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Circulation. 1995;91:2961-2971

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(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

Jacquelynn J. Cook, PhD; Gary R. Sitko, BS; Bohumil Bednar, PhD; Cindra Condra, MS; Michael J. Mellott, MS; Dong-Mei Feng, PhD; Ruth F. Nutt, PhD; Jules A. Shafer, PhD; Robert J. Gould, PhD; Thomas M. Connolly, PhD

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.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
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 {approx}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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Thrombin plays an important role in promoting arterial thrombosis through diverse activities.1 It is a procoagulant molecule, promoting its own formation by activating the zymogen form of several clotting factors and by catalyzing the cleavage of fibrinogen to fibrin.2 It also directly activates platelets and a variety of other cells via cell surface receptors.3 A thrombin receptor has been cloned from the human megakaryocytic DAMI cell line4 and from hamster CCL39 fibroblasts.5 This receptor belongs to the family of 7-transmembrane receptors whose activation signal is transduced by a guanine nucleotide binding protein. A novel mechanism of activation of this receptor has been proposed in which thrombin cleaves the receptor to create a new amino terminus that can then act as a tethered ligand to activate the receptor.6 Peptides as short as 5 residues derived from the sequence of the new amino terminus are able to fully activate naive cells transfected with the clone for the thrombin receptor. These receptor-derived peptides can also fully activate human platelets.7 8 9 10 11 12

Damaged vessel walls and exposed mural thrombi are sites of thrombin generation and of residual adsorbed thrombin.13 Thrombin is naturally inhibited by endogenous antithrombin III, and this reaction is enhanced by heparin14 ; however, heparin is not always efficacious in animal models of thrombosis.15 16 17 18 19 The lower efficacy of heparin may reflect its inability to catalyze the inactivation of clot-bound thrombin by antithrombin III. There are also other inhibitors of thrombin including small molecule catalytic site inhibitors,20 the leech protein hirudin,21 and hirulog, a hybrid comprised of the C-terminal domain of hirudin and the active site–directed inhibitor D-Phe-Pro-Arg.22 Hirudin and these small molecule inhibitors have been shown to be antithrombotic, eliminating thrombus formation in animal models of deep arterial injury, arteriovenous shunts, electrolytic injury, venous stasis, disseminated intravascular coagulation, and restenosis after percutaneous transluminal coronary angioplasty.15 16 17 18 19 23 24 25 26 However, a potential limitation to the use of hirudin or the low-molecular-weight inhibitors is that at a concentration that is effective in these models, primary hemostasis could be compromised since clotting time or template bleeding time are prolonged.16 17 24 25 The direct thrombin inhibitors, recombinant hirudin (r-hirudin) and Hirulog, have also demonstrated efficacy in preliminary human trials. r-Hirudin showed benefit over heparin in angiographic improvement of the arterial lesion in unstable angina,27 and Hirulog used as adjunctive therapy to streptokinase significantly improved the early patency rate of the infarct-related artery.28 It is not known if efficacy of the thrombin inhibitors is due primarily to their prevention of thrombin-mediated fibrin formation or of platelet activation or if inhibition of both is required. The lack of a potent and selective inhibitor of each of these processes has not allowed this question to be addressed.

We prepared neutralizing antibodies against the cloned thrombin receptor, characterized their activity against human and monkey platelets, and evaluated their effect in the model29 of cyclic flow reductions (CFRs), which was modified for use in the carotid artery of the African Green monkey.30 In this model, under conditions of arterial stenosis and endothelial injury, platelets adhere to exposed subendothelium and blood flow gradually declines as an intravascular thrombus forms. This provided an animal model of arterial thrombosis for investigation of the in vivo role of thrombin-stimulated platelet activation. The effect of thrombin receptor blockade on CFRs, platelet aggregation, bleeding time, and clotting parameters was evaluated and compared with that of blockade of platelet aggregation with a glycoprotein IIb/IIIa inhibitor, MK-0852, and the thrombin inhibitor r-hirudin. The current results indicate an important role for the thrombin receptor in promoting thrombus formation in this model.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
down arrowDiscussion
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Materials
Peptide conjugation and protein G IgG purification kits were from Pierce; protein G and A sepharoses were obtained from Pharmacia; polyacrylamide gels were from Novex; peroxidase-labeled goat anti-rabbit IgG was from Cappel; the Western blot detection system was the ECL detection kit from Amersham; control rabbit IgG and human thrombin were from Sigma; and H2N-Gly-Pro-Arg-Pro-OH was from Bachem.

Antibody Preparation
New Zealand White rabbits were immunized with the 15-residue peptide KYEPFWEDEEKNESC-NH2 conjugated to either rabbit serum albumin or keyhole limpet hemocyanin (KLH) via the terminal cysteine. This receptor peptide corresponds to amino acid residues 51 to 64 of the thrombin receptor, TR,51-64 thought to represent a thrombin binding site31 plus an additional carboxy terminal cysteine for coupling. The bleeds were screened initially by ELISA assay against both conjugated peptides, nonconjugated peptides, and irrelevant peptides. IgG from the ELISA-positive sera were isolated using protein G kits and evaluated as inhibitors of thrombin-stimulated platelet aggregation. Immune IgG from the rabbit, which was the most potent inhibitor of platelet aggregation, was purified on a larger scale on both protein G and protein A sepharoses for use in the in vivo studies. The isolated IgGs were evaluated as inhibitors of thrombin-stimulated platelet aggregation and secretion.

