Study On The Development And Function Of A Monoclonal Antibody To FVⅢ-C2 Domain Inhibit FVⅢ Procoagulant Activity

Thrombotic diseases are life-threatening diseases that affect millions of people every year in the world. Thrombosis research is a major subject in the field of medical sciences. Many studies have reported that elevated Factor VIII ( FVIII ) level is an independent risk factor of thrombosis diseases. The potential mechanism is that high FVIII levels stimulate the formation of thrombin and result in increased platelet activation and fibrin formation and this may contribute to the development of large occlusive thrombi from the microthrombi initially formed on the damaged endothelium. It has been reported that low factor VIII levels protect against ischemic heart disease. Mortality due to ischemic heart disease is much lower in patients with hemophilia A than in the general male population, which may suggest that factor VIII is involved in the pathogenesis of arterial thrombosis. On the other hand, it has been observed that venous thrombosis is very rare in hemophilia A patients. So, it is possible that antithrombotic treatment may be completed by inhibiting FVIII activity.Monoclonal antibodies are currently used for the treatment of cancer, viruse infection and anti-graft rejection. It also represents a novel type of anticoagulant factor. The specificity and pharmacokinetics properties of such antibodies provide opportunities for novel approaches of the prevention and treatment of thrombotic diorders. The C2 domain possesses functionally important binding sites which involve interaction with other proteins such as vWF, factor Xa, and thrombin as well as PS membranes. In our study, a monoclonal antibody to FVIII-C2 domain was developed and its function was investigated. The study contains three parts: (I) Recombinant FVIII-C2 (rFVIII-C2) polypeptide was expressed in Escherichia coli (E.coli) and purified and its function was studied. (II): A murine antihuman FVIII-C2 domain monoclonal antibody (mAb) designated SZ-132 was developed by standard hybridoma technology and characterized. (III) The effect of SZ-132 on FVIII procoagulant activity was investigated. And the mechanism of SZ-132 inhibiting FVIII activity was elucidated by that SZ-132 inhibits rhFVIII-vWF, rhFVIII-PS and rhFVIII-platelets binding assays.Part I Expression of human factor VIII-C2 domain in Escherichia coliFactor VIII ( FVIII ) plays an important role in the intrinsic pathway of blood coagulation. The mature FVIII molecule is a heterodimer of 2332 amino acids which consists of homologous domains arranged in the order A1-A2-B-A3-C1-C2. Under normal physiology condition, FVIII through C2 domain noncovalently binds to von Willebrand factor ( vWF ) D'D3 domain which protects FVIII from inactivation by activated protein C. It is important that FVIII-C2 domain binding to phospholipid membrane for FVIII procoagulant activity. So, FVIII-C2 domain plays an important role in maintaining the stability and activity of FVIII. In our study, FVIII-C2 cDNA was amplified from the plasmid pDLZ6 containing hFVIII-cDNA by polymerase chain reaction (PCR) and was inserted into the prokaryotic expression vector pQE30. To produce recombinant FVIII C2 (rFVIIIC2), Escherichia coli transfected with the plasmid pQE30-FVIII-C2 were induced by isopropyl-D-thiogalactopyranoside (ITPG). The rFVIIIC2 was purified by Ni-NTA agarose column and verified on reduced 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot was used to identify the purified FVIII-C2 protein. The binding of rFVIII-C2 protein to von Willebrand factor ( vWF ) and phosphatidylserine ( PS ) was detected by enzemy linked immunoabsorbance assays (ELISA). The results showed that a fragment of 489 bp was amplified by PCR and the sequencing result was consistent with FVIII-C2 cDNA. Escherichia coli transfected with the plasmid pQE30-FVIII-C2 can express FVIII-C2 protein effectively after being induced by ITPG for 4-6 hours. The rFVIII-C2 protein reacted with anti-His antibody detected by western blot. The rFVIII-C2 protein can bind