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(Circulation. 1995;91:1427-1431.)
© 1995 American Heart Association, Inc.

Articles

Shear-Induced Platelet Aggregation Is Inhibited by In Vivo Infusion of an Anti–Glycoprotein IIb/IIIa Antibody Fragment, c7E3 Fab, in Patients Undergoing Coronary Angioplasty

Konstantinos Konstantopoulos, PhD; Suraj G. Kamat, MD; Andrew I. Schafer, MD; Eugenio I. Bañez, MD; Robert Jordan, PhD; Neal S. Kleiman, MD; J. David Hellums, PhD

From the Cox Laboratory for Biomedical Engineering (K.K., J.D.H.), Rice University, Houston, Tex; the Department of Medicine (S.G.K., A.I.S., N.S.K.) and Department of Pathology (E.I.B.), Baylor College of Medicine, Houston, Tex; and Centocor, Inc (R.J.), Malvern, Pa.

Correspondence to J. David Hellums, PhD, Cox Laboratory for Biomedical Engineering, Rice University, Houston, TX 77251-1892.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Elevated levels of shear stress such as those that occur in stenotic arterial vessels can directly activate and aggregate platelets and thus contribute to the pathogenesis of acute arterial thrombosis. This shear-induced platelet aggregation (SIPA) is mediated by von Willebrand factor binding to platelet membrane glycoprotein (GP) Ib and GPIIb/IIIa. The chimeric Fab fragment of the monoclonal antibody 7E3 (c7E3 Fab) that binds selectively to GPIIb/IIIa is under clinical evaluation in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). This study was undertaken to investigate the effects on ex vivo SIPA of c7E3 Fab administered to patients undergoing PTCA.

Methods and Results Six patients received aspirin (325 mg) and boluses of heparin (12 000 U) followed by c7E3 Fab 0.25 mg/kg. Blood collected from each patient before and after heparin treatment and at various time points after c7E3 Fab administration was subjected to laminar shear stress in a cone-and-plate viscometer. Flow cytometry was used to quantify the extents of platelet aggregation and of antibody binding to GPIIb/IIIa. Results indicate that c7E3 Fab injection resulted in a rapid, extensive blockade of GPIIb/IIIa receptors (98.6±0.2%) and a 50% inhibition of ex vivo platelet aggregation induced by shear stress. c7E3 Fab also completely abolished the formation of large platelet aggregates ("large" refers to particles >10 µm in equivalent sphere diameter), which are presumably the aggregates of greatest clinical significance. Partial reversibility of the inhibition was noted within 2 days after drug administration, but even after 1 week, platelet function had not been fully restored.

Conclusions This study demonstrates that c7E3 Fab is a potent inhibitor of SIPA, which may be an important mechanism of its beneficial effect in the treatment of arterial occlusive diseases and in the prevention of thrombotic complications of coronary artery disease after angioplasty.

Key Words: platelets • glycoproteins • platelet aggregation inhibitors • angioplasty

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Fluid mechanical forces can exert profound effects on blood cell function. It has been established that abnormally high levels of shear stress such as those that occur in arteries partially occluded by atherosclerosis or vascular spasm activate and aggregate platelets in the absence of exogenous added chemical agonists.^{1 2 3 4} Platelet aggregation induced by high shear stress depends on the availability of von Willebrand factor (vWF) and the presence of both glycoprotein (GP) Ib and GPIIb/IIIa on the platelet membrane. In particular, it has been postulated that at high-shear-stress conditions, the interaction of vWF with the GPIb/IX complex is the initial event leading to platelet activation that also triggers the binding of vWF to GPIIb/IIIa to induce platelet aggregate formation.^{1 2}

There is abundant evidence that the formation of platelet aggregates plays an essential role in the pathogenesis of acute arterial thrombosis. Platelet activation and aggregation have been implicated in a number of vascular diseases such as unstable angina, myocardial infarction, transient ischemic attack, and stroke.^{5 6 7} Thus, pharmacological modulation of platelet function is believed to be of clinical importance.

