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

Abstract

Background Heparin-induced thrombocytopenia (HIT) is typically caused by platelet-activating 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 heparin-dependent 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-linked-immunosorbent 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.

Antibody epitopes in vaccine-induced immune thrombotic thrombocytopaenia

“…Our results explain why some VITT samples tested in studies by Schultz et al 2 and Greinacher et al 1 were inhibited by therapeutic doses of heparin. Our findings suggest VITT antibodies cause platelet activation through a similar heparin-like mechanism by stabilizing complexes of PF4, aligning the Fc-portion in close proximity and crosslinking FcγRIIa receptors on platelets, similar to that of the monoclonal antibody, 1E12, which can activate platelets independent of heparin 15 .…”

“…Monoclonal antibodies against PF4, such as KKO 18 and 1E12 15 facilitate the formation of ultra-large complexes of PF4 on the platelet surface. Previous studies using antibodies from HIT patients 19 and monoclonal antibodies that resemble HIT antibodies 15 have demonstrated that the higher affinity of HI antibodies in some HIT patients can cluster PF4 tetramers and create platelet-activating immune complexes, in the absence of heparin.…”

“…Monoclonal antibodies against PF4, such as KKO 18 and 1E12 15 facilitate the formation of ultra-large complexes of PF4 on the platelet surface. Previous studies using antibodies from HIT patients 19 and monoclonal antibodies that resemble HIT antibodies 15 have demonstrated that the higher affinity of HI antibodies in some HIT patients can cluster PF4 tetramers and create platelet-activating immune complexes, in the absence of heparin. Similarly, we showed that anti-PF4 antibodies from VITT patients have comparable binding response and avidity to the antibodies from HIT patients, especially HIT patients with HI antibodies, implying they can cluster PF4 tetramers and create the same ultra-large platelet-activating immune complexes (Extended Data Figure 5).…”

Vaccine-induced immune thrombotic thrombocytopenia and cerebral venous sinus thrombosis post COVID-19 vaccination; a systematic review

“…HIT is caused by platelet-activating immunoglobulin G (IgG) antibodies against platelet factor 4 (PF4) complexed with heparin. This complex then binds to the platelet FcRγIIA receptors and causes platelet activation and formation of platelet microparticles [ 39 ]. These microparticles initiate the formation of blood clots and inducing a prothrombotic cascade, which consequently decreases platelet count and causes thrombocytopenia.…”

Conformation-Crooking CXCL4 to Unravel Autoimmune Heparin-Induced Thrombocytopenia

“…In the study by Vayne et al in this issue of Thrombosis and Haemostasis, 12 humanized monoclonal antibodies (humAbs) were raised against a complex of CXCL4 with heparin to serve as a model for the antibodies found in patients with autoimmune HIT. Since these antibodies contained a human Fc region, they behaved as HIT or autoimmune HIT antibodies, similar to those found in patients.…”