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(Stroke. 2000;31:1402.)
© 2000 American Heart Association, Inc.

Original Contributions

Integrin _IIbß₃ Inhibitor Preserves Microvascular Patency in Experimental Acute Focal Cerebral Ischemia

Presented in part at a meeting of the International Society of Thrombosis and Haemostasis, Washington, DC, August 14–21, 1999.

Takeo Abumiya, MD, PhD; Robert Fitridge, MD; Curt Mazur, MS; Brian R. Copeland, MD; James A. Koziol, PhD; Juerg F. Tschopp, PhD; Michael D. Pierschbacher, PhD Gregory J. del Zoppo, MD

From the Department of Molecular and Experimental Medicine (T.A., B.R.C., J.A.K., G.J.d.Z.), The Scripps Research Institute, La Jolla, Calif; the Department of Surgery (R.F.), University of Adelaide, The Queen Elizabeth Hospital, Woodville, Australia; and the Integra LifeSciences Corp (C.M., J.F.T., M.P.), Corporate Research Center, San Diego, Calif.

Correspondence to Gregory J. del Zoppo, MD, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Rd, MEM 132, La Jolla, CA 92037. E-mail grgdlzop{at}hermes.scripps.edu

	Abstract

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Background and Purpose—Platelets become activated and accumulate in brain microvessels of the ischemic microvascular bed after experimental focal cerebral ischemia. The binding of glycoprotein IIb/IIIa (integrin _IIbß₃) on platelets to fibrinogen is the terminal step in platelet adhesion and aggregation. This study tests the hypothesis that inhibition of platelet-fibrin(ogen) interactions may prevent microvascular occlusion after experimental middle cerebral artery occlusion (MCA:O).

Methods—TP9201 is a novel Arg-Gly-Asp (RGD)-containing integrin _IIbß₃ inhibitor. Microvascular patency after 3-hour MCA:O and 1-hour reperfusion within the ischemic and nonischemic basal ganglia was compared in adolescent male baboons who received high-dose TP9201 (group A: IC₈₀ in heparin, n=4), low-dose TP9201 (group B: IC₃₀ in heparin, n=4), or no treatment (group C: n=4) before MCA:O.

Results—After MCA:O, microvascular patency decreased significantly in group C. However, in the ischemic zones of groups A and B compared with group C, patencies were significantly greater in the 4.0- to 7.5-µm-diameter (capillary) and 7.5- to 30.0-µm-diameter vessels (2P<0.05). A dose-dependent increase in hemorrhagic transformation was seen in group A (3 of 4 animals) compared with group B (1 of 4 animals), and no hemorrhage was visible in group C (² analysis for trend, P<0.05).

Conclusions—Platelet activation contributes significantly to ischemic microvascular occlusion. Occlusion formation may be prevented by this RGD–integrin _IIbß₃ inhibitor at a dose that does not produce clinically significant parenchymal hemorrhage. The effect of microvascular patency on neuron recovery can now be tested.

Key Words: cerebral ischemia, focal • integrins • microcirculation • platelet glycoprotein GPIIb/IIIa complex

	Introduction

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The participation of activated platelets in ischemic stroke has been deduced from the observation of refractile bodies in retinal arteries^{1 2} and of platelet emboli in the cerebral vasculature during carotid endarterectomy³ and by direct visualization within the cerebral circulation.⁴ The common observation of platelet-fibrin thrombi on carotid artery atheromas in patients with symptoms of transient cerebral ischemia and the numerous reports describing platelet activation after stroke onset provide further indirect support for the participation of platelets in cerebral ischemia arising from large-artery injuries.^{5 6} However, the accumulation of platelets in target cerebral microvascular beds in response to focal ischemia and their contribution to further injury have received little attention.

In situ accumulation of platelets in the cerebral microvasculature after middle cerebral artery (MCA) occlusion (MCA:O) has been observed in the nonhuman primate^{7 8 9} and the Wistar rat.¹⁰ An electron micrographic study confirmed the accumulation of degranulated platelets and fibrin together within microvessels of the ischemic territory.⁹ The finding of polymorphonuclear leukocyte–fibrin–platelet aggregates within postcapillary venules in tissues displaying "no-reflow" has suggested the active participation of platelets and polymorphonuclear leukocytes in microvascular occlusion formation.⁹ The deposition of ¹¹¹In-labeled platelets in the ischemic basal ganglia of primates was significantly reduced by the combination of ticlopidine and heparin given before MCA:O.⁸ Those observations suggest the general hypothesis that platelet accumulation in the microvasculature after MCA:O contributes to microvascular no-reflow, local ischemia, and further neuron injury.

