Molecular Dynamics Simulation Of The Adhesion And Signal-transduction Regions Of Platelet Glycoprotein Iba

Glycoprotein Ibα(GPIbα) is the main component of glycoprotein Ib-IX-V complex, theplatelet adhesion receptor. Under high shear stress condition, this molecule induces the ad-hesion of platelets to the von Willebrand factor (VWF) bound on the subendothelial matrix,and the secretion and aggregation of platelets. Inhibiting the association of GPIbαwith VWFor inhibiting the transduction of the association signal may prevent thrombosis triggered byatherosclerosis but not disturb physiologic haemostasis. Thus, GPIbαis an ideal target ofantithrombotic drugs. A precondition for development of such antithrombotic drugs is thecomprehension of the interaction of GPIbαwith VWF and cytoskeleton and the transduc-tion of mechano-chemical signal in these processes. In the first part of this study, based onfiow molecular dynamics simulation of conformational transition of GPIbα’sβ-switch region,whichisthebindinginterfacewithVWF,theregulationofbindingofthetwomoleculesbytheconformational transition is discussed; In the second part, based on steered molecular dynam-ics simulation of forced rupture of the bond between GPIbα’s cytoplasmic carboxyl-terminaldomain and filamin A’s IgFLNa17 domain, the possibility of mechano-chemical signal trans-duction by this bond is discussed. Molecular dynamics simulation applied in this study is animportant technique widely used in computational biology, structural chemistry, and drug de-sign. The technique reproduces the physical behavior of a system with numerical simulation,and investigates characters of biologic macromolecules based on the simulation.Theβ-switch region of GPIbα’s extracellular GPIbαN domain directly binds VWF. Inthe complex, this region assumeβ-hairpin conformation. Blood fiow may accelerate the con- formational transition ofβ-switch towardsβ-hairpin, and enhance the afinity of GPIbαwithVWF. To elucidate the mechanism of the regulation and verify its universality, this study sim-ulated the conformation ofβ-switch with free dynamics and fiow molecular dynamics, whichincluded widetype and six mutants. The results demonstrate that the stable conformationof this unbound region is structureless loop in water without fiow, evenβ-hairpin sponta-neously changing into loop; however, in fiowing water, this region shows tendency of tran-sition towardsβ-hairpin. Gain-of-function mutations G233V, D235V, K237V, and M239Vaccelerate the transition towardsβ-hairpin; Loss-of-function mutations Q232V and A238Vimpede such transition. Further analysis shows that the impact of the amino acid sequenceon the conformational transition roots in the changes of hydrophobicity of paired resides oftheβ-hairpin, and also in the changes of fiexibility of the region; efiects of gain-of-functionmutations is limited within the residues nearby, afiecting formation of hydrogen bonds androtation of residues. The reason that loss-of-function Q232V impedes the conformationaltransition may be the introduction of unfavorable hydrophobic interactions to the transition,which makes the peptide stuck in the middle of the transition and unable to continue the nextstep. Quantitative analysis shows that gain-of-function mutation G233V, D235V, K237V,and M239V’s increased tendencies towardsβ-hairpin in fiow correlate with the reported im-proved binding of these mutants with VWF induced by ristocetin of low concentrations. Theconformational difierence of GPIbαin static water and fiow is important to the design oflesion-specific antithrombotic drug.GPIbαattached to cytoskeleton via its cytoplasmic carboxyl-terminal domain. The cy-toskeleton component that directly and consistently binds this domain is filamin A, namelyIgFLNa17, the 17 repeat of the ROD domain. In high shear stress environment, plateletsbear fast loading of pulling force at the moment of initial tethering, and GPIbαtransduces themechnical signal outside-in. How the link of GPIbαand cytoskeleton changes is still unclear.The reported afinity of the two molecules measured with isothermal titration calorimetry isnot large, insuficient to account for in vivo observation of the tethering of platelets to ves-sel walls. This study focuses on the intrinsic properties of the bond between GPIbαandIgFLNa17, simulates the forced rupture of the complex by steered molecular dynamics withdifierent loading conditions, and deduces the biological implication of the properties of the bond. In the simulation, the unbinding of the complex can be accompanied by unfoldingof IgFLNa17, which only occurs near amino-terminal but never carboxyl-terminal. Calcu-lated from molecular force spectroscopy, the spring constant of the proteins is from 100 to200pN/nm, suggesting that the rigidity of the molecular bond is small; the rupture force atloading rate 17pN/ns is from 221 to 416pN, suggesting the strength of the linkage betweenIgFLNa17 and GPIbαis of the similar order of magnitude of the linkage of filamin and cy-toskeleton main body. Based on the position of IgFLNa17 in the full-length filamin, theseproperties suggests that the bond between GPIbαand cytoskeleton is not fragile to force, andIgFLNa17 may induce the transduction of mechanical signal, regulating the activity of an-other platelet receptor integrin, and thus relevant to the activation of platelets in high shearstress sites of arteries.In conclusion, this study discusses the mechanical properties in the atomistic scale ofGPIbαas platelet adhesion receptor and its interaction with other molecules, and the resultspictureascenewithdetailsofGPIbαinducingplateletactivationinarteries:β-switchofGPIbαchanges into high afinity conformation, GPIbαwith changed conformation binds to VWF,tethering a platelet on a injured vessel wall, and at the same time transduce the mechanicalsignal to the cytoskeleton, which is transformed into chemical signal and transmitted, finallyactivating the platelet. Such details will be important to the design of antithrombotic agents.