IntroductionThe platelet ␣IIb␤3 integrin plays a central role in platelet adhesion and aggregation. [1][2][3] Thus, it can support platelet adhesion to immobilized fibrinogen even in the absence of exogenous activators. 4,5 Moreover, when activated, the ␣IIb␤3 heterodimer can bind soluble ligands, including fibrinogen and von Willebrand factor, which can span between platelets to form aggregates. 1,3,6,7 Loss of the receptor or its function on an inherited basis results in the hemorrhagic diathesis Glanzmann thrombasthenia, 8 and inhibitors of the receptor have proven effective in the prevention and treatment of coronary artery thrombosis. 9,10 Biochemical, molecular biologic, and crystallographic evidence indicate that ligands bind to a groove in ␣IIb␤3 that is at the intersection of the ␣IIb ␤ propeller domain and the ␤3 ␤A (I-like) domain. 11 Fibrinogen binds to ␣IIb␤3 via a carboxyl-terminal dodecapeptide sequence in its ␥ chain that contains both a positively charged Lys and a negatively charged Asp (HHLGGAKQAGDV). [12][13][14] The integrin also binds ligands containing the sequence Arg-Gly-Asp (RGD) or Lys-Gly-Asp (KGD), including von Willebrand factor 6,15 and snake venom-derived disintegrins. 16 The drugs eptifibatide and tirofiban, which are patterned after the KGD and RGD sequences, respectively, span the ␣IIb␤3 ligand binding groove with orientations similar to that of an RGD-containing peptide (cilengitide) in the related receptor ␣V␤3 17 ; thus, their positively charged groups interact with ␣IIb Asp224 and their negatively charged carboxyl groups contribute to the coordination of the metal ion in the ␤3 metal ion-dependent adhesion site (MIDAS). 11 Conformational changes in ␣IIb␤3 occur upon receptor activation, and additional changes occur after the binding of ligand to the receptor, leading to the exposure of ligand-induced binding sites (LIBS) that can be detected by LIBS-specific monoclonal antibodies (mAbs). [18][19][20][21] The binding of RGD peptides and both eptifibatide and tirofiban increase the binding of LIBS-specific mAbs. 22 Since ␣IIb␤3 may remain in its high-affinity conformation after dissociation of the competitive inhibitors, transient interactions of these compounds with the receptor may actually facilitate ligand binding by "priming" the receptor. 23 It has been postulated that this effect may have contributed to the increased mortality observed during treatment with orally active inhibitors of ␣IIb␤3 that were administered on a chronic basis. [24][25][26][27][28][29] Moreover, the conformational changes induced by all of the antagonists may contribute to the thrombocytopenia observed with these agents. 30 To identify novel small molecules capable of inhibiting the interaction of fibrinogen with ␣IIb␤3, we used high-throughput screening of several libraries of small molecules, testing the ability of the compounds to inhibit platelet adhesion to fibrinogen. We identified one compound with unique features that provide insights into ␣IIb␤3 structure and function. Methods Monoclonal...

We previously reported on a novel compound (Compound 1; RUC-1) identified by high-throughput screening that inhibits human ␣IIb␤3. RUC-1 did not inhibit ␣V␤3, suggesting that it interacts with ␣IIb, and flexible ligand/rigid protein molecular docking studies supported this speculation. We have now studied RUC-1's effects on murine and rat platelets, which are less sensitive than human to inhibition by Arg-Gly-Asp (RGD) peptides due to differences in the ␣IIb sequences contributing to the binding pocket. We found that RUC-1 was much less potent in inhibiting aggregation of murine and rat platelets. Moreover, RUC-1 potently inhibited fibrinogen binding to murine platelets expressing a hybrid ␣IIb␤3 receptor composed of human ␣IIb and murine ␤3, but not a hybrid receptor composed of murine ␣IIb and human ␤3. Molecular docking studies of RUC-1 were consistent with the functional data. In vivo studies of RUC-1 administered intraperitoneally at a dose of 26.5 mg/kg demonstrated antithrombotic effects in both ferric chloride carotid artery and laser-induced microvascular injury models in mice with hybrid h␣IIb/m␤3 receptors. Collectively, these data support RUC-1's specificity for ␣IIb, provide new insights into the ␣IIb binding pocket, and establish RUC-1's antithrombotic effects in vivo. (Blood. 2009;114:195-201) IntroductionWe previously published data on the identification of a novel inhibitor of ␣IIb␤3 (Compound 1; now referred to as RUC-1). 1 We speculated that it interacted exclusively with the ␣IIb portion of the Arg-Gly-Asp (RGD) binding site based on its specificity for ␣IIb␤3 compared with ␣V␤3 and molecular docking studies into the human ␣IIb␤3 headpiece suggesting that the positively charged piperazinyl nitrogen of RUC-1 interacts with the carboxyl group of D224 in ␣IIb and that the heterocyclic fused ring of RUC-1 interacts with one or more of the 3 aromatic residues that line the ␣IIb pocket. RUC-1 also is too short to span between D224 of ␣IIb and the ␤3 metal ion-dependent adhesion site (MIDAS) and lacks a carboxyl group to coordinate the MIDAS metal ion, which is an invariant feature of all other small molecule ␣IIb␤3 antagonists. [2][3][4] In the present study, we further tested whether RUC-1 demonstrates specificity for ␣IIb by taking advantage of known differences in the abilities of ␣IIb␤3 antagonists to inhibit ␣IIb␤3-mediated platelet aggregation in different species. Consistent with these data, we also found that RUC-1 could inhibit thrombus formation in vivo in transgenic mice expressing human (h) ␣IIb in complex with murine (m) ␤3, but not wild-type (WT) mice. Estimates of electrostatic and van der Waals interaction energies of RUC-1 docked into the crystal structure of human ␣IIb␤3 or molecular models of rat ␣IIb␤3, mouse ␣IIb␤3, or hybrid human ␣IIb/mouse␤3 were consistent with the functional data. In aggregate, these data have important implications for understanding the structure of the ␣IIb binding pocket and the potential antiplatelet effects of ␣IIb-specific ␣IIb␤3 antagonists. Me...