to vWF and PS in a dose-dependent manner which demonstrated that there were binding sites of vWF and PS in FVIIIC2 domain. Part II Development and characterization of monoclonal antibody SZ-132 to FVIII-C2 domainMany studies have reported that elevated FVIII levels are an independant risk factor of thrombosis and it has also been found that decreased FVIII levels prevent patients from ischemic heart disease. In the hemophilia A patients, the morbidity of ischemic heart disease was significantly lower than normal which implies that FVIII involves in the pathogenesis of arterial thrombosis. On the other hand, it has been observed that venous thrombosis is very rare in hemophilia A patients. So, it is possible that antithrombotic treatment may be completed by inhibiting FVIII activity. The specificity and pharmacokinetics properties of monoclonal antibodies provide opportunities for novel approaches of the prevention and treatment of thrombotic disorders. So far, monoclonal antibodies developed as anticoagulant agents target three coagulation factors: tissue factor, factor IX and factor VIII. The C2 domain of FVIII contributes to FVIII binding to vWF and to phospholipids (PLs) and thereby determines the stability in plasma and the activity of FVIII. In our study, the Balb/c mice were immunized by rFVIII-C2 protein. Murine antihuman FVIIIC2 mAb was developed by standard hybridoma technology. An anti-FVIIIC2 mAb producing hybrioma clone was obtained and was designated SZ-132. It bound specifically to rhFVIII and rFVIIIC2 by ELISA. Immunoblotting indicated that SZ-132 reacted with rhFVIII and rFVIIIC2 protein. These experiments indicated that SZ-132 was FVIIIC2-specific mAb.Part III Study on function of monoclonal antibody SZ-132 to FVIIIC2 domain inhibiting FVIII procoagulant activityIt has been reported that monoclonal antibodies to FVIII could inhibit FVIII procoagulant activity and these antibodies could inhibit the formation of thrombosis in animal model of thrombosis. The capacity of SZ-132 to inhibit plasma FVIII activity was tested in a coagulation assay. The antibody SZ-132 inhibited FVIII activity in a dose-dependent manner when the concentration of SZ-132 was 0-25μg mL-1. When the concentrations were above 25μg mL-1, the FVIII activity was completely inhibited. The calculated IC50 for inhibiting FVIII activity was 9.96μg mL-1. APTT prolonged significantly when the concentrations of SZ-132 were between 0-25μg mL-1, when the concentrations were above 25μg mL-1, further prolongation of APTT was unobvious. To explore the mechanism of SZ-132 inhibiting FVIII activity, the effect of SZ-132 on rhFVIII-vWF and rhFVIII-PS was assessed by ELISAs. SZ-132 inhibited the binding of rhFVIII to vWF and PS in a dose-dependent manner. The concentrations of IC50 of rhFVIII binding to vWF and PS were 10.71 and 9.88μg mL-1, respectively. In the effect of SZ-132 on rhFVIII-vWF binding assay, when the concentrations of SZ-132 were 0-100μg/ ml, the values of OD at 490 nm were (3.28±0.45)-(0.24±0.12); In the effect of SZ-132 on rhFVIII-PS binding assay, when the concentrations of SZ-132 were 0-100μg/ ml, the values of OD at 490 nm were (3.25±0.50)-(0.41±0.14). The results explained the mechanism that SZ-132 inhibited FVIII procoagulant activity and also suggested that the sites for FVIII binding to vWF or PS have some overlap. The inhibition of SZ-132 on rhFVIII binding to immobilized platelets was tested in ELISA. The results show that SZ-132 inhibited rhFVIII binding to platelets in a dose-dependent manner. When the concentrations of SZ-132 were 0-100μg/ ml, the values of OD at 490 nm were (2.65±0.25)-(0.56±0.10). SZ-123 has no effect on rhFVIII-platelets binding. These results demonstrated that SZ-132 could inhibit rhFVIII binding to platelets. Above all, the mAb SZ-132 that directed against FVIII-C2 domain could inactivate FVIII procagulant activity, and the mechanisms of which may be that SZ-132 could inhibit FVIII binding to vWF and PS. The result that SZ-132 could inhibit FVIII binding to platelets implied that SZ-132 had potential antithrombotic effect.