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