The final common step in platelet aggregation, regardless of the stimulus, involves the interaction of adhesive proteins such as fibrinogen and vWF with platelet membrane GPIIb/IIIa. This ligand-receptor interaction is blocked by synthetic peptides containing the Arg-Gly-Asp (RGD) sequence, as well as by monoclonal antibodies directed against GPIIb/IIIa. The chimeric Fab fragment of the monoclonal antibody 7E3^{8 9} (c7E3 Fab), a specific antagonist of the GPIIb/IIIa complex, is a potentially useful antithrombotic agent that is currently being evaluated for clinical use in the setting of percutaneous transluminal coronary angioplasty (PTCA). A prospective, randomized, double-blind trial recently reported that ischemic complications of coronary angioplasty and atherectomy were reduced by use of this monoclonal antibody.¹⁰

The purpose of this study was to investigate the effects of injection of c7E3 Fab into patients undergoing PTCA on ex vivo platelet aggregation induced by shear stress in a cone-and-plate viscometer. A whole-blood flow cytometric methodology was used to quantify shear-induced platelet aggregation and the antibody binding to platelets. The advantage of this methodology is that it requires no washing or centrifugation steps that may induce artifactual platelet activation and permits the study of platelets in the presence of other blood elements.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Monoclonal Antibodies
c7E3 Fab⁹ (Centocor, Inc) is a mouse-human antibody derived from the murine monoclonal antibody 7E3. This antibody fragment contains murine variable and human constant regions. It has the advantage of retaining the specificity of the murine monoclonal but is a human isotype, thus minimizing the chance of an anti-isotype response. c7E3 Fab is directed against the human platelet GPIIb/IIIa receptor and effectively inhibits the binding to platelet GPIIb/IIIa of fibrinogen and other adhesive proteins such as vWF, fibronectin, and vitronectin. c7E3 Fab was supplied as a filtered, sterile, nonpyrogenic 2-mg/mL solution in 0.01 mol/L sodium phosphate/0.001% polysorbate 80, pH 7.2. For selected in vitro studies described below, the antibody was biotin-labeled by use of established methods.¹¹

6D1 is a mouse monoclonal IgG1 antibody (kindly supplied by Dr Barry S. Coller, Mount Sinai School of Medicine, New York) that binds selectively to the platelet membrane GPIb.¹² 6D1 was conjugated with fluorescein isothiocyanate (FITC)¹¹ to a final fluorescein/protein molar ratio ranging from 2 to 4.2. The fluorescently labeled antibody, 6D1-FITC, was used to identify platelets and platelet aggregates in whole blood by flow cytometry. 10E5 (provided by Dr Coller), an IgG2a monoclonal antibody directed against the platelet membrane GPIIb/IIIa,¹³ was biotin-labeled¹¹ and used in selected studies as described below. Phycoerythrin-streptavidin (Southern Biotechnology Associates) was used to detect biotin-c7E3 Fab or biotin-10E5 bound to platelets.

Patients
Six patients 51 to 68 years old referred for PTCA and enrolled in a phase 1 clinical trial of c7E3 Fab were studied. Patients' clinical characteristics are summarized in Table 1. None of the patients had any history of aspirin or heparin hypersensitivity or known allergy to murine proteins.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of Patients in Study Group

Medical Treatment
All patients were treated with 325 mg aspirin 2 to 6 hours before and 12 to 24 hours after administration of c7E3 Fab. Patients also received an intravenous bolus of 12 000 U heparin 15 minutes before the study medication, sufficient to achieve activated clotting times of 300 to 400 seconds during the angioplasty. c7E3 Fab was then administered as an intravenous bolus injection of 0.25 mg/kg body wt, and angioplasty was completed within 1 hour thereafter. Following the angioplasty, patients received a continuous infusion of heparin at a rate of 800 U/h for at least 6 hours adjusted to maintain a partial thromboplastin time of approximately twice the control value.

Blood was collected into vacuum tubes containing sodium citrate (0.38% wt/vol) from each patient before (baseline) and after heparin treatment, as well as 2 minutes, 2 hours, and 24 hours after c7E3 Fab injection. Blood samples in two patients were also obtained at various time points within 1 week after drug administration.

Viscometric–Flow Cytometric Studies
Whole blood was exposed to controlled levels of shear stress by a cone-and-plate viscometer (Ferranti-Shirley 781, Ferranti Electric Inc). The apparatus consists of a stationary platen and a rotating cone (both of stainless steel) that impose a uniform shear motion to the entire sample.³ The cone diameter is 7 cm and its angle 0.33°, thus providing a gap between the cone and the plate that ranges from 30 µm at center to 150 µm at outside.

