Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain

• Published in: Archives of biochemistry and biophysics Vol. 271; no. 2; pp. 346 - 358
• Main Authors: Puri, Rajinder N., Zhou, Fengxin, Bradford, Harlan, Hu, Chang-Jun, Colman, Roberta F., Colman, Robert W.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.06.1989; Elsevier
• Subjects: Adenosine - analogs & derivatives; Adenosine - pharmacology; Adenosine Diphosphate - pharmacology; Affinity Labels; Antipain - pharmacology; Biological and medical sciences; Blood coagulation. Blood cells; Blood Platelets - drug effects; Blood Platelets - metabolism; Calpain - antagonists & inhibitors; Calpain - blood; Calpain - pharmacology; Electrophoresis, Polyacrylamide Gel; Fibrinogen - metabolism; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods, hemostasis, fibrinolysis; Humans; Leupeptins - pharmacology; Molecular and cellular biology; Platelet Aggregation - drug effects; Platelet Aggregation Inhibitors - pharmacology; Platelet Membrane Glycoproteins - blood; Protease Inhibitors - pharmacology; Thrombin - pharmacology; Aggregation; Human; Blood coagulation; Platelet; Calcium; Radiolabelling; Gel electrophoresis; Enzyme; Coagulation factor; Biological receptor
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/0003-9861(89)90284-1

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 M r 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 Ca 2+ 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(β-aminoethyl ether) N,N′-tetraacetic acid (chelator of Ca 2+) inhibited platelet aggregation and cleavage of aggregin in [ 3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10–20 μ m, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca 2+. 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.