Evolving Paradigms in Laboratory Biomarkers of Fibrinolysis Phenotypes and Association with Post-Traumatic Vascular Thrombosis

Abstract

Traumatic tissue injury triggers blood coagulation to stanch bleeding and concomitant blood clot lysis to restore vascular patency. Approximately, 40% of trauma cases potentially present with trauma-induced coagulopathy that may coexist with clot dissolution or fibrinolysis. Laboratory test results of fibrinolysis biomarkers stratify fibrinolytic phenotypes into hyperfibrinolysis, physiological, hypofibrinolysis, and fibrinolytic shutdown. However, often, there is incongruence between laboratory findings and clinical presentation of bleeding or vascular thrombosis. Increasingly, it is becoming clear that laboratory findings transiently depend on the timing of blood sampling. The spectrum of evolving fibrinolysis phenotypes, a component of nature’s adaptation to wound healing that ranges from initial promotion of blood fluidity to subsequent thrombosis, presents a clinical diagnostic dilemma with regard to the timing of antifibrinolytics or anticoagulants intervention. This chapter will review the available literature on post-traumatic fibrinolytic phenotypes, diagnostic challenges, evolution over time, clinical outcomes following therapeutic interventions, and association with vascular thrombosis.

1. Introduction

1.1 Clinical presentation of fibrinolysis in trauma

Post-trauma fibrinolysis is one of the causes of uncontrolled diffuse bleeding from injured areas despite surgical repair. The tissues rich in fibrinolytic activity are commonly urothelium such as the prostate, tonsils, uterus and placenta, kidneys, and urogenital system [1]. There is failure to stabilize forming blood clots, or alternatively, clot breakdown at level of the capillary bed resulting in diffuse bleeding involving uninjured sites and is extremely difficult to stop with mechanical interventions. Also, oozing from puncture sites and operative sites, excessive bruising, petechial hemorrhages, and expanding hematomas are not attributable to major vascular injury. The defining characteristics are generalized diffuse oozing from wounds, clot formation followed by dissolution, delayed bleeding post trauma, surgery, or procedures such as dental extractions.

Early observations on the fibrinolytic activity were in cadaveric blood obtained immediately from dead animals or victims of sudden violent death where blood remained liquid without clotting, inhibited clotting of plasma from normal living donors when mixed, or caused dissolution of clots formed from pure thrombin and fibrinogen. Others observed that plasma during surgery of certain organs formed clots in normal way but soon dissolved, unlike samples collected before operation that formed clots that remained solid for indefinitely for weeks.

1.2 Physiology of fibrinolysis

Fibrinolysis occurs when circulating inactive plasminogen is converted to active plasmin that breaks down fibrin to fibrin soluble products. The activator for plasminogen is tissue plasminogen activator (t-PA) released from injured endothelium or urokinase plasminogen activator (u-PA) from urothelium. Other activators of the fibrinolytic system, though playing minor roles under physiological conditions, are lipoproteins, Kallikrein-kinin system of the contact pathway of intrinsic coagulation [2].

The system is regulated by limiting the reaction complexes on fibrin or cellular surfaces [3, 4]. When bound to cross-linked fibrin, t-PA, plasminogen, and fibrin, they form a trimolecular complex, but while on endothelial surface, they form a tetramer made of annexin-2 as the linker to cellular surface and S100A10 as the receptor for plasminogen in addition to t-PA and plasminogen. On urothelium, urokinase substitutes for t-PA and is anchored to the surface by urokinase plasminogen activator receptor (u-PAR) [2]. In this arrangement, Annexin-2 and u-PAR act as cofactors to impede inactivation of surface-bound plasmin from circulating inhibitors. In solution or liquid phase plasminogen activator inhibitor- 1&- 2 (PAI-1 &2) complexes with t-PA to limit its interaction with fibrin, α2-antiplasmin (α2-AP) to inhibit active free plasmin to prevent indiscriminate action in circulation [2].

1.3 Activation of fibrinolysis In trauma

Fibrinolysis is activated in trauma by-products of tissue injury, development of hypo perfusion or shock state, and occlusive thrombi [5].

1.3.1 Relation to markers of tissue injury

Increased fibrinolysis is positively correlated with injury severity and endothelial injury markers such as syndecan-1 [6, 7].

1.3.2 Relation to shock state

Fibrinolysis is associated with and correlates with severity of shock states [8, 9]. Due to the induction of metabolic acidosis in shock and the fact that in vitro acidification precipitates euglobulin fractions [10, 11] and that acidification inactivates PAI-1 in solution [12], it is presumed that low pH causes fibrinolysis. However, this position is not in tandem with other empirical observations since such as (a) plasmin fibrinolytic activity is optimal at neutral pH and activity decreases at low pH [10, 13, 14, 15, 16, 17], (b) paradoxically, in vivo experimental infusion of lactic acid in laboratory animals was associated with microvascular thrombosis [18]. The discrepancy between the two positions can be accounted for by the fact that although acidosis diminishes plasminogen activator inhibitor -1 (PAI-1) activity, while enhancing the activators in vitro, this effect is minimal in vivo t since it is counterbalanced by metabolomics produced during anaerobic metabolism in shock that promote broad fibrinolysis in overall [19, 20, 21].