Platelet-fibrin interactions are mediated by platelet glycoprotein (GP) IIb/IIIa (integrin _IIbß₃), which becomes activated upon platelet activation.^{11 12} Integrin _IIbß₃ interacts with fibrinogen in an arginine-glycine-aspartic acid (RGD)-dependent manner.^{11 12} Integrin _IIbß₃ receptor activation followed by fibrinogen ligation is the common terminal step of several identified cascades of steps leading to platelet activation,¹³ suggesting that inhibition of this receptor-ligand interaction may provide potent antiplatelet effects and the potential for therapeutic intervention. Plow and colleagues^{11 14} have demonstrated that RGD-containing peptides inhibit the binding of fibrinogen to thrombin-stimulated platelets via the integrin _IIbß_3. The Fab fragment of the humanized monoclonal antibody c7E3 inhibits platelet aggregation and platelet thrombus formation and reduces reocclusion after coronary artery angioplasty by binding to the integrin subunit ß₃.¹⁵ Inhibition of platelet integrin _IIbß₃ and/or vascular integrin _vß₃–mediated events most probably underlies those responses.¹³

TP9201 is a synthetic RGD-containing cyclic nonapeptide with a molecular mass of 1136 Da.¹⁶ It selectively blocks platelet integrin _IIbß₃ (and not _Vß₃) by competitive dose-dependent inhibition of the interaction of the receptor with fibrinogen in platelets activated by a variety of agonists. In vivo, TP9201 has been shown to inhibit thrombus formation on injured carotid arteries in a canine model.¹⁷ In other animal models of acute in situ coronary artery thrombosis, TP9201 contributed to an increased frequency of recombinant tissue plasminogen activator–associated coronary artery reflow¹⁸ and inhibited reocclusion after endothelial injury.¹⁹ Furthermore, in baboons, TP9201 has been shown to inhibit platelet accumulation on a Dacron graft mounted in an exteriorized arteriovenous shunt.²⁰ In all models, in vivo efficacy could be achieved at IC₈₀ to IC₉₀ or less as determined in heparinized plasma and, in most cases, without an increase in cutaneous bleeding time.^{18 20} This feature may be advantageous for any potential role of a GPIIb/IIIa inhibitor in cerebral ischemia when complications due to hemorrhage are severe.

The hypothesis tested by the present study states that platelet activation contributes substantially to the obstruction of the brain microvasculature after experimental MCA:O and that inhibiting the integrin _IIbß₃–fibrin(ogen) interaction can increase microvascular patency. A 3-arm, dose-sequential, placebo-controlled study of 2 dose rates of TP9201 was undertaken to test the effect of this integrin _IIbß₃ inhibitor on microvascular patency and the risk of intracerebral hemorrhage when begun immediately before MCA:O. TP9201 significantly inhibited microvascular occlusion, but a dose-related increase in clinically significant intracranial hemorrhage was observed.

	Materials and Methods

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All procedures in the present study were approved by the Institutional Animal Research Committee and were performed in accordance with the standards published by the National Research Council (The Guide for Care and Use of Laboratory Animals) and the US Department of Agriculture and Animal Welfare Act. In compliance with these standards, every effort was made to ensure that the animals were free of pain or discomfort. Before undergoing the experimental procedures, all animals were clinically normal and free of apparent infection or inflammation and showed no neurological deficits.

Integrin _IIbß₃ Inhibitor TP9201
TP9201 is a synthetic cyclic nonapeptide with the composition acetyl-L-cysteinyl-L-asparaginyl-L-propyl-arginylglycyl-L-aspartyl-O-methyl-L-tyrosyl-L-arginyl-L-cysteinamide, cyclic 1- to 9-sulfide. It was synthesized in an automated fashion (model 430A, Applied Biosystems) and purified by high-performance liquid chromatography as previously described.¹⁶ Purity was >98%. TP9201 inhibits platelet aggregation in citrated human plasma at an IC₅₀ of 0.2 µmol/L.^{18 19} The peptide was dissolved in 0.9N NaC1 at an initial concentration of 10 mg/mL and then diluted in saline to the desired infusion concentration. The agent was delivered intravenously with a loading bolus (5 mL), followed by continuous infusion (30 mL/h). The pharmacokinetic and pharmacodynamic half-life values of TP9201 in baboons were identical (60 to 70 minutes). The plasma level required for inhibition of ex vivo platelet aggregation was in agreement with plasma levels required for in vitro platelet aggregation in platelet-rich plasma (PRP) or whole blood.²⁰

Cohorts
Twelve adolescent male baboons (Papio anubis/cynocephalus) were used in the present study. Each animal served as his own control in the initial pharmacokinetic studies (see below), which preceded the interventional study. For the dose-sequential interventional study, the animals were distributed to 3 experimental groups: (1) animals that received TP9201, 675 µg/kg bolus+12 µg/kg per minute infusion (group A, n=4); (2) animals that received TP9201, 170 µg/kg bolus+3 µg/kg per minute infusion (group B, n=4); and (3) a control group that received saline vehicle only (group C, n=4).