Production of Peptides and Other Platelet Inhibitors
Thrombin receptor peptides were synthesized as previously described.32 The crude peptide products were purified by preparatory high-performance liquid chromatography on a DELTA-PAK C18 column. Fractions containing product of at least 99% purity were combined and characterized for amino acid composition after 6N HCl acid hydrolysis. Peptides synthesized as carboxyl terminal amides are indicated by the symbol (-NH2). The fibrinogen receptor antagonist MK-0852 was prepared as described,33 and r-hirudin was expressed in Saccharomyces cerevisiae yeast and isolated as previously described.34

In Vitro Assessment of Antiplatelet Activity
Platelet Aggregation and Secretion
Blood was collected from healthy human volunteers free of aspirin and other drugs for at least 8 days into 3.8% trisodium citrate anticoagulant (1:10). The erythrocytes and white cells were sedimented from the whole blood and separated from the platelet-rich plasma by centrifugation. Platelets were isolated from the plasma by differential centrifugation and then were washed in a modified Tyrode's buffer (5 mmol/L HEPES, 0.3 mmol/L NaH2PO4, 3 mmol/L KCl, 134 mmol/L NaCl, 5 mmol/L glucose, 2 mmol/L MgCl2, 12 mmol/L NaHCO3, pH 6.5) containing 1 mmol/L EGTA, 20 mg/mL apyrase, and 3.5 mg/mL bovine serum albumin as previously described.35 The final platelet suspension was at 2x108 platelets per milliliter in the same buffer at pH 7.4 without EGTA.

Characterization of the effect of the thrombin receptor IgG on platelet aggregation was carried out in 96-well plates (an assay developed by B. Bednar, C. Condra, R.J. Gould, and T.M. Connolly to measure platelet aggregation in a 96-well microplate reader). Briefly, the washed platelets with 0.2 mg/mL human fibrinogen were incubated with the IgG for 30 minutes at room temperature. Thrombin was added to the platelets, and they were agitated. Platelet shape change and aggregation were monitored as a decrease or increase in light transmittance, respectively, in a Vmax microplate reader. The amount of light transmittance (extent of aggregation) or the rate of change of light transmittance (rate of aggregation) of the samples incubated with the immune IgG was compared with that of both untreated control samples and samples treated with preimmune IgG. Secretion was monitored as the release of platelet-dense granule [3H]-serotonin in response to thrombin, as previously described,35 with a minor modification. The current assays were carried out in 100 µL in 96-well plates rather than in tubes, and correspondingly smaller aliquots were used for radioactivity determinations.

Aggregation of platelets from African Green monkeys was carried out in platelet-rich plasma as previously described35 or in a whole blood assay36 according to the following procedure. Blood was collected into 3.8% trisodium citrate anticoagulant (1:10). For the in vitro studies, 0.4 mL of blood was incubated for 30 minutes with immune IgG or vehicle at room temperature. The treated blood was then transferred to a stirring (1000 rpm) aggregometer at 37°C and incubated for 3 minutes with 4 mmol/L H2N-Gly-Pro-Arg-Pro-OH peptide to prevent thrombin-mediated fibrin polymerization.37 Thrombin was added to the stirred sample, and after an additional 4 minutes, the cuvette was removed and a platelet count was determined with an automated hematology analyzer (Serono-Baker Diagnostics). A decrease in the number of single platelets was used to measure the extent of platelet aggregation. Preliminary results with this method were comparable to those seen with the measurement of platelet aggregation by light transmittance in platelet-rich plasma. Ex vivo platelet aggregation was carried out using the same method as described for in vitro aggregation; the response to increasing concentrations of thrombin (5 to 100 nmol/L) was tested at each time point.

In Vivo Assessment of Antiplatelet Activity
All procedures used in this study were conducted according to the principles of the American Physiological Society and were approved by the Institutional Animal Care and Use Committee at Merck Research Laboratories, West Point, Pa.

