Molecular architecture and platelet-activating properties of small immune complexes assembled on intact heparin and their possible involvement in heparin-induced thrombocytopenia

Abstract

Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin leading to a reduction in circulating platelets with an increased risk of thrombosis. It is precipitated by polymerized immune complexes consisting of pathogenic antibodies that recognize a small chemokine platelet factor 4 (PF4) bound to heparin, which trigger platelet activation and a hypercoagulable state. Characterization of these immune complexes is extremely challenging due to the enormous structural heterogeneity of such macromolecular assemblies and their constituents (especially heparin). We use native mass spectrometry to characterize small immune complexes formed by PF4, heparin and monoclonal HIT-specific antibodies. Up to three PF4 tetramers can be assembled on a heparin chain, consistent with the results of molecular modeling studies showing facile polyanion wrapping along the polycationic belt on the PF4 surface. Although these assemblies can accommodate a maximum of only two antibodies, the resulting immune complexes are capable of platelet activation despite their modest size. Taken together, these studies provide further insight into molecular mechanisms of HIT and other immune disorders where anti-PF4 antibodies play a central role.

Reverse Engineering of a Pathogenic Antibody Reveals the Molecular Mechanism of Vaccine-Induced Immune Thrombotic Thrombocytopenia

“…Our work focused on characterizing the molecular architecture of immune complexes whose formation is precipitated by PF4. While characterization of immune complexes containing multiple antigen and antibody molecules remains challenging due to their large size and heterogeneity, native MS has been recently demonstrated to be capable of providing meaningful information on such macromolecular assemblies. − High molar excess of PF4 over RVT1 in aqueous solutions at near-physiological pH and ionic strength gives rise to mass spectra dominated by an abundant ionic signal corresponding to the 2:1 antigen/antibody complexes but shows no signal indicative of the presence of large immune complexes in solution (the blue trace in Figure ). However, adjusting the antibody/antigen ratio in solution to a near-equimolar level (3.5 μM each) results in a dramatic change in the appearance of the mass spectrum, which now displays abundant ionic signals representing PF4-polymerized antibodies (RVT1 2 PF4, RVT1 2 PF4 2 and RVT1 3 PF4 2 ) in addition to the uncomplexed antibody (the red trace in Figures and S3 in Supporting Information).…”

“…Both native MS analysis and molecular modeling studies of RVT1 provide a strong indication that the properties shared by all VITT-associated anti-PF4 antibodies are due to the high concentration of acidic amino acid residues within the paratope (Figure ). The resulting high-density negative charge basin localized within the antigen-binding region allows this antibody to interact effectively with the heparin-binding sites of PF4, where high-density positive charge is localized. , As PF4 in its native tetrameric state exhibits a contiguous polycationic regiona high positive charge-density equatorial belt circumscribing the entire proteinits recognition by the VITT antibody does not require any conformational changes within PF4. This is a major point of distinction between VITT and HIT, where heparin binding to the protein triggers a conformational change that allosterically creates a neoepitope at a remote site, thereby enabling the PF4 recognition by pathogenic antibodies .…”

Reverse engineering of a pathogenic antibody reveals the molecular mechanism of vaccine-induced immune thrombotic thrombocytopenia

“…Both native MS analysis and molecular modeling studies of RVT1 provide a strong indication that the properties shared by all VITT-associated anti-PF4 antibodies are due to the high concentration of acidic amino acid residues within the paratope (Figure 4). The resulting highdensity negative charge basin localized within the antigen-binding region allows this antibody to interact effectively with the heparin-binding sites of PF4, where high-density positive charge is localized (36,41). As PF4 in its native tetrameric state exhibits a contiguous polycationic regiona high positive charge-density equatorial belt circumscribing the entire protein -its recognition by the VITT antibody does not require any conformational changes within PF4.…”