Anti-PF4 (heparin-independent)/PF4 complex induces allosteric activation of integrins αIIbβ3 and αvβ3, a potential mechanism of vaccine-induced thrombotic thrombocytopenia (VITT) and autoimmune diseases

The classical immune-mediated heparin-induced thrombocytopenia (HIT) is induced by autoantibody against platelet-factor 4 (PF4)/heparin complex. Vaccine-induced thrombotic thrombocytopenia (VITT) and autoimmune HIT (aHIT) are induced by anti-PF4 in a heparin-independent manner. Activation of platelet integrin IIb{beta}3 is a key event that leads to IIb{beta}3 binding to fibrinogen and platelet aggregation, but is not involved in current models of HIT or VITT. Anti-PF4 (heparin-independent) is also detected in autoimmune diseases (e.g., SLE). However, the role of anti-PF4 in these diseases is unknown. Previous studies showed that several pro-inflammatory chemokines potently activated integrins by binding to the allosteric site (site 2). PF4 is known to be inhibitory since it inhibits angiogenesis and tumor growth. Here we describe that PF4 was predicted to bind to site 2 of IIb{beta}3 by docking simulation, but did not activate it. However, PF4/anti-PF4 mAb (RTO, heparin-independent) complex potently activated it at biological concentrations of PF4 (<1 g/ml), but anti-PF4/heparin (KKO) did not. This indicates that RTO changed the phenotype of PF4. We generated PF4 mutants defective in site 2 binding to integrin by introducing mutations in the predicted site 2 binding site of PF4. A PF4 mutant/RTO complex was defective in activating integrins. Furthermore, this PF4 mutant acted as an antagonist of PF4/RTO-induced integrin activation. We obtained similar results with vascular integrin v{beta}3. We propose that a potential mechanism, in which PF4/RTO complex binds to site 2 and activates integrins and triggers thrombocytopenia or autoimmune diseases. The inhibitory PF4 mutant may have potential as a therapeutic.