Keywords
fibrinolysis - biomarkers - standardization - thrombolytics - antifibrinolytics
Introduction
A simplified outline of fibrinolysis is presented in [Fig. 1] and more detailed reviews can be found elsewhere.[1]
[2] By necessity, in response to vascular damage, coagulation must be rapid to reduce
the dangers of bleeding and prevent entry of pathogenic microorganisms. Subsequently,
fibrinolysis takes place slowly under normal circumstances, over hours and days as
vessels are repaired. These considerations explain why there are many relatively simple,
rapid, well-standardized tests to measure blood clotting, which can identify defects
in coagulation pathways. However, fibrinolysis is more difficult to measure for diagnostic
purposes and methods are more cumbersome, so the role of fibrinolysis in the hemostatic
balance may be underestimated.
Fig. 1 Outline of the fibrinolysis system. The pathway of fibrin formation and degradation
is shown by heavy arrows. Fibrin is a substrate (surface) for reactions and a substrate (target for enzymes)
for plasmin.[1] Fibrin degradation products (FDP) are heterogeneous in size[88] and expose binding sites for D-dimer antibodies. Enzymes are in ovals and include
the plasminogen activators, tissue plasminogen activator (tPA) and urokinase plasminogen
activator (uPA). tPA activity is stimulated by binding to fibrin, where the finger
domain is dominant,[11]
[89] whereas uPA is generated from an inactive zymogen scuPA (not shown) by the action
of plasmin. Serpin inhibitors, PAI-1 and α2-antiplasmin (α2-AP) form inactive complexes to reduce fibrinolysis. Thrombin-activatable fibrinolysis
inhibitor (TAFI, also known as procarboxypeptidase U, or CBP2 gene product) is activated
by thrombin (and plasmin, not shown), to the active form (TAFIa). This enzyme modifies
fibrin (shown as Fibrin′) to remove C-terminal lysines, which is less effective at
binding plasminogen and plasmin and more resistant to lysis. TAFIa is thermally unstable
and degrades to an inactive form, shown as TAFIa′. Other components that are involved
include α2-macroglobulin, a broad specificity inhibitor and thrombomodulin which has a role
in regulating the activation of TAFI.[90] Additional factors that can impair fibrinolysis include variants of fibrinogen such
as γ′-fibrinogen which affects clot architecture and fibrinogen-binding sites to make
more resistant clots[91] and FXIII which creates a more resistant clot by cross-linking fibrin chains and
α2-AP to fibrin.[92]
[93] The incorporation of cells into clots can also delay fibrinolysis. Platelets cause
clot retraction and release PAI-1[94]; and red blood cells can interact with fibrin,[95] and also become compressed during clot retraction to form an impermeable barrier
which delays clot lysis.[96] PAI-1, plasminogen activator inhibitor 1.
Fibrinolysis assays are needed to study antifibrinolytic therapy and also in the development
and quality control of thrombolytic drugs. Diverse approaches are available to assess
fibrinolysis in healthy or sick populations to identify factors that may be involved
in regulation or dysregulation, and hyperfibrinolysis or fibrinolysis resistance.
The National Institute for Biological Standards and Control (NIBSC) is a World Health
Organization (WHO) collaborating center with the responsibility to generate, store
and distribute biological standards. A major part of our portfolio covers diagnostic
and drug-related standards in hemostasis, including fibrinolysis[3] and reviews on the ways in which our biological standards can be used have been
published elsewhere.[4]
[5]
Assays for Thrombolytic Proteins
Assays for Thrombolytic Proteins
Thrombolytics such as tissue plasminogen activator (tPA) or urokinase plasminogen
activator (uPA) are serine proteases that transform plasminogen into plasmin. Microbial
plasminogen binding and activating proteins such as streptokinase and staphylokinase
have no intrinsic protease activity but in practice, experimentally their reaction
kinetics look like other plasminogen activators and they can be analyzed in the same
way (although the details of the kinetic mechanisms may be complicated[6]). In practice, investigations on enzyme mechanism and regulation, or determination
of specific activity or concentration will be performed in vitro in purified systems
of proteins, or in plasma-based systems and can be optimized over a chosen thrombolytic
enzyme range to give the most robust results. Activities of fibrinolytic proteins
are often determined relative to the WHO International Standard as a primary calibrator.[3] Examples are available in publications of the development of fibrinolysis as per
WHO International Standards (e.g., Locke et al[7]).