Surgical Preparation
African Green monkeys of either sex weighing 3.4 to 6.7 kg were sedated with ketamine HCl (10 mg/kg IM) and anesthetized with sodium pentobarbital (12.5 mg/kg IV). After intubation for mechanical ventilation with room air and vessel cannulation (right femoral artery for continuous monitoring of hemodynamic parameters and blood collection, right femoral vein for supplemental anesthesia, and left femoral vein for compound administration), animals were surgically prepared for CFRs. Supplemental anesthesia was administered intravenously as needed (0.2 to 0.4 mL, 65 mg/mL sodium pentobarbital). A 4-cm segment of the left carotid artery was isolated and instrumented from proximal to distal with a flow probe (Doppler to measure blood flow velocity or electromagnetic, Carolina Medical Instruments, to measure blood flow), a Lexan or silver clip constrictor, and a snare ligature. Physiological parameters were recorded on a model 7D polygraph (Grass Medical Instruments). The vessel lumen was constricted such that mean blood flow was reduced an average of 60%, and the phasic pattern was nearly abolished; endothelial damage was induced by repetitively pinching the vessel from the outside. The accumulation of platelet aggregates in the vessel lumen was observed as a gradual reduction in blood flow. When flow reached its lowest level, the platelet plug was mechanically dislodged, and carotid arterial blood flow was restored. When using the silver clip, the clip was adjusted if necessary to ensure that the maximal blood flow that was established at the start of the protocol was not exceeded. The cyclical pattern of flow reductions was repeated for the duration of the protocol or until disrupted by an effective antithrombotic agent. Beginning at 60 minutes after administration of each agent, the vessel was redamaged at 15-minute intervals by pinching with a hemostat to ensure exposure of thrombogenic subendothelium. CFRs were monitored for 120 minutes after administration of each agent to determine the duration of in vivo antithrombotic effect.

Experimental Protocol
After instrumentation, the vessel was allowed to equilibrate for a minimum of 30 minutes before initiating CFRs. After 15 minutes of consistent CFRs, the vehicle was administered, and 15 minutes later, the test treatment was begun (time 0). MK-0852 (3 µg/kg+1 µg · kg-1 · min-1 or 10 µg · kg+3 µg · kg-1 · min-1) and r-hirudin (0.5 mg/kg+0.015 mg · kg-1 · min-1) were both given as a bolus followed by a 60-minute infusion, and the control IgG and the thrombin receptor antibody IgG 9600 were given as a bolus (10 mg/kg). A blood sample was taken before the initiation of CFRs for platelet aggregation and activated partial thromboplastin time (APTT) measurements, and control bleeding time was determined. During CFRs, these same parameters were measured after the administration of the vehicle and at 30-minute intervals (for 120 minutes) after the intravenous bolus infusion of the test compound: r-hirudin, MK-0852, thrombin receptor antibody (IgG), or control rabbit IgG. This protocol is summarized in Fig 1Down. Ex vivo platelet aggregation was performed as described above.



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  		Figure 1. Experimental protocol. Cyclic flow reductions (CFRs), vehicle infusion, and experimental treatment were initiated at the times indicated. MK-0852 and recombinant hirudin (rHirudin) were administered as a bolus+infusion, and the thrombin receptor and control antibodies were given as a bolus at the doses indicated in "Methods." Blood samples were taken for platelet aggregation and activated partial thromboplastin time, and bleeding time was measured at the following time points: -60, -5, +1, +30, +60, +90, and +120 minutes.

Bleeding Time Determinations
Template bleeding times were measured with a SIMPLATE bleeding time device (Organon Teknika Corporation). A sphygmomanometer was placed on the upper arm and inflated to 40 mm Hg; uniform incisions were made on the muscular area of the forearm, and the duration of bleeding was measured to a maximum of 15 minutes.

Activated Partial Thromboplastin Time
APTT values were determined as an indication of the function of the intrinsic clotting factors using an automated clot timer (Electra 900, Medical Laboratory Automation) and commercially available reagents (American Dade) according to the protocol provided by the supplier.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
down arrowReferences
 
Antibody Production and Characterization
A series of four rabbits were immunized with thrombin receptor peptide TR51-64 linked to either rabbit serum albumin or KLH. Evaluation for immune titer by ELISA revealed the production of antibodies against both conjugated and free peptide (data not shown). No reaction was observed with a peptide from the tethered ligand region of the receptor or if preimmune serum was used. The immune IgGs were isolated from several of the bleeds, and their ability to inhibit thrombin-stimulated platelet aggregation was evaluated. The most potent inhibitor of aggregation of the various immune IgGs was isolated from rabbit 9600. This antibody (IgG 9600) was used for most of the subsequent studies. The IgG 9600 decreased both the rate and final extent of thrombin-stimulated aggregation of human platelets in buffer, as shown in Fig 2ADown. This inhibition was dependent on the concentration of IgG and required an incubation of at least 20 minutes with the platelets. The IC50 for IgG 9600 to inhibit the final extent of platelet aggregation at 5 minutes after thrombin stimulus in this assay was 20 µg/mL. This IgG was next tested as an inhibitor of thrombin-stimulated platelet secretion. Unlike many platelet agonists, thrombin promotes direct, aggregation-independent secretion. IgG 9600 blocked platelet secretion to 1 nmol/L thrombin, which alone evoked a nearly maximal response. The IC50 for this inhibition was {approx}8 µg/mL IgG, as shown in Fig 2BDown. This antibody also inhibited the aggregation of human platelets in plasma, while aggregation and secretion induced by other agonists including ADP, collagen, and the thrombin receptor peptide SFLLR-NH2, as well as thrombin catalytic activity, were not inhibited by this antibody (data not shown). Western blot analysis of human platelets with IgG 9600 identified an {approx}64-kD protein as the prominent band, as shown in Fig 3Down. The size of this band is larger than that expected for the deduced amino acid composition of this receptor from the cDNA but is similar to that reported by others using a polyclonal38 or a monoclonal39 antibody to a similar peptide from the same thrombin-binding exosite region of the thrombin receptor.



