Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorder associated with a severe prothrombotic state. We investigated whether neutrophils and neutrophil extracellular traps (NETs) contribute to the development of thrombosis in HIT. Using an endothelialized microfluidic system and a murine passive immunization model, we show that HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice, endothelial adherence is enhanced immediately downstream of nascent venous thrombi, after which neutrophils undergo retrograde migration via a CXCR2-dependent mechanism to accumulate into the thrombi. Using a microfluidic system, we found that PF4 binds to NETs, leading them to become compact and DNase resistant. PF4-NET complexes selectively bind HIT antibodies, which further protect them from nuclease digestion. In HIT mice, inhibition of NET formation through Padi4 gene disruption or DNase treatment limited venous thrombus size. PAD4 inactivation did affect arterial thrombi or severity of thrombocytopenia in HIT. Thus, neutrophil activation contributes to the development of venous thrombosis in HIT by enhancing neutrophil-endothelial adhesion and neutrophil clot infiltration, where incorporated PF4-NET-HIT antibody complexes lead to thrombosis propagation. Inhibition of neutrophil endothelial adhesion, prevention of neutrophil chemokine-dependent recruitment of neutrophils to thrombi, or suppression of NET release should be explored as strategies to prevent venous thrombosis in HIT.
Kandace Gollomp, Minna Kim, Ian Johnston, Vincent Hayes, John Welsh, Gowthami M. Arepally, Mark Kahn, Michele P. Lambert, Adam Cuker, Douglas B. Cines, Lubica Rauova, M. Anna Kowalska, Mortimer Poncz
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