The time-average levels of shear stress in the normal arterial circulation are in the range of 10 to 30 dynes/cm² and may reach or even exceed levels of 200 to 400 dynes/cm² in partially obstructed vessels.^{14 15 16} In this work, whole blood was subjected to pathological shear rates of 3000 and 4000 s^-1 (shear stresses of 140 and 185 dynes/cm², respectively) for 30 seconds. Controls included blood specimens that were introduced into the viscometer but were not exposed to shear stress. In the circulatory system, shear exposure is repetitive, but for much shorter time intervals per exposure than the 30 seconds used in this work. However, the results of a study comparing repetitive with continuous shear stresses for the same total exposure time showed that platelet stimulation was at least as high in repetitive exposure as in continuous exposure.¹⁷

Aliquots of control and sheared blood were then fixed with 1% formaldehyde in Dulbecco's PBS (Sigma Chemical Co) and incubated with saturating concentrations of 6D1-FITC and 10E5-biotin or c7E3 Fab-biotin for 20 minutes in the dark. After an additional 15-minute incubation with a saturating concentration of phycoerythrin-streptavidin, specimens were diluted with 1% formaldehyde and analyzed in a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems). The light scatter and fluorescence channels were set at logarithmic gain.

The flow cytometric analysis used in this work has been described elsewhere in detail.¹⁸ In brief, platelets were distinguished from the other blood cells on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Data acquisition was carried out on each sample for a set period (usually 100 seconds). This procedure has the advantage that flow cytometric analyses of the same volumes for both the control (unsheared) and sheared blood specimens are achieved. As a result, platelet aggregation can be accurately quantified and expressed as the disappearance of single platelets.

Binding to platelets of 6D1-FITC and of the biotin-labeled antibodies 10E5 and c7E3 Fab was calculated by measuring the mean fluorescence intensity of at least 10 000 platelets in whole-blood specimens. At each experimental state, the FITC and phycoerythrin-streptavidin fluorescence values were normalized with the postheparin/pre-c7E3 Fab infusion fluorescence levels used as a reference. Binding of monoclonal antibodies detected by flow cytometry and expressed as mean fluorescence intensity has been shown to correlate with that detected by a radiometric method.^{19 20} Since the in vivo administration of c7E3 Fab to patients inhibits the subsequent in vitro binding of the biotin-labeled c7E3 Fab to platelets,^{21 22} the decrease in the phycoerythrin-streptavidin fluorescence values after the drug administration correlates with the percentage of GPIIb/IIIa receptors occupied by the drug. GPIIb/IIIa receptor blockade studies were also performed with radiometric assays.^{22 23} Free plasma concentrations of c7E3 Fab were determined by an ELISA with an affinity-purified preparation of rabbit anti-murine 7E3 variable-region antibodies for capture and a biotin-labeled conjugate of the rabbit antibody preparation for detection.²²

Statistical Analysis
The data are expressed as mean±SEM unless otherwise specified. Statistical significance of differences between means was determined by single-factor ANOVA. If means were shown to be significantly different, multiple comparisons by pairs were performed by the Tukey test.²⁴ Probability values of P<.05 were selected to indicate statistical significance.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Hematologic Parameters
Platelet counts were evaluated in all patients before (baseline) and after heparin administration, as well as 30 minutes and 24 hours after injection of c7E3 Fab. Neither heparin nor c7E3 Fab affected platelet counts significantly. Hematocrits and hemoglobin levels measured 24 hours after c7E3 Fab bolus injections were slightly lower than those at the baseline (35.4±3.2% versus 39.1±1.2% and 12.1±1.0 versus 13.6±0.7 g/dL, respectively).

The effects of heparin and c7E3 Fab administration to patients undergoing PTCA on the availability of GPIIb/IIIa receptors on the platelet surface are presented in Fig 1A. Injection of 12 000 U heparin into patients produced a slight increase in the ex vivo binding of biotin-labeled c7E3 Fab to platelets. However, bolus injection of c7E3 Fab at a dose of 0.25 mg/kg resulted in rapid and nearly complete occupation of GPIIb/IIIa receptors (98.6±0.2% at 2 minutes after injection), with little recovery at 24 hours. The GPIIb/IIIa blockade was still pronounced 4 days later, and even after 1 week, the percentage of free GPIIb/IIIa binding sites had returned to only 65% of the preinfusion level. Antibody binding to GPIIb/IIIa detected by flow cytometry is in relatively good agreement with that detected by the radiometric assay (Fig 1B).

