Activation of calpain I in thrombin-stimulated platelets is regulated by the initial elevation of the cytosolic Ca2+ concentration.
The source and concentration of Ca2+ required to activate calpain I were investigated in thrombin-stimulated platelets. The concentration of cytosolic free Ca2+ ([Ca2+]i) was measured in platelets containing fura-2-AM, and exhibited a biphasic response after stimulation with 0.05, 0.1 or 0.5 NIH units of thrombin/ml. An initial transient elevation, which was predominantly dependent upon Ca2+ released from the internal stores into the cytosol, peaked at 15 s after stimulation, and a secondary sustained elevation, which was due to Ca2+ influx, was observed following the initial elevation. Calpain I was present at about 540 ng/10(8) unstimulated platelets, as measured by immunoblotting using rabbit anti-(human calpain I) IgG. Calpain I was activated 10 s after thrombin stimulation, as determined by the appearance of the 78 kDa and 76 kDa forms on immunoblots. The activation ratio of calpain I was calculated as the amount of the 78 + 76 kDa forms as a percentage of the total (80 + 78 + 76 kDa), and was influenced by the extent of the initial transient [Ca2+]i elevation after stimulation. An initial increase in [Ca2+]i of 300 nM was required to achieve the maximal activation (60%) of calpain I, and half-maximal activation occurred at 160 nM- Ca2+]i. These results suggest that the activation of calpain I in platelets is regulated by the initial elevation in Ca2+]i after thrombin stimulation, and does not necessarily require a Ca2+ influx.
Ishii
H
H
Department of Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
Suzuki
Y
Y
Kuboki
M
M
Morikawa
M
M
Inoue
M
M
Kazama
M
M
D000328:Adult; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D002154:Calpain / Q000097:blood* / Q000302:isolation & purification; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D006801:Humans; D015151:Immunoblotting; D005779:Immunodiffusion; D066298:In Vitro Techniques; D007700:Kinetics; D013917:Thrombin / Q000494:pharmacology*
D005456:Fluorescent Dyes; C049925:fura-2-am; D004533:Egtazic Acid; D013917:Thrombin; D002154:Calpain; D002118:Calcium; D016257:Fura-2