Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy caused by antibodies to complexes of platelet factor 4 (PF4) and heparin. Pathogenic antibodies to PF4/heparin bind to and activate cellular FcγRIIA on platelets and monocytes to propagate a hypercoagulable state culminating in potentially life-threatening thrombosis. Research has helped better understand the mechanisms underlying PF4/heparin immunogenicity, disease susceptibility, and clinical manifestations of disease. Insights from clinical and laboratory findings have also recently been harnessed for disease prevention. Neutrophil extracellular traps (NETs), DNA structures released by neutrophils in response to diverse stimuli, are a somewhat unknown commodity as it relates to HIT. Dr. Jose Perdomo and others sought to explore this further, to determine if HIT immune complexes induce NETs formation and to evaluate the potential role of NETs in the pathogenesis of HIT.

As Dr. Perdomo stated, HIT was reconstituted ex vivo in a microfluidics system and in vivo in FcΥRIIa+/hPF4+ transgenic mice. NETs formation was evaluated by detection of extracellular DNA, citrullinated histone H3, and myeloperoxidase using confocal microscopy, Western blotting, and flow cytometry.

Based on the team’s findings, HIT immune complexes induce NETs formation, which is essential for the development of thrombosis. Specifically, NETs markers were present in HIT patients’ sera and neutrophils. HIT immune complexes activate neutrophils via FcγRIIa and through platelets/neutrophil interactions mediated by P-selectin/PSGL-1 and induce NETosis. Ex vivo, HIT thrombi are rich in platelets, neutrophils, extracellular DNA, and citrullinated histone H3. The lack of neutrophils were found to prevent thrombus formation. Purified neutrophils treated with HIT IgG formed thrombi containing NETs. HIT mice demonstrated markers of NETosis in plasma and in thrombi. Treatment of HIT mice with DNase I or the NETs inhibitor GSK484 led to a substantial decrease in thrombosis. Importantly, no thrombi were detected in NETs-deficient hFcΥRIIa+/hPF4+/PAD4-/- mice. Dr. Perdomo noted that these observations suggest a new concept of the pathogenesis of thrombosis in HIT and, as such, are clinically relevant.

“The key finding of this work is that neutrophils, and not platelets, are essential for thrombus formation in HIT. Therefore, our research suggests that inhibition of NETosis would be more effective in reducing the pro-thrombotic state present in patients with HIT.”

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