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Mechanisms and Extent of Thrombin Generation in Trauma-Induced Coagulopathy

Thomas Orfeo PhDUniversity of VermontFig. 1. This figure represents our current understanding of trauma-induced coagulopathy.

Gonzalez E et al. Scandinavian Journal of Surgery 2014;103:89-103Copyright by Finnish Society of SurgeryThis figure represents our current understanding of trauma-induced coagulopathy. On the left-hand column are those intrinsic and acquired factors that further potentiate TIC, including acidosis and hypothermia which were initially described with coagulopathy as the bloody vicious cycle. Hypoxia derived from hemorrhagic shock and tissue injury appears to be synergistic in driving TIC and activation of protein-C.DAMPS: damage-associated molecular pattern molecules; sCD40L: CD40 soluble platelet ligand; aPC: activated protein-C; tPA: tissue plasminogen activator; PAI: plasminogen activator inhibitor; TLR: toll-like receptor.Two topicsIntroduce a revised model of normal hemorrhage control that incorporates RBC contributions to thrombin generation and a proposed role for meizothrombin a relatively anticoagulant form of thrombin

Variability in the support of thrombin generation by transfusion products: studies characterizing thrombin generation by PRBCs

Standard model:-thrombin and normal hemostasisanticoagulantprocoagulantFVFVa FVIIIFVIIIa FbgnFn TAFITAFIa FXIFXIa PCAPC PltPlt* FXIIIFXIIIa CellularMigrationGrowthSecretionActivation-ThrombinBlood exposed to tissue factorHEMORRHAGECONTROLINJURYPlateletsFibrinogen-ThrombinProthrombinase assembly and thrombin output in blood from healthy individualsSite% of total prothrombin activationForms of thrombinPlatelets*50 60%-IIa onlyRBCs**30 40%-IIa and meizo-IIaLipoproteins/other10%-IIa and meizo-IIa* Wood JP, et al. Blood 2011** Whelihan MF, et al. Blood 2012Revised Model:Prothrombinase Assembly Sites

-IIa and mIIa-IIa onlyPS expressing RBCsActivated platelets

Lee CJ, et al. J Thromb Haemost 2008RBCs, meizothrombin, normal hemorrhage control?Injury-IIamIIa-IIaHemorrhage controlActivatedPlateletsRedBlood CellsStructure/Function:-thrombin vs meizothrombinmeizothrombinBSSCatalytic DomainABa-thrombinSSF2AF2SSF1F1prothrombinmembrane bindingdomain-Thrombin

-Thrombin vs. meizothrombinStructure/Function:-thrombin vs meizothrombinMeizothrombin is less procoagulant than -thrombinPC activation: mIIa-Tm IIa-TmMeizothrombin binds to membrane surfaces, -thrombin does notMeizothrombin reacts more slowly with AT and heparin-AT complexes longer functional half-lifeThrombin dynamics:When is meizothrombin produced and how can it contribute to hemostasis?Prothrombin activation (no flow)

Bradford HN, Krishnaswamy S. J Biol Chem 2012

Evaluating prothrombin activation under flowSyringe PumpCapillary96-Well PlateProthrombin inflowing phasePhospholipid coatedprothrombinaseCollagen coatedprothrombinase +activated plateletsORmeizothrombin-thrombinProthrombin activation under flowProthrombinase assembled on phospholipids*Prothrombinase assembled on activated platelets**

40-50% meizothrombin50-60%-thrombin100% -thrombin* Haynes LM, et al. Biophys J 2011** Haynes LM, et al. J Biol Chem 2012shear = 100 s-1shear = 100 s-1

Modified from Whelihan MF, Mann KG. Thromb Res 2013Fig. 1. This figure represents our current understanding of trauma-induced coagulopathy.

Gonzalez E et al. Scandinavian Journal of Surgery 2014;103:89-103Copyright by Finnish Society of SurgeryHistonesPS on RBCSemeraro F et al. Journal of Thrombosis and Haemostasis. 2014This figure represents our current understanding of trauma-induced coagulopathy. On the left-hand column are those intrinsic and acquired factors that further potentiate TIC, including acidosis and hypothermia which were initially described with coagulopathy as the bloody vicious cycle. Hypoxia derived from hemorrhagic shock and tissue injury appears to be synergistic in driving TIC and activation of protein-C.DAMPS: damage-associated molecular pattern molecules; sCD40L: CD40 soluble platelet ligand; aPC: activated protein-C; tPA: tissue plasminogen activator; PAI: plasminogen activator inhibitor; TLR: toll-like receptor.Topic 2Characterize variability between transfusion units with respect to their support of thrombin generation

Variation in thrombin generation

Global assays of thrombin generation display significant variation among healthy individuals

Some healthy individuals have thrombin generation phenotypes similar to those with hemorrhagic disordersSources of variationNormal range variation in plasma coagulation factor compositionPlatelet subpopulationsRed blood cells?ConsequencesVariability between transfusion unitsVariation and consequences22Packed RBCs: Inter-unit variability, aging, thrombin dynamicsDefine the variability in the capacity of RBCs from different donors to support prothrombinase (Drs. Klein and West, Dept. of Transfusion Medicine NIH)prior to storageacross the storage interval Study outdated PRBCsdevelop informative markers

Packed RBCs

Performance criteria42 days storageLess than 1% cell death 75% survival of transfused RBCs after 24h

Changes in PRBCs during storageShape change: discoid to spherical with projectionMembrane uptake of plasticizer (DEHP) used in collection bagsstabilizes membrane, reduces cell deathLoss of membrane lipids/proteinsAlterations in structural proteinsDysregulation of Na+/K+ homeostasisDepletion of 2,3 diphosphoglycerateresults in increased hemoglobin oxygen affinity and decreased tissue oxygenationAcidification due to glycolysislactic acid accumulation

NYBC RBC units over a 3 month period (95000 total units)

Characterization of 44 day old PRBC units (CP2D-AS3)PRBCUnitpH%LysisCell CountUnwashedPRBCDebrisWashedPRBCPTaseROTEMNo ActivatorFACSROTEMNo ActivatorPTasePTaseFACSWashing StepTAT/mTATTAT/mTATTAT/mTAT

mIIa100s-1Endothelial cell membraneTmmIIaEPCRPCaPCFVIIIaFVaEndothelial CellExtravascular CellActivatedPlateletRed BloodCellFibrin NetworkVenousValveTmAT-IIaHSPCwith ATATModified from Whelihan MF, Mann KG. Thromb Res 2013Histone modifiedRed Blood CellTransfusedRed Blood Cell