Affinity modulation of the platelet integrin alpha IIb beta 3 by alpha-chymotrypsin: a possible role for Na+/Ca2+ exchanger.
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.
Shiraga
M
M
Second Department of Internal Medicine, Osaka University Medical School, Japan.
Tomiyama
Y
Y
Honda
S
S
Kashiwagi
H
H
Kosugi
S
S
Handa
M
M
Ikeda
Y
Y
Kanakura
Y
Y
Kurata
Y
Y
Matsuzawa
Y
Y
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D015764:Bepridil / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D002918:Chymotrypsin / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D010042:Ouabain / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D000644:Quaternary Ammonium Compounds; D019831:Sodium-Calcium Exchanger; C041470:3',4'-dichlorobenzamil; D010042:Ouabain; D015764:Bepridil; D000584:Amiloride; D005340:Fibrinogen; D012964:Sodium; D002918:Chymotrypsin; D000254:Sodium-Potassium-Exchanging ATPase; C027917:tetramethylammonium; D002118:Calcium