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  		Figure 2. Plots show inhibition of thrombin-induced aggregation and secretion of human platelets by thrombin receptor IgG. Washed human platelets were incubated with 0.2 mg/mL fibrinogen and the indicated concentration of IgG 9600 for 30 minutes at room temperature, stimulated with 1 nmol/L human thrombin, and aggregation (A) and secretion (B) were monitored as described in "Methods." A is from one experiment representative of four separate experiments; B is mean±SEM (n=3). Control secretion is 74%.



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  		Figure 3. Western blot of the thrombin receptor: 25 µg human (H) or African Green monkey (M) platelet protein solubilized in SDS buffer was run on 8% polyacrylamide gel electrophoresis gels (Novex). The protein samples were transferred to nitrocellulose and blotted with 70 µg preimmune (lanes 1 and 2) or immune (lanes 3 and 4) IgG from rabbit 9600. A peroxidase-labeled anti-rabbit IgG from Cappel and an ECL detection kit from Amersham were used for detection.

Since the major goal of our studies was to use the antibody as a receptor-specific tool in in vivo studies, it was necessary to determine if it cross-reacted with platelets from other species commonly used in thrombosis studies. As human thrombin receptor–derived ligand peptides are able to promote full aggregation of platelets only from primates and guinea pigs,32 40 41 we first studied the interaction of this antibody with platelets from these species. Antibody 9600 identified a protein in African Green monkey whole platelet homogenates in Western blotting that was the same size as that from human platelet homogenates, as shown in Fig 3Up. This antibody also inhibited thrombin-stimulated platelet aggregation in African Green monkey platelet-rich plasma (Fig 4ADown). As with human platelets, this IgG had no effect on the platelet aggregation response to ADP, collagen, or a thrombin receptor activating peptide (TRAP) (Fig 4ADown). IgG 9600 was next tested to see if it inhibited thrombin-induced aggregation of African Green monkey platelets in whole blood. As shown in Fig 4BDown, this antibody completely inhibited aggregation induced by up to 100 nmol/L thrombin. In contrast to this result with monkey platelets, antibody 9600 did not block the aggregation of platelets from the guinea pig. Therefore, the species cross-reactivity in Western blots and in the functional assays suggested the use of antibody IgG 9600 in a thrombosis model in the African Green monkey.



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  		Figure 4. Graphs show aggregation of African Green monkey platelets. A, Platelets in plasma were stimulated by thrombin, ADP, collagen, and thrombin receptor activating peptide (TRAP) and aggregation-monitored as in "Methods." Inhibition of thrombin-induced aggregation by 30 µg/mL of the thrombin receptor antibody IgG 9600 (+) is shown along with the control response (-) of these platelets to thrombin. Aggregation responses to 20 µmol/L ADP, 10 µg/mL collagen, and 3 µmol/L TRAP are shown in the presence of 30 or 60 µg/mL IgG 9600 (+). Results are from one experiment in duplicate and are representative of two separate experiments. B, IgG (30 µg/mL) was incubated with citrated whole blood from the African Green monkey in the presence of 4 mmol/L Gly-Pro-Arg-Pro (to prevent thrombin-mediated fibrin polymerization) followed by stimulation with thrombin as described in "Methods." Platelet aggregation was determined by decrease in whole blood platelet count. Results are from one experiment in triplicate and are representative of three separate experiments.

In Vivo Model of Thrombosis in the African Green Monkey
IgG 9600 was compared with a potent and specific platelet inhibitor, MK-0852,33 and with the active site–directed thrombin inhibitor r-hirudin in a model of CFRs in the African Green monkey (Table 1Down). The GPIIb/IIIa receptor antagonist was studied for comparison, since it had previously demonstrated efficacy in a variety of thrombosis models including the model of CFRs and the more severe electrolytic injury model, both in the canine coronary artery.42 For the purposes of the present study, MK-0852 was tested with two dosing regimens: 10 µg/kg IV bolus+3 µg · kg-1 · min-1 for 60 minutes (10+3, n=3) and 3 µg/kg IV bolus+1 µg · kg-1 · min-1 for 60 minutes (3+1, n=3). At the higher dose, CFRs were abolished immediately after bolus administration and remained inhibited throughout the infusion in all animals, as shown in Fig 5Down. The baseline pattern of CFRs returned in an average of 51 minutes after the end of the infusion. Although the ex vivo platelet aggregation response to thrombin was only modestly reduced at the 1-minute postbolus time point (data not shown), it was completely inhibited at 60 minutes (end) into the infusion of MK-0852 (Fig 5Down). At the 120-minute time point (60 minutes after the end of the infusion), the ex vivo response to thrombin was similar to the pretreatment response, which corresponded with the return of intravascular thrombus formation (CFRs). The administration of a lower dose of MK-0852 (3+1) resulted in an initial prolongation of the CFR cycle length, and only by the end of the 60-minute infusion were CFRs abolished (data not shown). At the end of the lower-dose MK-0852 infusion, ex vivo thrombin-induced platelet aggregation was only partially inhibited in response to 20, 50, and 100 nmol/L thrombin; the response was about 50% of the response to these concentrations of thrombin in the pretreatment sample. In comparison, at the same time point, the response to thrombin was completely inhibited by the higher dose of MK-0852. CFRs returned in 27 minutes after the end of the lower-dose MK-0852 infusion, which is more rapid than with the higher dose (51 minutes).


