An antibody against the exosite of the cloned thrombin receptor inhibits experimental arterial thrombosis in the African green monkey.
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.


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 approximately 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.
Cook
J J
JJ
Department of Pharmacology, Merck Research Laboratories, West Point, Pa 19486, USA.
Sitko
G R
GR
Bednar
B
B
Condra
C
C
Mellott
M J
MJ
Feng
D M
DM
Nutt
R F
RF
Shafer
J A
JA
Gould
R J
RJ
Connolly
T M
TM
D000818:Animals; D000906:Antibodies / Q000627:therapeutic use*; D000917:Antibody Formation; D002522:Chlorocebus aethiops; D010974:Platelet Aggregation / Q000187:drug effects; D018179:Receptors, Thrombin / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D013927:Thrombosis / Q000188:drug therapy*
D000906:Antibodies; D018179:Receptors, Thrombin; D011994:Recombinant Proteins