The phospholipase C/protein kinase C pathway is involved in cathepsin G-induced human platelet activation: comparison with thrombin

Abstract

Cathepsin G, an enzyme released by stimulated polymorphonuclear neutrophils, and thrombin are two human proteinases which potently trigger platelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involvement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in cathepsin G-induced activation was investigated and compared with stimulation by thrombin. Exposure of 5-[14C]hydroxytryptamine-labelled platelets to cathepsin G, in the presence of acetylsalicylic acid and phosphocreatine/creatine kinase, induced platelet aggregation and degranulation in a concentration-dependent manner (0.1-3.0 microM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 microM) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumulation of phosphatidic acid. In addition cathepsin G, like thrombin, initiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chain and pleckstrin, a substrate for PKC, to levels similar to those observed in platelets challenged with thrombin. Inhibition of PKC by GF 109203X, a specific inhibitor, suppressed platelet aggregation and degranulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced by cathepsin G was shown to result, as for thrombin, from both mobilization of internal stores and Ca2+ entry across the plasma membrane. These findings provide evidence that cathepsin G stimulates the PLC/PKC pathway as potently as does thrombin, independently of thromboxane A2 formation and ADP release, and that this pathway is required for platelet functional responses.

Anti-neutrophil cytoplasmic antibody (ANCA) in malaria is directed against cathepsin G

“…Its deleterious effect may be mediated through direct catalytic activity on cellular and extracellular matrix, by induction of other mediators of inflammation through enhanced expression or decreased clearance [26][27][28][29][30][31][32][33][34], or through modulation of platelet [35][36][37] and lymphocyte activity [38], with resultant injury to endothelial and epithelial barriers, neutrophil migration, and thrombogenesis.…”

Human Neutrophil Elastase Proteolytically Activates the Platelet Integrin αIIbβ3 through Cleavage of the Carboxyl Terminus of the αIIb Subunit Heavy Chain

“…The standard linear regression analysis was applied to correlate the different parameters using the Statview 512 ϩ software (BrainPower Inc., Calabasas, CA). (11,16), cathepsin G alone added to platelet suspensions at the optimal concentration of 550 nM acts as a strong platelet agonist inducing extensive platelet aggregation (83.5 Ϯ 5.6%, n ϭ 5), accompanied by a marked exocytosis of intracellular granules as judged by the release of 5-[ 14 C]HT from dense granules (76.2 Ϯ 1.6%, n ϭ 5; Fig. 1, panels A and B).…”

“…The peptide FPQPPVNPLKVDWGL (using the single-letter code for amino acids), corresponding to the sequence 827-841 of the ␣ IIb subunit heavy chain, was synthesized by Neosystem Laboratoire (Strasbourg, France). All other reagents were obtained as indicated in Si-Tahar et al (16).…”

“…The platelet-rich plasma was isolated by centrifugation of blood at 180 ϫ g for 20 min and incubated with 5-[ 14 C]HT (0.05 mCi/ml) for 30 min at 37°C. For protein phosphorylation and phospholipid metabolism studies, platelets were labeled with [ 32 P]phosphoric acid as described previously (16). Then, labeled platelets were washed by two successive centrifugations (1,600 ϫ g, 10 min) and resuspended in Tyrode's buffer (composition, mM: NaCl, 137.0; KCl, 2.68; NaHCO 3 , 11.9; NaH 2 PO 4 , 0.42; CaCl 2 , 2.0; MgCl 2 , 1.0; glucose, 5.5; Hepes, 5.0; and bovine serum albumin, 0.35%, pH 7.4) supplemented with PGI 2 (0.25 M) and heparin (50 units/ml).…”

“…Cathepsin G and NE were purified as described previously (16), using a two-step chromatographic procedure (aprotinin-Sepharose affinity and CM-Trisacryl ion-exchange). The purity of the neutrophil proteinases was assessed by SDS-PAGE.…”