Application of a plasmin generation assay to define pharmacodynamic effects of tranexamic acid in women undergoing cesarean delivery

Abstract

Essentials Tranexamic acid (TXA) is an antifibrinolytic drug used to reduce bleeding. Assaying plasmin generation (PG) in plasma detects clinically relevant TXA levels in vitro and ex vivo. 3.1-16.2 µg/mL TXA half-maximally inhibits PG in plasma from women undergoing cesarean delivery. PG velocity shows the strongest dose-relationship at low TXA concentrations (≤10 µg/mL). ABSTRACT: Background Tranexamic acid (TXA) is used to reduce bleeding. TXA inhibits plasmin(ogen) binding to fibrin and reduces fibrinolysis. TXA antifibrinolytic activity is typically measured by clot lysis assays; however, effects on plasmin generation (PG) are unclear due to a lack of tools to measure PG in plasma. Aims Develop an assay to measure PG kinetics in human plasma. Determine effects of TXA on PG and compare with fibrinolysis measured by rotational thromboelastometry (ROTEM). Methods We characterized effects of plasminogen, tissue plasminogen activator, fibrinogen, and α₂ -antiplasmin on PG in vitro. We also studied effects of TXA on PG in plasma from 30 pregnant women administered intravenous TXA (5, 10, or 15 mg/kg) during cesarean delivery. PG was measured by calibrated fluorescence. PG parameters were compared with TXA measured by mass spectrometry and ROTEM of whole blood. Results The PG assay is specific for plasmin and sensitive to tissue plasminogen activator, fibrin(ogen), and α₂ -antiplasmin. Addition of TXA to plasma in vitro dose dependently prolonged the clot lysis time and delayed and reduced PG. For all doses of TXA administered intravenously, the PG assay detected delayed time-to-peak (≤3 hours) and reduced the velocity, peak, and endogenous plasmin potential (≤24 hours) in plasma samples obtained after infusion. The PG time-to-peak, velocity, and peak correlated significantly with TXA concentration and showed less variability than the ROTEM lysis index at 30 minutes or maximum lysis. Conclusions The PG assay detects pharmacologically relevant concentrations of TXA administered in vitro and in vivo, and demonstrates TXA-mediated inhibition of PG in women undergoing cesarean delivery.

Keywords: fibrin; fibrinolysis; plasmin; pregnancy; tranexamic acid.

FIGURE 1

The PG assay is specific for plasmin and sensitive to rtPA, fibrin(ogen), and α₂-antiplasmin in human plasma. A, Plasma was mixed with tissue factor (TF), phospholipids, and recombinant tissue plasminogen activator (rtPA) (reaction wells), or α₂-macroglobulin/plasmin complex (calibrator wells). Reactions were initiated by automatically dispensing fluorogenic substrate and CaCl₂ to each well. B, Fluorescence was monitored over time, and a PG curve was derived mathematically, yielding PG parameters: lag time, time to peak (TtPeak), velocity, peak, and endogenous plasmin potential (EPP). C, PG in normal pooled plasma mixed with plasminogen-deficient plasma. D, PGin normal pooled plasma; reactions were triggered with the indicated concentrations of rtPA. E, PG in normal pooled plasma mixed with fibrinogen-deficient plasma. F, PG in normal pooled plasma mixed with α₂-antiplasmin-deficient plasma. Panels C-F show representative curves from three independent experiments.

FIGURE 2

TXA inhibits PG in vitro. Normal pooled plasma was diluted 1:2 and clotted in the presence of tissue factor, phospholipids, rtPA, TXA, and CaCl₂. A, Clot formation and fibrinolysis measured by turbidity, representative curves. B, Clot lysis time from turbidity assays. C, PG, representative curves. PG parameters: (D) lag time, (E) TtPeak, (F) velocity, (G) peak, and (H) EPP. Dots show means and standard deviations from four to five independent experiments. *P < .05, **P < .01, ***P < .001, and ****P < .0001

FIGURE 3

TXA inhibits PG ex vivo. PG was measured in plasmas from women who received (A-E) 5, (F-J) 10, or (K-O) 15 mg/kg TXA. Bars indicate medians, each dot represents a separate subject. *P < .05, **P < .01, ***P < .001, and ****P < .0001

FIGURE 4

PG parameters correlate with plasma TXA concentration. TXA concentrations measured by mass spectrometry were correlated with (A) PG TtPeak, (B) PG velocity, (C) PG peak, (D) PG EPP, (E) ROTEM LI30, and (F) ROTEM ML. The TXA concentration-effect relationship for each parameter was characterized using a population modeling approach. Each dot represents a separate sample.