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

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.