1. TACTIC PROJECT 2: THE ROLE OF FACTOR XI IN TIC LEAD INVESTIGATOR: SAULIUS BUTENAS (UNIVERSITY OF VERMONT) CO-INVESTIGATOR: JAMES H MORRISSEY (UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN) PHILADELPHIA, SEPTEMBER 10, 2014 a

2. The Coagulation Cascade Intrinsic Pathway Factor XII Prekallikrein HMW Kininogen “Surface” Factor XIa HMW Kininogen Membrane Ca 2+, Zn 2+ Intrinsic Tenase AT-III IIa XIa Va i VIIIa i XIIa IXa Extrinsic Pathway Factor VIIa Tissue Factor Membrane Ca 2+ Factor IXa Factor VIIIa Membrane Ca 2+ Factor Xa Factor Va Membrane Ca 2+ Thrombin Thrombomodulin Membrane Ca 2+ Fibrin Clot Formation Extrinsic Tenase Prothrombinase Cross-Linked Fibrin Clot Soluble Fibrin Peptides TFPI AT-III PAI-1 AT-III TAFIa Plasmin APC  2 -AP AT-III XIIIa Fibrin IIa Xa IXa TFPI AT-III Red = Enzymes Yellow = Inhibitors Vascular Injury Tissue Factor IIa Va VIIIa

3. FXIa-Initiated Clotting of Whole Blood Initiator Concentration

4. Assays for FXIa  Clotting and thrombin generation (TGA) assays were developed.  Both assays are based on the response of contact pathway-inhibited (corn trypsin inhibitor; CTI) plasma to the inhibitory anti-FXI monoclonal antibody.  No exogenous initiator of thrombin generation is added.  No thrombin generation/clot formation is observed in plasma from healthy individuals

5. Study #1 (In collaboration with Dr. M. Park from Mayo Clinic)  Multiple time-points from 98 patients:  Burn 34  Blunt 47  Penetrating 17 Clotting assay was used for the quantitation of FXIa Supported by DoD and PO1 HL46703

10. *ISS – injury severity score. **Average ± SD for patients with FXIa ≥ 10 pM. Detectabiliy limit of FXIa in this clotting time-based assay is 10 pM. S. Butenas, M. Park, K. Mann (unpublished data). Active Factor XIa at Admission Injury ISS* > 25ISS ≤ 25 XIa Frequency(pM)**Frequency(pM) Burn13/19 (68%)38±363/15 (20%)47±7 Blunt19/31 (61%)39±287/16 (44%)37±26 Penetrating 3/7 (43%)55±651/10 (10%)160

11. Conclusion #1  The occurrence of FXIa correlates with the severity of trauma

13. Study #2 (In collaboration with Dr. J. Shupp from Washington Burn Center)  56 Burn patients  463 time-points  Up to 20 time-points per patient from 0 to 504 hours (3 weeks) Thrombin Generation assay was used for the quantitation of FXIa Supported by Systems Biology grant

18. Conclusion #2  Of 56 burn patients analyzed:  62% had TF and 100% had FXIa at at least one time-point.  TF activity was observed in 21% and FXIa in 90% time-point samples.

19. Study #3 (In collaboration with Dr. K. Freeman from FAHC, Burlington VT)  Multiple time-point plasma samples from 66 patients (no burn patients; 187 time-points)  56% of them had TF and 94% had FXIa at at least one time-point.  TF activity was observed in 30% and FXIa in 79% time-point samples. Thrombin Generation assay was used for the quantitation of FXIa Supported by Systems Biology grant

20. Final Conclusions  The majority of trauma patients have circulating FXIa, which correlates with trauma severity.  FXIa concentration over observation time varies in a wide range.  The frequency of FXIa in burn patients is higher than in other trauma patient categories  FXIa could be a potential marker of trauma severity