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.
Harmon
J T
JT
Cell Biology Laboratory, American Red Cross, Rockville, Maryland 20855.
Jamieson
G A
GA
D001792:Blood Platelets / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D010447:Peptide Hydrolases; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000502:physiology*; D018179:Receptors, Thrombin; D012701:Serotonin / Q000097:blood; D012706:Serratia marcescens / Q000201:enzymology; D013917:Thrombin / Q000502:physiology*
D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D012701:Serotonin; D010447:Peptide Hydrolases; D013917:Thrombin