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

Abstract

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 degrees 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.