HIT-IgGs induce a heparin-dependent pro-thrombotic phenotype in a novel endothelialized microfluidic disease model

Introduction Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction to heparin. Heparin/PF4/IgG complexes have been reported to activate platelets, neutrophils, and possibly endothelial cells, resulting in thrombocytopenia, hypercoagulability and thromboembolic complications. While the impact of anti-PF4/heparin antibodies on blood cell activation has been extensively studied, the role of endothelial cells in HIT-associated thrombosis remains underexplored. In particular, the interactions between HIT-induced procoagulancy and endothelial cells under flow conditions is not completely elucidated. In this report, we investigated how HIT antibodies induce thrombosis in an endothelialized microfluidic system.

Fig. 1 HIT-IgG together with prophylactic-dose heaprin induce thrombosis on pre-activated endothelial cells; Representative images of thrombi formed by whole blood incubated with HIT-IgG in absence and under 0.2 IU/mL unfractionated heparin (UFH). Endothelial cells were primed with TNF-α and perfused with whole blood under a shear stress of 10 dyn/cm² for 10 minutes. Overlay of brightfield and fluorescent channel image. Platelets: green, Calcein-AM. Scale bar: 50 µm. [if !supportLineBreakNewLine]

Method Microfluidic channels were coated with monolayers of human umbilical vein endothelial cells (HUVECs). Cells were primed with low-dose TNF-α, before perfusion with whole blood samples. Unstimulated or Thrombin Receptor Activator Peptide 6 (TRAP-6) activated whole blood was perfused at a venous shear rate. The HIT-thrombosis model was established and tested utilizing monoclonal anti-PF4/heparin antibodies. In brief, whole blood was pre-incubated with unfractionated heparin (UFH, 0.2 IU/mL or 100 IU/mL). Anti-PF4/heparin antibodies were introduced to the whole blood mixture and incubated for 30 minutes at 37°C, under rotation. Whole blood was recalcified and perfused over unstimulated or primed endothelial cells at a venous shear stress. Thrombus formation was recorded over time.

Fig. 2 HIT-IgG together with prophylactic-dose heaprin induce thrombosis on pre-activated endothelial cells; Maximal thrombus surface area coverage of TNF-α primed endothelial cells, perfused with unstimulated, TRAP-6 activated or HIT-like IgG/heparin challenged whole blood. HIT-like IgG did not increase thrombus formation in absence of heparin. Addition of low-dose heparin exerted a strong pro-thrombotic effect, that was fully reversible with a super-therapeutic concentration of heparin (HIT-IgG vs. HIT-IgG+0.2 IU/mL UFH, Thrombus SAC in%, mean±SD: 0.93±0.82% vs. 11.10±6.45%, p=0.0118, HIT-IgG vs. HIT-IgG+100 IU/mL UFH: 11.10±6.45% vs. 0.00±0.00%, p=0.0066).

Results The endothelialized microfluidic model successfully captures sub-thrombotic conditions under venous shear, activated platelets induced a procoagulant shift and three-dimensional thrombi. We applied our thrombosis model to investigate thrombus formation induced by HIT mimicking monoclonal antibodies. We observed that the anti-heparin/PF4 antibody-dependent platelet activation predicts the thrombotic response in the microfluidic system: HIT antibodies in absence of heparin did not exert a prothrombotic effect on activated endothelial cells. Co-incubation with 0.2 IU/mL UFH induced thrombosis, embolization of thrombi and channel occlusion only when endothelial cells were primed with TNF-α. The pro-thrombotic effect of HIT antibodies was fully reversed at the super-therapeutic dose of 100 IU/mL UFH.

Conclusion HIT antibodies induce thrombosis and embolization in a system emulating physiological environment. For the first time, we present a comprehensive thrombosis model that incorporates the enhanced thrombogenicity of HIT-mimicking antibodies in presence of heparin. We show that our thrombosis model incorporates both endothelial- and blood-based modulation of thrombosis. Primed endothelial cells and antibody-induced procoagulancy trigger thrombosis in a concerted fashion, allowing the investigation of underlying mechanisms and possible preclinical evaluation of potential inhibitors of HIT-thrombosis ([Fig. 1], [Fig. 2]).

Conflict of Interest

The authors declare no conflict of interests.

Publication History

Article published online:
26 February 2024