Intravenous immunoglobulin prevents thrombosis in an endothelialized disease model of heparin-induced thrombocytopenia

Introduction: Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction to heparin, associated with increased risk of thromboembolic complications. Pathogenic antibodies form immunogenic complexes with PF4 and heparin, which activate platelets and leukocytes mainly through the low-affinity IgG Fc receptor FcγRIIa. Intravenous immunoglobulins (IVIG) have been used as a therapeutic for HIT and are believed to alleviate thrombocytopenia and reduce thrombosis risk, possibly through competition with pathogenic IgG. Yet the anti-thrombotic effects of IVIG in HIT remain underexplored. The aim of our study was to investigate the effect of IVIG on thrombus formation in a disease model of HIT.

Method: Microfluidic channels were coated with a confluent monolayer of human umbilical vein endothelial cells (HUVECs). Cells were primed with low-dose TNF-α to induce a limited endothelial activation. Whole blood was preincubated with unfractionated heparin (0.2 IU/mL), with or without IVIG. The monoclonal anti-PF4/heparin HIT-like antibody (K070) was introduced to the blood mixture. Blood was recalcified and perfused over HUVECs at a venous shear stress. Thrombus structure and growth dynamics were investigated by immunofluorescence microscopy time series ([Fig. 1]).

Fig. 1 The HIT-like mAB K070 induces heparin-dependent thrombosis only on pre-injured endothelial cells A: Whole blood, or whole blood with low-dose heparin (0.2 IU/mL) was co-incubation incubated with K070 mAB for 15 min and perfused over buffer-treated or TNF-α (1 ng/mL) pre-stimulated endothelial cells at a venous shear stress of 10 dyn/cm². B: Endothelial cells were pre-stimulated with 1 ng/mL TNF-α and perfused at a shear stress of 10 dyn/cm² with whole blood, incubated with K070 without heparin, low dose (0.2 IU/mL), or supratherapeutic doses of UFH (100 IU/mL). Representative brightfield microscopy images after 10 min of perfusion. Scale bar: 200 µm.

Fig. 2 High-dose IVIG inhibits K070-mediated multicellular thrombus formation A: Whole blood, or whole blood with heparin (0.2 IU/mL) was pre-incubated with IVIG prior to addition of K070 and perfusion over pre-stimulated endothelial cells. Representative immunofluorescence images, showing platelets (CD41, blue), phosphatidylserine (Annexin, green), fibrin (magenta) and leukocytes (CD45, red). 10x magnification. Scale bar: 200 µm. B: Channel area covered by indicated thrombus constituents in% of total channel area. Analysis by One-Way ANOVA, corrected for multiple comparisons by Tukey’s.*p<0.05,**p<0.01, only significant comparisons shown, n=3 biological replicates.

Results: Monoclonal HIT-IgG induced thrombus formation in presence of prophylactic dose heparin, only when endothelial cells were challenged with TNF-α. HIT thrombi were enriched with fibrin, phosphatidylserine, and leukocyte aggregations, differing markedly from the two-dimensional platelet-fibrin rich adhesions formed in absence of pathogenic HIT IgG. We observed that thrombi formed on platelet adhesions, and recruited leukocytes into the three-dimensional thrombus structure. Pre-treatment of blood with IVIG inhibited the HIT thrombus formation. Cellular adhesions under IVIG resembled the two-dimensional phenotype in absence of pathogenic HIT IgG, and improved blood flow through microfluidic channels ([Fig. 2]).

Conclusion: We demonstrate that HIT-IgG induce thrombosis on minimally activated endothelial cells in a heparin-dependent manner. HIT thrombi were enriched phosphatidylserine-bearing platelets and incorporated leukocyte aggregates, indicative of the immune-thrombotic phenotype of HIT. Pre-treatment of blood with IVIG prevented thrombus formation. In conclusion, our endothelialized disease model of offers a versatile tool to study HIT thrombosis, and the pre-clinical investigation of pharmacological interventions.

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Artikel online veröffentlicht:
13. Februar 2025