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(Circulation. 1995;91:1354-1362.)
© 1995 American Heart Association, Inc.

Articles

Comparative Real-Time Effects on Platelet Adhesion and Aggregation Under Flowing Conditions of In Vivo Aspirin, Heparin, and Monoclonal Antibody Fragment Against Glycoprotein IIb-IIIa

Nancy A. Turner, BS; Joel L. Moake, MD; Suraj G. Kamat, MD; Andrew I. Schafer, MD; Neal S. Kleiman, MD; Robert Jordan, PhD; Larry V. McIntire, PhD

From the Cox Laboratory for Biomedical Engineering, Rice University, Houston, Tex (N.A.T, J.L.M., L.V.M.); the Department of Medicine, Baylor College of Medicine, Houston, Tex (J.L.M., S.G.K., A.I.S., N.S.K.); and Centocor, Inc, Malvern, Pa (R.J.).

Correspondence to Joel L. Moake, MD, Cox Laboratory for Biomedical Engineering, Rice University, PO Box 1892, Houston, TX 77251.

	Abstract

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Background A real-time in vitro system of human platelet thrombosis under arterylike flowing conditions similar to those produced in vivo by angioplasty would be useful for the evaluation of potential antiarterial thrombotic agents in association with in vivo trials. Aspirin, heparin, and the chimeric monoclonal antibody antigen-binding fragment 7E3 (c7E3 Fab) directed against platelet glycoprotein (GP) IIb-IIIa have been used in attempts to delay or prevent thrombotic reocclusion of coronary arteries after angioplasty. We compared the effects of these agents administered in vivo on GPIb-mediated platelet adhesion to von Willebrand factor (vWF)/collagen type I (as in atherosclerotic subendothelium) and on subsequent GPIIb-IIIa–fibrinogen/vWF–mediated platelet aggregation under flowing conditions analogous to those in constricted coronary arteries.

Methods and Results Citrated whole blood containing mepacrine-labeled platelets from patients and healthy donors was perfused for 1 minute at an abnormally elevated shear rate of 1500 seconds^-1 (arterial wall shear stress of 50 to 60 dynes/cm²) at 37°C over collagen I/vWF. The number of adherent fluorescent platelets was quantified every 15 seconds with a low-light-level video camera and epifluorescent microscopy. After 5 healthy donors had ingested 975 mg aspirin, platelet adhesion was unaffected in the aspirin-treated blood compared with the control blood in all experiments (10 of 10), and subsequent aggregation was unchanged in most runs (8 of 10). The blood of 3 aspirin-treated patients undergoing angioplasty was analyzed before and after a 12 000-U heparin injection and 2 minutes, 2 hours, and 24 hours after infusion of 0.25 mg/kg of c7E3 Fab. In these patients, the bolus of heparin did not inhibit either platelet adhesion to collagen I/vWF or subsequent aggregation. In contrast, there was >50% inhibition of platelet aggregation 2 minutes after the infusion of c7E3 Fab in all 3 patients, and inhibition persisted in 2 of the 3 patients at 2 hours and 24 hours after c7E3 Fab.

Conclusions In contrast to aspirin or heparin, the in vivo injection of c7E3 Fab considerably reduces platelet aggregate formation mediated by the binding of fibrinogen, vWF, or some other ligand to platelet GPIIb-IIIa under conditions of abnormally increased shear stress analogous to those in narrowed coronary arteries. Platelet adherence to collagen I/vWF is not affected. This study describes an in vitro model of arterial injury (similar to angioplasty) that uses human blood to compare directly, in real time, the precise relative effects of aspirin, heparin, and c7E3 Fab on platelet adhesion and subsequent aggregation.

Key Words: aspirin • heparin • glycoproteins • von Willebrand factor • blood flow

	Introduction

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In small arteries partially obstructed by atherosclerosis or vasospasm, fluid shear stresses may increase many times above the normal time-average level of about 20 dynes/cm² and may lead to platelet-mediated thrombosis.^{1 2 3} These abnormally elevated fluid shear stresses in the circulation are imposed on the surfaces of blood cells, where they may produce alterations in the structure, exposure, or clustering of membrane-associated molecules.

