Study On The Inhibition Of Mab SZ-123to VWF A3Domain On The Human VWF Binding To Collagen Type Ⅰ, Type Ⅲ And Type Ⅳ And Its Function Of Anti-platelet Adhesion

Background:In recent years, the thrombotic diseases have become one of the most common causes ofdeath and the incidence rate is still rising with the China’s aging population increasing. Arterialthrombosis has been widespread concern for its acute attack, high morbidity and high mortalityin thromboembolic diseases. Anti-artery thrombosis therapies mainly used the drugs ofanti-platelet therapy. The first generation anti-thrombus drugs such as aspirin, ticlopidine andclopidogrel were applied widespreadly in clinic. Aspirin can block the arachidonic acidpathway, ticlopidine and clopidogrel can selectively inhibit adenosine diphosphate (ADP)which binds to its platelet receptor. But the two types of drugs are prone to cause reinfarctionphenomena in interventional treatment. The second generation antithrombus drugs includingReopro and SZ-21are the GPⅡb/Ⅲa antagonist, which mainly blocks the final link inplatelet thrombus formation and platelet aggregation in the final path. However, some patientsmay develop serious bleeding complications. Zhao Y previously reported a specific murineanti-human vWF-A3domain monoclonal antibody SZ-123, which was focused on pathologicthrombus formation-platelet adhesion. Mab SZ-123not only prevents platelet aggregation, butalso blocks the formation of pathological thrombosis by inhibiting platelet adhesion, which canreduce bleeding side reaction.Objective:(1) The first purpose of this study is to investigate the inhibition of the anti-vWF A3domain mAb SZ-123on the binding of human vWF to collagen type Ⅰ, Ⅲ, Ⅳ fromhuman placenta.(2) The second purpose of this study is to investigate the inhibition of SZ-123on theadhesion of human platelet to collagen typeⅠ, Ⅲ and Ⅳ from human placenta by in vitroantithrombotic model (Flow Chamber) which can imitate vascular shear forces,temperature and other environment. It is also to study that mAb SZ-123has a goodantithrombotic effect. The experimental data will provide the theory foundation for the future development of antithrombotic drugs.Methods:(1) First, human collagen typeⅠ, Ⅲ and Ⅳ was immobilized onto microtiterplate wells. After that, normal human plasma and mAb SZ-123was added and incubated.Then, a Rabbit Anti-human polyclonal anti-vWF antibody conjugated with horseradishperoxidase (HRP) was added. Finally, visualisation was obtained withtetramethylbenzidine (TMB) and the absorbance was determined with microplate reader.(2) Human collagen type Ⅰ, Ⅲ and Ⅳ were coated onto coverslips respectively.Passing the night with incubation at4℃. Non-specific binding sites were blocked with2%BSA-PBS after washed by saline. Perfusions were carried out with whole blood(anticoagulant with low-molecular-weight heparin,40U/ml)，which were incubated withSZ-123or SZ-34for10min at37℃, respectively. The platelet-coated surface was rinsedtwice with saline, then evaluated platelet adhesion with a light microscope in FlowChamber under37℃,1000S-1shear force conditions. The adhesion rate of platelets wasmeasured and calculated by an image analysis system using SlideBook software analysis.Results:(1) The collagen binding test showed that mAb SZ-123inhibited plasma vWFbinding to collagen type Ⅰ, Ⅲ and Ⅳ in a dose-dependent manner in vitro. The inhibitionrates of mAb SZ-123were95%,96%and41%，respectively.(2) The Flow Chamber experiments showed that mAb SZ-123could inhibit theadhesion of platelet to collagen typeⅠ, Ⅲ and Ⅳunder high shear conditions at37℃.Conclusions:(1) MAb SZ-123can inhibit human platelet adhering to collagen typeⅠ, Ⅲ and Ⅳ.(2) The inhibition of mAb SZ-123on the binding of vWF to collagen type Ⅰ, Ⅲ issignificantly higher than that of type Ⅳ collagen.(3) MAb SZ-123has a good anti-platelet adhesion function and a good antithromboticeffect under high shear conditions. This will provide the theory foundation for the futuredevelopment of antithrombotic drugs and thrombosis mechanism.