Stimulation of human platelet Ca(2+)-ATPase and Ca2+ restoration by calpain.
To clarify the possible role of calpain (calcium activated neutral protease; EC 3.4.22.17) in Ca2+ homeostasis of human platelets, we investigated the effects of cell permeable calpain inhibitors, calpeptin and E-64d (EST), on the restoration of cytoplasmic Ca2+ ([Ca2+]i) in both Fura-2 and aspirin (ASA) loaded platelets. Although neither calpeptin (30 microM) nor EST (250 microM) altered the increase of [Ca2+]i in thrombin (1 U/ml) stimulated platelets, both calpain inhibitors delayed the decrease of [Ca2+]i back towards the basal level. These observations suggested that calpain might be involved in Ca2+ restoration. Then, the activity of Ca(2+)-ATPase was examined in thrombin (2 U/ml) stimulated platelets. Thrombin produced a rapid rise in Ca(2+)-ATPase activity by 2-fold at 8 s of incubation, which then returned to below the basal activity within 2 min. Calpeptin inhibited transient Ca(2+)-ATPase activation induced by thrombin in a dose related manner. Ca(2+)-ATPase of isolated platelet membranes was digested by purified human platelet calpain-I and Ca(2+)-ATPase activity was investigated. With a short incubation (8-15 s), Ca(2+)-ATPase activity was increased about 2-fold and then it decreased below the basal level at longer incubations or at a higher calpain/membrane ratio. The initial rate of Ca2+ uptake was also increased by about 2-fold with a short incubation (8-15 s). For molecular characterization of the Ca(2+)-ATPase, the formation of the enzyme-phosphate complex (EP) was investigated. The membrane bound intact 105 kD Ca(2+)-ATPase was converted by calpain to a fragment of approximately 50 kD.(ABSTRACT TRUNCATED AT 250 WORDS)
Ariyoshi
H
H
Department of Surgery II, Osaka University Medical School, Fukushima, Japan.
Shiba
E
E
Kambayashi
J
J
Sakon
M
M
Kawasaki
T
T
Yoshida
K
K
Mori
T
T
D001241:Aspirin / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002451:Cell Compartmentation; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006706:Homeostasis; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000187:drug effects*; D015539:Platelet Activation / Q000187:drug effects; D013268:Stimulation, Chemical; D013917:Thrombin / Q000494:pharmacology
D004151:Dipeptides; D009097:Multienzyme Complexes; C071482:calpeptin; D000001:Calcimycin; D013917:Thrombin; D002154:Calpain; D000252:Calcium-Transporting ATPases; D007930:Leucine; C108192:aloxistatin; D001241:Aspirin; D002118:Calcium