FAST: a 5-minute assay for measuring fibrinolytic activation

In this issue of Blood Advances, Ilich et al¹ address a critical unmet need for the rapid assessment of endogenous fibrinolytic activation in patients with severe bleeding. They report a novel plasmin generation assay—the Fibrinolytic Activity Screening Test (FAST)—that enables near–real-time measurement of plasmin-generating capacity in plasma.¹ Plasmin, the principal effector enzyme of fibrinolysis, is generated from its zymogen, plasminogen, through a tightly regulated activation system coordinated in space and time. Dysregulated plasmin activity drives hyperfibrinolysis and is a major contributor to uncontrolled hemorrhage.

FAST quantifies plasmin activity as the percentage change in fluorescence signal over 5 minutes relative to baseline. Using this assay, Ilich et al compared plasma from patients with postpartum hemorrhage (PPH) with and without acute obstetric coagulopathy (AOC), a condition associated with poor maternal and fetal outcomes.¹ Previous studies showed that plasma from patients with AOC exhibited a fourfold increase in plasmin-generating capacity compared with severe PPH without AOC (P < .0002), accompanied by extensive fibrinogen degradation and a 36-fold elevation in D-dimer levels.²

Current diagnostics for AOC rely on laboratory measurement of plasmin–antiplasmin (PAP) complexes, limiting their utility for early clinical decision-making. FAST addresses this gap: healthy pregnant controls showed FAST activity (∼111%) comparable to nonpregnant controls, whereas AOC samples displayed markedly elevated activity (mean, 263.6%) that correlated strongly with PAP complexes and D-dimer levels.¹ Together, these findings demonstrate FAST’s capacity to detect severe fibrinolytic activation in near real-time.

FAST is founded on 3 key principles. First, poly-lysine acts as a surrogate for fibrin-associated lysine residues, enabling efficient formation of tissue plasminogen activator (tPA)–plasminogen complexes in the absence of clot formation.³ Second, low concentrations of sodium dodecyl sulfate and Triton X-100 functionally suppress endogenous inhibitors of tPA and plasmin—including plasminogen activator inhibitor-1 (PAI-1) and α2-antiplasmin—thereby unmasking intrinsic plasmin–generating capacity.⁴ Third, tPA activity is enhanced at temperatures above 37°C and under mildly alkaline conditions, which together optimize plasminogen activation.^5,6

Operationally, FAST employs a straightforward workflow: 1 part plasma is mixed with 2 parts reaction premix, incubated at 42°C for 5 minutes, and plasmin generation is quantified (see figure). Under these defined conditions, the measured signal predominantly reflects endogenous tPA–mediated plasminogen activation.¹

Schematic of the FAST plasmin activity assay. One part PPP is combined with 2 parts reaction premix containing 0.2% sodium dodecyl sulfate, 0.2% Triton X-100, Boc-Glu-Lys-Lys-AMC substrate (final concentration 600μM), and 0.1% poly-L-lysine in HEPES-buffered saline (pH 7.5). Plasmin activity is recorded in a pre‑heated plate reader (Synergy H1, BioTek, USA) at 42 °C for 5 minutes (excitation/emission: 360/460 nm), with readings taken every minute. FAST activity is expressed as the percentage change in fluorescence from baseline at the 5-minute time point. This figure was created with BioRender.com. Law, R.H. (2025) https://BioRender.com/8iqi6hh. PPP, platelet-poor plasma; RFU, relative fluorescence units.

FAST is highly sensitive to physiological variation. In healthy controls, activity ranged from 91.4% to 119.5%, with modestly higher values observed in women than in men.¹ The assay also detected venostasis-induced fibrinolytic activation, with posttourniquet samples exhibiting significantly increased activity compared with pretourniquet samples.¹

Importantly, FAST activity was unaffected by therapeutic concentrations of tranexamic acid (10-1000 μg/mL), a key advantage given its widespread first-line use in bleeding management.⁷ It was similarly robust in the presence of clinically relevant concentrations of hirudin, dabigatran, fondaparinux, rivaroxaban, and unfractionated heparin,¹ supporting its applicability in supporting its robustness in plasma samples containing therapeutic anticoagulants. FAST activity correlated strongly with PAP complexes and D-dimer levels,¹ but not with rotational thromboelastometry (ROTEM) parameters, consistent with prior evidence that global coagulation assays lack sensitivity to early hyperfibrinolysis and fibrinogen depletion.8, 9, 10

Despite its strengths, FAST has several limitations that merit consideration. The assay does not resolve the individual contributions of endogenous serine protease inhibitors—such as α2-antiplasmin or plasminogen activator inhibitor-1—nor does it account for other coagulation components, including fibrinogen or factor V, that influence overall fibrinolytic balance. In its current implementation, FAST is performed on plasma rather than whole blood, making plasma separation the principal time-limiting preanalytical step and highlighting whole-blood adaptation as an important future direction.¹ Preanalytical variables, including anticoagulant selection and freeze–thaw cycles, can also affect assay performance and therefore require careful standardization of sample handling.¹ Finally, because localized endothelial release of tissue plasminogen activator may exceed systemic levels, FAST may underestimate fibrinolytic activation in clinical contexts characterized by pronounced spatial compartmentalization.¹

FAST has immediate potential for the diagnosis of AOC and for identifying patients who may benefit from plasmin-inhibitory therapy. Beyond obstetric bleeding, the assay may be applied to investigate fibrinolytic activation in trauma, severe burns, viral hemorrhagic infections (eg, Ebola, Lassa, dengue, and yellow fever), and cancer-associated fibrinolysis. In these contexts, FAST could help delineate the contribution of dysregulated fibrinolysis to disease progression and support more informed therapeutic stratification.

Fibrinolytic activation evolves rapidly during acute bleeding, making timely detection essential. FAST offers a rapid and practical approach for assessing plasmin-mediated fibrinolytic activation, supporting near–real-time clinical decision-making in high-risk settings such as AOC. Future development should prioritize adaptation to whole blood and stabilization of reagent formulations to facilitate bedside implementation. With further clinical validation, FAST has the potential to become a valuable tool for investigating pathological fibrinolysis and guiding targeted therapeutic interventions across a broad range of bleeding and thromboinflammatory disorders.

Conflict-of-interest disclosure: The authors declare no competing financial interests.