A Novel Fibrinolysis Resistance Capacity Assay Can Detect Fibrinolytic Phenotypes in Trauma Patients

Abstract

Background To evaluate residual fibrinolysis resistance activity (FRA) in plasma, a detergent-modified plasma clot lysis assay time (dPCLT) was established in which α2-antiplasmin (A2AP) and plasminogen activator inhibitor type 1 (PAI-1) are inactivated without impacting protease activity. We applied this novel assay to severely injured trauma patients’ plasma.

Material and Methods Tissue-type plasminogen activator (tPA)-induced plasma clot lysis assays were conducted after detergents- (dPCLT) or vehicle- (sPCLT) treatment, and time to 50% clot lysis was measured (“transition midpoint”, T _m). Residual FRA was then calculated as ([sPCLT T _m] - [dPCLT T _m]/[sPCLT T _m]) x100% = Δ T_m PCLT (%). Assay results were compared to rapid thromboelastography (TEG) LY30, tPA TEG LY30, and plasma fibrinolysis biomarkers in polytrauma patients’ plasma (N=43).

Results Δ T_m PCLT(%) in normal plasma (N=5) was 63.0 ± 8.3 whereas in A2AP-depleted plasma was -19.1 ± 1.3%, Plasmin-antiplasmin (PAP) complex increased after complete lysis of sPCLT, whereas that in dPCLT was negligible in normal plasma. In trauma plasma, significant correlations between Δ T_m PCLT and active PAI-1 (r = 0.85, p<0.0001), PAP complex (r = -0.85, p<0.0001), free A2AP (r = 0.66, p<0.0001), total A2AP levels (r = 0.52, p=0.001) and tPA TEG LY30 (r = -0.85, p<0.0001) were found. dPCLT in hyperfibrinolysis patients diagnosed by tPA TEG was significantly shorter than those with low fibrinolysis [10.2 ± 6.4 minutes versus 20.2 ± 2.1 minutes, p=0.0006].

Conclusion Hyperfibrinolysis after trauma is significantly related to exhaustion of FRA, and our novel assay appears to quickly assess this state and may be a useful clinical diagnostic after additional validation.

Key Points

• We established a new clot lysis assay to measure residual fibrinolysis resistance activity after inactivating PAI-1 and A2AP by detergents without impacting protease function.

• This novel clot lysis assay unmasked the mechanism of hyperfibrinolysis after trauma as exhaustion of fibrinolysis resistance activity, and appeared useful in quickly identifying these patients.

