Macromolecular Substrate Recognition in Blood Coagulation

$449,661P01FY2007HLNIH

Children'S Hosp Of Philadelphia, Philadelphia PA

Investigators

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Abstract

The specifipcroteolytiactivatiosntepsof bloodcoagulationarccatalyzedby scrineprotcinasetshatarehomologous toeach otherand tothearchetypalserincprotcinascosf digestionI.n many cases,explanationfsorthemolecularbasis of thedistinctimvaecromolccularsubstratscpecificitoyf thecoagulationreactionrsemain incomplete.Evidence suggeststhatprothrombinactivatiobny theprothrombinascornplcxr,esultfsrom interactionastan extended macromolccularrecognitiosnite(Exositeso)n theenzyme followedby activcsiteinteractionpsriorto cleavageand productrcleascW.c willuseprothrombinactivatiocnatalyz_ by theprothrombinasecomplex as a paradigm for specifimcacromolccularsubstratrcecognitioannd cleavageby coagulationcomplexes to investigatehebasisforits substratscpecificiatnyd functionU.sing fluorescenstubstratacnalogs,bindingand stoppedflow kineticmeasurements by resonanceenergytransfewrillbc used toinvestigatcexositcbindingand providea completekineticand thermodynamic descriptionf thestcpwiscinteractiontshatleadto substratrcecognitionand cleavage.Wc willtesthe hypothesisthatextendedsurfacesinfactorXa playa major roleinexositc-dcpcndcnstubstratreecognitionby prothrombinascT.hese structurafleatureswillbc exploredwithexositcprobesthatbindXa and selectiveliynhibit proteinsubstratbcinding.Using thebindingfootprinotf cxositc-directperdobesas a guidelinew,c willprovidefull biochemicalprooffortheseideasby preparingrecombinantXa derivativewsith a selectivdefectin cxositc-depcndcnt substratrcecognitiownithinprothrombinascW.c willinvestigatehehypothesisthatheorderedaccessibiliotfythetwo bonds inprothrombinby prothrombinasearisesfrom thecombined constraininegffectosf cxositctetherinagnd membrane bindingby thesubstratcT.hese bindinginteractionasrcproposedtoboth facilitatned constraibnond presentatioand thespecifiactionofprothrombinascon prothrombin.Inthelastseriesof studiesw,c willinvestigate thehypothesesthatheinitiaelxositetetherinignteractiowniththesubstratoevercomes both cnthalpiacnd cntropic limitationosf activesitedockinginteractiontsherebyieldingvastimprovements incatalytircateand leadingtothe orderedcleavageofprothrombin.We believethatheapproachescontainedinthisproposalwillprovideunanticipated insightsintothefunctionof thecoagulationenzymes and a new perspectivoen themechanism of actionof prothrombinascon itsbiologicaslubstrateT.he delineationf thedeterminantosf macromolecularsubstratscpecificity willlikelysuggestnovelapproachesfortherapeutitcargetinogf thesereactionisnthromboticand vasculardisease states.

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