Lactadherin and procoagulant activities of red blood cells in cyclosporine induced thrombosis.
BACKGROUND
The side effects of cyclosporine therapy include thromboembolic complications. However, the mechanisms underlying the hypercoagulable state induced by cyclosporine are not fully understood. Cyclosporine binds to red blood cells (RBCs) with a high affinity in circulation and alters the membranes of RBCs. Therefore, we propose that such alterations in RBCs membranes play a role in cyclosporine-induced coagulopathy and this disorder may be rectified by lactadherin, a phosphatidylserine binding protein.


METHODS
RBCs from healthy adults were treated with various concentrations of cyclosporine. Procoagulant activity of the RBC membrane was measured by the single stage recalcification time and confirmed by detection of tenase and thrombin assembly through enzymatic assays. Inhibition assays of coagulation were carried out in the presence of lactadherin, annexin V or antitissue factor. Phosphatidylserine exposure was detected by flow cytometry and confocal microscopy through binding with fluorescein isothiocyanate (FITC)-labeled lactadherin as well as FITC annexin V.


RESULTS
RBCs treated with cyclosporine demonstrated increased procoagulant activity. Cyclosporine treatment markedly shortened the clotting time of RBCs ((305 +/- 10) seconds vs (366 +/- 15) seconds) and increased the generation of intrinsic factor Xase ((7.68 +/- 0.99) nmol/L vs (2.86 +/- 0.11) nmol/L) and thrombin ((15.83 +/- 1.37) nmol/L vs (4.88 +/- 0.13) nmol/L). Flow cytometry and confocal microscopy indicated that cyclosporine treatment induced an increased expression of phosphatidylserine on the RBC membrane. Lactadherin was more sensitive in detecting phosphatidylserine exposure of the RBC membrane than annexin V. The modulating effect of procoagulant activity was concomitant with and dependent on phosphatidylserine exposure. Blocking of phosphatidylserine with lactadherin effectively inhibited over 90% of FXa generation and prothrombinase activity and prolonged coagulation time.


CONCLUSIONS
Procoagulant properties of RBCs membranes resulting from phosphatidylserine exposure may play an important role in cyclosporine-induced thrombosis. Lactadherin can be used as a sensitive probe for phosphatidylserine detection. Its high affinity for phosphatidylserine may provide a new approach for the treatment of cyclosporine induced thrombogenic properties.
Zheng
Yi-ning
YN
Department of Haematology, First Affiliated Hospital of Harbin Medical University, Heilongjiang, China.
Yu
Hong-juan
HJ
Hou
Jin-xiao
JX
Lu
Cheng-fang
CF
Zhou
Jin
J
D000328:Adult; D000818:Animals; D017304:Annexin A5 / Q000737:chemistry; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology*; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry*; D018613:Microscopy, Confocal; D008894:Milk Proteins / Q000737:chemistry*; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D013927:Thrombosis / Q000139:chemically induced* / Q000378:metabolism
D017304:Annexin A5; D008562:Membrane Glycoproteins; D008894:Milk Proteins; C106215:PAS-6-7 glycoprotein, Bos taurus; D010718:Phosphatidylserines; D016572:Cyclosporine