Biology subjects

takada, y.

Publications and source records attributed to takada, y..

3 recordsLinked to original sources

bioRxiv2022

Pro-inflammatory chemokines CCL5, CXCL12, and CX3CL1 bind to and activate platelet integrin αIIbβ3 in an allosteric manner.

Previous studies showed that pro-inflammatory chemokines CX3CL1 and CXCL12 bound to the allosteric binding site (site 2) of integrins and allosterically activated integrins, in addition to the classical ligand-binding site (site 1). We showed that CCL5 also bound to site 2, in addition to site 1, and activated soluble integrin v{beta}3. Platelet integrin IIb{beta}3, a fibrinogen receptor, is critical for hemostasis and thrombus formation and activation of IIb{beta}3 is a key event for thrombus formation. Activation of IIb{beta}3 is known to be mediated exclusively by inside-out signaling. We studied if IIb{beta}3 can be allosterically activated. We discovered that CCL5, CXCL12, and CX3CL1 are new ligands for IIb{beta}3. Notably they enhanced the binding of monovalent ligand to soluble IIb{beta}3 in 1 mM Ca2+ by binding to site 2. They activated cell-surface IIb{beta}3 on CHO cells quickly (half maximal response <1 min) and at low concentrations (1-10 ng/ml) compared to soluble IIb{beta}3, probably because chemokines bind to cell surface proteoglycans. Notably, activation of IIb{beta}3 by the chemokines was several times more potent than 1 mM Mn2+. Since CCL5 and CXCL12 are stored in platelet granules and rapidly transported to the surface upon platelet activation, we hypothesized that they are released from the granules and allosterically activate IIb{beta}3 by binding to site 2. Transmembrane CX3CL1 on activated endothelial cells likely mediates platelet-endothelial interaction by binding to and activating IIb{beta}3. Also, over-produced chemokines during inflammation may trigger IIb{beta}3 activation, which is a possible missing link between inflammation and thrombosis.

biochemistry↗

bioRxiv2022

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.

immunology↗

bioRxiv2022

The C-type lectin domain of CD62P (P-selectin) is an integrin ligand.

CD62P (P-selectin) is confined to the inside of platelets and endothelial cells, and is translocated to the surface upon activation of platelets or endothelial cells. In current models, CD62P recognizes sialyl-Lewis X on PSGL-1 and mediates rapid rolling of leukocyte over vascular surfaces during the initial steps in inflammation. Docking simulation using integrin v{beta}3 as a target predicted that the C-type lectin domain of CD62P is a potential integrin ligand. It has not been tested if CD62P binds to integrins. Here we describe that the lectin domain of CD62P specifically bound to soluble integrins v{beta}3, IIb{beta}3, 4{beta}1 and 5{beta}1. Known inhibitors of CD62P-PSGL-1 interaction did not suppress the binding of the lectin domain to integrins. We found that the R16E/K17E mutation in the predicted integrin-binding interface of the lectin domain strongly inhibited CD62P binding to IIb{beta}3 and v{beta}3 in 1 mM Mn2+. R16E/K17E is outside of the glycan binding site. Mutating Glu-88 to Asp (the E88D mutation) in the lectin domain, which is known to strongly disrupt glycan binding, only slightly affected integrin binding, indicating that glycan binding and integrin binding sites are distinct. Also, the lectin domain of CD62P supported cell adhesion in a cation-dependent manner. CD62P-integrin interaction is potentially important since integrins are widely expressed compared to PSGL-1, which is limited to leukocytes. These findings indicate that CD62P-integrin interaction plays potentially important role in a wide variety of cell-cell interaction in addition to CD62P-glycan interaction.

cell biology↗