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

Abstract

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.

Ex vivo evaluation of erythrocytosis‐enhanced platelet thrombus formation using the cone and plate(let) analyzer: effect of platelet antagonists

“…This was accompanied by the release of increased proaggregatory activity in the cell‐free, postshear supernatant, suggesting the presence of increased platelet recruitment potential. Indeed, cell–cell interactions between platelets and erythrocytes can significantly alter platelet reactivity, through modulation of both platelet activation and recruitment (Stormorken, 1971; Born et al , 1976; Born & Wehmeier, 1979; Schmid‐Schonbein et al , 1981; Reimers et al , 1984).…”

Mechanisms of hemolysis‐associated platelet activation

“…Aliquots of the samples were taken prior to and after stirring, to test for platelet count and hemolysis. Platelet aggregation was assessed by examining the percentage decrease in counts of individual platelets, as described previously [39,40]. Platelet counts were performed in the core laboratory at Wake Forest Baptist Medical Center.…”

“…A ratio of platelet count to RBC count was recorded before and after infusion to control for any dilution resulting from the infusate. Platelet aggregation was assessed by examining the percentage decrease in platelet count to RBC ratio [39,40]. Whole blood counts were performed by the Wake Forest Institute for Regenerative Medicine, using a Siemens' Advia 120 hematology system.…”

Red blood cells: the forgotten player in hemostasis and thrombosis

“…This review will briefly summarize what is currently known about the involvement of RBCs in hemostasis and thrombosis and its underappreciated importance. RBCs increase blood viscosity because of a rise in hematocrit, an increase in RBC aggregation or a decrease in RBC deformability (increasing flow resistance) Pro [2][3][4][5] Conversely, anemia is associated with low blood viscosity and bleeding tendency as a result of reduced platelet margination toward endothelium and enhanced NO availability Anti [2][3][4][5] RBCs undergo shear-dependent reversible aggregation mediated by plasma proteins (mainly fibrinogen and immunoglobulins) and/or local osmotic gradient Pro [14][15][16][70][71][72][73][74] RBCs with increased rigidity occlude small vessels Pro [11,12] Deformability of RBCs reduces frictional resistance to flow Anti [8,[11][12][13]] RBCs maintain biconcave shape and a high surface-to-volume ratio as a result of cytoskeleton and water/ions balance Pro or anti [5] RBCs migrate to the center of blood flow and push platelets toward the endothelium (margination) in a hematocrit-and shear-dependent manner Pro [59][60][61] Effects on platelet reactivity RBCs increase platelet adhesion and aggregation by release of ADP and thromboxane A 2 Pro [66,67] RBCs form aggregates with platelets via adhesive molecules (ICAM-4 and fibrinogen with aIIbb3)…”