Effects of cathepsin G pretreatment of platelets on their subsequent responses to aggregating agents

Cathepsin G, a proteolytic enzyme from activated leukocytes, can interact w ith platelets during inflammation and thrombosis. Platelets that have been exposed to cathepsin G in thrombi may recirculate if they are freed during fibrinolysis. To determine whether some of the subsequent functions of such platelets would be impaired, we investigated the responses of cathepsin G- pretreated platelets to agonists that they would encounter in the circulati on. Suspensions of washed human platelets were labeled with [C-14]serotonin and resuspended in Tyrode-albumin solution (with 2 mM Ca2+ and apyrase), A fter 15 minute incubation with 400 nM cathepsin G at 37 degrees C, 52+/-3% of [C-14]serotonin had been released, and glycoprotein Ib was degraded. The platelets were washed and resuspended in fresh medium to remove cathepsin C and released materials. Ristocetin-induced agglutination was abolished, i ndicating that the binding site for von Willebrand Factor on glycoprotein I b had been removed, Aggregation and release of residual [C-14]serotonin in response to 0.1-1.0 U/mL thrombin was blocked or greatly reduced by the cat hepsin G pretreatment, This inhibition is probably largely due to cleavage by cathepsin G of some of the protease-activated receptors at the C-termina l side of Ser(42) so that the tethered ligand is lost. Pretreatment with ca thepsin C did not affect responses to ADP or a low concentration of platele t-activating factor in the presence of fibrinogen, indicating that receptor s for these agonists were unaffected and that the function of the fibrinoge n receptor, GPIIb/IIIa was unchanged. Responses to cathepsin G, the thrombi n receptor-activating peptide SFLLRN, collagen, or the thromboxane A(2) mim etic U46619 were partially inhibited, even in the presence of added fibrino gen. Platelet adhesion to a collagen-coated surface was 51+/-7% inhibited, which may indicate cleavage of a collagen receptor or receptors; this may p artly account for strong inhibition of collagen-induced aggregation and rel ease of granule contents; additionally, as shown by inhibition of responses to U46619, the function of the thromboxane A(2) receptor may be compromise d. Thus, although cathepsin G activates platelets, if they recirculate afte r interaction with it, their subsequent adhesion to damaged vessel walls, a ggregation, and release of granule contents induced by thrombin and collage n will be diminished. (C) 1999 Elsevier Science Ltd. All rights reserved.