Arterial platelet thrombus formation under conditions of elevated fluid shear stresses may be initiated by platelet adhesion and aggregation from rapidly flowing blood onto the exposed subendothelium of atherosclerotic arteries and arterioles containing collagen and von Willebrand factor (vWF), with subsequent platelet aggregation. It has previously been demonstrated that vWF becomes rapidly insolubilized on the exposed subendothelium of human arteries,⁴ as well as on collagenous components of the vessel wall,^{5 6} and that this precedes and augments platelet adhesion.⁷ It has also been shown⁸ that platelet accumulation on human artery subendothelium can be inhibited by monoclonal antibodies against either glycoprotein (GP)Ib or GPIIb-IIIa. These previous studies used morphometric or radioactive techniques to evaluate the total accumulation of platelets on exposed surfaces after adhesion and subsequent aggregation had occurred, rather than during the course of these events.

We combined a parallel-plate perfusion chamber with a computerized epifluorescence video microscopy system to observe in real time and quantify separately the adhesion and subsequent platelet aggregation of human platelets in whole blood flowing under conditions of abnormally elevated shear stress over type I fibrillar collagen. (vWF can bind to collagen types I, II, and VI.^{9 10} Collagen type I predominates in atherosclerotic arterial subendothelium.¹¹ ) The precise sequence of interactions among vWF, GPIb, and GPIIb-IIIa during platelet adhesion and subsequent aggregation has been studied previously by this direct real-time observation and computerized epifluorescence video microscopy system.¹² Adhesion at elevated shear rates in our system is the result of the adsorption of large vWF multimers onto collagen I and the binding of platelet GPIb to the insolubilized vWF. Aggregation occurs subsequently and requires the binding of ligands, including vWF via its RGD binding domain, to GPIIb-IIIa. Platelet adhesion to collagen I/vWF in flowing whole blood is inhibited by two potentially useful arterial antithrombotic agents: polymeric aurin tricarboxylic acid,^{13 14} which binds to vWF and inhibits its attachment of GPIb, and a recombinant vWF fragment¹⁵ (rvWF^445-733) that binds to platelet GPIb (in the absence of any modulator) and blocks attachment of vWF multimers. In our model system, we investigated the effects of arterial antithrombotic agents that are either already in clinical use (aspirin, which blocks platelet fatty acid cyclooxygenase) or under evaluation in clinical trials (a chimeric Fab fragment of monoclonal antibody 7E3 directed against platelet GPIIb-IIIa, c7E3 Fab).^{16 17 18}

	Methods

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"Shear" refers to the relative parallel motion between adjacent fluid planes during flow. The difference in the velocities between layers of blood at various distances from the vessel wall determines the local shear rate, expressed in cm · s^-1 · cm^-1, or inverse seconds (s^-1). Fluid shear stress in dynes/cm² is a measure of the force required to produce a certain rate of flow of a viscous liquid and is proportional to the product of shear rate and blood viscosity.

Aspirin Study of Healthy Donors
Blood from five healthy donors was collected into sodium citrate (0.38%) for perfusion experiments before and 1 hour after they ingested aspirin. The aspirin dose was 975 mg to ensure rapid and near-complete in vivo blockade of platelet cyclooxygenase. Blood from each donor was used for 10 runs before and after aspirin ingestion. Donor platelet-rich plasma was tested for aggregation response to 1.25 mmol/L arachidonic acid before (72% to 82% light transmission) and after (2% to 4% light transmission) in vivo aspirin treatment.

c7E3 Fab
c7E3 Fab^{16 17 18} (Centocor, Inc) is a chimeric human/mouse Fab fragment derived from the murine monoclonal 7E3 antibody that binds selectively to the platelet GPIIb-IIIa receptor. The antibody specificity is contained in the mouse heavy- and light-chain variable regions. The remaining constant regions are human. c7E3 Fab is supplied as a sterile 2-mg/mL solution and is prefiltered through a 0.22-µm low protein binding filter before infusion.¹⁸

Angioplasty Patients
Perfusion experiments were performed with citrated blood obtained from three patients undergoing elective coronary angioplasty enrolled in c7E3 Fab phase 1 clinical trials. No patient was allergic to aspirin, heparin, or murine proteins. Each received 325 mg aspirin 2 to 6 hours before and 12 to 24 hours after angioplasty. Heparin (12 000 U) was injected 15 minutes before infusion of 0.25 mg/kg c7E3 Fab, which was previously determined to bind to 87% to 100% of platelet GPIIb-IIIa receptors and inhibit ADP-induced platelet aggregation by 90%.^{18 19 20} Angioplasty was performed within 1 hour thereafter by standard techniques. Blood for flow experiments was collected before heparin injection (baseline), 15 minutes after heparin, and then 2 minutes, 2 hours, and 24 hours after c7E3 Fab infusion. Platelet counts were determined before heparin, before c7E3 Fab, and 30 minutes, 2 hours, and 24 hours after c7E3 Fab infusion. Hematocrits were measured before the angioplasty and 24 hours after the c7E3 Fab infusion. Approval was obtained from the Institutional Review Board for these studies. Informed consent was provided according to the Declaration of Helsinki.

