5'-p-Fluorosulfonylbenzoyl adenosine (FSBA), a nucleotide analog of ADP, has been
shown to inhibit ADP-induced shape change, aggregation and exposure of fibrinogen
binding sites concomitant with covalent modification of a single surface membrane
polypeptide of Mr 100,000 (aggregin). Since thrombin can aggregate platelets which
have been modified by FSBA and are refractory to ADP, we tested the hypothesis that
thrombin-induced platelet aggregation might involve cleavage of aggregin. At a low
concentration of thrombin (0.05 U/ml), platelet aggregation, exposure of fibrinogen
receptors and cleavage of aggregin in FSBA-modified platelets did not occur, indicating
ADP dependence. In contrast, incubation of [3H]FSBA-labeled intact platelets with
a higher concentration of thrombin (0.2 U/ml) resulted in cleavage of radiolabeled
aggregin, aggregation, and exposure of fibrinogen binding sites. Under identical conditions,
aggregin in membranes isolated from [3H]FSBA-labeled platelets was not cleaved by
thrombin. Thrombin-induced platelet aggregation and cleavage of aggregin were concomitantly
inhibited by a mixture of 2-deoxy-D-glucose, D-gluconic acid 1,5-lactone, and antimycin
A. These results suggest that thrombin cleaves aggregin indirectly by activating an
endogeneous protease. Thrombin is known to elevate intracellular Ca2+ concentration
and thereby activates intracellular calcium dependent thiol proteases (calpains).
In contrast to serine protease inhibitors, calpain inhibitors including leupeptin,
antipain, and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (chelator
of Ca2+) inhibited platelet aggregation and cleavage of aggregin in [3H]FSBA-labeled
platelets. Leupeptin, at a concentration of 10-20 microM, used in these experiments,
did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape
change, or the rise in intracellular Ca2+. Purified platelet calpain II caused aggregation
of unmodified and FSBA-modified platelets and cleaved aggregin in [3H]FSBA-labeled
platelets as well as in isolated membranes. The latter is in marked contrast to the
action of thrombin on [3H]FSBA-labeled membranes. Thus, thrombin-induced platelet
aggregation may involve intracellular activation of calpain which proteolytically
cleaves aggregin thus unmasking latent fibrinogen receptors, a necessary prerequisite
for platelet aggregation.