View larger version (28K): [in this window] [in a new window]   Figure 1. Bar graphs showing availability of glycoprotein IIb/IIIa receptors on platelet surface before and after c7E3 Fab treatment as determined by (A) flow cytometry (n=4) and (B) a radiometric method (n=3). Values are mean±SEM. Data points at 2 days, 4 days, and 7 days after c7E3 Fab administration represent the mean values of two determinations. *P<.05 vs postheparin samples. PE indicates phycoerythrin-streptavidin.

The monomodal curves (Fig 2) that represent the ex vivo binding of biotin-labeled c7E3 Fab to platelets suggest that there is a dynamic equilibrium between the antibody molecules and the platelet GPIIb/IIIa receptors. Therefore, 4 and 7 days after infusion of c7E3 Fab to patients, this agent binds to a limited number of receptors of the entire platelet population (demonstrating transfer of the antibody molecules from previously circulating platelets to newly released platelets) rather than saturating the receptors of a limited platelet population as would have been the expected result of platelet clearance from the circulation.

View larger version (20K): [in this window] [in a new window]   Figure 2. Representative fluorescence histograms of ex vivo biotin-c7E3 Fab binding to platelets. Once platelets were distinguished from the other blood cells as described in "Methods," biotin-c7E3 Fab binding was quantified by analysis of the platelets for phycoerythrin fluorescence. At least 10 000 platelets were analyzed to generate each curve. Curve A represents biotin-c7E3 Fab binding to platelets in blood obtained after heparin treatment; curves B, C, and D represent antibody binding to platelets in blood obtained from the same patient 2 hours, 4 days, and 7 days after drug administration.

The plasma levels of c7E3 Fab increased immediately upon treatment of patients with the study medication (Fig 3). However, these increases were transient and returned to baseline levels a few hours after the infusion.

View larger version (31K): [in this window] [in a new window]   Figure 3. Bar graph showing time course of the free plasma levels of c7E3 Fab (ng/mL) after drug administration in six patients as determined by an ELISA. Plasma levels <19.5 ng/mL are below the sensitivity of the assay.

The effects of heparin and c7E3 Fab infusions on ex vivo platelet aggregation induced by shear stress are illustrated in Fig 4. The extent of platelet aggregation after heparin treatment was not significantly different from that of the baseline at both the stress levels studied. On the contrary, c7E3 Fab significantly inhibited, by about 50%, the extent of shear-induced platelet aggregation as shown in Fig 4. The inhibitory effects of c7E3 Fab were even more pronounced when expressed in terms of the formation of large platelet aggregates. The latter were defined as particles >10 µm in equivalent sphere diameter as estimated by the use of uniform microbeads of known size. c7E3 Fab completely abolished the formation of large platelet aggregates (Fig 5). The inhibition of both platelet aggregation and large platelet aggregate formation was still evident at 1 day, and even a week later there was some reduction in these responses.

View larger version (28K): [in this window] [in a new window]   Figure 4. Bar graph showing aggregation induced by shear stress in the absence of added chemical agonist. Whole blood was exposed to a pathological stress level of 4000 s-1 in a cone-and-plate viscometer. The percent platelet aggregation is defined in terms of the disappearance of platelet-sized particles identified in a flow cytometer by a fluorescein isothiocyanate–conjugated antibody to platelet membrane glycoprotein receptor Ib. Values are mean±SEM (n=4). Data points at 2 days, 4 days, and 7 days after c7E3 Fab administration represent the mean value of two determinations. *P<.025 vs postheparin samples.

View larger version (27K): [in this window] [in a new window]   Figure 5. Bar graph showing large platelet aggregate formation induced by a pathological stress level of 4000 s-1 for the same experiments as in Fig 4. Large aggregates are defined as particles >10 µm in equivalent sphere diameter as estimated by the use of uniform microbeads of known size. The number of large aggregates is normalized for an initial platelet count of 300 000/µL. *P<.01 vs postheparin samples.

The extensive GPIIb/IIIa receptor blockade by in vivo infusion of c7E3 Fab did not affect the binding of 10E5, which binds to GPIIb/IIIa but to an epitope distinct from that of 7E3,⁸ nor did it interfere with the binding of 6D1 (Table 2).

