Recombinant ancylostoma caninum anticoagulant peptide-5 （rAcAP5） has been reported to inhibit thrombin-activatable fibrinolysis inhibitor （TAFIa） activity and have thrombolytic effect. The present study was to scr...Recombinant ancylostoma caninum anticoagulant peptide-5 （rAcAP5） has been reported to inhibit thrombin-activatable fibrinolysis inhibitor （TAFIa） activity and have thrombolytic effect. The present study was to screen a strain expressing high-yield of rAcAP5 and to assess its thrombolytic effect on embolic middle cerebral artery occlusion （MCAO） model in rats. Codons encoding for AcAP5 were optimized. Six expression plasmids and eleven E. coli strains with different characteristics were used, a total of 66 recombinant expression strains were generated and the one with the highest yield was selected to express rAcAP5, which was purified through anion- and cation-exchange chromatography. The purity of rAcAP5 and its molecular weight were determined by HPLC and mass spectrometry, respectively. The thrombolytic effect of rAcAP5 was evaluated on embolic MCAO model in rats; regional cerebral blood flow （rCBF） was monitored with a Laser-Doppler flowmetry to test the occlusion and recanalization of MCA. The highest yield recombinant strain was C2566H/pTYB 1-rAcAP5. AcAP5 （28 mg） with 90% of purity was obtained from 1 L of cell culture. In rat embolic MCAO model, vehicle （normal saline） treatment did not change the rCBF, while treatment with rAcAP5 （50-200 μg/kg, i.v.） increased the rCBF in a dose-dependent manner. In conclusion, we prepared and characterized the rAcAP5 peptide and revealed its thrombolytic effect in embolic MCAO model and our results suggested that this peptide had the potential to be used as a thrombolytic agent.展开更多
Ancylostoma caninum anticoagulant peptide 5 （AcAP5） has been reported as an FXa inhibitor. We found that this peptide showed potent inhibitory activity on activated thrombin-activatable fibrinolysis inhibitor （TAFI...Ancylostoma caninum anticoagulant peptide 5 （AcAP5） has been reported as an FXa inhibitor. We found that this peptide showed potent inhibitory activity on activated thrombin-activatable fibrinolysis inhibitor （TAFIa）, which protects the fibrin clot against lysis. This study investigated the effects of recombinant AcAP5 （rAcAP5） on fibrinolytic activity in vitro and on thrombolytic activity in vitro and in vivo. In addition, euglobulin lysis time （ELT）, fibrinogen content and fibrin degradation product （FDP） in normal rat plasma were all determined to evaluate the influence ofrAcAP5 on fibrinolytic activity in circulation blood. TAFIa activity was detected by colorimetry; fibrinolysis in vitro was determined with turbidimetry. Thrombolysis in vitro was measured by thrombus weight reduction; thrombolysis in vivo was conducted by arteriovenous shunt method with thrombus weight reduction. ELT, fibrinogen content and FDP were detected using routine method, rAcAP5 concentration-dependently inhibited TAFIa activity in vitro with an IC50 of 63.7 nmol/L, rAcAP5 （5-40 nmol/L） significantly accelerated urokinase-induced clot lysis of rabbit plasma and shortened fibrinolysis time （P〈0.01）. rAcAP5 at 1.5 ktmol/L enhanced the reduced weight of thrombus induced by urokinase （P〈0.05）, and it also reduced thrombus weight （P〈0.05） in vitro when used alone. In an arteriovenous shunt thrombolytic model, rAcAP5 （50-200 μg/kg, i.v.） dose-dependently enhanced the reduced weight of the thrombus, rAcAP5 at effective doses failed to influence ELT, fibrinogen contents and FDP in normal rat circulation plasma. In conclusion, rAcAP5 is a thrombolytic peptide, which may be attributed to its TAFIa inhibition, rAcAP5 may only exert thrombolytic activity on the thrombus site but not influence the fibrinolysis activity and fibrinogen in circulation plasma. These findings suggest that rAcAP5 is a potential thrombolytic candidate agent.展开更多