2. Laboratory measurement of fibrinolysis activity

2.1 Biomarkers of fibrin degradation

Plasmin sequentially degrades fibrin into several degradation products varying in molecular weights that have been quantified as evidence of fibrinolysis [22, 23].

D-dimers (DD), the terminal degradation products of ligated fibrin, are considered an indirect evidence of fibrin formation and its subsequent lysis [24]. The commonest method of measurement is ELISA-based, and several assays are available giving results in D-dimer units (DDU) or fibrinogen equivalent units (FEU). It is commonly used for exclusion of DVT with sensitivity of 96–100% and specificity of 40–70% [24]. However, its utility as a measure of plasmin fibrinolysis activity post trauma has come into question due to (a) non-plasmin sources such as from neutrophil elastase (NE) [25, 26] and (b) high variability owing to altered hemodynamic effects on hepatic clearance [27].

2.2 Assay of individual protein components of activators and inhibitors

Levels of proteins acting as activators and inhibitors of the fibrinolytic system are usually measured using immunological techniques, using free and complexed antigen, and enzyme chromogenic assays for activity levels in plasma. The quantity of protein levels varies with sex, age and diurnal variation, and status of catecholamine activation. Trauma increases the concentration and half-lives of these proteins [28]. Amongst the clinically available tests are for t-PA, PAI-1, PAI-2, alpha-2 antiplasmin (α2-AP), and the plasmin-antiplasmin (PAP) complex. Notably, plasmin levels are difficult to measure due to rapid inactivation in plasma [28].

2.3 Plasma global functional assays

Euglobulin clot lysis test (ECLT): This involves separation of platelet-free plasma (PFP), dilution with distilled water, and precipitation of globulin fraction to obtain euglobulin that is then acidified using acetic acid, centrifuged, and precipitate obtained redissolved in buffer solution. The acidified precipitate is rich in t-PA but poor in inhibitors such as PAI1, α2-AP, and TAFI [29]. Clot formation is initiated by recalcification and addition of thrombin in a test tube. Fibrinolytic activity is determined by observing the time taken to clot dissolution. Because of its dependence on fibrinogen levels, this test is insensitive in low fibrinogen states [30].

Fibrin plate lysis test: In this method, developed by Astrup and Mullertz [31], thrombin is added to fibrinogen solution following which euglobulin fraction, containing residual fibrin FXII and residual plasminogen, is added to start the reaction. The fibrinolysis is determined by measuring the diameter of lytic area or photometrically in a microplate reader [30]. This method is of historical importance and is currently rarely used currently due to its lengthy nature.

2.4 Whole blood viscoelastic hemostatic assays (VHA)

The viscoelastic platforms detect changes in viscosity of blood against the wall in a rotating pin and cup after coagulation has been triggered. While thromboelastolastography (TEG) or thrombelastometry (ROTEM) of the commercially available instruments are commonly deployed in trauma, Sonoclot has not found wide application. Commercial instruments for these assays differ in mechanics, triggering agents, and nomenclature of machine readouts [29]. These methods are hugely popular in the point-of-care diagnosis of active and ongoing fibrinolysis, unlike the molecular assays that are time-consuming and are dependent on plasma clearance and give indication of dynamic changes in blood. TEG and ROTEM display graphically the evolution against time (onset of clot formation, rate of fibrin build-up, development of maximum clot strength, and thereafter its reduction indicating clot dissolution) [32]. In either of the systems, fibrinolysis is indicated by the percentage reduction in area under the curve as lysis % (Ly %) assuming MA remains constant) on TEG, or the percentage of clot remaining after MCF as Lysis index % (LI %), or as maximum lysis % (ML%) on ROTEM at 30 minutes and 60 minutes [33]. For fibrinolytic phenotypes stratification, while the cutoff for TEG parameters has been classified as hyperfibrinolysis (Ly 30 > 3%), physiologic (Ly 30 0.81–2.9%), and shutdown (Ly 30 < 0.8%) [34]; the cutoff for ROTEM parameters are physiologic (ML 3–15%), shutdown (ML <3%), and hyperfibrinolysis (ML > 15%) [35]. When a particular pattern of severe fibrinolysis termed ‘death diamond’ is present, the likelihood of hemorrhage and mortality is high [36, 37].

