CALRETICULIN, AN ANTITHROMBOTIC AGENT WHICH BINDS TO VITAMIN-K-DEPENDENT COAGULATION-FACTORS, STIMULATES ENDOTHELIAL NITRIC-OXIDE PRODUCTION, AND LIMITS THROMBOSIS IN CANINE CORONARY-ARTERIES

Coagulation Factor IX/IXa has been shown to bind to cellular surfaces, and Factor IXa expresses its procoagulant activity by assembling into the intrinsic Factor X activating complex (Factors IXa/VIIIa/X), whic h also forms on membrane surfaces. This led us to identify cellular pr oteins which bind Factor IX/IXa; an approximate to 55-kDa polypeptide was purified to homogeneity from bovine lung extracts based on its cap acity to bind I-125-Factor IX in a dose-dependent and saturable manner . From protein sequence data of the amino terminus and internal peptid es, the approximate to 55-kDa polypeptide was identified as calreticul in, a previously identified intracellular calcium binding protein. Rec ombinant calreticulin bound vitamin K-dependent coagulation factors, I -125-Factor IX, I-125-Factor X, and I-125-prothrombin (K-d values of a pproximate to 2.7, 3.2, and 8.3 nM, respectively), via interaction wit h its C-domain, although it did not affect the coagulant properties of these proteins. I-125-Calreticulin also bound to endothelial cells in vitro (K-d approximate to 7.4 nM), and mouse infusion studies showed an initial rapid phase of clearance in which calreticulin could be loc alized on the vascular endothelium. Exposure of endothelial cells to c alreticulin led to dose-dependent, immediate, and sustained increase i n the production of nitric oxide, as measured using a porphyrinic micr osensor. In a canine electrically induced thrombosis model, intracoron ary infusion of calreticulin (n = 7) prevented occlusion of the left c ircumflex coronary artery in a dose-dependent manner compared with veh icle-treated controls (n = 5). These results indicate that calreticuli n interacts with the endothelium to stimulate release of nitric oxide and inhibit clot formation.