial cells and firmly sequestered in an active form by the underlying ECM. Once thrombin is generated, it may bind to the ECM, being protected from inhibition and functionally active. The bound enzyme may participate locally in cellular invasion as well as in cell attachment and growth.