Mechanisms and Extent of Thrombin Generation in Trauma-Induced Coagulopathy Thomas Orfeo PhD University of Vermont
Mechanisms and Extent of Thrombin Generation in
Trauma-Induced Coagulopathy
Thomas Orfeo PhD
University of Vermont
Fig. 1. This figure represents our current understanding of trauma-induced coagulopathy.
Gonzalez E et al. Scandinavian Journal of Surgery 2014;103:89-103
Copyright © by Finnish Society of Surgery
Two topics
• Introduce 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:α-thrombin and normal hemostasis
anticoagulant procoagulant
FV→FVa FVIII→FVIIIa
Fbgn→Fn
TAFI→TAFIa
FXI→FXIa PC→APC
Plt→Plt*
FXIII→FXIIIa
Cellular
Migration
Growth
Secretion
Activation
α-Thrombin
Blood exposed to tissue factor
HEMORRHAGECONTROL
INJURY
Platelets
Fibrinogen
α-Thrombin
Prothrombinase assembly and thrombin output in blood from
healthy individuals
Site% of total prothrombin
activationForms of thrombin
Platelets* 50 – 60% α-IIa only
RBCs** 30 – 40% α-IIa and meizo-IIa
Lipoproteins/other 10% α-IIa and meizo-IIa
* Wood JP, et al. Blood 2011** Whelihan MF, et al. Blood 2012
Revised Model:Prothrombinase Assembly Sites
α-IIa and mIIa
α-IIa only
PS expressing RBCs
Activated platelets
Lee CJ, et al. J Thromb Haemost 2008
RBCs, meizothrombin, normal hemorrhage control?
Injury
α-IIamIIaα-IIa
Hemorrhage control
ActivatedPlatelets
RedBlood Cells
Structure/Function:α-thrombin vs meizothrombin
meizothrombin
B
S S
Catalytic Domain
A B
a-thrombin
S S
F2 A
F2
S S
F1
F1
prothrombin
membrane bindingdomain
α-Thrombin
α-Thrombin vs. meizothrombin
Structure/Function:α-thrombin vs meizothrombin
• Meizothrombin is less procoagulant than α-thrombin
• PC activation: mIIa-Tm ≥ αIIa-Tm• Meizothrombin binds to membrane
surfaces, α-thrombin does not• Meizothrombin reacts more slowly with AT
and heparin-AT complexes – longer functional half-life
Thrombin 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 flow
Syringe Pump
Capillary
96-Well Plate
Prothrombin inflowing phase
Phospholipid coatedprothrombinase
Collagen coatedprothrombinase +activated platelets
OR
meizothrombin α-thrombin
Prothrombin activation under flow
Prothrombinase assembled on phospholipids*
Prothrombinase assembled on activated platelets**
40-50% meizothrombin
50-60%α-thrombin
100% α-thrombin
* Haynes LM, et al. Biophys J 2011** Haynes LM, et al. J Biol Chem 2012
shear = 100 s-1
shear = 100 s-1
Modified from Whelihan MF, Mann KG. Thromb Res 2013
Fig. 1. This figure represents our current understanding of trauma-induced coagulopathy.
Gonzalez E et al. Scandinavian Journal of Surgery 2014;103:89-103
Copyright © by Finnish Society of Surgery
Histones
ΔPS on RBC
Semeraro F et al. Journal of Thrombosis and Haemostasis. 2014
Topic 2
• Characterize 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 disorders
• Sources of variation• Normal range variation in plasma coagulation factor
composition• Platelet subpopulations• Red blood cells?
• Consequences• Variability between transfusion units
Variation and consequences
Packed RBCs: Inter-unit variability, aging, thrombin dynamics
• Define the variability in the capacity of RBCs from different donors to support prothrombinase (Drs. Klein and West, Dept. of Transfusion Medicine NIH)– prior to storage– across the storage interval
• Study outdated PRBCs—develop informative markers
Packed RBCs
• Performance criteria—42 days storage– Less than 1% cell death – 75% survival of transfused RBCs after 24h
Changes in PRBCs during storage
• Shape change: discoid to spherical with projection• Membrane uptake of plasticizer (DEHP) used in
collection bags– stabilizes membrane, reduces cell death
• Loss of membrane lipids/proteins• Alterations in structural proteins• Dysregulation of Na+/K+ homeostasis• Depletion of 2,3 diphosphoglycerate
– results in increased hemoglobin oxygen affinity and decreased tissue oxygenation
• Acidification due to glycolysis– lactic acid accumulation
NYBC RBC units over a 3 month period (95000 total units)
Characterization of 44 day old PRBC units (CP2D-AS3)
PRBCUnit
pH
%Lysis
Cell Count
UnwashedPRBC
Debris
WashedPRBC
PTase
ROTEMNo Activator
FACS
ROTEMNo Activator
PTase
PTase
FACS
Was
hing Ste
p
αTAT/mTAT
αTAT/mTAT
αTAT/mTAT
mIIa
100s-1
Endothelial cell membrane
Tm mIIaEPCR
PCaPC
FVIIIa
FVa
Endothelial Cell
Extravascular Cell
ActivatedPlatelet
Red BloodCell
Fibrin Network
VenousValve
Tm
ΨΨ Ψ ΨΨΨAT
α-IIa
ΨHSPC
with AT
AT
Modified from Whelihan MF, Mann KG. Thromb Res 2013
Histone modifiedRed Blood Cell
TransfusedRed Blood Cell