Acceleration of Ca2+ ionophore-induced arachidonic acid liberation by thrombin without the proteolytic action toward the receptor in human platelets.
We investigated the regulation of arachidonic acid liberation catalyzed by group-IV cytosolic phospholipase A2 (cPLA2) in human platelets upon stimulation with thrombin through interaction with protease-activated receptor-1 (PAR-1) or glycoprotein Ib. Leupeptin, a protease inhibitor, completely inhibited thrombin-induced arachidonic acid liberation and Ca2+ mobilization, with inhibition of its protease activity. However, preincubation with thrombin in the presence of leupeptin potentiated Ca2+ ionophore-induced arachidonic acid liberation. The preincubation did not affect the intracellular Ca2+ level or cPLA2 activity in response to ionomycin. Human leukocyte elastase, which cleaves glycoprotein Ib, did not inhibit the enhancement of arachidonic acid liberation by thrombin in the presence of leupeptin. However, the effect of thrombin with leupeptin was abolished by a peptide corresponding to residues 54-65 of hirudin (hirudin peptide), which impairs the binding of thrombin to PAR-1. Furthermore, Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, which binds to platelets but has no protease activity, also enhanced Ca2+ ionophore-induced arachidonic acid liberation. In contrast, trypsin with leupeptin did not mimic the effect of thrombin with leupeptin, and furthermore trypsin-induced arachidonic acid liberation was insensitive to hirudin peptide. On the basis of the present results, we suggest that thrombin may accelerate cPLA2-catalyzed arachidonic acid liberation through non-proteolytic action toward PAR-1 but not toward glycoprotein Ib in co-operation with the proteolytic action leading to Ca2+ mobilization.
Akiba
S
S
Department of Pathological Biochemistry, Kyoto Pharmaceutical University, Japan. akiba@mb.kyoto-phu.ac.jp
Kawauchi
T
T
Sato
T
T
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000991:Antithrombins / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology; D019272:Leukocyte Elastase / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D000991:Antithrombins; D006629:Hirudins; D007476:Ionophores; D007976:Leupeptins; D010446:Peptide Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D016718:Arachidonic Acid; D015759:Ionomycin; D010741:Phospholipases A; D054467:Phospholipases A2; D019272:Leukocyte Elastase; D014357:Trypsin; D013917:Thrombin; C032854:leupeptin; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone; D002118:Calcium