Structural Mass-Spectrometric Description of Immune Complexes in Vaccine-Induced Immune Thrombocytopenia and Thrombosis

Antibodies binding the heparin-binding domain of platelet factor 4 (PF4) cause a severe prothrombotic condition first identified as vaccine-induced immune thrombocytopenia and thrombosis (VITT). Reverse-engineered recombinant antibodies (rAbs) generated from mass-spectrometry-based sequencing of patient sera recapitulate the functional characteristics of VITT patient serum. However, the structure of the antigen interface and the architecture of the immune complexes are unknown. Here we utilize structural mass spectrometry to describe the immune complexes formed by three rAbs. Employing cross-linking mass spectrometry (XLMS) with multiple linkers, we identified interacting paratope-epitope residues and determined that the PF4 binding occurs primarily via light-chain interactions with the PF4 C-terminus. Structural modeling corroborated interprotein links and predicted the formation of a tetravalent immune complex, satisfying observed cross-links and existing epitope information. Native MS demonstrated lower-order complexes and tetravalent assemblies, supporting the modeling predictions. Our study provides structural insights into the pathogenesis of VITT and related PF4 disorders and highlights the potential for complementary structural MS and modeling to study challenging protein–protein interactions.