Model Preparation
Preparation of the nonhuman primate model of right MCA occlusion and reperfusion and surgical implantation of the MCA occlusion device (PS Medical) have been described previously in detail.⁷ Anesthesia was undertaken with isoflurane by assisted ventilation (5% induction) and maintained with the same agent (1.5% to 2.0%). A Silastic balloon device was placed around the MCA and secured by titanium hooks by a transorbital approach. The proximal terminus was accessible under the scalp by a local cutdown procedure. Full recovery was routinely achieved 1 to 2 hours after completion of the surgery. Subsequently, all animals were allowed a 7-day procedure-free interval before entry into the experimental protocol and displayed normal neurological function during that interval.²¹ Before MCA:O, the animals (n=12) had a mean hematocrit of 38.7±2.8% and hemoglobin of 12.6±0.7 g/dL, platelet count of 427.3±72.7/µL, and total leukocyte count of 10.1±3.6/µL, which were not significantly different from their naive state.

Experimental Procedures
Occlusion of the right MCA in the awake animal was accomplished by inflation of the extrinsic MCA balloon with compression of the artery. After a 3-hour MCA:O occlusion, the MCA territory was reperfused for 1 hour after balloon deflation. The experiments were terminated 60 minutes after MCA balloon deflation by pressure-perfusion fixation with a chilled carbon tracer. Perfusion fixation consisted of an initial perfusion flush under antithrombotic and isosmotic conditions to wash out all blood elements and a subsequent isosmotic tracer-fixative perfusion. At the time of experiment termination, with the animals under thiopental sodium anesthesia (15 mg/kg infusion) and assisted ventilation, the thorax was opened, and the descending aorta and inferior vena cava were clamped. The left ventricle was rapidly cannulated and perfused with chilled flush solution containing 25 g/L BSA (Sigma Chemical Co), 2000 U/L heparin, and 6.7 µmol/L sodium nitroprusside (Fisher Scientific) in Plasmalyte (Baxter Healthcare) adjusted to 340 mOsm/L with NaCl, pH 7.4, at pressures of 180 to 210 mm Hg for 3 minutes. This was immediately followed by fixation with chilled carbon suspension/fixative solution consisting of india ink (1:1 [vol/vol], Pelikan Fount India, Pelikan AG) in Plasmalyte/paraformaldehyde (2% final concentration)/glutaraldehyde (0.5% final concentration) for up to 17 minutes. High mean arterial perfusion pressures were chosen to maximize vascular patency.

Specimen Preparation
The exposed brain was immersed in alcohol-formaldehyde-acid solution (87% ethanol, 10% formaldehyde, and 3% glacial acetic acid [vol/vol]) for 1 week. The fixed brain was sectioned in the coronal plane at 1-cm intervals and immersed for another week in alcohol-formaldehyde-acid solution to achieve complete intravascular gelation of the carbon tracer. Tissue blocks (1.0 cmx1.0 cmx0.2 cm) from stereoanatomically identical sites of the left and right basal ganglia and from the left (normal) temporal lobe were embedded in glycol methacrylate (Polysciences, Inc), sectioned to 10-µm thickness, stained with basic fuchsin/methylene blue, and examined by light microscopy for the presence of india ink–filled (patent) microvascular structures. All specimens from all animals demonstrated complete continuous carbon impactions of microvessels.^{9 22 23}

Quantitative Analyses
Sections were analyzed with the aid of a computerized video-imaging system consisting of an image system unit connected in-line with a Hamamatsu C2400 Newvicon NTSC video camera (Hamamatsu Photonics) staged vertically on the light microscope (VIDAS, Kontron and Carl Zeiss). The minimum transverse diameters of india ink–filled microvessels in 90 nonoverlapping 526.1-µmx491.4-µm fields at x200 optical magnification (25 mm²) were processed in each section. The sections were taken at 30-µm intervals from stereoanatomically identical sites of both basal ganglia. Identical numbers of fields were analyzed until >1000 vessels were counted in the left control basal ganglia. Reproducibility and reliability data have been reported previously.^{9 22} In the case of specimens with hemorrhage, regions of interest were taken at the hemorrhage border. The extent of reflow is expressed as the ratio of the number of microvessels containing carbon in the ischemic basal ganglia to those in the control basal ganglia. Microvessels are defined as vessels <100-µm minimum diameter and are represented as 4 discrete vessel size classes as previously published.^{22 23}

