Characterization of New Monoclonal PF4-Specific Antibodies as Useful Tools for Studies on Typical and Autoimmune Heparin-Induced Thrombocytopenia

International audienceBackground Heparin-induced thrombocytopenia (HIT) is typically caused by plateletactivating immunoglobulin G (IgG) antibodies (Abs) against platelet factor 4 (PF4) complexed with heparin (H). Much less frequent "autoimmune" HIT is distinguished from typical HIT by platelet activation without heparin and the presence of both anti-PF4/H and anti-PF4 IgG. We developed three murine monoclonal anti-PF4 Abs with a human Fc-part, 1E12, 1C12, and 2E1, resembling autoimmune HIT Abs. Objectives To characterize 1E12, 1C12, and 2E1 in comparison to the heparindependent monoclonal anti-PF4/H Abs 5B9 and KKO, and polyclonal Abs from patients with typical HIT (group-2) and autoimmune HIT (group-3). Methods Interactions of Abs with PF4 and PF4/H were studied by enzyme-linkedimmunosorbent assay, single-molecule force spectroscopy, isothermal titration calorimetry, and dynamic light scattering. Serotonin release assay and heparin-induced platelet activation assay were used to assess platelet activation. The binding sites of monoclonal Abs on PF4 were predicted in silico (MAbTope method). Results 1C12, 1E12, and 2E1 displayed higher affinity for PF4/H complexes than 5B9 and KKO, comparable to human group-3 Abs. Only 1C12, 1E12, 2E1, and group-3 Abs formed large complexes with native PF4, and activated platelets without heparin. The predicted binding sites of 1C12, 1E12, and 2E1 on PF4 differed from those of KKO and 5B9, but were close to each other. 2E1 exhibited unique bivalent binding, involving its antigen recognition site to PF4 and charge-dependent interactions with heparin. Conclusion 1C12, 1E12, and 2E1 are tools for studying the pathophysiology of autoimmune HIT. 2E1 provides evidence for a new binding mechanism of HIT Abs

Multicentre evaluation of 5B9, a monoclonal anti-PF4/heparin IgG mimicking human hit antibodies, as an internal quality control in hit functional assays: communication from the isth ssc subcommittee on platelet immunology.

Functional tests for the diagnosis of heparin-induced thrombocytopenia (HIT) exhibit variable performance. We evaluated in a multicentre study whether 5B9, a monoclonal anti-PF4/heparin IgG mimicking human HIT antibodies, could be used as an internal quality control. 5B9 was sent to eleven laboratories in seven countries, and six initial concentrations ranging from 10 to 400 μg/ml were tested by heparin-induced platelet activation assay (HIPA), serotonin release assay (SRA), platelet aggregation test (PAT), flow cytometry (FC) or heparin-induced multiple-electrode aggregometry (HIMEA). Each method was evaluated in three different laboratories, using experimental procedures identical to those usually applied for the diagnosis of HIT, by testing platelets from 10 different healthy donors. the procedures used varied among the laboratories, particularly when platelet-rich plasma and whole blood were employed. Nevertheless, positive results were obtained with at least 100 μg/ml of 5B9 for most donors tested by all centres (except one) performing HIPA, SRA, or HIMEA. FC and PAT results were more heterogeneous. FC results from one centre that used washed platelets pre-incubated with PF4 were positive with all donors at 50 µg/mL 5B9, but at least 200 μg/ml of 5B9 were required to activate cells with most donors tested using PAT. this study confirms that HIT functional tests are not well standardized and exhibit variable sensitivity for the detection of platelet-activating antibodies. However, 5B9 is a potentially useful tool to standardize functional tests, to select responding platelet donors, and consequently to improve the performance of these assays and comparability between laboratories