Neutrophil accumulation and NET release contribute to thrombosis in HIT

Abstract

Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorderassociated with a severe prothrombotic state. We investigated whether neutrophils andneutrophil extracellular traps (NETs) contribute to the development of thrombosis in HIT. Usingan endothelialized microffuidic system and a murine passive immunization model, we showthat HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice,endothelial adherence is enhanced immediately downstream of nascent venous thrombi, afterwhich neutrophils undergo retrograde migration via a CXCR2-dependent mechanism to accumulateinto the thrombi. Using a microffuidic system, we found that PF4 binds to NETs, leading them tobecome compact and DNase resistant. PF4-NET complexes selectively bind HIT antibodies, whichfurther protect them from nuclease digestion. In HIT mice, inhibition of NET formation throughPadi4 gene disruption or DNase treatment limited venous thrombus size. PAD4 inactivationdid aflect arterial thrombi or severity of thrombocytopenia in HIT. Thus, neutrophil activationcontributes to the development of venous thrombosis in HIT by enhancing neutrophil-endothelialadhesion and neutrophil clot infltration, where incorporated PF4-NET-HIT antibody complexes leadto thrombosis propagation. Inhibition of neutrophil endothelial adhesion, prevention of neutrophilchemokine-dependent recruitment of neutrophils to thrombi, or suppression of NET release shouldbe explored as strategies to prevent venous thrombosis in HIT.