Neurological Outcomes
Neurological function was assessed according to a well-described quantitative (100-point) scale weighted toward unilateral motor function loss.^{21 24}

Platelet Aggregation, Rationale, and Pharmacodynamic Studies
To rationally test the effect of the inhibitor on microvascular thrombosis, each naive animal was screened for platelet aggregation responses at baseline and after TP9201 exposure before selecting the appropriate TP9201 dose rate for each animal for the MCA:O studies. Blood for the platelet aggregation studies was collected in tubes containing citrate (0.38%) or heparin (5 IU/mL) by separate venipunctures. In vitro platelet aggregation studies were performed in triplicate for each naive animal. PRP and platelet-poor plasma were prepared by centrifugation of 10 mL whole blood at room temperature. The platelet count was adjusted to 2x10⁵/µL with autologous platelet-poor plasma. For in vitro and ex vivo platelet aggregation studies, PRP was incubated with 10 µmol/L ADP, and the activation was followed for 3 minutes with the aid of a Lumi-aggregometer (model 400VS, Chrono-Log Corp). To derive the inhibitory concentrations (eg, IC₅₀), concentration-response curves were generated by adding different concentrations of TP9201 to adjusted PRP from each naive animal in in vitro platelet aggregation experiments. Subsequently, on the basis of the in vitro studies, each naive animal received 1 of 2 dose rates of TP9201 to determine the exact dose rate that would achieve the 2 target platelet inhibitory effects (IC₃₀ and IC₈₀). Whole-blood samples were obtained at 0, 1, 2, 3, and 4 hours of infusion for the ex vivo aggregation studies. The degree of inhibition is expressed as the percentage of control values in the absence of TP9201 determined at maximal amplitudes of activation, with the mean of 3 baseline measurements set as 100%.

Bleeding Time Measurements
The bleeding time was measured by use of an automated template device (Simplate II, General Diagnostics).²⁵ Baseline bleeding times were determined at 1 hour before infusion and were repeated during the infusions at 1, 2, and 3 hours and before the end of the infusion (4 hours).

Plasma Levels of TP9201 and Pharmacokinetic Studies
TP9201 levels were determined to correlate ex vivo platelet inhibition with the in vitro inhibition curves. The plasma assays were performed by trifluoroacetic acid extraction of plasma followed by reversed-phase high-performance liquid chromatography on a C4 protein column as described previously.²⁶ Citrated blood samples (5 mL) were collected at baseline and 1, 2, 3, and 4 hours after the start of the TP9201 infusion. Samples were frozen immediately after collection and stored at -70°C before processing.

Statistical Analysis
Data are expressed as literal values or as mean±SD. Data were analyzed by using either paired or unpaired Student t test (2-tailed), ANOVA (with repeated measures), and other nonparametric tests, where appropriate. Statistical significance was generally set at 2P<0.05, except where noted.

	Results

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Hemostatic Parameters and Neurological Status
Alterations in hematologic parameters were selective (Table), most especially in total leukocyte count, which increased significantly within 1 hour of MCA:O in all 3 groups, as previously reported.^{22 24} There was no change in hematocrit or platelet count after MCA:O. After MCA:O, the neurological score changed from baseline (100) and varied within each group, consistent with the individual neurological status. A single animal in group A displayed a slight deficit after MCA device placement that persisted to MCA:O (postsurgical score 85 compared with 100 in the remainder). That animal suffered a symptomatic hemorrhage (Figure 5).

View this table: [in this window] [in a new window]   Table 1. Hematologic Data

View larger version (23K): [in this window] [in a new window]   Figure 5. Intracerebral hemorrhage by 4 hours after MCA:O. In group A, 3 of 4 animals displayed large symptomatic parenchymal hematomas (PH); in group B, 1 of 4 animals displayed a hemorrhagic infarction (HI); and in group C (control), no hemorrhage (nil) was visible (P=0.036).

