Effect of recombinant hirudin on cytosolic Ca2+ concentrations using different platelet stimulators.

Cytosolic Ca2+ concentration, [Ca2+]i, and aggregation behavior of hirudinized platelet suspensions were examined under stimulation with thrombin, adenosine diphosphate, platelet-activating factor, collagen and the calcium ionophore, ionomycin. A rise in [Ca2+]i, a key step in platelet activation, was only inhibited by recombinant hirudin r-hirudin using the agonist thrombin. All other stimulators including ionomycin were not affected by r-hirudin. If hirudinized blood was gel-filtered before platelet stimulation, thrombin induced the same response as citrated platelet suspensions. Aggregation studies and calcium influx using various stimulators showed good correlation: only thrombin-induced aggregation was completely inhibited by r-hirudin. These results support the hypothesis that hirudin inhibits platelet function by thrombin binding and not by direct interaction with platelets.