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.