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  		Table 1. Effects of MK-0852, r-Hirudin, IgG 9600, and Control IgG on Cyclic Flow Reductions



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  		Figure 5. Graphs show effects of MK-0852 on cyclic flow reductions and ex vivo thrombin-induced platelet aggregation. Results shown represent in vivo and ex vivo effects at 60 minutes after the initiation of treatment (10 µg/kg bolus+3 µg · kg-1 · min-1 for 60 minutes), which corresponds to the end of the infusion. Platelet aggregation was determined by the decrease in whole blood platelet count in response to increasing concentrations of thrombin (5 to 100 nmol/L).

The effect of direct thrombin inhibition was also investigated in this model using r-hirudin (0.5 mg/kg+0.015 mg · kg-1 · min-1 for 60 minutes). CFRs were immediately abolished after the administration of the bolus in all four monkeys, remained inhibited during the infusion, and returned to the baseline pattern of cyclical flow reduction in an average of 93 minutes (n=4) after the end of the infusion. Ex vivo platelet aggregation induced by all concentrations of thrombin was inhibited at 1, 30, and 60 minutes after the r-hirudin bolus. The ex vivo response to thrombin partially returned by 120 minutes (60 minutes after the end of the infusion), as aggregation was observed in response to the highest thrombin concentrations tested (50 and 100 nmol/L). Fig 6Down shows a representative blood flow tracing demonstrating the inhibition of CFRs after r-hirudin administration and the inhibition of thrombin-induced aggregation at the end of the 60-minute infusion.



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  		Figure 6. Graphs show effects of recombinant hirudin (rHirudin), a direct thrombin inhibitor, on cyclic flow reductions and ex vivo thrombin-induced platelet aggregation. Results shown represent in vivo and ex vivo effects at 60 minutes after the initiation of treatment (0.5 mg/kg bolus+0.015 mg · kg-1 · min-1 for 60 minutes), which corresponds to the end of the infusion. Platelet aggregation was determined by the decrease in whole blood platelet count in response to increasing concentrations of thrombin (5 to 100 nmol/L).

The demonstration of efficacy with the GPIIb/IIIa antagonist MK-0852 confirmed the contribution of platelets to the development of CFRs and the inhibitory effect of r-hirudin identified a role for thrombin in this model of intravascular thrombosis. However, the effect of selective blockade of the platelet thrombin receptor remained to be investigated. IgG 9600 as an intravenous bolus at 10 mg/kg clearly showed activity in all four monkeys in which it was tested. It immediately and completely inhibited CFRs in three of the four animals and reduced by 50% the CFR frequency in the final monkey. Fig 7Down shows baseline CFRs and the immediate abolition of this intravascular event in response to the thrombin receptor antibody in a representative monkey. In the three animals in which CFRs were abolished, there was no return of thrombus accumulation, as evidenced by flow reduction for 120 minutes after the bolus was given despite repeated vessel redamage over the last 60 minutes. In addition, the 50% reduction in CFR frequency in the fourth monkey persisted for the entire 120 minutes of observation. Ex vivo thrombin-induced platelet aggregation was completely inhibited at 30, 60, 90, and 120 minutes after the administration of the bolus at all concentrations of thrombin (5 to 100 nmol/L). The mean ex vivo platelet aggregation response to thrombin (n=4) is also shown in Fig 7Down for samples taken at the 60-minute postbolus time point. Normal rabbit IgG had no significant effect on any of the parameters measured (see Fig 8Down). In addition, after the vehicle infusion at the start of each experiment, no changes were observed in CFR pattern or frequency, and the ex vivo response to thrombin was not altered. Table 1Up summarizes the effect of all treatments on CFRs.



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  		Figure 7. Graphs show effects of IgG 9600 on cyclic flow reductions and ex vivo thrombin-induced platelet aggregation. Results shown represent in vivo and ex vivo effects at 60 minutes after the initiation of treatment (10 mg/kg IV bolus). Platelet aggregation was determined by the decrease in whole blood platelet count in response to increasing concentrations of thrombin (5 to 100 nmol/L).



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  		Figure 8. Graphs show effects of control IgG on cyclic flow reductions and ex vivo thrombin-induced platelet aggregation. Results shown represent in vivo and ex vivo effects at 60 minutes after the initiation of treatment (10 mg/kg IV bolus). Platelet aggregation was determined by the decrease in whole blood platelet count in response to increasing concentrations of thrombin (5 to 100 nmol/L).