Structural data of integrin ␣IIb␤3 have been interpreted as supporting a model in which: 1) the receptor exists primarily in a "bent," low affinity conformation on unactivated platelets and 2) activation induces an extended, high affinity conformation prior to, or following, ligand binding. Previous studies found that "clasping" the ␣IIb head domain to the ␤3 tail decreased fibrinogen binding. To study the role of ␣IIb extension about the genu, we introduced a disulfide "clamp" between the ␣IIb thigh and calf-1 domains. Clamped ␣IIb␤3 had markedly reduced ability to bind the large soluble ligands fibrinogen and PAC-1 when activated with monoclonal antibody (mAb) PT25-2 but not when activated by Mn 2؉ or by coexpressing the clamped ␣IIb with a ␤3 subunit containing the activating mutation N339S. The clamp had little effect on the binding of the snake venom kistrin (M r 7,500) or ␣IIb␤3-mediated adhesion to immobilized fibrinogen, but it did diminish the enhanced binding of mAb AP5 in the presence of kistrin. Collectively, our studies support a role for ␣IIb extension about the genu in the binding of ligands of 340,000 and 900,000 M r with mAb-induced activation but indicate that it is not an absolute requirement. Our data are consistent with ␣IIb extension resulting in increased access to the ligand-binding site and/or facilitating the conformational change(s) in ␤3 that affect the intrinsic affinity of the binding pocket for ligand.The platelet ␣IIb␤3 receptor plays an important role in both hemostasis and thrombosis (1). Ligand binding to ␣IIb␤3 is controlled by an activation process that affects the conformation of the receptor and ligand binding, in turn, can also affect the conformation of the receptor (2). Several different conformations of ␣IIb␤3 have been identified based on inferences from biochemical analyses (3), studies employing monoclonal antibodies (4 -7) and electron microscopy (8 -10), comparison of the crystal structures of the liganded ␣IIb␤3 headpiece (11) and the unliganded complete ectodomain (12), and analysis of the unliganded and liganded ectodomain of the related ␣V␤3 receptor (13,14). Receptor extension about the regions encompassing the thigh, genu, and calf-1 domains of ␣IIb and the plexin-semaphorin-integrin (PSI), 2 integrin epidermal growth factor-1 (IEGF-1), and IEGF-2 domains of ␤3 or comparable regions of other integrin receptors has been proposed to play an important role in receptor activation (12,(15)(16)(17)(18), but there is uncertainty about whether this conformational change occurs prior to or after ligand binding (19 -21). Thus, "cross-clasping" the ␣IIb headpiece ␤-propeller domain to the ␤3 IEGF-4 domain in the tail region via a newly engineered disulfide bond prevented the binding of fibrinogen induced by activating mAb in concert with the activating divalent cation Mn 2ϩ , and a similar effect was observed with cross-clasped ␣V␤3 (16). In both cases the loss of ligand binding could be rescued by reducing the cross-clasped receptors with dithiothreitol (DTT). In contr...

A collection of αIIbβ3 integrin receptor antagonists possessing a unique MIDAS metal ion displacement mechanism of action is presented. Insight into these agents’ structure-activity relationships, binding modality, and pharmacokinetic and pharmacodynamic profiles highlight the potential of these small molecule ion displacement ligands as attractive candidates for clinical development.

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