Ca+2 mobilization and fibrinogen binding of platelets refractory to adenosine diphosphate stimulation.
The mechanism of adenosine diphosphate (ADP)-induced refractoriness was explored with iodine 125-labeled fibrinogen and the fluorescent Ca+2 indicator quin-2-tetraacetoxymethyl ester (quin-2). Gel-filtered platelets were rendered refractory by incubation (30 minutes, 22 degrees C) with either 10 mumol/L ADP alone or ADP and 125I-labeled 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 at various times during incubation showed that fibrinogen binding was normal after 1 minute but decreased to 50% in 30 minutes. According to Scatchard analysis, this decreased binding was attributed to decreased fibrinogen receptor availability, not decreased receptor affinity. Moreover, 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 not accompanied by a rise in quin-2 fluorescence or exposure of additional fibrinogen receptors. Stimulation of platelets with thrombin, however, led to a rise in quin-2 fluorescence, exposure of additional fibrinogen receptors, and 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 minutes with ADP and 125I-fibrinogen also showed an 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, however, 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 not accompanied by increased fibrinogen binding or quin-2 fluorescence and failed to elicit significant platelet aggregation.(ABSTRACT TRUNCATED AT 400 WORDS)
Peerschke
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D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000634:Aminoquinolines; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000097:blood*; D005456:Fluorescent Dyes; D006801:Humans; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012701:Serotonin / Q000097:blood; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D000634:Aminoquinolines; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D012701:Serotonin; D000244:Adenosine Diphosphate; C035739:Quin2; D002118:Calcium