Effect of Inhibitors on Hematologic Parameters
Bleeding times and clotting times expressed as APTT are summarized for all experimental groups in Table 2Down. These values represent measurements from the 60-minute post–drug administration (bolus or end of infusion) time point and represent the peak effect for each treatment and correspond to the time period of observed in vivo intravascular effects. The higher dose of the fibrinogen receptor antagonist MK-0852 (10+3) caused a prolongation of bleeding time of 6.5-fold, while the APTT was not affected by this treatment. With the low dose of MK-0852 (3+1), the bleeding time was only slightly increased (1.5-fold), and the APTT was not altered. Immediately after the initial bolus of r-hirudin, APTT was at least eightfold greater than baseline. However, APTT values rapidly decreased to a fourfold elevation at 5 minutes after the bolus and were maintained at that level for the duration of the infusion. Bleeding time was only slightly prolonged, 1.4-fold above baseline, at 60 minutes after the end of the r-hirudin infusion. With antibody 9600, at the 60-minute postbolus time point, bleeding time was only moderately elevated (twofold) and APTT was not affected. Normal rabbit IgG had no effect on bleeding time or clotting time.


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  		Table 2. Effects of MK-0852, r-Hirudin, IgG 9600, and Control IgG on Bleeding Time and Activated Partial Thromboplastin Time

Although MK-0852, r-hirudin, and control IgG had no significant effect on whole blood platelet count, a gradual decrease was observed after the intravenous bolus administration of IgG 9600. The mean platelet count±SEM (1000/µL) before administration of this antibody was 279±11, and the postdose values were as follows: 30 minutes, 256±14; 60 minutes, 233±15; 90 minutes, 230±10; and 120 minutes, 225±10 (n=4). Although the 17% and 19% decreases at 90 and at 120 minutes, respectively, were only modest, they were statistically different (P<.05, ANOVA followed by Dunnett's multiple comparison) from the baseline platelet count. The platelet counts during the initial observation of the in vivo antithrombotic effect promoted by IgG 9600, at 30 and 60 minutes after the administration of the antibody, however, were not significantly reduced from pretreatment values.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
Thrombin is a potent activator of platelets, promoting shape change, secretion of granular contents, synthesis and secretion of thromboxane A2, and platelet-platelet interactions.1 3 Because of its potent stimulatory effect on platelets and the localization of thrombin to a developing thrombus, thrombin is likely to contribute significantly to thrombogenesis and the resultant vascular occlusion.43 The role of thrombin in occlusive disorders is supported by the results of clinical studies that demonstrate the efficacy of direct thrombin inhibitors in unstable angina and as adjunctive therapy to thrombolysis.27 28 A mechanism by which thrombin activates its functional receptor on platelets and other cells has recently been proposed.4 6 The cleavage of the N-terminal portion of the receptor by this serine protease creates a new amino terminus that functions as a tethered ligand and thereby activates the receptor. Synthetic peptides (5 to 14 amino acids), the sequences of which were derived from this new amino terminus, have been shown to mimic the effect of thrombin on platelets.7 8 9 10 11 12 Stimulation of platelets by thrombin can be prevented either by the direct inhibition of thrombin catalytic activity or by the blockade of the cell surface thrombin receptor. Antagonism of the thrombin receptor has been demonstrated in vitro using antibodies directed against the tethered ligand or thrombin-binding exosite portion of the thrombin receptor.38 39 Inhibition of this receptor with a weak antagonist showed only limited efficacy in an animal model.44 Therefore, previous efforts to demonstrate the in vivo effect of inhibition of this receptor were limited by the tools available.

The antibody against the cloned thrombin receptor that was used in the current study inhibited both secretion and aggregation of human platelets stimulated by thrombin but not by ADP, collagen, or a thrombin receptor agonist peptide. Thrombin-induced platelet aggregation was inhibited by this antibody in several primate species, including the African Green monkey, but not in a variety of nonprimate species. Therefore, a model of arterial thrombosis in the African Green monkey was selected for this study. In this model, CFRs are experimentally induced in stenosed and deendothelialized carotid arteries. This repetitive pattern of flow reduction has been characterized both histologically29 and pharmacologically45 46 as a predominantly platelet-dependent phenomenon. Adherent platelets present on the damaged endothelium and platelet aggregates comprise a significant portion of the formed thrombi in the lumen of the constricted and injured vessel.47 48 Interruption of CFRs has been shown in the canine coronary artery with numerous antiplatelet agents including aspirin, prostacyclin, thromboxane synthetase inhibitors, thromboxane A2 receptor antagonists, the 5-HT antagonist ketanserin, and the glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitor MK-0852.42 48 Efficacy was also demonstrated with the monoclonal antibody directed against the platelet GPIIb/IIIa receptor 7E3 in the carotid artery49 50 and by thromboxane receptor antagonism in the renal artery30 51 of the cynomolgus monkey. The thrombin inhibitor MCI-9038 was reported to interrupt CFRs early (30 minutes) but not late (3 hours) after induction in the dog.52 Hirulog, a synthetic, hirudin-based, thrombin-inhibitory peptide, prevented thrombotic arterial occlusions in the carotid artery of the pig.19 However, therapeutic anticoagulant levels of the antithrombin III–dependent thrombin inhibitor heparin were less effective in inhibition of CFRs than direct thrombin inhibitors.19 46 Although both platelet inhibitors and thrombin inhibitors have demonstrated efficacy in this model, it has not yet been shown if blockade of the specific interaction between thrombin and platelets would also be effective.

