| The prevailing theory about blood coagulation is the two-stage principle, which emphasizes that the coagulation process is initiated by the tissue factor pathway in the human body. A number of experimental and clinical studies have shown that the imbalance of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) is closely related with the cause and the development of thrombosis diseases. The study direction of our lab is the modulation of TF pathway and its clinical significance. The present work focuses on the following two aspects: 1. Experimental research of monoclonal antibody against human TFPI and its application. TFPI has been recently identified as a physiological anticoagulant protein, Which plays a critical role in the inhibition of coagulation initiated by TF. In view of the fact that the assay for TFPI was not established and there was no report of studying TFPI in the country, it is undoubtedly beneficial to the experimental and clinical studies of TFPI to prepare TFPI monoclonal antibody, to explore its binding site, and to research into the method for TFPI antigen measurement and its application. 2. Regulation of TF expression in vascular endothelial cells by endogenous nitric oxide. Under normal circumstances, endothelial cells do not express TF; however, with endothelial cell activation or frank dysfunction, TF expression can be significantly induced. Nitric oxide (NO) is a cellular signaling molecule that plays a major role in a variety of important biological processes, including the prevention of vascular thrombosis, yet , up to now its role in the regulation of TF expression remains unknown. NO is synthesized from L-arginine by NO synthase (NOS), and cellular NO production is absolutely dependent on the availability of L-arginine. The expression of TF in vascular endothelial cells induced by lipopolysaccharide (LPS) or cytokines is commonly associated with the pathogenesis and development of disseminated intravascular coagulation (DIC). Apparently, it is very important to reveal the novel biological activity of NO and its antithromboticmechanism through investigating the effect of endogenous NO generated from L-arginine on TF expression in the vasculature.The following are the abstracts of these two studies:I. Preparation and characterization of monoclonal antibody against tissue factor pathway inhibitorObjective: To prepare monoclonal antibody (McAb) against human tissue factor pathway inhibitor (TFPI) and to use it for measurement of TFPI by ELISA, and to evaluate the effects of the McAb on prothrombin time (PT) and activated partial thromboplastin the (APTT). Methods: Twice intrasplenic immunization of BALB/c mice with TFPI-bound nitrocellulose paper followed by an intraperitoneal boost with TFPI raised two hybridoma cell lines, 4F8 and 4F4, by hybridoma technique. The 4F8McAb was purified from ascites by a simple procedure of caprylic acid precipitation. The McAb was well-characterized and conjugated with horseradish peroxidase (HRP) by a modified periodate oxidation procedure. Furthermore, the 4F8 McAb was added to normal and factor IX deficient plasma for observations of dilute PT and APTT. Results: Two hybridomas (4F4,4F8) purified secreting McAb against TFPI were established. The Ig class and subclass of the McAb from 4F8 was IgGi. Immunoblotting indicated that the 4F8 McAb recognized a single band of TFPI with a molecular weight of 34.8KD. The McAb was also proved to shorten markedly the dilute thromboplastin coagulation time of factor IX-deficient and normal plasma. Based on the immunological reactions of 4F8 McAb with full length TFPI and TFPIuiei and the difference of procoagulant effect of this McAb on PT and APTT, the recognizing site may be at Kl domain. The results of Sandwich ELISA using the HRP labeled McAb showed that the mean TFPI in normal human plasma is 103.2 11.5 g/L, being similar to that measured with TFPI ELISA kit from America Diagnostica Inc.(98.4 10.3 g/L)(r=0.92). Conclusions: McAb 4...
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