CA-2+ MOBILIZATION AND FIBRINOGEN BINDING OF PLATELETS REFRACTORY TO ADENOSINE-DIPHOSPHATE STIMULATION

  • 1 January 1985
    • journal article
    • research article
    • Vol. 106  (2) , 111-122
Abstract
The mechanism of ADP-induced refractoriness was explored with 125I fibrinogen and the fluorescent Ca+2 indicator quin-2-tetraacetoxymethyl ester (quin-2). Gel-filtered platelets were rendered refractory by incubation (30 min 22.degree. C) with either 10 .mu.mol/L ADP alone or ADP and 125I fibrinogen. During the incubation period, platelets incubated with ADP alone showed an initial increase in quin-2 fluorescence, which gradually returned to baseline levels. Addition of 125I-fibrinogen to aliquots of the platelet suspension of various times during incubation showed that fibrinogen binding was normal after 1 min but decreased to 50% in 30 min. According to Scatchard analysis, this decreased binding was attributed to decreased fibrinogen receptor availability, not decreased receptor affinity. Similar numbers of glycoprotein (GP)IIb-IIIa complexes remained available on platelets before and after incubation, as judged by the ability of a monoclonal antibody (10E5) directed against a complex specific epitope on GPIIb or IIIa to bind to control and refractory platelets. After incubation, platelets aggregated poorly in response to restimulation with ADP, although the amount of fibrinogen they bound (50% of normal) was sufficient to aggregate control platelets. Platelet restimulation with ADP was unaccompanied by a rise in quin-2 fluorescence or exposure of additional fibrinogen receptors. Stimulation of platelets with thrombin, led to a rise in quin-2 fluorescence, exposure of additional fibrinogen receptorsand enhanced aggregation. Restimulation of platelets with epinephrine also increased fibrinogen receptor exposure and restored the ability of platelets to aggregate, but was accompanied by barely detectable changes in quin-2 fluorescence similar to those observed with epinephrine-treated control platelets. Platelets incubated for 30 min with ADP and 125I fibrinogen also showed on initial rise in quin-2 fluorescence, which returned to baseline levels during incubation, but the amount of platelet-bound fibrinogen, normal at the onset, remained quantitatively unchanged. Much of this fibrinogen no longer dissociated from platelets in the presence of ethylenediaminetetraacetic acid or apyrase, suggesting that a different type of platelet-fibrinogen interaction had developed. Restimulation of these platelets with ADP was unaccompanied by increased fibrinogen binding or quin-2 fluorescence and failed to elicit significant platelet aggregation. Aggregation was enhanced by stimulating platelets with epinephrine, when additional fibrinogen receptors were exposed. The response of these platelets to thrombin was not measured because the samples would have clotted. Fibrinogen bound to platelets during their initial stimulation with ADP evidently remains quantitatively unchanged as long as ADP is present, but the ability of unoceupied receptors to bind fibrinogen decreases with time. ADP-induced refractoriness is accompanied by a block in intraplatelet Ca+2 mobilization and an inability of bound fibrinogen to support aggregation.