Increased urokinase and consumption of α2 -antiplasmin as an explanation for the loss of benefit of tranexamic acid after treatment delay

Abstract

Essentials Delayed treatment with tranexamic acid results in loss of efficacy and poor outcomes. Increasing urokinase activity may account for adverse effects of late tranexamic acid treatment. Urokinase + tranexamic acid produces plasmin in plasma or blood and disrupts clotting. α₂ -Antiplasmin consumption with ongoing fibrinolysis increases plasmin-induced coagulopathy. SUMMARY: Background Tranexamic acid (TXA) is an effective antifibrinolytic agent with a proven safety record. However, large clinical trials show TXA becomes ineffective or harmful if treatment is delayed beyond 3 h. The mechanism is unknown but urokinase plasminogen activator (uPA) has been implicated. Methods Inhibitory mechanisms of TXA were explored in a variety of clot lysis systems using plasma and whole blood. Lysis by tissue plasminogen activator (tPA), uPA and plasmin were investigated. Coagulopathy was investigated using ROTEM and activated partial thromboplastin time (APTT). Results IC₅₀ values for antifibrinolytic activity of TXA varied from < 10 to > 1000 μmol L^-1 depending on the system, but good fibrin protection was observed in the presence of tPA, uPA and plasmin. However, in plasma or blood, active plasmin was generated by TXA + uPA (but not tPA) and coagulopathy developed leading to no or poor clot formation. The extent of coagulopathy was sensitive to available α₂ -antiplasmin. No clot formed with plasma containing 40% normal α₂ -antiplasmin after short incubation with TXA + uPA. Adding purified α₂ -antiplasmin progressively restored clotting. Plasmin could be inhibited by aprotinin, IC₅₀ = 530 nmol L^-1 , in plasma. Conclusions Tranexamic acid protects fibrin but stimulates uPA activity and slows inhibition of plasmin by α₂ -antiplasmin. Plasmin proteolytic activity digests fibrinogen and disrupts coagulation, exacerbated when α₂ -antiplasmin is consumed by ongoing fibrinolysis. Additional direct inhibition of plasmin by aprotinin may prevent development of coagulopathy and extend the useful time window of TXA treatment.

Keywords: alpha-2-antiplasmin; fibrinolysis; hemorrhage; tranexamic acid; urokinase type plasminogen activator.

Figure 1

Plasma clot lysis with tissue plasminogen activator (tPA) or plasmin and inhibition by tranexamic acid (TXA). Plasma clots contained 2.5 nmol L⁻¹ tPA or plasmin as shown with a range of TXA concentrations, and times to 50% lysis were determined. Panel A shows representative raw data for clot lysis time‐courses for clots containing 2.5 nmol L⁻¹ tPA. Panel B shows analysis of lysis profiles as extension of time to 50% lysis at each TXA concentration used to calculate an IC₅₀ = 150 μmol L⁻¹ (± 20 μmol L⁻¹ as the standard error [SE] of the fit). Means and standard deviations [SDs] of duplicates are shown. Panel C summarizes lysis extension results as single‐point estimates for clot lysis curves where plasmin has been incorporated into the clots in place of tPA at the concentrations shown. Estimates of IC₅₀ values ranged from 4 to 41 μmol L⁻¹ TXA (SE for fitting was 0.3 μmol L⁻¹ at the lowest K_D up to 18 μmol L⁻¹ at the higher IC₅₀ values).

Figure 2

Inhibition by tranexamic acid (TXA) of euglobulin clot lysis with single chain urokinase plasminogen activator (scuPA) or scuPA and tissue plasminogen activator (tPA) or plasmin. Clots formed from euglobulin contained plasminogen activator or plasmin and a range of TXA concentrations, and times to 50% lysis were determined. Panel A shows representative raw data for clots containing 5 nmol L⁻¹ scuPA and Panel B clots contain 5 nmol L⁻¹ scuPA + 0.6 nmol L⁻¹ tPA. Panel C shows single‐point estimates of extension of time to 50% lysis with increasing TXA concentrations at the plasmin concentrations shown. K_D estimates from these data were in the range 600–1900 μmol L⁻¹ (standard error [SE] of fitting 31–90 μmol L⁻¹).