The simplest methods for following proteolytic activity involve optical monitoring
of amidolytic substrates made of peptides linked to a chromophore or fluorophore.
These types of substrates are not so useful when used directly on an enzyme of interest,
but are valuable to study linked reactions that generate plasmin, for example. Active-site
titration of serine proteases is a subgroup of chromogenic/fluorogenic assays and
is useful to establish molar concentrations of active enzymes, including thrombolytic
enzymes and thrombin.[8] Early fibrin-based methods for measuring plasminogen activator activity used fibrin
plates, but have been superseded by microtiter plate-based methods.[9] This approach can be adapted to internal lysis (in which plasminogen activator is
mixed with fibrinogen, plasminogen and thrombin to form a clot that is subsequently
lysed evenly throughout), or external lysis (where activator is added to the top of
a preformed clot). Internal lysis is related to normal hemostasis, while superficially
added activator more closely reflects the situation during thrombolytic therapy. It
is possible to combine fibrin-based methods and chromogenic substrate-linked measurement
of plasminogen activation to precisely follow plasmin generation in the presence of
fibrin.[10]
[11] In this way, rates of plasmin generation in SI units (e.g., pM/s) for tPA, uPA and
streptokinase can be compared in the same format. Comparison of WHO assigned international
units (IU) for these activators is not so useful as the units are unrelated. The long-established
European Pharmacopoeia method for assaying alteplase (tPA) activity,[12] in which clot lysis time is determined by passage of a ball through the clot in
a tube or release of trapped bubbles from the fibrin network, is simple and reliable.
More recently, the “Halo” method has been published that uses a small volume of whole
blood, clotted in a ring or halo around the edge of a microtiter-plate well.[13] Lysis is followed by monitoring increasing absorbance as the clot breaks down and
products released into solution.
Although it is relatively simple to generate reproducible time courses of data to
study the activity of thrombolytic enzymes using these methods, the analysis of the
resulting data is the next challenge. It is common in fibrinolysis assays to report
lysis times, usually as time to 50% lysis, but how this is calculated is not always
explained. Alternatively, zymogen activation rates determined with chromogenic substrates
require the use of time squared plots, which can be tedious to generate and analyze.
To standardize calculations of lysis times and zymogen activation rates and improve
reproducibility, several online apps have been developed and published in association
with ISTH/SSC (International Society on Thrombosis and Haemostasis/Scientific and
Standardization Committee) Subcommittee on Fibrinolysis.[14]
[15] These apps are freely available and run in a computer browser without downloading
any software (see also Longstaff[16] for summaries, links to apps and detailed instructions).
Diagnostic Methods
Functional Tests
Functional tests begin with the problem mentioned above that fibrinolysis without
any stimulation is slow. To speed up the process, it is common to either add tPA to
plasma to stimulate the generation of plasmin, or to remove inhibitors, by for example
preparing the euglobulin fraction from plasma. A long-established functional method
for measuring fibrinolysis in subject samples is to determine euglobulin clot lysis
times (ECLTs), and this approach has been reviewed previously.[5]
[17]
[18] The general approach is time-consuming and difficult to automate. The euglobulin
fraction is reported to have a greater than 90% reduction in α2-antiplasmin, but there is also significant depletion of plasminogen activator inhibitor
1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI).[19] It is observed that the ECLT is strongly influenced by PAI-1 (and free tPA) concentrations.
Recently an updated method has been published[20] where samples received added fibrinogen and ovalbumin to increase the clot turbidity.
These authors also explored a fibrinolysis resistance test using added tPA which identified
samples with high free active PAI-1, but also showed some sensitivity of lysis times
to TAFI levels.
