Probing Chemical and Conformational Differences in the Resting and Active Conformers of Platelet Integrin αIIbβ3

Abstract

Integrin ␣ IIb ␤ 3 is the fibrinogen receptor that mediates platelet adhesion and aggregation. The ligand binding function of ␣ IIb ␤ 3 is "activated" on the platelet surface by physiologic stimuli. Two forms of ␣ IIb ␤ 3 can be purified from platelet lysates. These forms are facsimiles of the resting (Activation State-1 or AS-1) and the active (Activation State-2 or AS-2) conformations of the integrin found on the platelet surface. Here, the differences between purified AS-1 and AS-2 were examined to gain insight into the mechanism of activation. Four major findings are put forth. 1) The association rate (k 1 ) between fibrinogen and the integrin is a key difference between AS-1 and AS-2. 2) Although the divalent ion Mn 2؉ enhances the ligand binding function of AS-1, this ion is unable to convert AS-1 to AS-2. Therefore, its effect on integrin is unrelated to activation. 3) Peptide mass fingerprints indicate that the chemical structure of AS-1 and AS-2 are virtually identical, calling into question the idea that post-translational modifications are necessary for activation. 4) The two forms of ␣ IIb ␤ 3 have significant conformational differences at three positions. These include the junction of the heavy and light chain of ␣ IIb , the divalent ion binding sites on ␣ IIb , and at a disulfide-bonded knot linking the amino terminus of ␤ 3 to the cysteine-rich domain. These observations indicate that integrin is activated by a series of specific conformational rearrangements in the ectodomain that increase the rate of ligand association.

Probing Conformational Changes in the I-like Domain and the Cysteine-rich Repeat of Human β3 Integrins following Disulfide Bond Disruption by Cysteine Mutations

“…Indeed, the expressed ␤ 3 Ala 177 , ␤ 3 Ala 273 , and ␤ 3 Tyr 598 mutants, when studied under nonreducing conditions by SDS-PAGE, exhibited a slower than normal electrophoretic mobility as compared with wild type ␤ 3 , a result consistent with that observed for other ␤ 3 cysteine mutations, such as C457Y (41) or C542R (42). It is noteworthy that this difference in electrophoretic mobility was not observed for the purified resting (AS-1) and active ␣ IIb ␤ 3 (AS-2) receptors, reported to differ in the number and position of unpaired cysteines (10). The three mutations investigated here had a clear inhibitory effect on ␣ IIb ␤ 3 biosynthesis, processing, and cell surface exposure.…”

“…On the other hand, the Cys 232 -Cys 273 bond, which does not exist in the ␤ 4 integrin subunit, is in close proximity to the hexapeptide 275 VGSDNH 280 in the ␤ 3 subunit that confers species restricted heterodimer assembly to ␣ IIb ␤ 3 (40). And finally, Cys 598 is located in the conserved motif II of the fourth CRR and is part of a small tryptic fragment of the fourth CRR (residues 581-600) that has recently been identified to contain unpaired cysteines (10). By mutating the cysteine residues Cys 177 or Cys 273 into alanine and Cys 598 into tyrosine, we disrupted each of the three cysteine loops by creating free sulfhydryls.…”

“…Evidence has also been provided that mutational introduction of disulfide bonds into the ␣ L ␤ 2 integrin I domain locks this receptor in an open ligand binding or closed nonbinding conformation (22,23). And finally, Yan et al (10) have recently demonstrated that a small number of cysteines, located within the epidermal growth factor-like cysteine-rich repeats (CRR) of the ␤ 3 subunit, are unpaired and exhibit the properties of a redox site that could be involved in integrin activation. This ␣ IIb ␤ 3 activation appears to be controlled by a protein-disulfide isomerase activity (24,25), recently shown to be an intrinsic activity of integrin ␣ IIb ␤ 3 (26).…”

“…Although the molecular basis for the conformational changes associated with integrin receptor function modulation is still elusive, evidence has recently been provided that sulfhydryls may contribute to receptor function and that thiol bond reshuffling could represent a possible mechanism responsible for the conformational changes necessary for integrin activation (10). Blocking of free sulfhydryls on the platelet surface inhibits platelet adhesion to collagen, fibronectin, and fibrinogen (11), platelet aggregation following ADP stimulation (10), and adhesion of Mn 2ϩ -or antibody-activated platelets (11), suggesting that free sulfhydryls play an important role in the process of integrin-ligand interaction and that sulfhydryl blockers could act downstream of the conversion of the integrin high affinity state, possibly on the process of ligand binding itself.…”

“…Blocking of free sulfhydryls on the platelet surface inhibits platelet adhesion to collagen, fibronectin, and fibrinogen (11), platelet aggregation following ADP stimulation (10), and adhesion of Mn 2ϩ -or antibody-activated platelets (11), suggesting that free sulfhydryls play an important role in the process of integrin-ligand interaction and that sulfhydryl blockers could act downstream of the conversion of the integrin high affinity state, possibly on the process of ligand binding itself. On the other hand, mild reducing agents, such as dithiothreitol, can increase the ligand binding function of ␣ IIb ␤ 3 as well as other integrins, a method commonly used to activate recombinant integrins in transfected nucleated cells (12)(13)(14)(15)(16)(17).…”