Lactadherin and procoagulant activities of red blood cells in cyclosporine induced thrombosis

Background The side effects of cyclosporine therapy include thromboembolic complications.However,the mechanismsunderlying 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 inRBCs membranes play a role in cyclosporine-induced coagulopathy and this disorder may be rectified by lactadherin,aphosphatidylserine binding protein.Methods RBCs from healthy adults were treated with various concentrations of cyclosporine.Procoagulant activity of theRBC membrane was measured by the single stage recalcification time and confirmed by detection of tenase and thrombinassembly through enzymatic assays.Inhibition assays of coagulation were carried out in the presence of lactadherin,annexinV or antitissue factor.Phosphatidylserine exposure was detected by flow cytometry and confocal microscopy through bindingwith fluorescein isothiocyanate (FITC)-labeled lactadherin as well as FITC annexin V.Results RBCs treated with cyclosporine demonstrated increased procoagulant activity.Cyclosporine treatment markedlyshortened the clotting time of RBCs ((305±10) seconds vs (366±15) seconds) and increased the generation of intrinsic factorXase ((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 cytometryand confocal microscopy indicated that cyclosporine treatment induced an increased expression of phosphatidylserine on theRBC membrane.Lactadherin was more sensitive in detecting phosphatidylserine exposure of the RBC membrane thanannexin V.The modulating effect of procoagulant activity was concomitant with and dependent on phosphatidylserineexposure.Blocking of phosphatidylserine with lactadherin effectively inhibited over 90% of FXa generation andprothrombinase activity and prolonged coagulation time.Conclusions Procoagulant properties of RBCs membranes resulting from phosphatidylserine exposure may play animportant role in cyclosporine-induced thrombosis.Lactadherin can be used as a sensitive probe for phosphatidylserinedetection.Its high affinity for phosphatidylserine may provide a new approach for the treatment of cyclosporine inducedthrombogenic properties.