Based on results of previous studies with antiplatelet and antithrombin agents, we selected doses of MK-0852, the platelet GPIIb/IIIa antagonist, and r-hirudin that either completely inhibited ex vivo ADP-induced platelet aggregation42 or elicited a three- to fourfold increase in APTT,19 respectively. These levels were previously shown to be necessary to achieve in vivo antithrombotic activity in animal models of arterial thrombosis. The administration of both MK-0852 and r-hirudin resulted in the inhibition of intravascular thrombus formation. The in vivo efficacy demonstrated by MK-0852 and r-hirudin therefore established that both platelet aggregation and thrombin activity play a significant role in the intravascular event that results in the cyclical pattern of flow reduction in this model. The thrombin receptor antibody IgG 9600 provided us with a specific tool to investigate the potential contribution of the platelet thrombin receptor, as opposed to all thrombin-mediated events, to arterial thrombus formation in the monkey. This thrombin receptor antibody showed complete and immediate abolition of CFRs in three of the four monkeys. Activity was also evident in the fourth animal, as CFR frequency was reduced by 50%. IgG 9600 also prevented the return of thrombus formation in response to increasing intimal damage in all monkeys. In addition, this antibody completely inhibited the ex vivo platelet aggregation response to all concentrations of thrombin at all time points. These results demonstrate that the thrombin receptor on platelets plays a significant role in the thrombotic event that leads to flow reduction in this model.

The relation between the effects on CFRs and ex vivo platelet aggregation was not the same for all agents tested. The inhibition of the ex vivo response to thrombin was more pronounced in the higher-dose MK-0852 group than in the lower-dose group at the end of the infusion (60 minutes). Platelet aggregation in response to lower thrombin concentrations (5 to 20 nmol/L) was inhibited by both dosing regimens, but the higher concentrations of thrombin (50 and 100 nmol/L) were only completely inhibited in the higher-dose MK-0852 group. However, CFRs in all animals in both groups were abolished at this same time point (60 minutes). This suggests that thrombin may not be present locally in the artery at the concentrations represented by the higher concentrations on the thrombin curve or that some intermediate effect by MK-0852 on the inhibition of thrombin-induced platelet aggregation is sufficient to prevent the intravascular event. Since the thrombin concentration in a human thrombus that develops over an extended period of time has been determined to be approximately 100 nmol/L,53 it is conceivable that the local thrombin concentration in the thrombus formed acutely in this model might be <100 nmol/L. A similar dissociation of the effect on the inhibition of CFRs and on ex vivo aggregation was previously reported with antibodies directed against the platelet GPIIb/IIIa receptor.49 Abolition of platelet thrombus formation in the carotid artery of the cynomolgus monkey was achieved at doses that did not abolish ex vivo platelet aggregation.49 Alternatively, ex vivo aggregation in response to thrombin can be inhibited without in vivo efficacy in the model of CFRs in the African Green monkey carotid artery. Other antibodies were raised against the thrombin-binding exosite and the tethered ligand region of the cloned thrombin receptor that were less potent inhibitors of in vitro thrombin-induced human platelet aggregation than IgG 9600. In preliminary experiments, two of these other antibodies were administered to the African Green monkey at the same dose as the dose of IgG 9600 used in the current studies. Although they reduced ex vivo thrombin-induced platelet aggregation, they did not inhibit CFRs in the carotid artery (data not shown). The relationship between ex vivo platelet aggregation and in vivo CFR production remains to be established.

