Structural Transition of Cellular Integrins and Applications Thereof

Versiti Blood Health, Inc., Milwaukee WI

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Abstract

SUMMARY - Integrins are α/β heterodimeric cell surface receptors, which, via their ability to bind ligands through extracellular domains and to recruit a series of effector proteins to cytoplasmic tails, regulate diverse biological processes and play critical roles in many human diseases. The normal biological functions of integrins, like αIIbβ3- mediated hemostasis, hinge on a meticulously controlled equilibrium between activated and resting states. Inappropriate activation of αIIbβ3 in platelets causes thrombosis, making it a validated anti-thrombotic target. αIIbβ3 recognizes its ligands through an RGD (Arg-Gly-Asp) or RGD-like sequence. RGD-mimetic drugs are employed as inhibitors of αIIbβ3 for antithrombosis treatment. In its resting state, αIIbβ3 adopts a bent conformation. Activation of αIIbβ3 occurs rapidly when platelets are stimulated by agonists like ADP and thrombin, or by RGD-mimetic drugs like eptifibatide. Upon activation, αIIbβ3 undergoes a significant long-range conformational rearrangement, marked by an extension and opening of the headpiece, and a separation of the leg domains. This conformational activation is crucial for high-affinity ligand binding. Returning αIIbβ3 to its resting state, or deactivation, is essential for maintaining the appropriate function of αIIbβ3. While structural studies have unveiled various static conformations of integrins, encompassing inactive, intermediate, and active states, the precise mechanism through which the α and β subunits, each comprising multiple domains, collaboratively orchestrate the reversible large-scale structural transitions between the active and inactive states remains elusive. RGD-mimetic drugs, like eptifibatide, elicit a high-affinity active integrin conformation. This activation must be reversible, as the sustained active state post-drug dissociation could potentially lead to thrombocytopenia and, in certain cases, paradoxically increase the risk of thrombosis in patients. Platelet antagonists, such as P2Y12 and PAR-1 inhibitors, are commonly prescribed as antithrombotic drugs. However, the influence of these antiplatelet medications on the activation status of αIIbβ3 remains unknown. A more comprehensive understanding of the structural basis for integrin "pacification" could propose novel strategies for antiplatelet treatment. Our recent studies have developed innovative reagents and protocols for investigating the reversible conformational changes of αIIbβ3 on the platelet surface and in solution. Aim 1 of this grant aims to scrutinize the molecular mechanisms behind these reversible large-scale conformational changes in αIIbβ3. The acquired information will be utilized to explore novel therapeutic strategies aimed at attenuating integrin function by regulating its conformation. αIIbβ3 is among the most targeted receptors by allo- and auto-antibodies in alloimmune and drug- induced thrombocythemia. Our recent studies propose previously unrecognized mechanisms that contribute to the pathogenesis of antibody binding, potentially influencing the conformation and function of αIIbβ3. Aim 2 of this grant will utilize innovatively designed integrin constructs and newly available alloantibodies and autoantibodies of human or mouse origin to elucidate the structural basis for antibody binding and its impact on integrin function.