Recombinant production and evaluation of a multifunctional haemostatic fusion protein

[摘要] Platelets and coagulation both play a pivotal role in thrombosis, one of themajor life-threatening diseases in our society. We have thus experienced adrastic increase in the development of potent and secure antithrombotic,antiplatelet and fibrinolytic agents during the past decade. Recently, muchresearch has been devoted to the development of chimeric proteins, wherehaemostasis is simultaneously targeted at different levels. For the purpose ofthis study, such a chimera, named PLATSAK, was designed. A 29 amino acidantithrombotic and antiplatelet peptide, comprising three inhibitory regions, waslinked to staphylokinase via a cleavable factor Xa recognition sequence. Theoverall activities of PLATSAK should include inhibition of thrombin, preventionof platelet aggregation and activation of fibrinolyis.The gene encoding PLATSAK was expressed in E. coli cells under controlledconditions. PLATSAK was produced as a strongly expressed protein of 18 kDaand was purified form native E. coli proteins using metal affinitychromatography. In vitro analysis of PLATSAK activity revealed stronginhibition of thrombin and potent fibrin degradation. However, no effect onplatelet aggregation could be observed. Several attempts at producing morepotent antiplatelet variants were unsuccessful.According to its in vitro activity, PLATSAK appeared to be a potent novelhaemostatic agent and was prone to be evaluated in an in vivo system. The invivo activity of PLATSAK was evaluated by assessing its effect on plateletdeposition in a baboon model of arterial and venous thrombosis. Dacronvascular graft segments and expansion chambers, inserted as extensions intopermanent femoral arteriovenous shunts, were used to simulate arterial andvenous thrombosis, respectively. PLATSAK (3.68 mg/kg) was administered asa bolus. Platelet deposition onto the graft surface and in the expansionchamber was imaged in real time with a scintillation camera as the deposition of111ln-labelled platelets. After two hours, platelet deposition in the graft segments and expansion chambers was inhibited by 50% and 85% respectivelywhen compared to control studies. The aPTT was lengthened to >120 seconds.Interestingly, the level of FOP in plasma did not increase after administration ofPLATSAK. These results demonstrate that PLATSAK effectively inhibitedplatelet deposition in both arterial- and venous-type thrombosis an animalmodel. This is in contrast to the lack of the antiplatelet activity of PLATSAK invitro. This illustrates that in vitro platelet aggregation results can not be directlyapplied to an in vivo situation.In summary, the recombinant production of a multifunctional haemostatic fusionprotein, PLATSAK, was successful. In vitro PLATSAK showed significantantithrombin and fibrinolytic activity, but trivial antiplatelet activity. In vivostudies revealed that PLATSAK is a potent antithrombin and also preventedplatelet deposition on thrombogenic material. The strong immuun response ofPLATSAK however needs to be investigated and a variant with a weakimmunogenic nature needs to be produced.

[发布机构] University of the Free State