CROVIDISIN, A COLLAGEN-BINDING PROTEIN ISOLATED FROM SNAKE-VENOM OF CROTALUS VIRIDIS, PREVENTS PLATELET-COLLAGEN INTERACTION

By means of liquid chromatography consisting of gel filtration, anioni c exchange, and C8 reverse-phase HPLC, a selective inhibitor of collag en-induced platelet aggregation, named crovidisin, has been purified t o homogeneity from the venom of Crotalus viridis snake. Crovidisin is a single-chain, 53-kDa protein with a selective inhibitory activity on collagen-induced aggregation of human washed platelets without affect ing those elicited by thrombin, sodium arachidonate, and ADP. Partial sequencing of tryptic digests of crovidisin reveals that partial seque nce of crovidisin appears to be identical to that of catrocollastatin, a collagen antagonist occurring in the venom of Crotalus atrox snake. Crovidisin dose-dependently blocked aggregation of human washed plate lets triggered by 5 and 10 mu g/ml of collagen with IC50 of 0.17 and 0 .47 mu M, respectively. Not only platelet aggregation but also release reaction, thromboxane formation, and increase of intracellular Ca2+ l evel of platelets in response to collagen were all completely abolishe d by crovidisin. In the presence of crovidisin, the Mg2+-dependent adh esion of platelets to collagen was diminished in a dose-dependent mann er, while the glycoprotein IIb/IIIa-mediated platelet-fibrinogen inter action was unaffected. When collagen was pretreated with crovidisin an d followed by three washes with phosphate-buffered saline, the antiadh esion activity of crovidisin was unaffected. In addition, collagen fib ers emitted fluorescence after incubation with fluorescein isothiocyan ate conjugated crovidisin, indicating that crovidisin binds directly t o collagen fibers. In conclusion, crovidisin blocks the interaction be tween platelets and collagen fibers through its binding to collagen fi bers, resulting in the blockade of collagen-mediated platelet function s such as adhesion, release reaction, thromboxane formation, and aggre gation. (C) 1997 Academic Press