Lysophosphatidic acid induces thrombogenic activity through phosphatidylserine exposure and procoagulant microvesicle generation in human erythrocytes.
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.


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, PKCzeta, 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.


CONCLUSIONS
These results suggested that LPA-exposed erythrocytes could make an important contribution to thrombosis mediated through PS exposure and procoagulant microvesicle generation.
Chung
Seung-Min
SM
College of Pharmacy, Seoul National University, Seoul 151-742, Korea.
Bae
Ok-Nam
ON
Lim
Kyung-Min
KM
Noh
Ji-Yoon
JY
Lee
Moo-Yeol
MY
Jung
Yi-Sook
YS
Chung
Jin-Ho
JH
D000293:Adolescent; D000328:Adult; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D013927:Thrombosis / Q000503:physiopathology*
D008246:Lysophospholipids; D008565:Membrane Proteins; D010718:Phosphatidylserines; D011493:Protein Kinase C; C032881:lysophosphatidic acid; D002118:Calcium