Effects of a novel antiplatelet agent in mural thrombogenesis on collagen-coated glass

Abstract

A parallel plate flow chamber and an epifluorescence video microscopy system were used to investigate the inhibitory effect of a novel antiplatelet agent (GT-12), a carbamoylpiperidine congener, on surface platelet aggregation and on the kinetics of thrombus growth induced by collagen-coated glass under controlled flow. Both macroscopic and microscopic measurements revealed that increasing concentrations of the drug correspondingly decreased the reaction rate between platelets at the surface, thereby reducing thrombus rate of growth at the surface. Because of decreased platelet/platelet adhesion, there was some embolization of the larger thrombi near the inlet of the reactive surface. In the presence of GT-12, average thrombus size and number of platelets per thrombus were both strikingly lowered. In addition, the net rate of growth of individual thrombi decreased to zero after short exposure times (about 60 seconds), in sharp contrast to controls. In contrast to chlorpromazine, GT-12 was effective in inhibiting platelet aggregation and thrombus rate of growth at relatively low concentrations (less than 100 mumol/L) in whole blood. The drug's effectiveness relative to controls in impeding platelet/platelet interactions was found to increase with decreasing incubation time and increasing perfusion time.

Mathematical analysis of mural thrombogenesis. Concentration profiles of platelet-activating agents and effects of viscous shear flow

“…In other words, embolization of such small protrusions may occur only at relatively high wall shear rate. In fact, this correlates well with the previous experimental observations from our laboratory (19,24), that occasional embolization of the larger thrombi (10-20 ,um high) formed on collagen-coated glass occurred at wall shear rates >1,500 s-'. It is likely, however, that as the size of the aggregate increases, the embolizing stresses may become functions of both the wall shear rate and the thrombus size.…”

“…It should be pointed out that the downstream maximum is always larger than the upstream one, which can be explained as follows: some of the molecules released from the upstream part or the top of the thrombus are carried along by convection in the direction of the downstream vortex, increasing the local concentration there. This theoretical result correlates well with previous experimental observations made in our laboratory (19,24,36). Indeed, it was postulated that "fresh" platelets arriving at the site of aggregation become activated as they pass through a "cloud" of platelet-activating agents surrounding the thrombus, and therefore adhere preferentially at the downstream edge of the aggregate.…”

“…Heparin-anticoagulated whole blood with fluorescent-labelled platelets, prepared as previously reported (19), was aspirated by a syringe pump (Harvard Apparatus Co., Inc., South Natick, MA, Model 935) for two and a half minutes at a controlled shear rate of 100 s-', 800 s-', or 1,500 s-1 from a test tube into a polycarbonate parallel-plate flow chamber, described in detail elsewhere.20…”

“…These data were then used in the calculations of fluxes of platelet-activating substances in the mathematical model (see Appendix C). The local visualization of mural thrombogenesis was performed at the location of maximum macroscopic platelet accumulation on the collagen-coated surface, as determined by a microphotometric measurement system, described previously (19). At the low wall shear rates of 100 s-' and 800 s-', maximum platelet density occured near the beginning of the collagen coating because of platelet diffusion-limited processes downstream.…”

A novel flow cytometric analysis for platelet activation on immobilized von Willebrand factor or fibrillar collagen

“…Whether platelet accumulation remains mural and limited, or evolves into an occlusive thrombus after endothelial cell denudation or atherosclerotic plaque rupture, is likely to depend on the nature and extent of exposed thrombogenic material. In vitro flow experiments have shown that platelets may adhere individually and separately, form small clumps, or cohere into large aggregates on surfaces coated with different purified matrix proteins [1–4]. For example, platelets adhere as singlets or form a few small platelet aggregates on fibrinogen‐, fibrin‐, or von Willebrand factor (VWF)‐coated surfaces, whereas they form large multiplatelet thrombi on fibrillar collagen I‐coated surfaces.…”