The phospholipase C/protein kinase C pathway is involved in cathepsin G-induced human platelet activation: comparison with thrombin.
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.
Si-Tahar
M
M
Unité de Pharmacologie Cellulaire, Unité Associée Institut Pasteur/INSERM U285, Institut Pasteur, Paris, France.
Renesto
P
P
Falet
H
H
Rendu
F
F
Chignard
M
M
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D056649:Cathepsin G; D002403:Cathepsins / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism*; D012697:Serine Endopeptidases; D012701:Serotonin / Q000378:metabolism; D013917:Thrombin / Q000502:physiology*; D010738:Type C Phospholipases / Q000378:metabolism*
D010743:Phospholipids; D012701:Serotonin; D011493:Protein Kinase C; D010738:Type C Phospholipases; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D013917:Thrombin; D002118:Calcium