Abstract
The nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) is a potent irreversible inhibitor of ADP-mediated platelet activation. Utilizing this compound, the role of ADP in epinephrine-mediated platelet activation was evaluated. Pretreatment of platelets with FSBA under conditions producing covalent incorporation was able to completely block epinephrine-stimulated aggregation of human platelets. In addition, the exposure of latent fibrinogen-binding sites by epinephrine was also inhibited in platelets modified by FSBA. The inhibition of epinephrine-mediated activation of the cells was time dependent, reflecting the need for covalent modification of the ADP receptor by FSBA. The inhibitory effect of FSBA was not due to effects on the affinity of binding methyl [3H]yohimbine or the number of platelet alpha 2-adrenergic receptors. Studies of the effect of epinephrine on the ability of ADP to protect against FSBA incorporation demonstrated that epinephrine can increase the affinity of ADP for its receptor 10-fold without affecting the total amount of FSBA covalently bound. This effect of epinephrine is mediated through the alpha 2-adrenoreceptor since the effect can be reversed by the competitive antagonist, methyl yohimbine. These results suggest that promotion of platelet aggregation and the exposure of fibrinogen receptors by epinephrine is dependent on ADP. The mechanism by which epinephrine renders low concentrations of ADP effective appears to be mediated by an increased avidity of the ADP receptor for the nucleotide.
Highlights
From the $Thrombosis Research Center and Hemutology-OncologySection, Department of Medicine, Temple UniversitySchool of Medicine, Philadelphia, Pennsylvania 19140 and the $Department of Chemistry, University of Delaware, Newark, Delaware 19716
Studies from our laboratory have identified a membrane protein (A& 100,000)on the surface of intact platelets which is specificallyand uniquely covalently labeled by 5‘-p-fluorosulfonylbenzoyl adenosine (FSBA)
Inhibition of Epinephrine-inducedPlateletActivation of FSBA-Gel-filtered platelets were treated withFSBA in DMF or DMF alone in the presence of adenosine deaminase to obviate any effects of adenosine arising from the degradation ofFSBA
Summary
FSBA was not due to effects on the affinityof binding support a role for ADP in epinephrine-mediated platelet methyl r3H]yohirnbine or the number of platelet cuzadrenergic receptors. Incorporation into intact platelets is linear over 5 min using These resultsindicates that epinephrine increases the ability the dialysis procedure described under “Materials and Meth- of the receptor to bind ADP.
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