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 journalArticle

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
Publication statusPublished - 1996 Oct 1
Externally publishedYes

Fingerprint

Platelets
Integrins
Blood Platelets
Modulation
Chymotrypsin
Amiloride
Bepridil
Fibrinogen
Inhibitory Concentration 50
Platelet Aggregation
Chemical activation
Ristocetin
Agglomeration
Agglutination
Ouabain
alpha-chymotrypsin
Adenosine Triphosphatases
Monoclonal Antibodies
Assays
Molecules

ASJC Scopus subject areas

  • Hematology

Cite this

Shiraga, M., Tomiyama, Y., Honda, S., Kashiwagi, H., Kosugi, S., Handa, M., ... Matsuzawa, Y. (1996). Affinity modulation of the platelet integrin α(11b)β3 by α- chymotrypsin: A possible role for Na+/Ca2+ exchanger. Blood, 88(7), 2594-2602.

Affinity modulation of the platelet integrin α(11b)β3 by α- chymotrypsin : A possible role for Na+/Ca2+ exchanger. / Shiraga, Masamichi; Tomiyama, Yoshiaki; Honda, Shigenori; Kashiwagi, Hirokazu; Kosugi, Satoru; Handa, Makoto; Ikeda, Yasuo; Kanakura, Yuzuru; Kurata, Yoshiyuki; Matsuzawa, Yuji.

In: Blood, Vol. 88, No. 7, 01.10.1996, p. 2594-2602.

Research output: Contribution to journalArticle

Shiraga, M, Tomiyama, Y, Honda, S, Kashiwagi, H, Kosugi, S, Handa, M, Ikeda, Y, Kanakura, Y, Kurata, Y & Matsuzawa, Y 1996, 'Affinity modulation of the platelet integrin α(11b)β3 by α- chymotrypsin: A possible role for Na+/Ca2+ exchanger', Blood, vol. 88, no. 7, pp. 2594-2602.
Shiraga M, Tomiyama Y, Honda S, Kashiwagi H, Kosugi S, Handa M et al. Affinity modulation of the platelet integrin α(11b)β3 by α- chymotrypsin: A possible role for Na+/Ca2+ exchanger. Blood. 1996 Oct 1;88(7):2594-2602.
Shiraga, Masamichi ; Tomiyama, Yoshiaki ; Honda, Shigenori ; Kashiwagi, Hirokazu ; Kosugi, Satoru ; Handa, Makoto ; Ikeda, Yasuo ; Kanakura, Yuzuru ; Kurata, Yoshiyuki ; Matsuzawa, Yuji. / Affinity modulation of the platelet integrin α(11b)β3 by α- chymotrypsin : A possible role for Na+/Ca2+ exchanger. In: Blood. 1996 ; Vol. 88, No. 7. pp. 2594-2602.
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AU - Honda, Shigenori

AU - Kashiwagi, Hirokazu

AU - Kosugi, Satoru

AU - Handa, Makoto

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AU - Kurata, Yoshiyuki

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N2 - 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.

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