The Antidote For A Class Of New Anti-coagulant(DTI) And The Exploration Of The Mechanism Of TM-dentritic Cells Interaction

The coagulation cascade is usually the first responder at the site of a challenge,triggered by exposure of tissue factor, finally resulting in a fibrin clot. The mostimportant part in the cascade is thrombin generation and thrombin cleavage offibrinogen to fibrin. Most of the coagulation disorders are associated with thrombindysfunctional. Thus, thrombin is the effective target for anticoagulant drugs. A classof new anti-coagulant has been developed recently, direct thrombin inhibitors（DTIs）. There are one parenteral DTI, argatroban, a small molecule, and an oralDTI that has been recently approved, dabigatran etexilate（Pradaxa）. This kind ofnew anti-coagulants has great potential to replace the current clinical practice forpatients who require long-term anticoagulation. Because it has minimal drug-drugand drug-food interactions compared to the current clinical anticoagulant warfarin,and it does not require routine monitoring by a clotting time assay. But the majorreason limit the widespread use of DTI comes from the safety concern. It shouldhave an effective antidote that can rapidly reverse the DTI effect in case of eitheracute bleeding or the urgent need for major invasive surgery. In this study, thrombinmutants were employed on identifying the antidote for DTIs.Three thrombin mutants （S195A, S195A/Y76A, S195A/Y76A/R101A） wasdesigned based on the structure-function relationship of thrombin. S195A containsa single mutation at the active site of thrombin with significantly reducedenzymatic activity. The data from in vitro coagulation assays suggest that S195Aact as an antagonist of both argatroban and dabigatran. This supports the hypothesisthat a thrombin mutant can function as an antidote for DTI. Because small molecule DTIs do not bind to the thrombin exosites, besides S195, two other mutation sites（Y76from exosite I and R101from exosite II） were involved into S195A/Y76Aand S195A/Y76A/R101A. So that the inactive thrombin mutant will not interferewith the normal actions of physiological thrombin in the blood circulation.Thrombinoscope data suggest that S195A/Y76A/R101A function as an anantagonist of DTI, while S195A/Y76A does not bind to diabigatran. This maybecause of that the active DTI binding site of S195A/Y76A is blocked due to thecombination of the two mutations. Thus, the antidote candidates for DTIs would beS195A and S195A/Y76A/R101A.More in vitro and in vivo experiments will be carried out and the outcome ofthis project will be a clinical candidate with efficacy, safety and bothpharamacokinetic and pharmacodynamic data suitable for advancing into the clinic.Thrombomodulin is another important protein in blood coagulation cascade.Despite its anticoagulant role in coagulation system, more and more recent studiesrevealed that thrombomodulin also function as an anti-inflammatory protein, andlectin domin is supposed to be responsible for that function. But the mechanisms oflectin domin regulates inflammatory responses is still not clear by now. DCs areantigen presenting cells that are generated in the bone marrow, migrate to organswhere they interrogate foreign antigens. There is a subset of DCs called BDCA3+DCs, which express TM. BDCA3+DCs are small minority of the total DCs but anincrease in their levels has been correlated with asthma, malaria and canceramongst other diseases. Bone marrow stem cells was seperated from mice, andinduced by cytokines to become bone marrow derived dentritic cells （BMDC）.There are1.9%of TM+DC in mice BMDC. Recombinant fusion proteins TM-mfc,lectin-mfc and mfc was constructed and purified for understanding the mechanismof TM-dendritic cell interaction. In this study, the novel UCOE vector was used toobtain the recombinant fusion protein, succesfully avoided post-transfection geneslience. aPC assay and HMGB1binding assay have shown that TM-mfc andlectin-mfc fusion proteins are functional as natural TM protein. The fusion proteinswas then incubated with bone marrow derived dentritic cells separately. FACs datasuggested that TM-mfc cause the dentritic cells to change from an immunogenic toa tolerogenic phenotype, but the role of lectin domin in this mechanism is not clear.Futher experiments is under way to detect the TM signaling pathway on dentriticcells.