Evidence for a Single Class of Thrombin-Binding Sites on Human Platelets

Douglas M. Tollefsen, Philip W. Majerus

Research output: Contribution to journalArticle

39 Scopus citations

Abstract

We previously demonstrated that human platelets bind a small number of thrombin molecules with high affinity and a larger number with relatively lower affinity (Tollefsen, D. M., Feagler, J. R., and Majerus, P. W. (1974), J. Biol. Chem. 249, 2646). in the present report, equilibrium binding studies using [125I]DIP-thrombin (diisopropylphosphorylthrombin) over the range of 0.0002–10 U/ml yield a Hill coefficient of 0.775. Measurements of the rate of dissociation of [125I]DIP-thrombin bound to platelets at relatively high affinity (0.04 U/ml added) indicate a much faster dissociation in the presence of 14 U/ml unlabeled DIP-thrombin (T½ - 1.0 min) than in its absence (T½= 140 min). [125I]DIPthrombin bound at low affinity (1.0 U/ml added) dissociates from platelets with a T½ = 1.7 min in the absence of unlabeled DIP-thrombin. These results suggest a negative cooperative interaction among receptor sites for thrombin; i.e., as thrombin binds to unoccupied sites, high-affinity receptors are apparently converted to low-affinity receptors. In an attempt to detect whether there is heterogeneity of thrombin receptors, [125I]DIP-thrombin was covalently cross-linked to intact platelets using 1 mM glutaraldehyde. A single complex (apparent molecular weight, 200 000) containing [125I]DIP-thrombin was formed throughout a range of thrombin concentrations in which both high- and low-affinity binding was observed. Since incorporation of [125I]DIP-thrombin into this complex did not occur in the absence of platelets and was inhibited by unlabeled thrombin, the complex may represent thrombin cross-linked to its receptor. We conclude that a single class of receptor sites can account for both high- and low-affinity binding of thrombin to platelets, although interaction between nonidentical sites cannot be excluded.

Original languageEnglish
Pages (from-to)2144-2149
Number of pages6
JournalBiochemistry
Volume15
Issue number10
DOIs
StatePublished - May 1 1976

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