Keywords

fibrinolysis inhibitors - fibrinolytic disorders - surgery - hypercoagulability

• References

• 1 Stafford JL. Fibrinolysis and intrinsic haemostasis. Br Med Bull 1964; 20: 179-184
• 2 Cesarman-Maus G, Hajjar KA. Molecular mechanisms of fibrinolysis. Br J Haematol 2005; 129 (03) 307-321
• 3 Fraser SR, Booth NA, Mutch NJ. The antifibrinolytic function of factor XIII is exclusively expressed through α₂-antiplasmin cross-linking. Blood 2011; 117 (23) 6371-6374
• 4 Collen D. Natural inhibitors of fibrinolysis. J Clin Pathol Suppl (R Coll Pathol) 1980; 14: 24-30
• 5 Foley JH, Cook PF, Nesheim ME. Kinetics of activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-catalyzed cleavage of C-terminal lysine residues of fibrin degradation products and removal of plasminogen-binding sites. J Biol Chem 2011; 286 (22) 19280-19286
• 6 Moore HB, Barrett CD, Moore EE, Pieracci FM, Sauaia A. Differentiating pathologic from physiologic fibrinolysis: not as simple as conventional thrombelastography. J Am Coll Surg 2024; 239 (01) 30-41
• 7 Moore HB, Moore EE, Liras IN. et al. Acute fibrinolysis shutdown after injury occurs frequently and increases mortality: a multicenter evaluation of 2,540 severely injured patients. J Am Coll Surg 2016; 222 (04) 347-355
• 8 Moore HB, Moore EE, Chapman MP. et al. Viscoelastic tissue plasminogen activator challenge predicts massive transfusion in 15 minutes. J Am Coll Surg 2017; 225 (01) 138-147
• 9 Callcut RA, Kornblith LZ, Conroy AS. et al; Western Trauma Association Multicenter Study Group. The why and how our trauma patients die: a prospective Multicenter Western Trauma Association study. J Trauma Acute Care Surg 2019; 86 (05) 864-870
• 10 Shakur H, Roberts I, Bautista R. et al; CRASH-2 trial collaborators. Effects of tranexamic acid on death, vascular occlusive events, and blood transfusion in trauma patients with significant haemorrhage (CRASH-2): a randomised, placebo-controlled trial. Lancet 2010; 376 (9734) 23-32
• 11 Guyette FX, Brown JB, Zenati MS. et al; STAAMP Study Group. Tranexamic acid during prehospital transport in patients at risk for hemorrhage after injury: a double-blind, placebo-controlled, randomized clinical trial. JAMA Surg 2020; 156 (01) 11-20
• 12 Li SR, Guyette F, Brown J. et al. Early prehospital tranexamic acid following injury is associated with a 30-day survival benefit: a secondary analysis of a randomized clinical trial. Ann Surg 2021; 274 (03) 419-426
• 13 Roberts I, Shakur H, Afolabi A. et al; CRASH-2 collaborators. The importance of early treatment with tranexamic acid in bleeding trauma patients: an exploratory analysis of the CRASH-2 randomised controlled trial. Lancet 2011; 377 (9771) 1096-1101 , 1101.e1–1101.e2
• 14 Moore HB, Moore EE, Gonzalez E. et al. Hyperfibrinolysis, physiologic fibrinolysis, and fibrinolysis shutdown: the spectrum of postinjury fibrinolysis and relevance to antifibrinolytic therapy. J Trauma Acute Care Surg 2014; 77 (06) 811-817 , discussion 817
• 15 Moore HB, Moore EE, Chapman MP. et al. Does tranexamic acid improve clot strength in severely injured patients who have elevated fibrin degradation products and low fibrinolytic activity, measured by thrombelastography?. J Am Coll Surg 2019; 229 (01) 92-101
• 16 Cardenas JC, Matijevic N, Baer LA, Holcomb JB, Cotton BA, Wade CE. Elevated tissue plasminogen activator and reduced plasminogen activator inhibitor promote hyperfibrinolysis in trauma patients. Shock 2014; 41 (06) 514-521
• 17 Chapman MP, Moore EE, Moore HB. et al. Overwhelming tPA release, not PAI-1 degradation, is responsible for hyperfibrinolysis in severely injured trauma patients. J Trauma Acute Care Surg 2016; 80 (01) 16-23 , discussion 23–25
• 18 Barrett CD, Maginot ER, Kemp KM. Fibrinolytic and functional fibrinogen diagnostics in trauma: a window of opportunity or a bridge to nowhere?. J Thromb Haemost 2023; 21 (10) 2705-2707
• 19 Moore EE, Moore HB, Gonzalez E, Sauaia A, Banerjee A, Silliman CC. Rationale for the selective administration of tranexamic acid to inhibit fibrinolysis in the severely injured patient. Transfusion 2016; 56 (Suppl. 02) S110-S114
