Procoagulant constituents from Cordyceps militaris

Cordyceps militaris,belongs to Clavicipitaceae family,was investigated for its chemical compounds,and six compounds were isolated and purified by silica gel column chromatography,Sephadex LH-20 and recrystallization,and their structures were elucidated by spectral techniques and physicochemical properties as ergosterol(1),adenosine(2),cordycepin(3),ergosterol peroxide(4),tetracosanoic acid 2,3-dihydroxypropylester(5),mannitol(6).Procoagulant activity was screened by assaying the activated partial thromboplastin time(APTT),prothrombin time(PT),thrombin time(TT)and fibrinogen(FIB)in vitro.The results indicated that ergosterol,adenosine,cordycepin,ergosterol peroxide and mannitol showed strong procoagulant activity.

Increased procoagulant activity of red blood cells in the presence of cisplatin

Background Cisplatin based chemotherapy is a well recognized risk factor for coagulation disorders and thrombosis. The pathophysiological mechanisms by which cisplatin promote thrombosis are not well understood. Methods Red blood cells （RBCs） were separated from peripheral blood of patients with breast cancer （n=10） and healthy adults （n=6） and treated with cisplatin. Coagulation time of RBCs was assessed by one step recalcification time and the productions of thrombin, intrinsic and extrinsic factor Xa were measured in the presence or absence of various concentrations of lactadherin. Exposed phosphatidylserine was stained with lactadherin and observed by confocal microscopy and flow cytometry. Results Neither fresh RBCs nor RBCs treated without cisplatin had potent procoagulant activity. Cisplatin treatment increased procoagulant activity of RBCs in a cell number- and concentration-dependent manner. Exposed phosphatidylserine was stained with lactadherin and after cisplatin treatment, strong fluorescence was revealed by confocal microscopy. Lactadherin bound RBCs from patients with breast cancer increased from （1.9＋0.5）% on control RBCs to （68.0±3.5）% on RBCs treated with 10umol/L cisplatin for 24 hours. Conclusions Cisplatin treatment increases procoagulant activity of RBCs, which have a strong association with exposure of phosphatidylserine. The increased procoagulant activity may contribute to the pathogenesis of thrombophilia during cisplatin based chemotherapy in breast cancer patients.

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）-Iabeled 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.

Effect of all-trans retinoic acid and arsenic trioxide on tissue factor expression in acute promyelocytic leukemia cells

study the effect of all trans retinoic acid (ATRA) and arsenic trioxide (As 2O 3) on tissue factor (TF) expression and procoagulant activity (PCA) of acute promyelocytic leukemia (APL) cells in vivo and in vitro Methods PCA from freshly isolated APL blasts from APL patients treated with ATRA or As 2O 3 was detected using a one stage clotting assay TF antigen was detected by ELISA and TF mRNA by RT PCR The maturation sensitive (NB4) or resistant subclones (NB4 R1) of the promyelocytic NB4 cell line, as well as U937 cells infected with pMSCV PML RARa treated with or without ATRA or As 2O 3, were also examined Results Both ATRA and As 2O 3 can down regulate the TF antigen, its mRNA transcription and membrane PCA of APL cells in vivo and in vitro, in a time dependent manner The TF antigen level in PML RARa+ U937 cells was significantly higher than that in U937 cells infected with retrovirus vector Both ATRA and As 2O 3 can also down regulate the TF antigen in U937 cells transfected with or without PML RARa Conclusion Tissue factor expression and PCA in APL cells may be down regulated by ATRA and As 2O 3 By down regulating TF expression, As 2O 3 might also be used to improve the DIC related hemorrhage in APL Our data indicate that elevated TF antigen in PML RARa+ U937 may be related to the fusion protein PML RARa The down regulating effect of ATRA and As 2O 3 on TF expression in U937 cells might not involve this fusion