Humanization Of An Anti-platelet GPIbα Monoclonal Antibody And Identification Of A Novel Active Small Molecule Based On GPIbα Structure

Part I: Engineering and characterization of a chimeric anti-platelet glycoprotein Ibαmonoclonal antibody and preparation of its Fab fragmentBackground Glycoprotein Ibα(GPIbα) is a platelet-specific membrane protein. It mediates platelets adhesion to collagen exposed at vascular injury site by binding to von Willebrand factor (VWF) in plasma. This process is crucial for arterial thrombus formation.Objective Genetic engineering of a chimeric anti-GPIbαmonoclonal antibody which blocks interaction between GPIbαand VWF, providing a novel antiplatelet drug candidate.Methods and results We used 5'-rapid amplification of cDNA ends (RACE) method to clone the variable cDNA sequences with signal peptide coding sequences from hybridoma cells expressing monoclonal antibody, SZ2, against platelets glycoprotein Ibα. The sequences were blasted in IMGT website after DNA sequencing. Result showed that the obtained sequences were accordant with sequences of antibody. Two pairs of primer were designed based on the sequence result for PCR amplification of the heavy and light chains. Then the PCR products were cloned into eukaryotic expression vector to form fusion gene with constant regions of IgG1 heavy chain and kappa chain respectively. The two plasmids were cotransfected into dihydrofolate reductase (dhfr)-deficient Chinese hamster ovary (CHO) cells and cells were grown in methotrexate contained medium to amplify the antibody genes to screen antibody high-producing cells. The expression level was measured by ELISA. We got a cell clone, 3G09, with its expression level at 2.5 pg/cell/day. Results of western blot showed a 150 kDa band under non-reducing condition and two bands at 50 kDa and 25 kDa under reducing condition, consistent with standard IgG. The chimeric antibody in supernatant was collected and purified with protein A column. The purified antibody was identified by being separated in SDS-PAGE followed by coomassie blue staining. 95 % purity was obtained. Flow cytometry analysis confirmed specific binding of the chimeric antibody to the GpIb-expressing CHO cells. To produce chimeric Fab fragment, the chimeric antibody was treated with papain. The reaction mix was then treated with protein A to remove the uncleaved intact antibody and Fc fragment. The purity of chimeric Fab reached 90 % after being assayed by SDS-PAGE and coomassie blue staining. Functional assay in vitro showed that the chimeric Fab fragment of SZ2 inhibited ristocetin-induced human platelet aggregation in a dose-dependent manner. The chimeric Fab also blocked platelets adhesion on immobilized VWF under flow condition.Conclusions These results demonstrated that the chimeric antibody was successfully engineered and suggested that the Fab of chimeric antibody against GpIbαis a promising and more suitable therapeutic antibody for prevention and treatment of arterial thrombosis.Part II: Identification of a novel glycoprotein Ibα-specific small-molecule agonist inducing platelet activation and aggregationBackground Interaction of glycoprotein (GP) Ibαwith Von Willebrand factor (VWF) plays a critical role in platelet adhesion and signal transduction forαIIbβ3 activation under condition of high shear stress.Objective To identify small-molecule compounds that inhibit VWF binding to GPIbαMethods and Results DOCK4.0 software was employed to dock compounds in SPECS database to VWF-binding site on GPIbαbase on the crystal structure of GPIbα. The 149 highest-scoring compounds were obtained for further bioassay and those with inhibitory activity of VWF binding to GPIbαwere investigated the effect on platelet activation and aggregation. One compound, we designate it as YC148, blocked ristocetin-induced plasma VWF binding to recombinant N-terminal fragment GPIbα. More interestingly, YC148 did not inhibit ristocetin-induced platelet aggregation; on the contrary, it induced platelet aggregation itself in the absence of exogenous modulators such as ristocetin and botrocetin. YC148 can also induce washed platelet aggregation. Flow cytometry results also showed that YC148 increased P-selectin expression on platelet membrane and promoted monoclonal antibody PAC-1 binding to platelet. YC148-induced platelet aggregation was partially inhibited by an anti-GPIbαmonoclonal antibody 6D1. Furthermore, platelets lacking the N-terminal fragment of GPIbαdid not aggregate in response to YC148 stimulation.Conclusion A novel exogenous small-molecule agonist was found to aggregate platelets through binding to GPIbα. It provides us a new tool for investigating platelet GPIb outside-in signaling pathway in platelet adhesion and aggregation. Furthermore, the structure of YC148 may provide a structural basis for developing new hemostatic drugs based on the inhibition of VWF-GPIb interaction.