Affinity modulation of the platelet integrin α(11b)β3 by α- chymotrypsin: A possible role for Na+/Ca2+ exchanger

Masamichi Shiraga, Yoshiaki Tomiyama, Shigenori Honda, Hirokazu Kashiwagi, Satoru Kosugi, Makoto Handa, Yasuo Ikeda, Yuzuru Kanakura, Yoshiyuki Kurata, Yuji Matsuzawa

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

Abstract

In the present study, we have investigated the mechanism of affinity modulation of α(IIb)β3 by chymotrypsin. We first confirmed that α- chymotrypsin could activate α(IIb)β3 (~7,000 molecules per platelet) without major intracellular signaling. However, we unexpectedly found that high concentrations of amiloride dose-dependently inhibited 125I- fibrinogen binding to the chymotrypsin-treated platelets, as well as the platelet aggregation (IC50 (50% inhibitory concentration] for fibrinogen binding, 530 μmol/L). In contrast, amiloride did not inhibit α(IIb)β3 activation induced by anti-α(IIb)β3 monoclonal antibody PT25-2 or AP5. To identify the pathway involved, the effects of alteration of Na+ gradient in platelets were examined. Lowering Na+ gradient by replacing extracellular Na+ with tetramethylammonium (TMA) increased the number of activated α(IIb)β3 by twofold, as assessed by fibrinogenbinding assay. The incubation of platelets with ouabain, a Na+/K+-adenosine triphosphatase (ATPase) inhibitor, further augmented α(IIb)β3 activation. These data suggested that a likely candidate for the pathway was Na+/Ca2+ exchanger. At 140 mmol/L [Na+](o). 45Ca2+ influx to the chymotrypsintreated platelets was twofold greater than that to nontreated platelets. Replacement of Na+ with TMA further increased the Ca2+ influx, and the increase was inhibited by amiloride dose-dependently. 3',4'-Dichlorobenzamil (DCB) and bepridil, relatively specific inhibitors of Na+/Ca2+ exchanger, also inhibited the chymotrypsin-induced α(IIb)β3 activation, and the IC50 values of these inhibitors for fibrinogen binding were 25 μmol/L and 52 μmol/L, respectively. Moreover, platelet aggregation induced by various physiologic agonists was inhibited by DCB or bepridil, while platelet agglutination by ristocetin was not. Our data newly suggest that Na+/Ca2+ exchanger operating in reverse mode may be directly involved in inside-out signaling that activates α(IIb)β3.

Original languageEnglish
Pages (from-to)2594-2602
Number of pages9
JournalBlood
Volume88
Issue number7
DOIs
Publication statusPublished - 1996 Oct 1

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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