Contemporary views indicate that proteins are ensembles of pre-existing populations that undergo significant structural fluctuation at room temperature. Thus, allosteric effectors act by shifting the equilibrium between these conformational and dynamic states. Consistent with these views, we found that purified llb 3, the platelet receptor for fibrinogen that mediates platelet aggregation, is an ensemble of active and inactive molecules. Further, using optical tweezers to measure the nanomechanics of fibrinogen binding to and unbinding from IIb 3, we found that active IIb 3 is present in a minimum of two inter-convertible conformations that differ in their affinity for fibrinogen and in the mechanical stability of the complexes they form with fibrinogen. Accordingly, we postulate that by modulating the active state of IIb 3 with allosteric inhibitors, it can be stabilized in its lower affinity conformation, impairing the formation of more mechanically-stable thrombi in regions of high shear, such as those present in stenotic arteries, but at the same time, preserving sufficient platelet function for ordinary hemostasis. Thus, the objectives of this project are to relate IIb 3 structure to its dynamic behavior, with the ultimate goal of developing novel allosteric IIb 3 inhibitors that attenuate platelet aggregation by preventing the formation of higher affinity, more mechanically stable, IIb 3-fibrinogen complexes. The Project consists of two Specific Aims.
In Specific Aim 1, we will identify the regions of active IIb 3 involved in its allosteric conversion from a lower affinity conformation to the higher affinity conformation that forms more mechanically stable complexes with fibrinogen and fibrin. To identify targets for allosteric inhibition, the effect of mutations in these regions on the lifetime and strength of IIb 3-fibrinogen bonds will be measured at the single molecule level using optical tweezers. We will then experimentally screen chemical libraries and computationally screen molecular data bases for potential allosteric inhibitors whose activity will be verified in vitro using the optical tweezers and in vivo using mouse thrombosis models. The relative contributions of fibrin and fibrinogen to the formation of platelet thrombi will also be addressed, as will the role of protein disulfide isomerase in allosteric IIb 3 regulation.
In Specific Aim 2, we will study the dynamic behavior of the IIb and 3 cytosolic domains, testing the hypothesis that IIb 3 activation by the cytoskeletal proteins talin-1 and kindlin-3 is a cooperative event;the association of either protein with its binding site on 3 cytosolic domain occurs at the thermodynamic expense of disrupting favorable 3-membrane binding interactions. Biophysical experiments will be performed to determine the order and synergy of talin and kindlin binding, the role of membrane phospholipids in the process, and whether the result obtained by studying IIb 3 can be extrapolated to other regulated integrins.
PROJECT 4: Relevance Fibrinogen and von Willebrand factor binding to the active form of the integrin IIb 3 is responsible for the platelet stickiness that quenches bleeding after trauma and causes the thrombi that complicate atherosclerosis. However, current intravenous IIb 3 inhibitors have limited clinical applicability and oral inhibitors were associated with excess mortality. The goals of this project are to determine how platelets regulate IIb 3 function and to use this information to design novel, effective, and safer IIb 3 inhibitors.
|Stalker, Timothy J; Welsh, John D; Brass, Lawrence F (2014) Shaping the platelet response to vascular injury. Curr Opin Hematol 21:410-7|
|Min, Sang H; Suzuki, Aae; Stalker, Timothy J et al. (2014) Loss of PIKfyve in platelets causes a lysosomal disease leading to inflammation and thrombosis in mice. Nat Commun 5:4691|