Objective. Beta 2 -glycoprotein I (␤ 2 GPI) is an important autoantigen in the antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiologic functions, since it displays both anticoagulant and procoagulant properties. We have previously reported that ␤ 2 GPI can directly bind thrombin, a key serine protease in the coagulation pathway. The present study was undertaken to examine the influence of ␤ 2 GPI on thrombin inactivation by the serpin heparin cofactor II (HCII). The effect of anti-␤ 2 GPI antibodies was also examined.Methods. HCII inactivation of thrombin was assessed using chromogenic and various platelet functional assays. The influence of intact and proteolytically cleaved ␤ 2 GPI and anti-␤ 2 GPI antibodies was determined in these systems.Results. ␤ 2 GPI protected thrombin against inactivation by HCII/heparin. Cleavage of ␤ 2 GPI at Lys 317 -Thr 318 abrogated its protective effect. Patient polyclonal IgG and murine monoclonal anti-␤ 2 GPI antibodies potentiated the procoagulant influence of ␤ 2 GPI in this system.Conclusion. These novel findings suggest that ␤ 2 GPI may regulate thrombin inactivation by HCII/ heparin. The observation that anti-␤ 2 GPI antibodies potentiate the protective effect of ␤ 2 GPI on thrombin in this system, thereby promoting a procoagulant response, may potentially delineate one of the pathophysiologic mechanisms contributing to the prothrombotic tendency in patients with APS.

Human CD48, a membrane-bound, glycosylphosphatidylinositol (GPI)-linked glycoprotein, is a potential tumour target for the treatment of leukaemias and lymphomas. CD48 is expressed on Tand B-cells, however <5% of CD34 + progenitor cells express CD48. A truncated, 45 kDa soluble form of the full length CD48 was expressed in Chinese hamster ovary (CHO) cells, and was shown to consist of a broad range of charge isoforms, with the most abundant isoforms between pI 4.5 and 5.0. The truncated form of CD48 was shown to bind to antibodies raised against native, GPI-linked CD48 by surface plasmon resonance analysis. A synthetic, human, scFv immunoglobulin gene library was screened against recombinant CD48 by phage display, and an scFv antibody fragment, (designated N2A) was isolated after four rounds of biopanning. N2A was reassembled as a human IgG1 human monoclonal antibody, expressed in CHO cells and the binding of IgG1-N2A to recombinant CD48 was confirmed by surface plasmon resonance. Flow cytometry studies of IgG1-N2A binding to Raji cells showed the specificity of N2A for GPI-linked CD48 was conserved, and presents the potential for IgG1-N2A as a lead antibody candidate for the treatment of white blood cell malignancies.

CD48 is a cell surface, glycosylphosphatidylinositol-linked glycoprotein, and a potential target for treatment of leukemia and lymphoma. Two anti-CD48 mAbs, murine HuLy-m3 and human IgG1-N2A, were compared in cellular assays using a human lymphoma cell line (Raji) for their ability to inhibit cell growth and induce apoptosis. In vitro studies revealed both HuLy-m3 and IgG1-N2A mAbs were able to induce potent growth inhibition, reflected by a reduction in viable cells of approximately 70% compared to controls after 90 h. Furthermore, Raji cells treated with IgG1-N2A showed evidence of apoptosis, including increased ethidium bromide uptake, cell shrinkage and chromosomal DNA degradation.