Pharmacodynamic Studies and Dose Derivation
Before device implantation, each naive animal participated in individual pharmacodynamic studies (Figure 1). On the basis of in vitro and ex vivo studies with a separate primate cohort,²⁰ each animal received a constant infusion of TP9201 to achieve full inhibition of platelet aggregation to ADP in citrate and either 70% to 80% inhibition (group A) or 20% to 30% inhibition (group B) of platelet aggregation in normocalcemic conditions (Figure 1). A significant early increase in bleeding time was observed within 2 hours of infusion in group A but not group B (Figure 1). A significantly greater inhibition of platelet aggregation in vitro with TP9201 and ex vivo during each TP9201 infusion in response to ADP was observed under low Ca²⁺ conditions (citrate) than under normocalcemic conditions (heparin) (Figure 2). Logistic regression fits for platelet aggregation inhibition and TP9201 concentrations indicated an IC₅₀ (citrate) of 0.11 µmol/L and an IC₅₀ (heparin) of 2.28 µmol/L (Figure 2). The dose rates chosen for each individual animal within the MCA:O cohorts A and B were based on these IC₅₀ values and the plasma TP9201 levels.

View larger version (27K): [in this window] [in a new window]   Figure 1. Dose finding in naive animals. Top, Inhibition of platelet aggregation ex vivo under heparin (open squares) and citrate (solid circles) conditions. Solid lines depict respective modeled inhibition curves based on those data. Bottom, Simultaneous bleeding times for both infusion groups from identical animals. Solid lines depict modeled bleeding times based on those data.

View larger version (22K): [in this window] [in a new window]   Figure 2. IC50 values of TP9201 for platelet aggregation in vitro. Both group A and group B data are presented. Citrate conditions are depicted as solid circles (group A), and heparin conditions are depicted as open circles (group A) and open squares (group B).

Microvascular Patency
After placement of the MCA devices, each awake animal received 675 µg/kg+12 µg/kg per minute TP9201 (group A), 170 µg/kg+3 µg/kg per minute TP9201 (group B), or vehicle (group C) before MCA:O and continuously for 3 hours of MCA:O through 1 hour of reperfusion (Figure 3). Steady-state TP9201 blood levels of 2.5 µg/mL or 0.62 µg/mL were reached within 10 to 60 minutes of initiating the infusions in group A and group B, respectively (Figure 3). The patterns of ex vivo platelet aggregation inhibition, bleeding times, and plasma levels were in good agreement with the in vitro aggregation data from the same groups of animals. The anticipated bleeding time increase was observed in group A; however, no change in bleeding time was seen in any group B animal.

View larger version (20K): [in this window] [in a new window]   Figure 3. TP9201 plasma levels obtained during 3 hours of MCA:O and 1 hour of reperfusion (R) in group A (circles) and B (squares) animals. Broken lines indicate the plasma levels predicted from the experiments in Figures 1 and 2 and separate data.

Compared with vehicle, both TP9201 regimens produced significant increases in microvascular patency in both the capillary (4.0 to 7.5 µm) and precapillary arteriole/postcapillary venule (7.5 to 30.0 µm) diameter categories by 1 hour of reperfusion (Figure 4). Variation in the number and fractional patency (relative reflow) of larger microvessels (eg, 50.0 to 100.0 µm and >100.0 µm) was due in part to the relatively small numbers of vessels in those categories. However, in each diameter category, the vascular patency in the low-dose group (group B) was as great as that in the high-dose group (group A).

View larger version (41K): [in this window] [in a new window]   Figure 4. Relative reflow (patency) within separate microvessel diameter categories in animals receiving TP9201 at IC80 (group A, solid bars), TP9201 at IC30, (group B, stippled bars), and no TP9201 (group C, open bars) before and during MCA:O. Cohorts A and B displayed normal patency, which was significantly increased compared with cohort C in the 4.0- to 7.5-µm-diameter (2P=0.01 and 0.02, respectively) and 7.5- to 30.0-µm-diameter (2P=0.02 and 0.002, respectively) categories. Other comparisons were not significantly different.

Hemorrhagic Transformation
A dose-dependent increase in hemorrhage was observed among the 3 groups (P=0.036) (Figure 5). In group A, 3 animals suffered large parenchymal hemorrhages, which contributed significantly to the early neurological deficit, whereas only 1 animal in group B displayed evidence of a hemorrhagic infarction. The contribution of this hemorrhage to outcome is uncertain. The remainder of the animals in all 3 groups had no evidence of carbon tracer extravasation.

Neurological Outcome
Measurable neurological deficits were observed within 5 to 10 minutes after MCA:O in each animal. Subsequently, no difference in the course of the animals was observed in group B compared with group C. Group A showed worse neurological scores compared with group B and group C, owing entirely to the contribution of large parenchymal hemorrhages in 3 animals (Figure 6).

