The serine-proteinase eathepsin G (CG) is a potent SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analyagonist of platelet aggregation inducing the release and surface sis. Each subunit has a modular organization typical of multiexpression of c~-granule adhesive proteins such as fihrinogen (Fg) functional adhesive proteins, and contains specific domains and thrombospondin-1 (TSP-1). Because Fg and TSP-1 are that support interactions of TSP-1 with cell surface glycopropotential substrates for the enzymatic activity of CG, we tein receptors, sulfated proteoglycans and membrane lipids, investigated the fate of these proteins during CG-induced platelet and various matrix proteins [4][5][6][7]. Regarding platelet aggregaaggregation using an immunoblot technique. Only a small tion, particular attention has been paid to the amino-terminal proportion of secreted Fg was proteolyzed by CG and platelet domain of TSP-1 which contains high-affinity binding site(s) aggregation was efficiently inhibited by anti-fibrinogen Fab for heparin, thus commonly designated as the heparin-binding fragments. In contrast, TSP-1 was extensively proteolyzed on domain (HBD) [7]. This domain can be cleaved by various aggregated platelets releasing in the milieu a fragment with M, ~ 28 000, corresponding to the amino-terminal heparinserine-proteinases, producing a fragment with Mr in the range binding domain (HBD). Several antibodies, directed against 25 000-35 000 [7,8]. A role for the HBD in platelet-to-platelet the cell-associated earboxy-terminal TSP-lf fragment interactions is currently supported by the following observa-(M, ~ 165 000) impaired the formation of stable macroaggretions: (i) some antibodies directed at epitopes within the HBD gates, indicating that this fragment may contribute to platelet as well as recombinant HBD polypeptides are potent inhibiaggregation in the absence of the HBD.tots of ct-thrombin-induced platelet aggregation, and can specifically inhibit the interaction of purified TSP-1 with ad-

SummaryAlthough plasmin can trigger strong platelet responses such as shape change and exocytosis of internal granules, limited platelet aggregation is induced by this proteinase, owing to its capacity to rapidly proteolyse secreted adhesive proteins. In this context, we have investigated the state of activation of the fibrinogen receptor, the integrin αIIbβ3, on platelets exposed to plasmin. Following incubation with plasmin at 37 °C, washing, and resuspension, platelets exhibit a moderate, low-velocity aggregation when stirred in the presence of fibrinogen. Optimum aggregability is observed when platelets have been exposed to plasmin activity of ≈0.5 CU/ml for 20 min, and aggregation is insensitive to the presence of antagonists such as prostaglandin (PG) E1 and apyrase. Plasmin-induced platelet aggregability is associated with the expression of active fibrinogen receptors on the cell surface, which, using a l25I-fibrinogen binding assay, can be quantified to ≈2,300 molecules per platelet. Exposure of active αIIbβ3 receptors appears to depend partially, but not totally on a metabolic activation and granule exocytosis at the time of incubation with plasmin. In contrast with a-thrombin, plasmin-induced activation of αIIbβ3 is sustained and cannot be reversed by exposure of platelets to PGE1. Immunoblotting analysis of the receptor subunits shows no extensive proteolytic modification of αIIbβ3 by plasmin, and only reveals a limited proteolysis of the aminoterminal domain of the αIIb subunit. In addition to their capacity to aggregate in the presence of fibrinogen alone, plasmin-treated platelets also show a potentiated aggregability in response to low doses of ADP. Thus, plasmin has the potential to activate the platelet fibrinogen receptor in such a way that it remains irreversibly available to fibrinogen on the surface of nonaggregated cells, a feature that may participate to pathological states of in vivo platelet hyperaggregability.

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