Preparation of Collagen-Coated Coverslips
Suspensions of 2 mg/mL type I acid-insoluble collagen fibrils from bovine Achilles tendon (Sigma Chemical Co) in 0.5 mol/L acetic acid, pH 2.8, were prepared, and collagen concentrations were determined by a modified Lowry analysis.²¹ Before assem- bly in the flow chamber,^{12 21 22 23} glass coverslips (No. 1, 24x50 mm; Corning, Inc) were coated with 200 µL fibrillar collagen solution spread over all but the first 15 mm of the slide length (coated area, 12.7x23 mm) and placed in a humid environment for 45 minutes, and the excess collagen was rinsed off with 10 mL 0.9% NaCl. The decrease in collagen concentration in the supernatant solutions was measured, and the collagen density on the glass surfaces was calculated to be 20 µg/cm².

Flow Chamber and Perfusion Studies
Before the perfusion experiments, blood was incubated at 37°C for 15 minutes. The fluorescent dye mepacrine (quinacrine dihydrochloride; Sigma) (10 µmol/L) was added to visualize the platelets. Mepacrine is concentrated in the dense granules of platelets and the granules of leukocytes and has no effect on normal platelet function at this concentration.²⁴ These two cell types are clearly distinguished by epifluorescence microscopy. Previous studies^{12 22 23} have determined that any fluorescence from within erythrocytes is quenched by hemoglobin; that leukocytes do not adhere to the collagen-coated surface at the relatively high shear rates used in our studies; and that platelet secretion of mepacrine from dense granules after adhesion is sufficiently limited in rate and extent that platelet fluorescence does not decrease detectably under our experimental conditions during adhesion and subsequent aggregation.

Control runs at the beginning and end of each experiment demonstrated the same extent of platelet accumulation on collagen I/vWF-coated slides. One side of a parallel-plate flow chamber, described in detail elsewhere,^{12 21 22 23} was formed by a collagen I–coated glass coverslip with a flow path height of 205 µm (determined by a silicon gasket). The flow chamber was assembled and filled with isotonic saline. A syringe pump (model 935, Harvard Apparatus) was used to aspirate blood through the flow chamber, displacing the saline, at a constant flow rate for 1 minute. The flow rate of 8.0 mL/min produced a 1500-s^-1 wall shear rate,²⁵ as in partially constricted arteries,^{1 2 3 25} and corresponds to a wall shear stress of approximately 50 to 60 dynes/cm². The blood was considered a newtonian fluid with a viscosity range of 3.5 to 4 cp²⁵ in laminar flow. The entire system was maintained at 37°C by a thermostatic air bath (model 279, Laboratory Products). The flow chamber was mounted on an inverted-stage microscope (Diaphot-TMD, Nikon) equipped with an epifluorescent illumination attachment (TMD-EF, Nikon), a x60 Fluor objective, a x5 projection lens (Nikon), and a silicon-intensified target video camera (model C1000, Hamamatsu) suitable for very low light levels. Epifluorescence illumination was used to directly visualize platelet adhesion and subsequent platelet aggregation throughout the perfusion period. The experiments were recorded in real time on a 0.5-in videocassette recorder (JVC model BR-3100U).

Evaluation of Platelet Adhesion and Subsequent Platelet Aggregation
Videotape images, focused 0.38 mm downstream from the glass–collagen I interface, were digitized and computer analyzed at 15-second intervals for 1 minute²⁶ (IC-300 Modular Image Processing Workstation, Inovision Corp). The number of individual platelet thrombi in the microscope field of 3.7x10⁴ µm² after 15 seconds of flow was defined as the measurement of platelet adhesion (ie, platelet-surface attachment) that initiates thrombus formation. As individual platelet thrombi merge into larger clumps at 30 and 60 seconds of flow, the number of thrombi decreases concomitantly. The number of platelets in each individual thrombus was calculated as the total intensity of each thrombus (area times average intensity) multiplied by a value determined by dividing the number of single platelets in the 15-second images by the sum of the total intensities of these single platelets.^{21 22 23} The extent of platelet aggregation (ie, platelet-platelet cohesion) that occurred subsequent to platelet adhesion was expressed as the percentage of thrombi in each category of increasing number of platelets. Three-dimensional representations of the platelet thrombi present at 15, 30, 45, and 60 seconds during the perfusion period were constructed and photographed from the computer screen.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Aspirin
Citrated whole blood from healthy donors, before and after the ingestion of 975 mg aspirin, was perfused at 1500 s^-1 for 1 minute over collagen I. Table 1 shows the number of platelet thrombi adherent to the collagen surface after 15, 30, 45, and 60 seconds of flow from five healthy donors before and after in vivo aspirin treatment. There was no reduction of GPIb-mediated¹² platelet adhesion to collagen I/vWF from the blood collected after aspirin ingestion compared with those samples collected before aspirin ingestion. In three of the runs, there was little change in the adherence of aspirin-treated platelets; however, in 13 of 20 time points analyzed, there was an actual increase in the number of adherent platelet thrombi from the aspirin-treated blood compared with the number from control blood samples (Fig 1).

