Affiliations: [a] The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin, USA | [b] Departments of Anatomy and Cell Biology, Microbiology, Pathology, and Pediatrics of The Medical College of Wisconsin, Milwaukee, Wisconsin, USA | [c] The Department of Pediatrics, University of Wisconsin, Madison, Wisconsin, USA
Note: [] Address requests for reprints to: Thomas J. Kunicki, Ph.D., The Blood Center of Southeastern Wisconsin, Inc., 1701 West Wisconsin Avenue, Milwaukee, Wisconsin 53233, USA.
Note: [] Dr. Furihata is currently located at Shinshu University Hospital, Matsumoto, Japan.
Note: [] Dr. Kekomaki is currently located at the Finnish Red Cross Blood Transfusion Service, Helsinki, Finland.
Abstract: Splenocytes from a patient with chronic, immune-mediated thrombocytopenic purpura (ITP) were transformed with Epstein-Barr virus. A stable lymphoblastoid cell line (LCL) derived from this transformation (2A3) produces IgM antibody reactive with platelet glycoprotein IIb. 2A3 was fused to the 6-thioguanine-resistant, ouabain-resistant, murine-human heteromyeloma cell line, F6. The resultant heterohybridomas were selected by growth in medium containing hypoxanthine/aminopterin/thymidine and ouabain. One hybridoma line, 2E7, produces high levels of IgM antibody (2 to 4 μg IgM/ml/24 hr/105 cells) reactive with glycoprotein IIb. 2E7 has been repeatedly subcloned by limiting dilution and has been maintained in continuous culture for 26 months. 2E7 binds to human platelets but not endothelial cells, as determined by flow cytometry, and does not react with platelets of patients with Glanzmann's thrombasthenia that lack IIb-IIIa. The epitope recognized by 2E7 is likely to be a contiguous peptide sequence since the antibody binds to the IIb heavy chain in immunoblot assays of denatured, reduced platelet protein. Treatment of intact platelets or purified IIb-IIIa with papain or chymotrypsin, but not SV8 protease, destroys the epitope. Thus, the 2E7 epitope may be at or very close to a site on IIb that is cleaved by these proteases. The expression of the 2E7 epitope is significantly affected by the presence of divalent cations. Treatment of intact platelets with EDTA at 37°C results in a three- to four-fold increase in the number of 2E7 molecules bound per platelet and an eight-fold increase in the affinity of the antibody. The binding of 2E7 to normal platelets does not inhibit any of the functions attributed to IIb-IIIa, such as fibrinogen-dependent platelet aggregation or clot retraction. 2E7 represents the first human monoclonal antibody reported to recognize an epitope on platelet glycoprotein IIb. The epitope is unique to IIb and not shared by other integrin alpha subunits.
Keywords: human monoclonal antibody, platelet, glycoprotein IIb, integrin, IgM
