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

Abstract

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 αIIbβ3 is a key event that leads to αIIbβ3 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 αIIbβ3 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 μg/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 αvβ3. 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.

FGF2 binds to the allosteric site (site 2) and activates αvβ3 integrin and FGF1 binds to site 2 but suppresses integrin activation by FGF2: a potential mechanism of anti-inflammatory action of FGF1

“…Previous studies showed that several integrin ligands that bind to site 2 and activates integrins bound to peptides from site 2 (of β1 and β3) (Fujita et al 2014, Fujita et al 2015, Fujita et al 2018, Takada et al 2021, Takada et al 2022, Yoko K Takada 2022). We found that cyclic peptides derived from site 2 of β3 bound to FGF2 at higher levels than control scrambled β3 peptide, indicating that FGF2 is required to bind to site 2 to activate αvβ3 (Fig.…”

FGF2 binds to the allosteric site (site 2) and activates integrin αIIbβ3 and FGF1 binds to site 2 but suppresses integrin activation by FGF2: A potential mechanism of anti-thrombotic action of FGF1

“…2d and 2e), which is consistent with the idea that FGF2 activates αIIbβ3 independent of inside-out signaling. Previous studies showed that several integrin ligands that bind to site 2 and activates integrins bound to peptides from site 2 (of β1 and β3) (Fujita et al 2014, Fujita et al 2015, Fujita et al 2018, Takada et al 2021, Takada et al 2022, Yoko K Takada 2022). We found that cyclic peptides derived from site 2 of β3 bound to FGF2 at higher levels than control scrambled β3 peptide, indicating that FGF2 is required to bind to site 2 to activate αIIbβ3 (paper submitted).…”

Virtual Screening of Protein Data Bank via Docking Simulation Identified the Role of Integrins in Growth Factor Signaling, the Allosteric Activation of Integrins, and P-Selectin as a New Integrin Ligand

“…Also, CD40L bound to site 2 of αIIbβ3 and allosterically activated αIIbβ3 without inside-out signaling. Two HIMG1 mutants, K143T and G144E, on the surface of trimeric CD40L suppressed CD40L-induced αIIbβ3 activation [ 50 ]. These findings suggest that CD40L binds to αIIbβ3 in a manner different from that of αvβ3 and α5β1 and induces αIIbβ3 activation.…”

“…We showed that PF4 binds to soluble integrin αIIbβ3 in cell-free conditions but did not activate this integrin at physiological PF4 concentrations (<1 μg/mL). Notably, an anti-PF4 (RTO)/PF4 complex potently activated soluble αIIbβ3 in a heparin-independent manner [ 50 ]. We propose that anti-PF4 changed the conformation of PF4 and strongly activates αIIbβ3 by binding to site 2 and results in the strong aggregation of platelets.…”

“…We have developed a PF4 mutant that does not induce αIIbβ3 and αvβ3 activation by mutating the site 2 binding site in PF4 (predicted by docking simulation). The PF4 mutant acted as an antagonist of the anti-PF4/PF4-induced activation of integrins in ELISA-type activation assays, indicating that it has potential as a therapeutic [ 50 ].…”