1. Mechanisms and Extent of Thrombin Generation in Trauma-Induced Coagulopathy Thomas Orfeo PhD University of Vermont

2. 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

3. 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

4. Standard model: α-thrombin and normal hemostasis anticoagulantprocoagulant 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 HEMORRHAGE CONTROL INJURY Platelets Fibrinogen α-Thrombin

5. Prothrombinase assembly and thrombin output in blood from healthy individuals Site % of total prothrombin activation Forms of thrombin Platelets*50 – 60%α-IIa only RBCs**30 – 40%α-IIa and meizo-IIa Lipoproteins/other10%α-IIa and meizo-IIa * Wood JP, et al. Blood 2011 ** Whelihan MF, et al. Blood 2012

6. 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

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

8. Structure/Function: α-thrombin vs meizothrombin

9. meizothrombin B SS Catalytic Domain AB  -thrombin SS F2A SS F1 prothrombin membrane binding domain

10. α-Thrombin

11. α-Thrombin vs. meizothrombin

12. 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

13. Thrombin dynamics: When is meizothrombin produced and how can it contribute to hemostasis?

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

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

17. 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

18. Modified from Whelihan MF, Mann KG. Thromb Res 2013

19. 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

20. Topic 2 Characterize variability between transfusion units with respect to their support of thrombin generation

21. 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

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

23. 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

24. Packed RBCs Performance criteria—42 days storage –Less than 1% cell death –75% survival of transfused RBCs after 24h

25. 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

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

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

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