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

INJURY
Standard model:
α-thrombin and normal hemostasis
Blood exposed to tissue factor
Platelets
α-Thrombin
Fibrinogen
HEMORRHAGE
CONTROL
PC→APC anticoagulant
Cellular
Migration
Growth
Secretion
Activation
Plt→Plt*
Fbgn→Fn
FXIII→FXIIIa
TAFI→TAFIa
FVIII→FVIIIa
FV→FVa
FXI→FXIa procoagulant
α-Thrombin

Prothrombinase assembly and thrombin output in blood from healthy individuals
Site
Platelets*
RBCs**
Lipoproteins/other
% of total prothrombin activation
50 – 60%
30 – 40%
10%
Forms of thrombin
α-IIa only
α-IIa and meizo-IIa
α-IIa and meizo-IIa
* Wood JP, et al. Blood 2011
** Whelihan MF, et al. Blood 2012

Revised Model:
Prothrombinase Assembly Sites
PS expressing RBCs
α-IIa and mIIa
Activated platelets
α-IIa only
Lee CJ, et al. J Thromb Haemost 2008

RBCs, meizothrombin, normal hemorrhage control?
Injury
Activated
Platelets
α-IIa mIIa
α-IIa
Hemorrhage control
Red
Blood Cells

membrane binding domain
F1 prothrombin
F2 Catalytic Domain
S S
F1 meizothrombin
F2 A
S S
B A a
-thrombin
B
S S

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

Prothrombin activation (no flow)
Bradford HN, Krishnaswamy S. J Biol Chem 2012

Evaluating prothrombin activation under flow
Prothrombin in flowing phase
Collagen coated prothrombinase + activated platelets
OR
Phospholipid coated prothrombinase
Syringe Pump
Capillary
96-Well Plate meizothrombin α-thrombin

Prothrombin activation under flow
Prothrombinase assembled on phospholipids*
Prothrombinase assembled on activated platelets** shear = 100 s -1
40-50% meizothrombin
50-60%
α-thrombin
100% α-thrombin shear = 100 s -1
* Haynes LM, et al. Biophys J 2011
** Haynes LM, et al. J Biol Chem 2012

Modified from Whelihan MF, Mann KG. Thromb Res 2013

Fig. 1. This figure represents our current understanding of trauma-induced coagulopathy.
Histones
ΔPS on
RBC
Semeraro F et al.
Journal of
Thrombosis and
Haemostasis.
2014
Gonzalez E et al. Scandinavian Journal of Surgery
2014;103:89-103
Copyright © by Finnish Society of Surgery

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

Variation and consequences
• Sources of variation
• Normal range variation in plasma coagulation factor composition
• Platelet subpopulations
• Red blood cells?
• Consequences
• Variability between transfusion units

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)

PRBC
Unit
Characterization of 44 day old
PRBC units (CP2D-AS3)
PTase
ROTEM
No Activator
αTAT/ mTAT
Washed
PRBC
FACS
Debris
PTase
ROTEM
No Activator
αTAT/ mTAT pH
%
Lysis
Cell
Count
Unwashed
PRBC PTase
αTAT/ mTAT
FACS

Histone modified
Red Blood Cell
Transfused
Red Blood Cell
FVa aPC
PC
Tm mIIa
EPCR
Endothelial cell membrane
FVIIIa
100s -1
α-IIa
Ψ
AT
Ψ Ψ
Tm mIIa Ψ Ψ Ψ
Endothelial
Cell
Extravascular
Cell
Activated
Platelet
Red Blood
Cell
Fibrin
Network
Venous
Valve
Ψ
AT
HSPC with AT
Modified from Whelihan MF, Mann KG. Thromb Res 2013