View larger version (28K): [in this window] [in a new window]   Figure 6. Neurological outcome. Group A demonstrated worse neurological scores than did groups B and C by 4 hours after MCA:O. This course was consistent with the significant contribution of symptomatic hemorrhage to the outcome of group A (see text).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Circulating platelets are activated and accumulate in cerebral microvessels within the ischemic territory after MCA:O.^{7 8 9 10} Fibrinogen and polymorphonuclear leukocytes can participate with activated platelets in microvascular deposits that lead to microvascular obstruction and contribute to the focal no-reflow phenomenon.^{22 23} It is postulated that microvascular obstruction, due in part to platelet activation and leukocyte adhesion, (1) is a consequence of MCA:O and (2) may lead to local ischemia and augmentation of brain cellular injury in primates. To address the first step of this postulate, we have demonstrated that microvascular patency can be restored by inhibiting the platelet integrin _IIbß₃–fibrinogen interaction before and during MCA:O. Two dose/concentrations of the well-characterized integrin _IIbß₃ inhibitor TP9201 encompassing the IC₃₀ and IC₈₀ of platelet aggregation (heparin) produced microvascular patency; however, symptomatic hemorrhage occurred only at the higher dose. A low risk of detectable hemorrhagic transformation accompanied the low dose/concentration. The present study confirms a role for platelet activation in functional microvascular obstruction generated during ischemic brain injury. A central implication of these experiments is that careful characterization of the precise target and hemorrhagic risk of the specific integrin _IIbß₃ (GPIIb/IIIa) inhibitor before its application to clinical ischemic stroke is categorically required for its use in acute brain ischemia to avoid excessive clinically significant parenchymal hemorrhage.

Activation of platelets within cerebral microvessels is a consequence of ischemia. These experiments corroborate our initial observation of platelet deposition within the ischemic basal ganglia in the nonhuman primate model.⁸ Inhibition of fibrin generation by heparin and blockade of the platelet ADP receptor by ticlopidine, in combination, reduced microvascular platelet deposition and occlusion formation. At least 3 further findings support the participation of platelets in microvascular occlusions after MCA:O in the nonhuman primate: (1) the time-dependent and monotonic increase in the number of microvessels displaying fibrin accumulation,²⁷ (2) the time-dependent accumulation of activated platelets within the ischemic microvasculature,⁷ and (3) the appearance of platelet-fibrin-leukocyte aggregates within ischemic cerebral microvessels, indicating activation of both cell types.⁹ Implied by those observations is the interaction of the platelet integrin _IIbß₃ receptor with fibrinogen and thrombin generation stimulating both fibrin formation and platelet activation. More recently, Garcia et al¹⁰ described the accumulation of platelets within microvessels in peripheral ischemic zones after MCA:O in the Wistar rat. However, it has remained unclear whether platelet accumulation per se could cause microvascular obstruction and hence brain tissue injury or whether the obstruction results from the tissue injury itself or both.

Given the redundancy of platelet activation pathways, inhibition of steps upstream from adhesion or aggregation produces a relative blockade of platelet aggregation.^{11 13 14} Hence, the inhibition by aspirin, a noncompetitive inhibitor of cyclooxygenase, and by ticlopidine, a thienopyridine noncompetitive ADP receptor antagonist, is relative and may be defeated by agonists other than thromboxane A₂ or ADP. The final step of platelet aggregation involves the interaction of the glycoprotein integrin _IIbß₃ with an RGD sequence on the 2 fibrinogen A chains.¹² This interaction is irreversible, and because the receptor is expressed only on platelets, it is specific. Inhibition of this integrin receptor–ligand interaction by potentially irreversible antiplatelet agents (eg, monoclonal antibodies) may be disadvantageous because of the risk of significant hemorrhage with pathologies in which platelet activation plays a role.^{28 29} This is of primary importance for central nervous system vascular processes in which antiplatelet agents have been shown to contribute to the risk of intracerebral hemorrhage.³⁰

A range of inhibitor subclasses that demonstrates relative species dependence with regard to fibrinogen binding blockade has been developed.^{31 32} Generally, significantly greater potency attends their inhibition of human and nonhuman primate platelet aggregation than that of dogs, rabbits, and rodents.³¹ Although the molecular natures of these species-related differences are not understood, they underscore the relevance of a nonhuman primate model to human clinical studies. The humanized Fab fragment of the monoclonal antibody (c7E3) has been shown to inhibit integrin _IIbß₃–dependent platelet aggregation, platelet-rich thrombus formation, and reocclusion after coronary artery angioplasty.¹⁵ Immunogenicity, thrombocytopenia, dose titration, and limitations in the management of hemorrhage associated with the platelet defect are concerns for use of this agent in cerebral nervous system injury.³³ There is no report testing this agent in experimental cerebral ischemia. Selected snake venom peptides (disintegrins), which are potent integrin _IIbß₃–blocking agents by virtue of competition for the RGD site on fibrinogen, have therapeutic potential.³⁴ Many such peptides are nonspecific, with high affinities for other receptors, including the integrins _vß₃, _vß₅, and ₅ß₁.^{35 36} Major side effects include their immunogenicity, significant alterations in hemostasis marked by increased bleeding times, and the potential for intracerebral and gastric hemorrhage.³⁷