Figure 3

Clot lysis in the halo format by tissue plasminogen activator (tPA) with blood or plasma showing inhibition by tranexamic acid (TXA). Panel A shows representative blood clot lysis time‐courses for a series of reactions that included 2.5 nmol L⁻¹ tPA and a range of TXA as shown. Panel B is the same arrangements, except clots were made from plasma containing 0.15 mg mL⁻¹ fluorescent fibrinogen. Panels C and D are the corresponding plots for lengthening of 50% lysis time with tPA (circles) or urokinase plasminogen activator (uPA) (squares) at each TXA with concentration in blood (C) and plasma (D), shown as means ± SD of triplicate wells.

Figure 4

Generation of plasmin from clots incubated with urokinase plasminogen activator (uPA) or tissue plasminogen activator (tPA) and tranexamic acid (TXA). Panel A used the plasma clot halo system where plasminogen activator, TXA and plasmin chromogenic substrate S‐2251 were added to preformed clots. Initial rates of plasmin generation were calculated from plots of absorbance vs. time squared 22 and the points shown are means ± standard deviations (SDs) of duplicate wells. Panel B shows similar data for plasma clots in the microtiter plate format used in Fig. 1, with 5 nmol L⁻¹ uPA incorporated into the clots (or 10 nmol L⁻¹ tPA as shown). Chromogenic substrate S‐2251 containing a range of TXA and aprotinin as shown was added to preformed clots (all points show plasminogen activation rates from single wells). The insert shows inhibition of peak plasmin generation by increasing aprotinin concentrations, with IC₅₀ = 530 ± 119 nmol L⁻¹ (± standard error [SE] of fitting).

Figure 5

Clotting is disrupted by urokinase plasminogen activator (uPA) and tranexamic acid (TXA) as assessed by ROTEM or activated partial thromboplastin time (APTT) methods. Panel A shows effects on ROTEM clot formation time following preincubation of blood with 2.5 nmol L⁻¹ tissue plasminogen activator (tPA) or 5 nmol L⁻¹ uPA with 400 μmol L⁻¹ TXA (all single‐point estimates). Panel B shows similar results for APTT with plasma. In both methods, pre‐incubation of up to 90 min with uPA + TXA extended clotting times, but only after many hours of pre‐incubation with tPA + TXA (similar to no additions). Panel C summarizes a series of pre‐incubation experiments using plasma with reduced α₂‐antiplasmin with various additions as shown. uPA or TXA alone had no effect but together abolished clotting after 25 min of pre‐incubation when α₂‐antiplasmin was 40% of normal. The sensitivity of this plasma to uPA + TXA was corrected by replacement of α₂‐antiplasmin. All APTT results shown as means of duplicate determinations ± range.

Figure 6

Digestion of fibrinogen by mixtures of urokinase plasminogen activator (uPA) and tranexamic acid (TXA). Panels A and B show the effects of activator, 5 nmol L⁻¹ uPA or 2.5 nmol L⁻¹ tissue plasminogen activator (tPA) with or without the addition of 400 μmol L⁻¹ TXA on fibrinogen in a purified system. Panel A is a trace showing absorbance changes from S‐2251 hydrolysis as a result of plasmin generation, from 200 nmol L⁻¹ plasminogen with 1 mg mL⁻¹ fibrinogen, in the presence of uPA + TXA, but not with tPA. The corresponding Coomassie stained SDS PAGE of reaction mixtures after 30 min illustrates that the generated plasmin is able to digest fibrinogen in the presence of TXA (the α, β, and γ chains of fibrinogen are annotated). Panels C and D replicate these results using V.I. plasma (with 0.4 U mL⁻¹ of α₂‐antiplasmin) under the same conditions used for Fig. 5(C). Plasmin was rapidly generated in the presence of 5 nmol L⁻¹ uPA and 400 μmol L⁻¹ TXA, as shown in panel C. This plasmin was able to substantially and rapidly destroy the normal level of 3.2 mg mL⁻¹ fibrinogen present in this plasma sample, as shown by the western blot using anti‐fibrinogen antibodies in panel D.