Structural and functional changes underlying activation of monocytes in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated disorder associated with thrombosis developing in response to anticoagulation with heparin. Monocytes targeted by HIT antibodies contribute to the prothrombotic state, but structural and functional alterations of the activated monocytes have not been described.

To study morphologic and functional changes in monocytes caused by HIT antibodies interacting with membrane-associated platelet factor 4 (PF4) in vitro.

THP-1, isolated human, or FcγRIIA-positive and FcγRIIA-negative mouse monocytes were incubated with recombinant human PF4 and/or anti-PF4/heparin antibodies followed by scanning electron microscopy and confocal microscopy.

Binding of PF4 to monocytes induced formation of "knobs" ∼150 nm in size that protruded from the cell surface. Addition of pathogenic HIT-like monoclonal antibodies (KKO) caused profound remodeling of the cell membrane and time-dependent formation and clustering of KKO/PF4/glycosaminoglycan complexes into large "blebs" ranging in size from 500 to 1200 nm. Dynamic confocal microscopy revealed formation of monocyte-derived microvesicles in response to PF4 and KKO. In contrast, RTO, a monoclonal antibody that blocks PF4 oligomerization and prevents thrombocytopenia/thrombosis in an animal HIT model, inhibited PF4-induced modification of monocyte surfaces. Comparing monocytes from transgenic mice expressing hFcγRIIA to wild-type mice lacking FcγRIIA indicated that bleb formation results from clustering of knobs caused by bivalent HIT antibodies through crosslinking of FcγRIIA.

Binding of pathogenic HIT antibodies to PF4-containing antigenic complexes assembled on the monocyte surface promotes large-scale plasma membrane remodeling as part of cell activation through the FcγRIIA receptors, resulting in the release of procoagulant microvesicles, which together may contribute to thrombosis in HIT.