View this table: [in this window] [in a new window]   Table 1. Platelet Adhestion to Collagen

View larger version (26K): [in this window] [in a new window]   Figure 1. Bar graph showing difference between the number of adherent platelet thrombi on the collagen I/von Willebrand factor surface from blood collected after aspirin (ASA) ingestion and the number of thrombi from samples before in vivo aspirin treatment at 15, 30, 45, and 60 seconds of flow. Citrated whole blood collected from five healthy donors before and after ingestion of 975 mg aspirin was perfused for 1 minute at 1500 s-1 and 37°C.

Fig 2 demonstrates the platelet aggregation subsequent to initial platelet adherence to collagen I/vWF surfaces¹² when blood from the same donors before (top) and after (bottom) aspirin ingestion was perfused for 1 minute at 1500 s^-1. The platelet thrombus size distribution is shown in Table 2. In 8 of 10 perfusion experiments, the extent of platelet aggregation subsequent to initial adhesion was similar in the blood samples obtained before and after aspirin treatment. A representative experiment is shown in Fig 2A and 2B.

View larger version (4K): [in this window] [in a new window]   Figure 2. Facing page. Three-dimensional computer-generated color representations (from videotape of real-time data) of platelet adhesion and subsequent aggregation on collagen I/von Willebrand factor from normal citrated whole blood collected before (top four panels) and after (bottom four panels) in vivo aspirin (ASA) treatment (975 mg) and perfused at 37°C for 1 minute at 1500 s-1. Relative increase in platelet accumulation is indicated by progression in color from orange to yellow peaks.

View this table: [in this window] [in a new window]   Table 2. Platelet Thrombus Size Distribution

Platelets and Hematocrits in Angioplasty Patients
Platelet counts from three patients undergoing elective coronary angioplasty were determined from blood samples collected before heparin anticoagulation; before c7E3 Fab infusion; and 30 minutes, 2 hours, and 24 hours after c7E3 Fab infusion. There was little change in platelet counts after heparin or c7E3 Fab in two of the three patients and <20% decrease in patient 3 (data not shown). Hematocrits were measured before the angioplasty procedures and at 24 hours after c7E3 Fab infusion, and they remained >37% (data not shown).

Heparin
The extent of GPIb-mediated platelet adhesion to collagen I/vWF¹² after perfusion at 1500 s^-1 for 1 minute in citrated whole-blood samples from aspirin-treated angioplasty patients changed little after a 12 000-U heparin injection. The resulting platelet adhesion, measured by the number of adherent platelet thrombi on the collagen I surface, is shown in Table 3 for one of the three patients studied. By comparing the first two columns of Table 3 (baseline and 15 minutes after heparin), we can see that there are only modest differences in the relative number of adherent platelet thrombi on the collagen I surface after heparin injection. Three-dimensional representations of the thrombi after 60 seconds of flow for this patient are shown in Fig 3.

View this table: [in this window] [in a new window]   Table 3. Platelet Adhesion to Collagen: Patient 1

View larger version (4K): [in this window] [in a new window]   Figure 3. Three-dimensional color representations of platelet adhesion and subsequent aggregation on collagen I/von Willebrand factor from citrated whole blood of a coronary angioplasty patient that was perfused at 37°C for 1 minute at 1500 s-1. The blood was collected at baseline, 15 minutes after 12 000 U heparin, and 2 minutes, 2 hours, and 24 hours after a 0.25-mg/kg infusion of c7E3 Fab (7E3). The 60-second time points are shown.