The results of the effect of the four different treatment groups on primary hemostasis indicate that in vivo antithrombotic activity can occur at doses that do not elevate template bleeding time. Although the higher dose of MK-0852 (10+3) produced a peak prolongation of bleeding time of 6.5-fold over baseline, the lower dose (3+1) was also fully efficacious, and bleeding time was only extended 1.5-fold. The maximum prolongation of bleeding time in the r-hirudin–treated group was 1.75-fold and was observed immediately after the initial loading bolus; APTT values suggest that plasma levels of r-hirudin peaked at this time point. During the interruption of CFRs by r-hirudin, bleeding time was not different from pretreatment. As mentioned above, the thrombin receptor antibody IgG 9600 showed complete inhibition of CFRs at a dose that increased bleeding time only twofold. In addition, there was no observation of excessive surgical bleeding or rebleeding from previous hemostatic plugs in the group of monkeys receiving antibody IgG 9600. Previous studies by other investigators using a GPIIb/IIIa antibody50 or a thromboxane receptor antagonist51 did not report bleeding time or had difficulty in obtaining reproducible results with forearm template bleeding time measurements. However, in the present study, pretreatment bleeding times were extremely consistent, as were peak bleeding times in response to the various treatments. Baseline bleeding times in all groups were 1.0 to 1.5 minutes, and these did not change during the administration of vehicle or control IgG. A significant effect was observed with the higher dose of the GPIIb/IIIa inhibitor MK-0852, and the time course of the prolongation of bleeding time correlated with the time course of the inhibition of ex vivo thrombin-induced platelet aggregation. With this higher dose of MK-0852, bleeding time was elevated 1.7-fold at 1 minute after the bolus and start of infusion, along with a slight inhibition of platelet aggregation. The effect on bleeding time and the inhibition of aggregation both increased at 30 minutes (4.7-fold increase in bleeding time) and again at 60 minutes (6.5-fold increase in bleeding time) after the start of the infusion and returned toward baseline 60 minutes after the end of the infusion (2.5-fold increase in bleeding time). In addition, the peak effect of r-hirudin on bleeding time occurred concomitantly with the peak effect on APTT. The effect of IgG 9600 on ex vivo platelet aggregation induced by thrombin was the same as that of the higher dose of MK-0852; however, the thrombin receptor antibody elicited a mean (n=4) maximal effect on bleeding time of 2.8-fold increase at 120 minutes after bolus administration. The results of these various treatments indicate that the procedure used for bleeding time measurement in this study was sensitive enough to identify actual effects on this measure of hemostasis and that the thrombin receptor antibody did not significantly alter this critical parameter. Similarly, in a subsequent study, the GPIIb/IIIa antibody mentioned above was shown to inhibit intravascular thrombus formation at a dose that had only modest effects on bleeding time using an alternate, reproducible method for determination of bleeding time.49

It is not known if the dose of IgG 9600 used in the present study is the minimally efficacious dose. Due to the limited supply of this antibody, we were not able to study a range of doses and therefore do not know if a lower dose would be sufficient for the activity observed. It is also impossible to predict if the efficacious dose, which did not suggest bleeding complications, would be sufficient for activity in other animal models. A higher dose of this antibody might be necessary for antithrombotic activity in more severe models, and the effect of other doses on primary hemostasis is unknown. Additional antibodies have been prepared, and these questions will be addressed in subsequent studies.

A moderate and gradual decrease (17% at 60 minutes and 19% at 120 minutes) in whole blood platelet count was observed over time after the bolus administration of the thrombin receptor antibody IgG 9600. This effect was not protocol related, as it was not observed in any other treatment groups. It also does not appear to be a nonspecific effect of IgG, since platelet count did not change in the control IgG group. The inhibition of CFRs occurred immediately after antibody administration; however, the platelet count was not significantly changed from pretreatment values until 90 minutes later. A reduction in platelet count, even to the level of thrombocytopenia, is not unprecedented by molecules that bind to platelet receptors. For example, administration of a monoclonal antibody against the platelet GPIIb/IIIa complex to the baboon resulted in a significant reduction in platelet number.54 In addition, administration of a von Willebrand fragment that binds to the platelet glycoprotein Ib receptor produced a transient but significant decrease in platelet number in the cynomolgus monkey.55 The effect on platelet number in the present study is less pronounced than that observed in those studies. The time course of the return of platelet count to normal after IgG 9600 administration is not known, since it was not observed by the end of this acute protocol. Thrombocytopenia could potentially present a bleeding risk to the patient; therefore, the safest antagonist of this receptor for clinical use would be one that did not typically reduce circulating platelet number.

In summary, an antibody that blocks the 7-transmembrane thrombin receptor was effective in prevention of intravascular thrombus formation in the model of CFRs, modified for use in the carotid artery of the African Green monkey. At an efficacious dose of IgG 9600, in which ex vivo platelet aggregation induced by thrombin was completely inhibited, this antibody did not alter coagulation time (APTT) and had little effect on bleeding time. MK-0852 and r-hirudin were also effective in this model in the absence of significant prolongation of bleeding time. These results demonstrate that blockade of the platelet thrombin receptor prevents arterial thrombus formation in a primate model of arterial thrombosis without altering hemostatic parameters and that efficacy can be achieved without inhibiting the enzymatic activity of thrombin for fibrinogen. Prevention of platelet secretion by blockade of the platelet thrombin receptor also gives this mechanism a potential advantage over inhibition of the GPIIb/IIIa receptor. Since thrombin contributes significantly to platelet recruitment at sites of vascular injury, blockade of the thrombin receptor on platelets is an attractive antithrombotic mechanism.


*    Acknowledgments
 
The authors would like to thank Dr William Schumacher of Bristol-Myers Squibb for sharing his expertise with the model of cyclic flow reductions in primates and Dr E. Dale Lehman for providing the r-hirudin used in these studies.

Received October 17, 1994; revision received November 28, 1994; accepted December 3, 1994.


*    References
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*References
 

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