Potentiation by red blood cells of shear-induced platelet aggregation: relative importance of chemical and physical mechanisms.

Evidence has been reported to indicate that red blood cells (RBCs) may potentiate platelet adherence and platelet aggregation (PAG) in different flow systems in vitro as well as hemostatic platelet plug formation in response to vascular injury. In this study, we demonstrate that RBCs enhance PAG induced by well-defined, low-intensity, uniform, laminar shear stress. Potentiation by RBCs of shear-induced PAG was associated with appreciable loss of adenine nucleotides from 14C-adenine-labeled RBCs, the extent of which increased with increasing RBC concentration. The concentrations of RBC-derived ADP measured in the medium after shear, as determined by both high pressure liquid chromatography and the luciferin/luciferase system, were within the range of concentrations of ADP which may trigger PAG or potentiate PAG induced by low concentrations of other platelet agonists in the aggregometer. To assess the relative contribution of chemical (ADP) and physical (platelet surface transport) mechanisms in the RBC-mediated potentiation of shear-induced PAG, aliquots of citrated platelet-rich plasma (C-PRP) were exposed to shear stress in the presence of untreated RBCs or RBCs exposed to an antihemolytic concentration (5 mumol/L) of the membrane stabilizing agent, chlorpromazine (CPZ). Potentiation of shear-induced PAG in the RBC-CPZ system was significantly less than that in the untreated RBC system. However, CPZ-induced reduction of PAG potentiation was associated with an increase rather than a decrease in loss of adenine nucleotides from RBC. Furthermore, shear-induced PAG in C-PRP as well as ADP- and collagen-induced PAG in C-PRP in the aggregometer was significantly inhibited by 5 mumol/L CPZ, indicating that the observed reduced potentiation of shear-induced PAG by RBCs in the presence of CPZ was due to a direct inhibitory effect of the drug on platelets rather than a reduction of shear-induced liberation of ADP from RBCs. When aliquots of C-PRP were exposed to shear stress in the presence of RBCs completely depleted of ADP by fixation in 1% glutaraldehyde, potentiation of PAG was approximately half of that observed with intact RBCs. These findings indicate that both RBC-derived ADP and RBC-mediated platelet surface transport are involved in the potentiation by RBCs of PAG induced by laminar shear stress.

Role of cytoplasmic and releasable ADP in platelet aggregation induced by laminar shear stress.

We examined the extent of lytic and sublytic platelet injury after exposure of platelets to shear stress and the role in shear-induced PAG of ADP liberated from platelets as a result of shear-induced platelet dense body release and/or platelet damage. Platelets in C-PRP or TAS were subjected to well-defined, laminar shear stress in a rotational viscometer, and PAG (loss of single, nonaggregated platelets), 14C-serotonin release, and loss from platelets of LDH and 51Cr were determined. Increased PAG with increasing shear stresses was associated with progressive loss of LDH and 51Cr. Loss of 51Cr was consistently in excess of that of LDH, indicating sublytic platelet injury, which was confirmed by electron microscopy. At the lowest shear stress used (50 dynes/cm2), PAG in C-PRP was observed in the absence of detectable loss of 51Cr or LDH. When platelets in TAS were sheared in the presence of CP/CPK, an enzyme system capable of removing extracellular ADP, PAG was only partially (approximately 40%) inhibited. However, when platelets were preincubated with CP/CPK and ATP (to saturate platelet ADP receptors), shear-induced PAG was almost completely suppressed. Similar results were obtained with PAG induced by collagen in the aggregometer. The findings indicate that (1) shear-induced PAG in this system may occur without measurable lytic or sublytic platelet damage and (2) ADP liberated from platelets as a result of shear-induced release or damage may represent the major if not sole mediator of shear-induced PAG.