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1199 Anti-PF4/Heparin Monoclonal Antibodies Induce Thrombus Formation in an Ex Vivo Endothelialized Microfluidic System

Program: Oral and Poster Abstracts
Session: 301. Vasculature, Endothelium, Thrombosis and Platelets: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
autoimmune disorders, Research, Fundamental Science, Bleeding and Clotting, Translational Research, Diseases, thrombocytopenias, Immune Disorders, thrombotic disorders, Adverse Events
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Andreas Witzemann, BS1*, Lisann Pelzl, PhD1*, Nina Wolska, PhD1*, Jan Zlamal1,2*, Jean Amiral3* and Tamam Bakchoul, MD PhD1,4

1Institute for Clinical and Experimental Transfusion Medicine, Medical Faculty, Tuebingen, Germany
2University Hospital of Tuebingen, Tuebingen, Germany
3Hyphen Biomed, Neuville Sur Oise, FRA
4Center for Clinical Transfusion Medicine GmbH, Tuebingen, Germany

Introduction: Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction to heparin. Antibodies against platelet factor 4 (PF4) or a heparin/PF4 complex 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 platelet, neutrophil and monocyte 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 dynamic conditions is not completely analyzed. In this report, we investigated how HIT antibodies induce thrombosis in an endothelialized microfluidic system.

Methods: Fibrinogen microfluidic channels were coated with confluent monolayer of human umbilical vein endothelial cells (HUVECs). Cells were primed with low-dose TNF-α to induce a sub-thrombotic endothelial activation, prior to perfusion of whole blood. Recalcified unstimulated or Thrombin Receptor Activator Peptide 6 (TRAP-6) activated whole blood was perfused at a venous flow rate. HIT-thrombosis model was established and tested with monoclonal anti-PF4/heparin antibodies in whole blood. In brief, whole blood was pre-incubated with unfractionated heparin (UFH, 0.2 IU/mL and 100 IU/mL). Next, anti-PF4/heparin antibodies were introduced to the whole blood mixture and incubated for 30 minutes. Whole blood was recalcified and perfused over resting or primed endothelial cells at a venous shear stress. Thrombus formation was recorded over time.

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 a HIT mimicking monoclonal antibody. We show that the heparin-dependent platelet activation predicts the thrombotic response in the microfluidic system: HIT antibodies in absence of heparin did not exert a pro-thrombotic effect on activated endothelial cells. Co-incubation with 0.2 IU/mL UFH induced thrombosis, embolization of thrombus material 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.

Conclusions: 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 established thrombogenicity of HIT-mimicking antibodies. We show that our thrombosis model incorporates both endothelial- and blood-based control of thrombosis. Primed endothelial cells and antibody-induced procoagulancy trigger thrombosis in a concerted fashion, allowing investigation of the underlying mechanisms and possible preclinical evaluation of potential inhibitors of HIT-thrombosis.

Figure 1, upper panel: 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. Lower panel: representative images of thrombi formed by HIT-like IgG in absence and under low or high-dose heparin. Platelets: green, Calcein-AM. Scale bar: 200 µm.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH