AIM: To further investagate the mechanism of thrombus formation and develop a new remedy of anti-thrombus formation. METHODS: The amplified DNA fragment of vWF-A1 domain was inserted into expression vector with 6?his taq (pQE-31), the recombinant expression vect or was transformed into E coli (strain M15) and induced by IPTG. The recombinant fragment, comprising residues 449-728 of mature vWF subunit, designate rvWF-A1. It was purified by Ni-NTA agarose column and renatured by Tris buffer containin g GSH and GSSG. FACS and platelet aggregometer were employed to analyse the rvWF -A1 function of binding to platelet glycoprotein Ib and inhibiting ristocetin-in duced platelet aggregation. RESULTS: The rvWF-A1 was expressed successfully in E coli, comin g up to 30% of total bacterial protein. Its purify was over 95% through Ni-NTA a garose. It was identified to have ability to bind to GPIb, its biologic activity to inhibit ristocetin-induced platelet aggregation was observed, and the inhibi tive rate was 84 7%. CONCLUSION: The above results indicated that high-level expressi on of rvWF-A1 was successfully achieved in E coli and rvWF-A1 may be an effectiv e antithromotic agent in preventing thrombus formation.

OBJECTIVETo prepare a single chain antibody (ScFv) of mAb SZ-21 against platelet GPIIIa for its future clinical application.

METHODSThe expression vector pET20b-SZ-21ScFv was constructed and the fusion protein was expressed in E. coli BL21 (DE3) PlysS. The activated fusion protein was obtained after a series of purification steps, including cell breakage, inclusion body solubilization, His-bind resin affinity chromatography and protein refolding.

RESULTSThe fusion protein yields were up to 21% of the total amount of bacteria protein. The ScFv fragment could inhibit ADP-induced platelets aggregation in a dose-dependent manner in vitro and the maximal inhibition rate was obtained at a concentration of 20 micro g/ml. It also reacted with endothelial cells as detected by flow cytometry. Moreover, the ScFv fragment was able to inhibit the binding of fibrinogen to platelet.

CONCLUSIONThe SZ-21ScFv fragment had the activity to inhibit platelets aggregation and the binding of fibrinogen to platelet, being potentially useful for the treatment of thrombotic diseases.

Antibodies, Monoclonal ; pharmacology ; Blood Platelets ; metabolism ; Endothelium, Vascular ; cytology ; Fibrinogen ; metabolism ; Humans ; Immunoglobulin Fragments ; pharmacology ; Platelet Aggregation ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Recombinant Fusion Proteins ; isolation & purification ; pharmacology