Glycosylation of high-affinity thrombin receptors appears necessary for thrombin binding.

Monosaccharide binding competition, lectin affinity chromatography, and glycosylation inhibitors have been used to determine if glycosylation plays a role in thrombin-receptor interactions. Mannose appeared to specifically inhibit thrombin binding to mouse embryo (ME) and hamster fibroblasts. Concanavalin A bound to antibody-purified receptor fractions, and was used as an affinity ligand to purify receptor fractions that retained thrombin binding activity. Cells treated with tunicamycin (6.25 ng/ml) for 24 h lost approximately 35% of their high-affinity thrombin binding sites, yet binding of receptor monoclonal antibody TR-9 was not affected, indicating that the receptor was present in the membrane, but unable to bind thrombin. Thus thrombin receptor glycosylation may be directly involved in thrombin binding.     

Platelet activation by thrombin in the absence of the high-affinity thrombin receptor

The receptor status of the moderate-affinity platelet binding site for alpha-thrombin has been established by treating platelets with Serratia marcescens protease under conditions causing cleavage of 95-97% glycoprotein Ib (2.5 micrograms for 30 min). High-affinity binding was lost under these conditions, but the platelets continued to show moderate-affinity binding (Kd1 = 16 +/- 5 nM; 930 +/- 300 sites/platelet) and low-affinity binding (Kd2 = 4.6 +/- 3 microM; 170,000 +/- 90,000 sites/platelet), in good agreement with the values previously obtained for moderate- and low-affinity binding in intact platelets [Harmon, J.T., & Jamieson, G.A. (1986) J. Biol. Chem. 261, 15928-15933]. Platelets treated with Serratia protease under these conditions were about 4-fold less sensitive to activation by alpha-thrombin, as measured by serotonin secretion. Crossover studies with analogues showed that binding of alpha-thrombin was compatible by both D-phenyl-alanyl-L-prolyl-L-arginine chloromethyl ketone treated thrombin and N alpha-p-tosyl-L-lysine chloromethyl ketone treated thrombin, and both analogues were capable of inhibiting activation of Serratia-proteolyzed platelets by alpha-thrombin. These studies establish that the moderate-affinity platelet binding site for alpha-thrombin is a receptor, occupancy of which is required for platelet activation in the absence of the high-affinity receptor.     

Cell receptors of thrombin

Modern notions on the structure, function and localization of thrombin receptors in different human and animal cells are considered. Molecular models of thrombin interaction with receptors are presented. The role these receptors play in the DNA synthesis regulation, proliferation and migration of cells is described. The functions of the thrombin-receptor complexes and mechanisms controlling the quantity of thrombin receptors are discussed.     

Effect of oligodeoxynucleotide thrombin aptamer on thrombin inhibition by heparin cofactor II and antithrombin

'Thrombin aptamers' are based on the 15-nucleotide consensus sequence of d(GGTTGGTGTGGTTGG) that binds specifically to thrombin's anion-binding exosite-I. The effect of aptamer-thrombin interactions during inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) and antithrombin (AT) has not been described. Thrombin inhibition by HCII without glycosaminoglycan was decreased approximately two-fold by the aptamer. In contrast, the aptamer dramatically reduced thrombin inhibition by >200-fold and 30-fold for HCII-heparin and HCII-dermatan sulfate, respectively. The aptamer had essentially no effect on thrombin inhibition by AT with or without heparin. These results add to our understanding of thrombin aptamer activity for potential clinical application, and they further demonstrate the importance of thrombin exosite-I during inhibition by HCII-glycosaminoglycans.