When normal plasma is used to investigate fibrinolysis, it is common to add significant
amounts of extraneous tPA (from 200 to 700 ng/mL, see Table 2 in Longstaff[18]). To generate a clot, CaCl2 is added as a minimum, and often thrombin and/or tissue factor (TF) with phospholipids
may also be used. Attempts have been made to establish a standardized method for this
procedure to improve reproducibility.[21] A commonly used output is 50% lysis time where lysis is followed optically, and
analysis can be facilitated by online apps.[14] A thorough review of data from clot lysis studies has been presented to explore
the relationship of fibrinolytic potential and risk for arterial and venous thrombosis.[22] Conclusions from many studies are not always strong or consistent in identifying
molecular risk factors in different populations. However, an important observation
is that hypofibrinolysis, especially in combination with hypercoagulability, can constitute
an increased thrombosis risk.
An interesting development in this area is methods to simultaneously measure generation
of both thrombin and plasmin during plasma clotting and lysis.[23] However, the methods proposed so far (reviewed in Longstaff[18]) have not become established, possibly because they are technically difficult to
perform and analyze and no commercial equipment or software is available, in contrast
to popular thrombin-generation platforms.
Antigen Assays
Antigen tests for plasma proteins involved in fibrinolysis are common and relatively
simple to perform, even in large population studies. Many studies have been organized
to investigate variations in circulating tPA, PAI-1 and TAFI, including free-active/inactive/latent
and inhibited tPA–PAI-1 complex forms, to look for associations with arterial or venous
thrombosis.[24]
[25]
Reduced levels of fibrinolysis inhibitors are not diagnosed as often as coagulation
deficiencies, but when found often lead to increased bleeding risk.[17] Alternatively, high circulating PAI-1 and TAFI may indicate a “fibrinolysis deficit”
and thrombosis risk. There are many large-scale population studies involving PAI-1
and TAFI, but results are not consistent.[26] Standardization of assay methods is poor in this area and it is not possible to
directly compare absolute values of analytes from studies using different methods
for PAI-1 antigen or activity or tPA antigen.[27]
[28] Generally, elevated PAI-1 is associated with cardiovascular disease, metabolic syndrome,
diabetes, obesity, senescence and as a prognostic marker for several cancers.[29] It is likely that harmonization of results from different methods could be improved
with common standards.[30]
[31] However, although there are WHO International Standards for tPA antigen in plasma
and PAI-1 activity, they are not so popular because they are labeled in IU, while
commercial methods report results in ng/mL. Unfortunately, the origin of the commercial
kit standards is not consistent, and each “ng” is different. To establish a firm basis
for reported ng, we at NIBSC are pursuing isotope dilution mass spectrometry using
13C-labeled recombinant proteins as a way of establishing real gravimetric concentrations
of plasma analytes. Work on TAFI and PAI-1 antigens is underway. In addition to these
considerations, pre-test processing issues further complicate PAI-1 measurements and
make tPA activity measurements unreliable. Factors such as diurnal and seasonal variations,[32] and release of PAI-1 from platelets during venepuncture must be considered.[33] Historically, a source of variation in TAFI assays has been the Thr325Ile polymorphism,
which affects TAFI activation and stability.[34]
[35] It has been proposed that TAFIa is a more important biomarker than zymogen,[36] but measurements require very sensitive methods.[37]
[38]
[39]
Interest in PAI-1 and TAFI as drug targets to modulate hemostasis has raised awareness
of the importance of robust assay methods.[40]
[41]
[42] The idea of inhibiting PAI-1 activity is interesting in the context of aging as
a mutation in the SERPINE1 gene resulting in around 50% reduced circulating PAI-1
is associated with longevity in animal models and a population of Amish in the United
States.[43]
Hyperfibrinolysis and Hypofibrinolysis
Hyperfibrinolysis and Hypofibrinolysis
Moore and colleagues[44] have attempted to define or clarify different types of pathological fibrinolysis
observed clinically, for instance in trauma but also in surgery and disseminated intravascular
coagulation (DIC). In addition to hyperfibrinolysis, there are varieties of fibrinolysis
resistance (classically identified in ECLT assays) including hypofibrinolysis (a failure
to trigger fibrinolysis after clotting) and fibrinolysis shutdown (where there is
a rebound of increased PAI-1 activity and antigen after the triggering of coagulation
and early release of tPA). The timing of fibrinolysis resistance and concepts such
as occult fibrinolysis provide further complications.[44]
Hyperfibrinolysis in acute situations such as trauma or surgery is a life-threatening