A number of small peptide and nonpeptide RGD mimetics, which selectively and reversibly block integrin _IIbß₃, some of which have been investigated clinically, have been synthesized.^{38 39 40} TP9201 was selected as a potential therapeutic candidate from a panel of RGD-containing peptide compounds because it does not prolong the bleeding time at doses that inhibit ex vivo platelet aggregation in humans, nonhuman primates, and dogs.^{18 19 20} It is less active by more than an order of magnitude in guinea pigs, rabbits, rats, and mice. TP9201 also competes with Ca²⁺ for binding to the integrin receptor. This property is conferred by the presence of a positive charge that is 2 residues C-terminal to the RGD sequence⁴¹ and implies increased inhibition of the fibrinogen–integrin _IIbß₃ interaction below physiological Ca²⁺ concentrations. The IC₃₀ (heparin) is achieved at a TP9201 concentration that produces complete inhibition of platelet aggregation to ADP in hypocalcemic conditions (IC₁₀₀ [citrate]) (Figure 2). The present studies are consistent with earlier results indicating that TP9201 plasma levels at IC₃₀ (heparin) are sufficient to achieve appropriate in vivo efficacy^{19 20} while avoiding the deleterious consequences of hemorrhagic transformation and increased bleeding times shown in the present study. Furthermore, in several models, complete large-artery patency could be achieved at IC₃₀ (heparin) measured ex vivo in heparinized plasma without bleeding time elevation.^{18 19 20} Finally, thrombocytopenia did not confound any experiments.

There are few data involving well-characterized focal cerebral ischemia models with highly specific integrin _IIbß₃ inhibitors. Strict species requirements have dictated that preclinical studies against the platelet receptor have been mostly limited to nonhuman primates.^{31 32} However, intravenous weight-adjusted doses of the organic integrin _IIbß₃ inhibitor SDZ GPI 562 produced a reduction in ischemic injury volume at 24 hours in one study with a murine MCA:O model.⁴² GPI 562 displayed an IC₅₀ of 11 µmol/L to ADP-induced aggregation of isolated murine platelets in heparin, 5-fold higher than that for isolated baboon platelets to TP9201 ex vivo (Figure 2). A salutary effect on injury volume correlated with the inhibition of ¹¹¹In-labeled platelet accumulation at 24 hours and a reduction in fibrin deposition. The latter findings are consistent with the effects of anti–tissue factor strategies during MCA:O in the nonhuman primate.²³ However, it could not be excluded that GPI 562 at the high doses used also affected the course of tissue injury by independent effects. Hemorrhage confounded the surgical procedures and increased dose-dependently at 24 hours with GPI 562.

The increased microvascular patency at the IC₃₀ and IC₈₀ of TP9201 confirms the participation of activated platelets in cerebral microvascular responses to MCA:O.⁹ Platelet activation and accumulation are in accord with the progressive accumulation of fibrin within target microvessels after MCA:O.^{23 27} The distribution of microvascular occlusions was heterogeneous within the ischemic territory, where patent vessels appeared adjacent to occluded ones. Whereas 10% of microvessels appear activated by 2 hours of MCA:O (eg, vascular endothelial growth factor and integrin _vß₃ expression),⁴³ loss of patency occurs in up to 70% of microvessels, mostly capillaries and postcapillary venules, by 4 hours of MCA:O. This suggests the rapid recruitment of vessels by acute and progressive microvascular occlusion in the ischemic bed.

