title: The glycocalicin portion of platelet glycoprotein Ib expresses both high and moderate affinity receptor sites for thrombin. A soluble radioreceptor assay for the interaction of thrombin with platelets.
abstract: A soluble radioreceptor assay has been developed to characterize thrombin receptor activities of the human platelet membrane. 125I-Thrombin was added to platelet membranes solubilized in 1% Triton X-100, and thrombin bound to platelet receptors was separated from free thrombin by precipitation with wheat germ agglutinin (WGA) in the presence of alpha 1-acid glycoprotein as carrier. Both high affinity binding (Ki, 0.09 nM; R1, 0.30 pmol/mg protein) and moderate affinity binding (K2, 38 nM; R2, 72 pmol/mg protein) were detected in the detergent-solubilized membrane preparations and these binding parameters were in excellent agreement with values previously determined using intact platelets (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). Using the soluble radioreceptor assay, both high and moderate affinity binding was detected in highly purified preparations of glycoprotein Ib (GPIb) and glycocalicin, and the binding isotherms were identical with those of the crude detergent-solubilized membrane preparation. Treatment of detergent-solubilized membranes with increasing concentrations of a monospecific polyclonal antibody to glycocalicin resulted in the stepwise depletion of GPIb and concomitant reductions of thrombin binding activity. These results demonstrate that both high and moderate affinity binding of thrombin to platelets is completely expressed in the glycocalicin portion of GPIb.
mesh_terms: D000818:Animals; D000906:Antibodies; D001792:Blood Platelets / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D017830:Octoxynol; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011092:Polyethylene Glycols; D011817:Rabbits; D011869:Radioligand Assay; D012995:Solubility; D013917:Thrombin / Q000378:metabolism*
chemical_list: D000906:Antibodies; D008565:Membrane Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; C023592:glycocalicin; D011092:Polyethylene Glycols; D017830:Octoxynol; D013917:Thrombin