situation requiring rapid tests to direct treatment such as plasma or clotting factor
replacements, or possibly with antifibrinolytics such as tranexamic acid (TXA). Instances
where markers of fibrinolysis are elevated include trauma, DIC (where there is a fibrinolytic
phenotype),[45] acute promyelocytic leukemia, liver damage, congenital abnormalities and surgical
procedures (reviewed in Kolev and Longstaff[17]). Common markers for ongoing fibrinolysis would be elevated D-dimer,[46] raised tPA or decreased PAI-1, reduced plasminogen, reduced α2-antiplasmin and elevated plasmin-α2-antiplasmin (P-AP) complexes. Assays for these proteins are time-consuming, with
the possible exception of point-of-care tests for D-dimer. However, D-dimer tests
are approved for excluding thrombosis and the accurate measurement of high D-dimer
levels is complicated by the low specificity of these tests and poor standardization.[47]
[48] Where hyperfibrinolysis is detected there is a high risk of death. For instance,
in trauma only a minority of patients display a hyperfibrinolysis phenotype but mortality
in this group is very high.[49]
[50] The underlying fibrinolysis imbalance is likely dominated by an increase in tPA
resulting in plasmin generation with concomitant consumption of inhibitors PAI-1 and
α2-antiplasmin.[51]
[52] The potential for hyperfibrinolysis to be associated with thrombotic complications
has been highlighted previously.[22]
Viscoelastic methods potentially have a role in diagnosing hyperfibrinolysis and fibrinolysis
resistance as they are capable of generating results more rapidly than other available
methods.[53] In particular, rapid thrombelastography (r-TEG) has been developed to speed up clotting
by stimulating both intrinsic and extrinsic coagulation using kaolin and TF activators,
and making tests available in cartridge form with potential for improved reliability.[54] The fundamentals of viscoelastic methods have been reviewed elsewhere[55]
[56] and involve the analysis of clot formation and lysis by physical measurement of
blood viscosity and clot strength. In practice, the common platforms rotational thromboelastometry
(ROTEM) and TEG provide an array of parameters from multiple variations of clotting
tests and there is no agreed way of implementing results from these tests, although
attempts are being made to develop optimized algorithms.[57]
[58] While the speed of testing is attractive, sensitivity and specificity may be an
issue. A study by Raza and colleagues tested samples from trauma patients and found
high P-AP complexes and D-dimer in samples where ROTEM did not detect ongoing fibrinolysis.[49] It has been proposed that occult (local) fibrinolysis or the earlier production
of long-lived D-dimer or P-AP complexes could be responsible for this discrepancy
between fibrinolysis biomarkers and viscoelastic methods.[44]
An interesting and controversial aspect of these discussions is how to use antifibrinolytic
therapies, particularly TXA to reduce bleeding in surgery[59] or trauma. Several large-scale clinical trials have demonstrated that TXA given
early in trauma is safe and effective[60] and pre-hospital treatment is recommended in Europe. In some quarters there are
concerns that adding antifibrinolytics to a situation where there may be fibrinolysis
resistance is potentially dangerous and could lead to thrombotic complications, including
widespread vascular microthrombosis, organ failure and death. Thus, it is proposed
that rapid testing, by viscoelastic methods, particularly rapid TEG, should be used
to target only those patients who would benefit from antifibrinolytic treatment. However,
several large clinical trials of TXA in trauma,[61] postpartum hemorrhage[62] and traumatic brain injury[63] observed no increases in thromboembolic complications,[60] and there is a lack of evidence of disperse microvascular thrombi.[64]
[65] On the other hand, the HALT-IT trial, which failed to show benefit of TXA in the
treatment of gastrointestinal bleeding, did observe an increased risk of venous thromboembolic
events (deep vein thrombosis plus pulmonary embolism) in the TXA treatment group.[66] The authors speculated that the increased risk may be due to disturbed hemostasis
in the liver cirrhosis and variceal bleeding patients that made up half the subjects
in the study, and/or the high dose of TXA used (4 g over 24 hours), which may also
explain an observed increase in seizures. The conclusions from many studies and systematic
reviews seem to be that more research is needed before there is sufficient confidence
in viscoelastic methods for routine diagnostic testing, although there are promising
signals of benefit in situations to reduce blood component use.[67]
[68]
[69] Hard evidence from randomized controlled trials to support the application of viscoelastic
methods in targeting antifibrinolytic therapy is lacking.[50]
[70] It is argued that without this evidence the established risks of TXA treatment delay
should outweigh theoretical but unproven risks of non-targeted treatment.