View this table: [in this window] [in a new window]   Table 2. In Vitro Binding of 10E5, 6D1, and c7E3 Fab Monoclonal Antibodies to Platelets Before and After c7E3-Fab Injection as Determined by Flow Cytometry

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
PTCA is now an established approach to revascularize cardiac muscle. However, this procedure causes injury to the coronary artery that signals the activation of the hemostatic mechanism. Platelet deposition and thrombus formation at the site of injury are associated with acute closure and restenosis.²⁵ Therefore, use of antiplatelet agents may be beneficial for the prevention of thrombotic complications after angioplasty. Current therapy includes aspirin and heparin, but to date these agents have not eliminated abrupt closure and its ischemic sequelae,^{26 27} presumably because they are not effective against platelet aggregation in response to all agonists. For instance, the platelet inhibitory effects of aspirin can easily be overcome by surges in catecholamines.²⁸ Furthermore, the elevated levels of fluid shear stress generated in stenosed coronary arteries can directly cause platelet activation and aggregation,^{1 2 3 4} and aspirin has been shown to be ineffective in inhibiting shear-induced platelet aggregation.^{2 18} A better clinical response may require inhibition of multiple pathways of platelet activation, and that is achieved by blocking the binding of cytoadhesive proteins to platelet membrane GPIIb/IIIa.

In this work, we investigated the effects on shear-induced platelet aggregation of c7E3 Fab, an anti-GPIIb/IIIa antibody, injected into patients undergoing elective PTCA. All patients were pretreated with aspirin (baseline control) and heparin. Ingestion of 325 mg aspirin by normal subjects, although it completely suppresses the cyclooxygenase pathway, has no effect on shear-induced platelet aggregation.^{2 18} Heparin administered to patients at a dose of 12 000 U also did not alter the extent of aggregation. Intravenous bolus injection of c7E3 Fab at a dose of 0.25 mg/kg to patients undergoing PTCA resulted in an immediate, extensive blockade of GPIIb/IIIa binding sites (98.6±0.2% at 2 minutes after injection). Partial reversibility was noted within 24 hours after drug administration. However, the reversibility proceeded slowly, and even 1 week later, platelet function had not been fully restored.

Despite the extensive GPIIb/IIIa receptor blockade caused by c7E3 Fab administration, the GPIb receptors remained available for binding of vWF, as probed by the anti-GPIb monoclonal antibody 6D1. This finding is in accord with results of a recent investigation that showed that injection of c7E3 Fab into patients did not affect platelet adhesion onto a collagen I surface under conditions of abnormally high arterial flow, a situation that is entirely dependent on GPIb-vWF interaction.²⁹ We have found that the GPIIb/IIIa blockade results in the essentially complete elimination of large platelet aggregate formation induced by pathological levels of shear stress. The availability of the GPIb receptors on the platelet surface may help explain why low levels of shear-induced platelet aggregation (due to the formation of small platelet aggregates) still occur even immediately after c7E3 Fab administration. Thus, it seems that c7E3 Fab alters platelet function in a way that decreases platelet aggregation without interfering with the GPIb-vWF interaction that is essential for platelet adhesion and primary hemostasis.

The results of this study indicate that c7E3 Fab is an effective inhibitor of shear-induced platelet aggregation. This is in agreement with the finding of a recent study²¹ in which F(ab')₂ of 7E3 antibody protected against platelet aggregation and thrombus formation caused by very high shear forces developed in severely stenosed monkey carotid arteries. Injection of the drug at a dose of 0.25 mg/kg to patients undergoing PTCA immediately suppressed aggregation by about 50% and completely abolished the formation of large platelet aggregates at the shear-stress levels studied. Partial reversibility of the inhibition was observed within 2 days after drug administration, but some inhibition remained even after 1 week. It is likely that some impairment in the shear-induced aggregation response persists until all the GPIIb/IIIa binding sites are free from the study agent.

In conclusion, c7E3 Fab has a rapid onset of action and is a potent inhibitor of shear-induced platelet aggregation. This finding may provide a mechanism for the beneficial effects of c7E3 Fab treatment in coronary artery disease¹⁰ in which elevated shear levels are operative and may contribute to our understanding of mechanisms for reducing thrombotic complications after angioplasty.

	Acknowledgments

 
This work was supported by the NIH under grants 5R37-HL-18584, 5P50-NS-23327, and HL-36045 and a grant from the Veterans Affairs Merit Review Board. We thank Dr B.S. Coller for supplying MoAbs.

	Footnotes

 
Guest Editor for this article was Dr Barry S. Coller, Mount Sinai Medical Center, New York, NY.

Received June 8, 1994; revision received September 27, 1994; accepted October 14, 1994.

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