Hemorrhagic transformation is a natural accompaniment of ischemic stroke and occurs in up to 65% of patients with carotid territory ischemia.^{43 44} Parenchymal hematomas, most often symptomatic, are associated with anticoagulation^{45 46} and less so with antiplatelet agents, including aspirin.³⁰ However, integrin _IIbß₃ inhibitors may produce substantial blockade of platelet aggregation and clinically significant peripheral hemorrhage.^{15 47} Significant parenchymal hemorrhage with early neurological deterioration at TP9201 plasma concentrations equivalent to IC₈₀ indicates the necessity of competent platelet activation to limit or prevent cerebral hemorrhage. Although the mechanisms of early hemorrhage are not discernible in the present study, this observation underscores the potent effects on hemostasis of GPIIb/IIIa inhibitors at unregulated doses to increase the risk of brain hemorrhage. Although uncertain, it cannot be excluded that the large vessels in group A were the sources of the parenchymal hematomas. The benign character of the single visible hemorrhage in group B suggests that the group A hemorrhages were most probably extensions of microvascular hemorrhages seen in the absence of antithrombotic treatment in this model.⁴⁸

The finding of complete microvascular patency with 30% inhibition of platelet aggregation and no substantial hemorrhage under conditions that produce microvascular occlusion indicates that only partial blockade of the platelet-fibrinogen interaction is required to produce patency. With near complete inhibition of the integrin _IIbß₃–fibrinogen interactions, clinically devastating intracerebral hemorrhage results, indicating that the characteristics of this inhibitor and its dose adjustment are critical to the generation of significant intracerebral hemorrhage. However, the present experiments also indicate that dose adjustments for efficacy and cerebral hemorrhagic risk must take into account the degree of platelet aggregation response to inhibitor levels, species considerations, Ca²⁺ concentration, and specific characteristics of the inhibitor. It is incorrect to assume that an IC₃₀ dose of another inhibitor of the integrin _IIbß₃–fibrinogen interaction is equivalent to the effect of TP9201 at its IC₃₀. Moreover, these experiments set the stage to test the second step of the hypothesis, that early inhibition of platelet aggregation that produces microvascular occlusion formation leads to a substantial decrease in neuron injury in a clinically relevant stroke model.

	Acknowledgments

 
This study was supported in part by grant R01 NS-26945 (Dr del Zoppo) of the National Institute of Neurological Disorders and Stroke and a grant from Integra LifeSciences Corp. We are very grateful for the expect technical assistance of Pearl Akamine, Jacinta Lucero, and James O. Tolley.

Received November 23, 1999; revision received January 19, 2000; accepted March 7, 2000.

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Editorial Comment

Presented in part at a meeting of the International Society of Thrombosis and Haemostasis, Washington, DC, August 14–21, 1999.

Patricia D. Hurn, PhD, Guest Editor

Department of Anesthesiology/Critical Care Medicine Johns Hopkins Medical Institutions Baltimore, Maryland

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Platelets have been implicated, but not proven, as injurious participants in ischemic stroke. These blood-borne elements are likely activated on transit through an ischemic microcirculatory bed, and abnormal platelet accumulation has been demonstrated within brain after both focal and global cerebral ischemic insults.^{R1 R2 R3} The molecular means by which platelet aggregation and degranulation are achieved have been extensively studied, although little is known about these mechanisms under complex ischemic conditions. The platelet glycoprotein (GP) GPIIb/IIIa is a member of the integrin family (integrin _IIbß₃) and the single most abundant species of GP on the platelet surface. This molecule is well recognized as a receptor for fibrinogen and mediates many cohesive platelet interactions. GPIIb/IIIa also actively participates in various types of intraplatelet signaling, for example with other GPs (GPIb/IX/V), leading to aggregation during shear stress.^R4 The prevailing hypothesis relative to stroke is that microvascular platelet and platelet product deposition contribute to secondary injury by occlusive vessel derecruitment and/or through amplification of inflammatory mechanisms. The findings presented by Abumiya et al demonstrate that preischemic administration of an anti-GPIIb/IIIa, antiplatelet agent can improve microvascular perfusion in a vessel size–dependent manner, but that precise dosing is imperative if hemorrhagic complications are to minimized. These impeccably performed experiments in a nonhuman primate model are of interest to clinician and scientist alike. The results offer some proof of principle that a highly specific platelet receptor inhibitor can improve postischemic vascular integrity (which may be important to ultimate outcome for flow-recipient tissue). Further, we gain evidence from this study to support a commonly held supposition that functional platelets are essential to prevent intracerebral hemorrhage during focal cerebral ischemia. The study was designed to establish safety and determine reasonable dose ranges for further study and is appropriately limited in conclusions drawn about efficacy as a therapeutic agent. It remains to be shown that improved perfusion during the first hour of reperfusion will alter histological and functional outcome in the intact animal. We anticipate with interest further studies to better understand the relative necessity of preserved versus inhibited platelet activation/aggregation in reducing neuronal damage without hemorrhagic sequelae.

Received November 23, 1999; revision received January 19, 2000; accepted March 7, 2000.

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