The platelet aggregation that occurs subsequent to platelet adhesion to collagen I is mediated by vWF and fibrinogen binding to platelet GPII-IIIa.^{12 27 28} Aggregation from the perfused citrated whole-blood samples of the angioplasty patients after the 12 000-U heparin injection was not different from the platelet aggregation observed in preheparin patient samples. This similar extent of platelet aggregation is shown by the three-dimensional representations of platelet thrombi in Fig 3; the percentage of deposited platelets in Fig 4; and by comparison of the percentage of thrombi containing different numbers of platelets in the baseline (control) sample and the sample collected 15 minutes after heparin injection in Table 4. For brevity, the three-dimensional representations and the distribution of thrombi are shown for only one of the three patients studied.

View larger version (86K): [in this window] [in a new window]   Figure 4. Bar graphs showing percent of platelets relative to baseline (100%) samples deposited on 3.7x104 µm2 of the collagen I/von Willebrand factor surface from the perfusion of citrated blood of angioplasty patients collected 15 minutes after injection of 12 000 U heparin and 2 minutes, 2 hours, and 24 hours after a 0.25-mg/kg c7E3 Fab infusion. The whole blood was perfused at 37°C for 1 minute at 1500 s-1. A, patient 1; B, patient 2; C, patient 3.

View this table: [in this window] [in a new window]   Table 4. Platelet Thrombus Size Distribution: Patient 1

c7E3 Fab
After heparin injection, citrated whole blood was collected from the three angioplasty patients 2 minutes, 2 hours, and 24 hours after the 0.25-mg/kg infusion of c7E3 Fab and was perfused over collagen I at 1500 s^-1 for 60 seconds. Quantification of the number of individual platelets and platelet thrombi attached to the surface at 15, 30, 45, and 60 seconds demonstrated that platelet adhesion after c7E3 Fab infusion either was unaffected over this time period (patient 2, data not shown) or was relatively increased compared with baseline samples (patient 1 in Table 3 and patient 3, data not shown). c7E3 Fab reduced total platelet accumulation and the number of platelets per thrombus (platelet aggregation) by >50% compared with experiments using patient blood samples obtained at baseline or after heparin injection (Figs 3 and 4, Table 4). In the experiments with the blood sample from patient 2 obtained 2 minutes after c7E3 Fab infusion, the largest thrombus size after 60 seconds of flow was reduced from 10 000 platelets per thrombus to 20 platelets per thrombus (data not shown). Blood samples obtained 2 minutes after c7E3 Fab infusion from the other two patients showed platelet thrombus size reductions of 5000 platelets per thrombus compared with baseline or postheparin samples after 60 seconds of perfusion onto collagen I at 1500 s^-1 (Table 4). In these same 60-second perfusion experiments, the samples collected 2 minutes after c7E3 Fab infusion had 66%, 99%, and 48% reduction in platelet deposition onto the collagen I surface, respectively, compared with patient baseline samples (Figs 3 and 4).

Blood samples obtained from the angioplasty patients 2 hours after c7E3 Fab infusion showed 65% to 73% bound GPIIb-IIIa receptors compared with the samples obtained after heparin injection (100% free receptors) by use of a ¹²⁵I-labeled c7E3 Fab platelet binding assay.¹⁸ Samples collected from the patients 24 hours after c7E3 Fab showed 47% to 60% bound GPIIb-IIIa receptors (data not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Platelet activation often occurs in association with percutaneous transluminal coronary angioplasty²⁹ and increases the likelihood of ischemic complications.³⁰ Reducing platelet deposition at sites of arterial injury after percutaneous transluminal coronary angioplasty may be necessary to prevent abrupt closure in narrowed atherosclerotic arteries with abnormally elevated levels of fluid shear stress. Aspirin and heparin provide only partial clinical protection from this potentially catastrophic complication.^{29 30 31 32 33 34} Specific platelet antagonists capable of inhibiting either platelet adhesion or aggregation at sites of intimal damage may be necessary to reduce the incidence of reocclusion in these vessels.

Our flow system is a model of blood flowing over arterial regions that have desquamated endothelial cells with exposed subendothelium or that have deeper layers of the arterial wall exposed to the flowing blood. The model allows real-time observation and quantification of platelet adhesion and subsequent aggregation in the presence of controlled levels of elevated shear stress. Under this system, c7E3 Fab reduces platelet deposition more effectively than either heparin or aspirin.

