Effects of leukocyte-derived cathepsin G on platelet membrane glycoprotein Ib-IX and IIb-IIIa complexes: a comparison with thrombin.
Cathepsin G is a serine, chymotrypsin-like protease released by activated polymorphonuclear leukocytes (PMN) that may act as a platelet agonist. The effect of this enzyme on platelet surface glycoproteins (Gp) Ib and IIb-IIIa was evaluated by means of a cytofluorimetric assay, using fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed at the alpha chain of Gp Ib (SZ2), at Gp IX or at the complex Gp IIb-IIIa (P2), and the fibrinogen-receptor-specific MoAb PAC-1. In human washed platelets, cathepsin G increased the binding of P2 and PAC-1, decreased the binding of SZ2, but only slightly affected the binding of anti-Gp IX. SZ2 binding decrease was more rapid in cathepsin G- than in thrombin-stimulated platelets, whereas the increase of P2 and PAC-1 binding occurred to a comparable extent with either agonist. In paraformaldehyde (PFA)-fixed and energy-depleted platelets, no effect on either Gp Ib or Gp IIb-IIIa complex was observed with thrombin. At variance, cathepsin G was still able to reduce binding of SZ2, whereas increased binding of P2 or PAC-1 antibodies was not observed. Triton X-100 permeabilization of cathepsin G-treated, PFA-fixed platelets did not restore SZ2 binding at variance with thrombin. Moreover, platelet incubation with cathepsin G resulted in the loss of ristocetin-induced agglutination in the presence of the von Willebrand factor and in the appearance of Gp Ib-derived proteolytic products in supernatants. After dissociation by EDTA pretreatment of surface Gp IIb-IIIa complexes, cathepsin G still induced increased binding of P2. Aspirin and an adenosine diphosphate scavenger system had only a slight but not significant effect on changes in antibody binding induced by cathepsin G. All these data would indicate that cathepsin G, like thrombin, interacts with platelet-surface Gp, inducing the exposure of the intracellular pool of the Gp IIb-IIIa complex with concomitant expression of a functional fibrinogen receptor. Moreover, it induces a loss of antigenic sites on Gp Ib, but the mechanism involved, a proteolytic cleavage of Gp Ib, is substantially different from that of thrombin. These changes, induced by a product of activated PMN, might reduce the reactivity of platelets to the subendothelium, while increasing their ability to undergo aggregation and release reaction.
Instituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy.
M
M
Molino
M
M
Di Lallo
N
N
Martelli
G
G
de Gaetano
C
C
Cerletti
D000244:Adenosine Diphosphate / Q000502:physiology; D000328:Adult; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D056649:Cathepsin G; D002403:Cathepsins / Q000494:pharmacology*; D006801:Humans; D009504:Neutrophils / Q000201:enzymology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D013917:Thrombin / Q000494:pharmacology*; D013928:Thromboxane A2 / Q000502:physiology
D000911:Antibodies, Monoclonal; D010980:Platelet Membrane Glycoproteins; D013928:Thromboxane A2; D000244:Adenosine Diphosphate; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D013917:Thrombin