Convulxin binding to platelet receptor GPVI: Competition with collagen related peptides

Convulxin (CVX), a potent platelet aggregating protein from the venom of th e snake Crotalus durissus terrificus, is known to bind to the platelet coll agen receptor, glycoprotein VI (GPVI). CVX binding to human platelets was i nvestigated by flow cytometry, using fluorescein labeled convulxin (FITC-CV X). Scatchard analysis indicated high and low affinity binding sites with K d values of 0.6 and 4 nM and Bmax values of 1200 and 2000 binding sites per platelet. FITC-CVX binding was inhibited by collagen related peptides (CRP s) comprising a repeated GPO sequence, namely GCO(GPO)(10)GCOGNH(2) and GKO (GPO)(10)GKOGNH(2), which also bind to receptor GPVI. These peptides (monom eric or cross-linked forms) gave a high affinity inhibition of 10-20% for c oncentrations between 10 ng/ml and 5 mu g/ml, followed by a second phase of inhibition at concentrations greater than 5 mu g/ml. It was shown also tha t the inhibition of FITC-CVX binding by Clips was independent on the time o f preincubation of platelets with CRPs, and the same percentage of inhibiti on was seen with various concentrations of convulxin. Confocal microscopy o f the distribution of FITC-CVX binding sites on platelets showed an homogen eous distribution of FITC-CVX bound to GPVI, although some limited clusteri ng may exist. (C) 2000 Academic Press.