We demonstrated previously that there are no significant differences in platelet adhesion or aggregation on collagen I/vWF at 1500 s^-1 from normal whole blood with either citrate, hirudin, or heparin used as anticoagulant.¹² Blood samples obtained into citrate from the angioplasty patients before and after heparin injection also showed a similar extent of platelet adhesion and aggregation. These observations imply that, although vWF monomers contain a binding site for heparin,^{35 36} the amount of heparin previously used in vitro (10 U/mL)¹² and used in vivo in this study (12 000 U IV) does not interfere with either the adsorption of vWF multimers onto collagen I^{9 36} or subsequent platelet adhesion and aggregation.

In most of the perfusion experiments comparing blood from healthy donors obtained before and after in vivo ingestion of 925 mg aspirin, there was little difference in platelet aggregation subsequent to initial platelet adhesion to collagen I/vWF. The number of small adherent platelet thrombi (<20 platelets per thrombus) from aspirin-treated blood, however, was almost always greater than the number of adherent platelet thrombi from the blood of healthy donors who had not taken aspirin. The explanation for this observation, which is compatible with observations reported by Grabowski,³⁷ is not known. There are no data, for example, on whether the phenomenon relates to a relative increase in lipoxygenase products in platelets with aspirin-inhibited cyclooxygenase. Our in vitro findings of relatively increased platelet adhesion, without inhibition of subsequent aggregation, under flowing conditions in aspirin-treated blood may indicate why aspirin is not a more effective inhibitor of restenosis after coronary angioplasty.³³

The angioplasty patients in the c7E3 Fab study received 325 mg aspirin between 2 and 6 hours before monoclonal antibody infusion and an additional 325 mg aspirin 12 to 24 hours later. Because there was little inhibitory effect on platelet aggregation in blood samples from healthy aspirin-treated donors who had ingested 925 mg aspirin and no inhibitory effect of heparin injection, it was possible for us to test the effect of c7E3 Fab on platelet adhesion and aggregation in our flow system using the blood of angioplasty patients who had received 325 mg aspirin and an injection of heparin before the c7E3 Fab infusion.

Injection of c7E3 Fab did not interfere with platelet adhesion to the collagen I surface (mediated by platelet GPIb interactions with vWF adsorbed onto the collagen I^{12 36 38} ) under abnormally elevated arterial flow conditions. In fact, there was an actual increase in the number of adherent single platelets and small adherent platelet thrombi in some of the experimental runs using blood exposed to c7E3 Fab. This finding may reflect the relative increase in the number of individual platelets available to attach to collagen during flow under conditions of inhibition of platelet-platelet cohesion (aggregation) by c7E3 Fab.

The presence of c7E3 Fab (which blocks GPIIb-IIIa interactions with vWF, fibrinogen, and fibronectin^{16 17} ) in patient blood samples reduced considerably the extent of platelet aggregation on collagen I/vWF subsequent to the initial platelet adhesion. There was an overall reduction in the sizes of platelet thrombi as well as in total platelet deposition. Maximal inhibition of platelet aggregation in the flow system was observed in blood samples collected 2 minutes after the infusion of c7E3 Fab. These inhibitory effects persisted in the blood collected 24 hours after c7E3 Fab infusion in patients 1 and 2. (A 24-hour sample was not collected from patient 3.)

In the EPIC study,³¹ a bolus followed by an extended 12-hour infusion of c7E3 Fab prevented recurrent ischemic events more effectively than a bolus alone. The greater efficacy of the c7E3 Fab bolus plus extended infusion may indicate the value of a longer period of interference with platelet aggregation in vivo in the hours or days after angioplasty.

Our flow system is designed to observe platelet adhesion and aggregation on collagen I/vWF in real time as a model for the exposure of subendothelium or deeper layers of the arterial wall (as produced by angioplasty). Human blood is used under controlled shear conditions analogous to those in constricted atherosclerotic or vasospastic arteries. The model can be used to screen the effectiveness of potential new antithrombotic agents that are ingested, injected, infused, or added ex vivo to normal blood at different dosage levels. The technique can also be used in association with clinical trials to evaluate the effects of different dosages of prospective new antithrombotic compounds.

	Acknowledgments

 
This work was supported by NIH grants HL-18672, HL-32200, NS-23327, and HL-18584, Robert A. Welch Foundation grant C-938, and grants from the Butcher Fund and the Advanced Technology Program of the State of Texas.

	Footnotes

 
Guest Editor for this article was Dr Joseph Loscalzo, Boston University Medical Center Hospital, Boston, Mass.

Received April 28, 1994; revision received August 19, 1994; accepted September 23, 1994.

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