• 20 Barrett CD, Moore HB, Vigneshwar N. et al. Plasmin thrombelastography rapidly identifies trauma patients at risk for massive transfusion, mortality, and hyperfibrinolysis: a diagnostic tool to resolve an international debate on tranexamic acid?. J Trauma Acute Care Surg 2020; 89 (06) 991-998
• 21 Urano T, Castellino FJ, Ihara H. et al. Activated protein C attenuates coagulation-associated over-expression of fibrinolytic activity by suppressing the thrombin-dependent inactivation of PAI-1. J Thromb Haemost 2003; 1 (12) 2615-2620
• 22 Suzuki Y, Sano H, Mochizuki L, Honkura N, Urano T. Activated platelet-based inhibition of fibrinolysis via thrombin-activatable fibrinolysis inhibitor activation system. Blood Adv 2020; 4 (21) 5501-5511
• 23 Bouton MC, Geiger M, Sheffield WP. et al. The under-appreciated world of the serpin family of serine proteinase inhibitors. EMBO Mol Med 2023; 15 (06) e17144
• 24 Urano T, Strandberg L, Johansson LB, Ny T. A substrate-like form of plasminogen-activator-inhibitor type 1. Conversions between different forms by sodium dodecyl sulphate. Eur J Biochem 1992; 209 (03) 985-992
• 25 Yan D, Urano T, Takada Y, Takada A. Dissociation of alpha 2-plasmin-inhibitor-plasmin complex and regeneration of plasmin activity by SDS treatment. Thromb Res 1993; 69 (06) 491-499
• 26 Moore HB, Moore EE, Gonzalez E. et al. Plasma is the physiologic buffer of tissue plasminogen activator-mediated fibrinolysis: rationale for plasma-first resuscitation after life-threatening hemorrhage. J Am Coll Surg 2015; 220 (05) 872-879
• 27 Moore HB, Moore EE, Gonzalez E. et al. Hemolysis exacerbates hyperfibrinolysis, whereas platelolysis shuts down fibrinolysis: evolving concepts of the spectrum of fibrinolysis in response to severe injury. Shock 2015; 43 (01) 39-46
• 28 Berkenpas MB, Lawrence DA, Ginsburg D. Molecular evolution of plasminogen activator inhibitor-1 functional stability. EMBO J 1995; 14 (13) 2969-2977
• 29 Barrett CD, Moore HB, Banerjee A, Silliman CC, Moore EE, Yaffe MB. Human neutrophil elastase mediates fibrinolysis shutdown through competitive degradation of plasminogen and generation of angiostatin. J Trauma Acute Care Surg 2017; 83 (06) 1053-1061
• 30 Urano T, Sano Y, Suzuki Y. et al. Evaluation of thrombomodulin/thrombin activatable fibrinolysis inhibitor function in plasma using tissue-type plasminogen activator-induced plasma clot lysis time. Res Pract Thromb Haemost 2024; 8 (04) 102463
• 31 Hardaway RM. The significance of coagulative and thrombotic changes after haemorrhage and injury. J Clin Pathol Suppl (R Coll Pathol) 1970; 4: 110-120
• 32 Gruen RL, Mitra B, Bernard SA. et al; PATCH-Trauma Investigators and the ANZICS Clinical Trials Group. Prehospital tranexamic acid for severe trauma. N Engl J Med 2023; 389 (02) 127-136
• 33 Spinella PC, Bochicchio K, Thomas KA. et al. The risk of thromboembolic events with early intravenous 2- and 4-g bolus dosing of tranexamic acid compared to placebo in patients with severe traumatic bleeding: a secondary analysis of a randomized, double-blind, placebo-controlled, single-center trial. Transfusion 2022; 62 (Suppl. 01) S139-S150
• 34 Collaborators H-IT. HALT-IT Trial Collaborators. Effects of a high-dose 24-h infusion of tranexamic acid on death and thromboembolic events in patients with acute gastrointestinal bleeding (HALT-IT): an international randomised, double-blind, placebo-controlled trial. Lancet 2020; 395 (10241): 1927-1936
• 35 Moore HB, Moore EE, Huebner BR. et al. Tranexamic acid is associated with increased mortality in patients with physiological fibrinolysis. J Surg Res 2017; 220: 438-443
• 36 Myers SP, Kutcher ME, Rosengart MR. et al. Tranexamic acid administration is associated with an increased risk of posttraumatic venous thromboembolism. J Trauma Acute Care Surg 2019; 86 (01) 20-27
• 37 Barrett CD, Neal MD, Schoenecker JG, Medcalf RL, Myles PS. Tranexamic acid in trauma: after 3 hours from injury, when is it safe and effective to use again?. Transfusion 2024; 64 (Suppl. 02) S11-S13