PF4-HIT antibody (KKO) complexes activate broad innate immune and inflammatory responses. Issue 159 (November 2017) - Haile, Lydia A.
Abstract: Introduction: Heparin-induced thrombocytopenia (HIT) is an immune-mediated complication of heparin anticoagulation therapy resulting in thrombocytopenia frequently accompanied by thrombosis. Current evidence suggests that HIT is associated with antibodies developed in response to multi-molecular complexes formed by platelet factor 4 (PF4) bound to heparin or cell surface glycosaminoglycans. These antibody complexes activate platelets and monocytes typically through FcγRIIA receptors increasing the production of PF4, inflammatory mediators, tissue factor and thrombin. The influence of underlying events in HIT including complex-induced pro-inflammatory cell activation and structural determinants leading to local inflammatory responses are not fully understood. Methods: The stoichiometry and complex component requirements were determined by incubating fresh peripheral blood mononuclear cells (PBMC) with different concentrations of unfractionated heparin (H), low molecular weight heparin (LMWH), PF4- and anti-PF4-H complex antibodies (KKO). Cytokine mRNA or protein were measured by qRT-PCR or Meso Scale Discovery technology, respectively. Gene expression profile analysis for 594 genes was performed using Nanostring technology and analyzed using Ingenuity Pathway Analysis software. Results and conclusions: The data show that antibodies magnify immune responses induced in PBMCs by PF4 alone or in complex with heparin or LMWH. We propose that following induction of HIT antibodies by heparin-PF4 complexes, binding of the antibodies to PF4 is sufficient to induce a local pro-inflammatory response which may play a role in the progression of HIT. In vitro assays using PBMCs may be useful in characterizing local inflammatory and innate immune responses induced by HIT antibodies in the presence of PF4 and different sources of heparins. FDA disclaimer: The findings and conclusions in this article are solely the responsibility of the authors and are not being formally disseminated by the Food and Drug Administration. Thus, they should not be construed to represent any Agency determination or policy. Highlights: Heparin-PF4-KKO complex requirements for immune responses were determined in PBMC. Cytokine mRNA and corresponding proteins were measured by qRT-PCR and ELISA. Gene profile analysis for 594 genes was generated using Nanostring Technology. Antibody and PF4 are required for full pro-inflammatory responses by the complexes. Links can be made between the pro-inflammatory effect and immunogenicity risks.