alpha-chymotrypsin and 3--4--dichlorobenzamil

The platelet integrin IIbbeta3 has become a new target for the treatment of pathological thrombosis. It becomes apparent that the affinity of IIbbeta3 for its ligands is dynamically regulated by inside-out signaling. However, the components that couple diverse intracellular signals to the cytoplasmic domains of IIbbeta3 remain obscure. Employing a chymotrypsin-induced IIbbeta3 activation model, we previously proposed the hypothesis that Na+/Ca2 + exchanger (NCX) may be involved in inside-out signaling (Shiraga et al: Blood 88:2594, 1996). In the present study, employing two unrelated Na+/Ca2+ exchange inhibitors, 3',4'-dichlorobenzamil (DCB) and bepridil, we investigated the role of NCX in platelet activation induced by various agonists in detail. Both inhibitors abolished platelet aggregation induced by all agonists examined via the inhibition of IIbbeta3 activation. Moreover, these inhibitors abolished IIbbeta3 activation induced by phorbol 12-myristate 13-acetate or A23187. On the other hand, neither of these inhibitors showed apparent inhibitory effects on protein phosphorylation of pleckstrin or myosin light chain, or an increase in intracellular calcium ion concentrations evoked by 0.1 U/mL thrombin. These effects of the NCX inhibitors are in striking contrast to those of protein kinase C inhibitor, Ro31-8220. Biochemical and ultrastructural analyses showed that NCX inhibitors, particularly DCB, made platelets "thrombasthenic". These findings suggest that the NCX is involved in the common steps of inside-out signaling through integrin IIbbeta3.

Topics: Amiloride; Bepridil; Blood Platelets; Blood Proteins; Calcimycin; Calcium; Chymotrypsin; Cytoplasmic Granules; Fibrinogen; Humans; Ion Transport; Myosin Light Chains; P-Selectin; Phosphoproteins; Phosphorylation; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Processing, Post-Translational; Serotonin; Signal Transduction; Sodium; Sodium-Calcium Exchanger; Tetradecanoylphorbol Acetate

In the present study, we have investigated the mechanism of affinity modulation of alpha IIb beta 3 by chymotrypsin. We first confirmed that alpha-chymotrypsin could activate alpha IIb beta 3 (approximately 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 mumol/L). In contrast, amiloride did not inhibit alpha IIb beta 3 activation induced by anti-alpha IIb beta 3 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 alpha IIb beta 3 by twofold, as assessed by fibrinogen-binding assay. The incubation of platelets with ouabain, a Na+/K(+)-adenosine triphosphatase (ATPase) inhibitor, further augmented alpha IIb beta 3 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 chymotrypsin-treated platelets was twofold greater than that to non-treated 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 alpha IIb beta 3 activation, and the IC50 values of these inhibitors for fibrinogen binding were 25 mumol/L and 52 mumol/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 alpha IIb beta 3.

Topics: Amiloride; Antibodies, Monoclonal; Bepridil; Biological Transport, Active; Calcium; Carrier Proteins; Chymotrypsin; Enzyme Inhibitors; Fibrinogen; Humans; Ouabain; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Binding; Quaternary Ammonium Compounds; Sodium; Sodium-Calcium Exchanger; Sodium-Potassium-Exchanging ATPase