Coronary artery disease is the leading cause of morbidity and mortality the United States and Western Europe. Thrombin plays a critical role in the pathogenesis of coronary artery disease at many levels. Although it is perhaps best recognized as the serine protease responsible for the cleavage of fibrinogen to fibrin of fibrinogen to fibrin during blood clotting, thrombin also functions for a diverse number of cellular activities. It is the most potent physiological stimulus for platelet aggregation known and there are recent data to suggest that thrombin is a pivotal mediator of arterial thrombosis in vivo. Also well-documented are thrombin's mitogenicity in vascular smooth muscle cells, fibroblasts and splenocytes and chemotactic properties for monocytes. It therefore appears that in addition to its critical role in thrombosis, thrombin may play an important role in regulating the inflammatory and repair processes that accompany arterial wall injury. The long term goal of this project is to identify the molecules and molecular mechanisms that mediate thrombin activation of platelets and other cells. Attainment of this goal may facilitate the rational design of drugs capable of specifically antagonizing pathological thrombosis and cellular activation by thrombin.
The specific aims of the work proposed herein are 1) to determine the importance of protease activity and specific thrombin structural determinants in cell and platelet activation, and other thrombin-specific functions, and 2) to obtain and characterize a cDNA clone for the thrombin receptor. The importance of thrombin's protease activity in signal transduction will be investigated using oligonucleotide-directed mutagenesis to create recombinant thrombin mutants that are catalytically inert but retain receptor binding ability. The structural domains that mediate thrombin signalling will be investigated using oligonucleotide-directed mutagenesis to delete or exchange those domains that differ from other serine proteases and therefore seem likely to be responsible for thrombin's unique spectrum of activity. The thrombin receptor will be isolated by the strategy of expression cloning in which microinjection of Xenopus oocytes with mRNA from thrombin-responsive cells confers thrombin responsiveness upon the oocytes. Alternative approaches to the isolation of the thrombin receptor are affinity purification of the receptor using a novel catalytically inert thrombin with unaltered binding affinity for its receptor and the cloning and expression of thrombin receptor candidates such as glycoprotein V.
| Hung, D T; Vu, T H; Nelken, N A et al. (1992) Thrombin-induced events in non-platelet cells are mediated by the unique proteolytic mechanism established for the cloned platelet thrombin receptor. J Cell Biol 116:827-32 |
