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Fibronectin modulates formation of PF4/heparin complexes and is a potential factor for reducing risk of developing HIT

Krystin Krauel, Patricia Preuße, Theodore E. Warkentin, Catja Trabhardt, Sven Brandt, Inga Jensch, Martin Mandelkow, Elke Hammer, Sven Hammerschmidt and Andreas Greinacher

Key points

  • Fibronectin disrupts PF4/heparin complexes and inhibits anti-PF4/heparin antibody mediated platelet activation in vitro.

  • Low plasma fibronectin is associated with increased anti-PF4/heparin antibody formation and increased breakthrough of HIT.

Abstract

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating anti-PF4/heparin antibodies. Platelet activation assays that utilize "washed" platelets are more sensitive for detecting HIT antibodies than platelet-rich plasma (PRP)-based assays. Moreover, heparin-exposed patients vary considerably with respect to risk of PF4/heparin immunization and−among antibody-positive patients−risk of subsequent "breakthrough" of clinical HIT with manifestation of thrombocytopenia. We used washed platelets and PRP, standard laboratory HIT tests and physicochemical methods, to identify a plasma factor interfering with PF4/heparin complexes and anti-PF4/heparin antibody-platelet interaction, thus explaining differences in functional assays. For investigating a modulating risk of PF4/heparin immunization and breakthrough of HIT, we also tested 89 plasmas from two serosurveillance trials. Fibronectin levels were measured in four patient groups exhibiting different degrees of heparin-dependent immunization and expression of HIT. The heat-labile plasma protein, fibronectin, inhibited PF4 binding to platelets in a dose-dependent fashion, particularly in washed (versus PRP) systems. Fibronectin also inhibited PF4/heparin binding to platelets, anti-PF4/heparin antibody binding to PF4/heparin complexes and anti-PF4/heparin antibody-induced platelet activation as a result of PF4/heparin complex disruption. In addition, plasma fibronectin levels increased progressively among the following 4 patient groups: enzyme-linked immunosorbent assay (ELISA)+/serotonin-release assay (SRA)+/HIT+ < ELISA+/SRA+/HIT– ~ ELISA+/SRA–/HIT– < ELISA–/SRA–/HIT–. Altogether, these findings suggest that fibronectin interferes with PF4/heparin complex formation and anti-PF4/heparin antibody-induced platelet activation. Reduced fibronectin levels in washed platelet assays helps explain greater sensitivity of washed platelet (versus PRP) assays for HIT. More importantly, lower plasma fibronectin levels could represent a risk factor both for PF4/heparin immunization and clinical breakthrough of HIT.

  • Submitted May 10, 2018.
  • Accepted December 10, 2018.