基金National Technology Graveness Special Purpose Fund(Grant No.2009ZX09301-010)
文摘Recombinant ancylostoma caninum anticoagulant peptide-5 （rAcAP5） has been reported to inhibit thrombin-activatable fibrinolysis inhibitor （TAFIa） activity and have thrombolytic effect. The present study was to screen a strain expressing high-yield of rAcAP5 and to assess its thrombolytic effect on embolic middle cerebral artery occlusion （MCAO） model in rats. Codons encoding for AcAP5 were optimized. Six expression plasmids and eleven E. coli strains with different characteristics were used, a total of 66 recombinant expression strains were generated and the one with the highest yield was selected to express rAcAP5, which was purified through anion- and cation-exchange chromatography. The purity of rAcAP5 and its molecular weight were determined by HPLC and mass spectrometry, respectively. The thrombolytic effect of rAcAP5 was evaluated on embolic MCAO model in rats; regional cerebral blood flow （rCBF） was monitored with a Laser-Doppler flowmetry to test the occlusion and recanalization of MCA. The highest yield recombinant strain was C2566H/pTYB 1-rAcAP5. AcAP5 （28 mg） with 90% of purity was obtained from 1 L of cell culture. In rat embolic MCAO model, vehicle （normal saline） treatment did not change the rCBF, while treatment with rAcAP5 （50-200 μg/kg, i.v.） increased the rCBF in a dose-dependent manner. In conclusion, we prepared and characterized the rAcAP5 peptide and revealed its thrombolytic effect in embolic MCAO model and our results suggested that this peptide had the potential to be used as a thrombolytic agent.
基金Original New Drug Fund of Peking University- Funder and National Technology Graveness Special Purpose Fund (Grant No.2009zx09301-010)
文摘Ancylostoma caninum anticoagulant peptide 5 （AcAP5） has been reported as an FXa inhibitor. We found that this peptide showed potent inhibitory activity on activated thrombin-activatable fibrinolysis inhibitor （TAFIa）, which protects the fibrin clot against lysis. This study investigated the effects of recombinant AcAP5 （rAcAP5） on fibrinolytic activity in vitro and on thrombolytic activity in vitro and in vivo. In addition, euglobulin lysis time （ELT）, fibrinogen content and fibrin degradation product （FDP） in normal rat plasma were all determined to evaluate the influence ofrAcAP5 on fibrinolytic activity in circulation blood. TAFIa activity was detected by colorimetry; fibrinolysis in vitro was determined with turbidimetry. Thrombolysis in vitro was measured by thrombus weight reduction; thrombolysis in vivo was conducted by arteriovenous shunt method with thrombus weight reduction. ELT, fibrinogen content and FDP were detected using routine method, rAcAP5 concentration-dependently inhibited TAFIa activity in vitro with an IC50 of 63.7 nmol/L, rAcAP5 （5-40 nmol/L） significantly accelerated urokinase-induced clot lysis of rabbit plasma and shortened fibrinolysis time （P〈0.01）. rAcAP5 at 1.5 ktmol/L enhanced the reduced weight of thrombus induced by urokinase （P〈0.05）, and it also reduced thrombus weight （P〈0.05） in vitro when used alone. In an arteriovenous shunt thrombolytic model, rAcAP5 （50-200 μg/kg, i.v.） dose-dependently enhanced the reduced weight of the thrombus, rAcAP5 at effective doses failed to influence ELT, fibrinogen contents and FDP in normal rat circulation plasma. In conclusion, rAcAP5 is a thrombolytic peptide, which may be attributed to its TAFIa inhibition, rAcAP5 may only exert thrombolytic activity on the thrombus site but not influence the fibrinolysis activity and fibrinogen in circulation plasma. These findings suggest that rAcAP5 is a potential thrombolytic candidate agent.
