Preparation and Charaterization of MoAb SZ118 and SZ118-scFv Against Platelet Glycoprotein VI.

GlycoproteinVI (GPVI ), one of the platelet collagen receptors, plays an important role in platelet activation and thrombosis. GPVI participates in platelet adhesion to exposed collagen under high shear rate condition, and leads to platelet activation and thrombus formation. GPVI is widely recognized as a requisite factor for the formation of platelet aggregates on the collagen surface under blood flow. There were growing evidences showed that impairment of GPVI function could result in a long-term antithrombotic protection and less bleeding side-effect, which potentialized that GPVI might be an interesting target for safe anti-adhesion and anti-thrombotic therapy. Recently monoclonal antibodies directed against GPVI were developed, which impaired the interaction of platelets and collagen. A human soluble recombinant GPVI (rGPVI), consisting of extracellular domain of GPVI, was expressed in E. coli. After immunizing Balb/C mice with the purified rGPVI, a monoclonal antibody, SZ118, was developed. Meanwhile the gene encoding for the light-and heavy-chain variable regions of SZ118 had been cloned by RT-PCR from murine hybridoma cells. After ligated into the vector pET20b(+), expression plasmid pET20b(+)-SZ118-scFv was contructed, and SZ118-scFv fragment was expressed in E.coli. The parallel plate perfusion chamber and platelet aggregometer were used to study the effects of SZ118 and SZ118-scFv on the platelet adhesion and aggregation to collagen. The citrated whole blood with or without SZ118 was perfused over the surface of type I fibrillar collagen (Chrono Log, at 80μg/ml) at high shear rate (1000 s⁻¹) for 5 min. The platelet aggregates were visualized by the phase-contrast microscope, the coverage rates of the platelet aggregates were measured. Monoclonal antibody SZ118 and SZ118-scFv could bind to human platelets exclusively by flow cytometry. SZ118 inhibited collagen-induced platelet aggregation in PRP in dose-dependent manner, nearly full inhibition being attained at the concentration of 110 ug•mL⁻¹. Similarly SZ118-scFv inhibited collagen-induced platelet aggregation with maximum inhitition rate 84.3%±5.6%. And also SZ118 and SZ118-scFv had part effect on convulxin-induced platelet aggregation, but having little effect on ADP. After treating the citrated whole blood with SZ118 or SZ118-scFv at final concentration 50μg/ml at 37° for 10 min, the platelet adhesion on the surface of collagen was markedly inhibited with the coverage rates of 1.03%±0.22%, respectively (22.7%±5.7% for control), which indicated that the platelets adhesion to collagen was inhibited by 95.5%by SZ118 and 68.3% by SZ118-scFv. It was concluded that the MoAb SZ118 inhibited platelet aggregation to collagen and platelet adhesion to immobilized fibrillar collagen under high shear rate condition.