Lysophosphatidic Acid Induces Thrombogenic Activity Through Phosphatidylserine Exposure and Procoagulant Microvesicle Generation in Human Erythrocytes

Abstract

Objective-Although erythrocytes have been suggested to play a role in blood clotting, mediated through phosphatidylserine (PS) exposure and/or PS-bearing microvesicle generation, an endogenous substance that triggers the membrane alterations leading to a procoagulant activity in erythrocytes has not been reported. We now demonstrated that lysophosphatidic acid (LPA), an important lipid mediator in various pathophysiological processes, induces PS exposure and procoagulant microvesicle generation in erythrocytes, which represent a biological significance resulting in induction of thrombogenic activity. Methods and Results-In human erythrocytes, LPA treatment resulted in PS exposure on remnant cells and PS-bearing microvesicle generation in a concentration-dependent manner. Consistent with the microvesicle generation, scanning electron microscopic study revealed that LPA treatment induced surface changes, alteration of normal discocytic shape into echinocytes followed by spherocytes. Surprisingly, chelation of intracellular calcium did not affect LPA-induced PS exposure and microvesicle generation. On the other hand, protein kinase C (PKC) inhibitors significantly reduced PS exposure and microvesicle generation induced by LPA, reflecting the role of calcium-independent PKC. Activation of PKC was confirmed by Western blot analysis showing translocation of calcium-independent isoform, PKC, to erythrocyte membrane. The activity of flippase, which is important in the maintenance of membrane asymmetry, was also inhibited by LPA. Furthermore, LPA-exposed erythrocytes actually potentiated the thrombin generation as determined by prothrombinase assay and accelerated the coagulation process initiated by recombinant human tissue factor in plasma. The adherence of erythrocytes to endothelial cells, another important feature of thrombogenic process, was also stimulated by LPA treatment. Conclusion-These results suggested that LPA-exposed erythrocytes could make an important contribution to thrombosis mediated through PS exposure and procoagulant microvesicle generation.

Stimulation of human red blood cells leads to Ca2+-mediated intercellular adhesion

“…As illustrated in Fig. 3b, during that is not related to the PS exposure [16,17]. Therefore, RBCs were stained with annexin-V-FITC conjugates in order to visualize PS exposure on the surface of RBCs.…”

“…The development of the HOT provides a tool for testing this hypothesis at the cellular level. Concentrations of 2.5 µM and 10 µM LPA were chosen because they seemed to be within the common range of concentrations used with other cell types [24,25] in addition to RBCs [17]. Moreover, this concentration is comparable to the local LPA concentration in the immediate surroundings of activated platelets, e.g., inside a blood clot [26,27].…”

Hemin-induced suicidal erythrocyte death

“…Eryptotic cells may further adhere to the vascular wall [3,17,28,60,72] and foster blood clotting [3,16,75]. Thus, phosphatidylserine-exposing erythrocytes may compromise the microcirculation.…”

Sphingomyelinase-induced adhesion of eryptotic erythrocytes to endothelial cells

“…Phosphatidylerine-exposing cells may activate coagulant enzymes (12) and adhere to the vascular wall (11), thus triggering thrombosis and thrombo-occlusive diseases (2,12,13,17,43,55,60). However, the mechanisms possibly underlying erythrocyte-endothelial cell interaction, especially in diseases with excessive eryptosis, are not well defined.…”