Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain.
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.
Puri
R N
RN
Thrombosis Research Center, Philadelphia, Pennsylvania.
Zhou
F X
FX
Bradford
H
H
Hu
C J
CJ
Colman
R F
RF
Colman
R W
RW
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000345:Affinity Labels; D000976:Antipain / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000097:blood* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000097:blood*; D011480:Protease Inhibitors / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
D000345:Affinity Labels; D007976:Leupeptins; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D011480:Protease Inhibitors; C055422:aggregin; D000976:Antipain; D000244:Adenosine Diphosphate; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D005340:Fibrinogen; D013917:Thrombin; D002154:Calpain; C032854:leupeptin; D000241:Adenosine