As TXA treatment is delayed, it becomes progressively less effective and after 3 hours
benefit is lost[60] and the dangers appear to be excess bleeding, not thrombosis. There are few studies
of uPA in pathological fibrinolysis, but Hijazi and colleagues[71] have identified the slow development of a peak of uPA after several hours in a mouse
model of brain injury, following the earlier rise and decay of tPA. We have shown
in vitro that TXA plus urokinase stimulates plasmin generation to aggravate consumption
of α2-antiplasmin, which is often reduced in trauma patients, to allow the fibrinolytic
system to go unchecked. The consequences are destruction of fibrinogen and clotting
factors, which could contribute to a bleeding pathology.[72] There are little data available on changes in uPA or uPA–PAI complexes in trauma
patients and currently tests for these analytes are poorly standardized so that estimates
of gravimetric or molar concentrations in patient plasma are unreliable.
Covid-19
Coagulopathy was soon observed to be a life-threatening complication of infection
with SARS-CoV-2 in hospitalized patients.[73]
[74] Venous thromboembolic events are noted in many patients in intensive care and a
high proportion of patients are diagnosed with pulmonary embolism.[75]
[76] Post-mortem investigations have identified widely distributed microvascular thrombi
in the lungs, heart, kidney, liver, skin and fat.[77] Biomarkers in severely ill patients include raised fibrinogen and D-dimer, which
can reach very high levels,[77]
[78] often >2 µg/mL, many times over the routine cut-off used to exclude a diagnosis
of thrombosis (0.5 µg/mL). D-dimer has been investigated as a marker to predict mortality
and manage patient care and direct anticoagulant treatment.[74]
[79] Reporting results and standardization of D-dimer testing protocols is an area of
concern in Covid-19 patients and is particularly important if it is to be linked to
patient care.[80] Given the prevalence of thrombotic complications in Covid-19 patients in intensive
care, it is not surprising that thrombolytic therapy with tPA is being considered
for seriously ill patients via the intravenous route[81] or in nebulizer form.[82] Other factors that may influence coagulation and fibrinolysis in Covid-19 patients
may include neutrophil extracellular trap (NET) formation, which stimulates coagulation
and retards fibrinolysis[83]
[84] and NETs have been observed in some early studies with samples from Covid-19 patients.[77]
[85]
[86] There is interest in changes in PAI-1 levels during Covid-19 infection, and there
are early reports of fibrinolysis resistance assessed by antigen studies and viscoelastic
methods.[85]
[87]
Conclusions
There is a long history of fibrinolysis research and assay method development, but
no simple direct methods to establish something like a fibrinolysis capacity or a
measurement of fibrinolysis resistance on par with prothrombin time or activated partial
thromboplastin time, for example. Many problems associated with poor standardization
and assay variability remain unresolved. Rapid universal methods would be useful in
situations such as trauma, DIC and surgery, but there is a need for large-scale randomized
trials to establish safety and efficacy. A better understanding is needed of changes
in fibrinolysis following infection with agents causing hemorrhagic diseases, and
the emergence of SARS-CoV-2 coagulopathy highlights the need for ongoing research
to improve the measurement of fibrinolysis.
