(Circulation. 1995;91:2961-2971.)
© 1995 American Heart Association, Inc.
Articles |
An Antibody Against the Exosite of the Cloned Thrombin Receptor Inhibits Experimental Arterial Thrombosis in the African Green Monkey
From the Departments of Pharmacology (J.J.C., G.R.S., M.J.M.), Biological Chemistry (B.B., C.C., J.A.S., R.J.G., T.M.C.), and Medicinal Chemistry (D.-M.F., R.F.N.), Merck Research Laboratories, West Point, Pa.
Correspondence to Jacquelynn J. Cook, PhD, WP42-300, Merck Research Laboratories, West Point, PA 19486.
Background Thrombin inhibitors have been shown to be efficacious in animal models of thrombosis and in initial human clinical trials. It is unknown if their efficacy is due to their prevention of thrombin-mediated fibrin formation or to an inhibitory effect on thrombin-stimulated platelet activation. Appropriate tools to address this question have not been available. Therefore, to evaluate the role of the platelet thrombin receptor in intravascular thrombus formation, a polyclonal antibody was raised against a peptide derived from the thrombin-binding exosite region of the cloned human thrombin receptor. This antibody serves as a selective inhibitor of the thrombin receptor for in vivo evaluation.
Methods and Results The immune IgG (IgG 9600) inhibited
thrombin-stimulated aggregation and secretion of human platelets. In
contrast, it had no effect on platelet activation induced by other
agonists including ADP, collagen, or the thrombin receptor–derived
peptide SFLLR-NH2. IgG 9600 also inhibited thrombin-induced
aggregation of African Green monkey (AGM) platelets. By Western blot
analysis, the IgG identified a protein of 
64 kD in homogenates
of both human and AGM platelets. The effect of thrombin receptor
blockade by this antibody on arterial thrombosis was evaluated in an in
vivo model of platelet-dependent cyclic flow reductions (CFRs) in the
carotid artery of the AGM. The intravenous administration of IgG 9600
(10 mg/kg) abolished CFRs in three monkeys and reduced CFR frequency by
50% in a fourth monkey. Ex vivo platelet aggregation in response to up
to 100 nmol/L thrombin was completely inhibited during the 120-minute
postbolus observation period in all four animals. There was a twofold
increase in bleeding time, which was not statistically different from
baseline, and ex vivo clotting time (APTT) was not changed. The
glycoprotein IIb/IIIa receptor antagonist MK-0852 and the thrombin
inhibitor recombinant hirudin also demonstrated inhibitory effects on
CFRs at doses that did not significantly prolong template bleeding
time. Control IgG had no effect on CFRs, ex vivo platelet aggregation,
bleeding time, or APTT.
Conclusions These results demonstrate that blockade of the platelet thrombin receptor can prevent arterial thrombosis in this animal model without significantly altering hemostatic parameters and suggest that the thrombin receptor is an attractive antithrombotic target.
Key Words: platelets • thrombosis • antibodies
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