TY - JOUR
T1 - Inhibition of calpain blocks platelet secretion, aggregation, and spreading
AU - Crocet, Kevin
AU - Flaumenhaft, Robert
AU - Rivers, Marc
AU - Furie, Bruce
AU - Furie, Barbara C.
AU - Herman, Ira M.
AU - Potter, David A.
PY - 1999/12/17
Y1 - 1999/12/17
N2 - Previous studies have indicated that the Ca2+-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced α-granule secretion (IC50 = 20 μM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC50 = 50 μM), and platelet spreading on glass surfaces (IC50 = 34 μM). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.
AB - Previous studies have indicated that the Ca2+-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced α-granule secretion (IC50 = 20 μM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC50 = 50 μM), and platelet spreading on glass surfaces (IC50 = 34 μM). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.
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U2 - 10.1074/jbc.274.51.36321
DO - 10.1074/jbc.274.51.36321
M3 - Article
C2 - 10593923
AN - SCOPUS:0033579525
SN - 0021-9258
VL - 274
SP - 36321
EP - 36327
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 51
ER -