CD40L Activates Platelet Integrin alpha IIb beta 3 by Binding to the Allosteric Site (Site 2) in a KGD-Independent Manner and HIGM1 Mutations Are Clustered in the Integrin-Binding Sites of CD40L

CD40L is expressed in activated T cells, and it plays a major role in immune response and is a major therapeutic target for inflammation. High IgM syndrome type 1 (HIGM1) is a congenital functional defect in CD40L/CD40 signaling due to defective CD40L. CD40L is also stored in platelet granules and transported to the surface upon platelet activation. Platelet integrin ffIIb fi3 is known to bind to fibrinogen and activation of ffIIb fi3 is a key event that triggers platelet aggregation. Also, the KGD motif is critical for ffIIb fi3 binding and the interaction stabilizes thrombus. Previous studies showed that CD40L binds to and activates integrins alpha v beta 3 and alpha 5 beta 1 and that HIGM1 mutations are clustered in the integrin-binding sites. However, the specifics of CD40L binding to alpha IIb beta 3 were unclear. Here, we show that CD40L binds to ffIIb fi3 in a KGD-independent manner using CD40L that lacks the KGD motif. Two HIGM1 mutants, S128E/E129G and L155P, reduced the binding of CD40L to the classical ligand-binding site (site 1) of alpha IIb beta 3, indicating that alpha IIb beta 3 binds to the outer surface of CD40L trimer. Also, CD40L bound to the allosteric site (site 2) of alpha IIb beta 3 and allosterically activated ffIIb fi3 without inside-out signaling. Two HIMG1 mutants, K143T and G144E, on the surface of trimeric CD40L suppressed CD40L-induced alpha IIb beta 3 activation. These findings suggest that CD40L binds to alpha IIb beta 3 in a manner different from that of ffvfi3 and alpha 5 beta 1 and induces ffIIbfi3 activation. HIGM1 mutations are clustered in ffIIbfi3 binding sites in CD40L and are predicted to suppress thrombus formation and immune responses through ffIIbfi3.