Treatment of thrombocytopenia and thrombosis in HIT in mice using deglycosylated KKO: a novel therapeutic?

Heparin-induced thrombocytopenia (HIT) is characterized by thrombocytopenia associated with a highly prothrombotic state due to the development of pathogenic antibodies that recognize human platelet factor 4 (hPF4) complexed with various polyanions. Although nonheparin anticoagulants are the mainstay of care in HIT, subsequent bleeding may develop, and the risk of developing new thromboembolic events remain. We previously described a mouse immunoglobulin G2bκ (IgG2bκ) antibody KKO that mimics the sentinel features of pathogenic HIT antibodies, including binding to the same neoepitope on hPF4-polyanion complexes. KKO, like HIT IgGs, activates platelets through FcγRIIA and induces complement activation. We then questioned whether Fc-modified KKO could be used as a novel therapeutic to prevent or treat HIT. Using the endoglycosidase EndoS, we created deglycosylated KKO (DGKKO). Although DGKKO retained binding to PF4-polyanion complexes, it inhibited FcγRIIA-dependent activation of PF4-treated platelets triggered by unmodified KKO, 5B9 (another HIT-like monoclonal antibody), and IgGs isolated from patients with HIT. DGKKO also decreased complement activation and deposition of C3c on platelets. Unlike the anticoagulant fondaparinux, injection of DGKKO into HIT mice lacking mouse PF4, but transgenic for hPF4 and FcγRIIA, prevented and reversed thrombocytopenia when injected before or after unmodified KKO, 5B9, or HIT IgG. DGKKO also reversed antibody-induced thrombus growth in HIT mice. In contrast, DGKKO was ineffective in preventing thrombosis induced by IgG from patients with the HIT-related anti-PF4 prothrombotic disorder, vaccine-induced immune thrombotic thrombocytopenia. Thus, DGKKO may represent a new class of therapeutics for targeted treatment of patients with HIT.

Recognition of PF4-VWF complexes by heparin-induced thrombocytopenia antibodies contributes to thrombus propagation.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by complexes between platelet factor 4 (PF4) and heparin or other polyanions, but the risk of thrombosis extends beyond exposure to heparin implicating other PF4 partners. We recently reported that peri-thrombus endothelium is targeted by HIT antibodies, but the binding site(s) has not been identified. We now show that PF4 binds at multiple discrete sites along the surface of extended strings of von Willebrand factor (VWF) released from the endothelium following photochemical injury in an endothelialized microfluidic system under flow. The HIT-like monoclonal antibody KKO and HIT patient antibodies recognize PF4-VWF complexes, promoting platelet adhesion and enlargement of thrombi within the microfluidic channels. Platelet adhesion to the PF4-VWF-HIT antibody complexes is inhibited by antibodies that block FcγRIIA or the glycoprotein Ib-IX complex on platelets. Disruption of PF4-VWF-HIT antibody complexes by drugs that prevent or block VWF oligomerization attenuate thrombus formation in a murine model of HIT. Together, these studies demonstrate assembly of HIT immune complexes along VWF strings released by injured endothelium that might propagate the risk of thrombosis in HIT. Disruption of PF4-VWF complex formation may provide a new therapeutic approach to HIT.

Fc-modified HIT-like monoclonal antibody as a novel treatment for sepsis.

Sepsis is characterized by multiorgan system dysfunction that occurs because of infection. It is associated with high morbidity and mortality and is in need of improved therapeutic interventions. Neutrophils play a crucial role in sepsis, releasing neutrophil extracellular traps (NETs) composed of DNA complexed with histones and toxic antimicrobial proteins that ensnare pathogens, but also damage host tissues. At presentation, patients often have a significant NET burden contributing to the multiorgan damage. Therefore, interventions that inhibit NET release would likely be ineffective at preventing NET-based injury. Treatments that enhance NET degradation may liberate captured bacteria and toxic NET degradation products (NDPs) and likely be of limited therapeutic benefit as well. We propose that interventions that stabilize NETs and sequester NDPs may be protective in sepsis. We showed that platelet factor 4 (PF4), a platelet-associated chemokine, binds and compacts NETs, increasing their resistance to DNase I. We now show that PF4 increases NET-mediated bacterial capture, reduces the release of NDPs, and improves outcome in murine models of sepsis. A monoclonal antibody KKO which binds to PF4-NET complexes, further enhances DNase resistance. However, the Fc portion of this antibody activates the immune response and increases thrombotic risk, negating any protective effects in sepsis. Therefore, we developed an Fc-modified KKO that does not induce these negative outcomes. Treatment with this antibody augmented the effects of PF4, decreasing NDP release and bacterial dissemination and increasing survival in murine sepsis models, supporting a novel NET-targeting approach to improve outcomes in sepsis.