Divalent Cations Define the Structural Requirements for αIIbβ3 Integrin Activation

Abstract 260

Platelet αIIbβ3 integrin undergoes structural rearrangements to increase the affinity for ligands. Two major changes in integrin three-dimensional structure are critical for this activation process. One is the straightening of integrin legs (extension), and the other is the swing out movement of the β3 hybrid domain (swing-out). In this study, we have found that this structural requirement for activation differs depending upon the divalent cations present. We previously reported that the swing-out-defective mutant αIIbD319C/β3V359C did not bind fibrinogen in the presence of Ca²⁺/Mg²⁺. However, the same mutant bound fibrinogen in the presence of Mn²⁺, when activated by monoclonal antibody PT25-2 or by forcing the integrin legs to extend by concomitant αIIbQ595NTT mutation. We engineered another mutation that was designed specifically to prevent the swing-out. Likewise, this β3G327C/β3V419C mutation blocked fibrinogen binding in the presence of Ca²⁺/Mg²⁺, but not in the presence of Mn²⁺. On the other hand, when integrin extension was specifically prevented by αIIbD464C/αIIbS728C mutation, fibrinogen binding was observed neither in the presence of Ca²⁺/Mg²⁺ nor in Mn²⁺. Thus, swing-out is not essential for activation in Mn²⁺, as long as integrin legs are in extended state, while both swing-out and extension are required in Ca²⁺/Mg²⁺. To explore the underlying mechanism originating this difference, three distinct cation-binding sites in the βA domain were mutated. Among the three sites, MIDAS is occupied by Mg²⁺, while ADMIDAS and LIMBS/SyMBS are occupied by Ca²⁺ in Ca²⁺/Mg²⁺ condition. When amino acid residues composing these sites were mutated to Ala, none of them bound fibrinogen, except for D126A and D127A in the presence of Ca²⁺/Mg²⁺. Notably, these mutations restored fibrinogen binding in the swing-out-defective mutant. Amino acid residues D126 and D127 compose ADMIDAS together with S123 and M335. Swing-out of the hybrid domain disrupts M335 from ADMIDAS. This allows Ca²⁺ to move toward MIDAS, bringing the β1-α1 loop closer to ligand to provide contact site. However, similar rearrangement of the cation/ligand binding sites has been shown to take place in Mn²⁺ without the swing-out. The results suggest partial disruption of ADMIDAS is the key event for activation in Ca²⁺/Mg²⁺, while it is not so in Mn²⁺. Our results may reconcile the dispute over the apparently contradicting findings in the crystal structure that integrin with a closed head with Mn²⁺ represents an active conformation as one with an open head with Ca²⁺/Mg²⁺.