ROTEM has been utilized to demonstrate high rates of hyperfibrinolysis in trauma patients [38]. Due to the increased incidences of hypofibrinolysis or fibrinolytic shutdown in trauma and surgery, the method has been modified to incorporate plasminogen activators to increase sensitivity [39]. Validation and standardization of ROTEM [40, 41] have shown good congruence with alternative methods for fibrinolysis measurements. However, great discrepancy has been reported between the VHA and molecular biomarkers of fibrinolysis in trauma [42].

3. Concepts in fibrinolysis

3.1 Local fibrinolysis

Fibrinolysis is initiated by localized tissue injury that leads to release of plasminogen activators in general circulation. Some tissues, such as endothelium, endometrium, tonsils, and urothelium, express high amounts of plasminogen activators [43], and therefore would be expected to exhibit localized clot lysis without systemic involvement. Indeed, surgical incisions or injury of these anatomical sites have been observed to evince characteristic fibrinolytic hemorrhage without systemic changes in biomarkers [44]. The process is aided by localized plasminogen receptor S100A10 and their endothelial receptor of Annexin-2 on vascular linings in vivo [45, 46]. Local fibrinolysis ensures vascular patency of vascular territories without affecting clots formed elsewhere, therefore, ensuring cardiovascular integrity [47].

3.2 Systemic fibrinolysis

In systemic fibrinolysis, there is evidence of fibrinolysis activity in the general circulation. Characteristic features include increased t-PA, PAP, and fibrin degradation products such as FDPs or D-dimers. Additionally, bleeding is generalized and not restricted to particular sites. It is often seen in DIC, severe trauma, circulatory hypoperfusion/ shock, or administration of thrombolytics [42, 48].

3.3 Primary fibrinolysis

This is the normal process that occurs during physiological conditions when there is optimal balance between the activators and the inhibitors that keep the vascular lumen patent [49]. Primary fibrinolysis implies a response arising in the absence of hypercoagulable or thrombotic state [50]. Usually, bleeding or thrombosis risks are only increased in case of congenital deficiency in one or more of the factors. It is rarely encountered in trauma.

3.4 Secondary fibrinolysis

This process is secondary to a pathological or disease process causing imbalance in the regulation of fibrinolysis process. Secondary fibrinolysis develops in response to intravascular thrombin generation and fibrin deposition [50]. The evident dysregulation could involve one or a combination of factors including alteration of rate of endothelial secretion, release of t-PA or alternative activators, or alteration of concentration of inhibitors such as PAI-1 and hepatic clearance of t-PA or other activators. Although many disease processes cause secondary fibrinolysis, in trauma tissue damage that is associated with shock and acidosis leads to t-PA secretion and delayed clearance that overwhelm the natural inhibitors leading to consequent hyperfibrinolysis. Furthermore, the procoagulant tissue factor that is released during tissue injury participates in initiation of coagulation, microthrombi formation initialization of cascade of events that lead to plasminogen activation.

3.5 Physiological fibrinolysis

The concept of physiological fibrinolysis is based on a balance between activators and inhibitors of fibrinolytic system leading to an equilibrium between activated and nonactivated states [51]. This is the ideal and naturally intended state during trauma response where vascular patency is maintained but without bleeding.

3.6 Pathological fibrinolysis

Although fibrinolysis is a protective mechanism geared toward to restoration of vascular patency, it can become excessive leading to hemorrhage or insufficient leading to thrombosis [50]. Majority fibrinolysis phenotypes in trauma belong to this group presenting as either hyperfibrinolysis or shutdown.

3.7 Fibrinolysis phenotypes in trauma

Using a combination of plasma protein assays and VHA, the fibrinolysis in trauma has been stratified into hyperfibrinolysis, physiological, and shutdown, reflecting outcomes related to organ failure or hemorrhage [34].

3.7.1 Hyperfibrinolysis

This is when fibrinolysis activation causes or sustains a bleeding tendency [50]. It is associated with severe injury, prolongation of routine coagulation tests, and greater mortality [38]. Moreover, it is the most lethal phenotype of lysis pattern in trauma resulting in high risks of hemorrhage and high mortality. The laboratory criteria characteristically show markedly elevated PAP and DD, as well as increased lysis on vHA [52]. The cause of in trauma is the markedly elevated t-PA that rapidly and massively activates plasminogen to plasmin overwhelming the inhibitory factors such as PAI-1 and alpha-2 antiplasmin [53, 54].

3.7.2 Physiological fibrinolysis

The laboratory evidence for physiological fibrinolysis includes a combination of normal or slightly elevated plasma PAP and DD together with normal or low lysis on VHA [52]. Although this phenotype has low mortality rates, patients usually require blood transfusion, suffer organ failure, or hemorrhage [34].

3.7.3 Occult fibrinolysis

In this, patients who have concealed evidence of fibrinolysis [55]. Characteristically, despite low lytic activity on VHA (low ROTEM %ML) patients exhibit paradoxically, discordant high plasma levels of fibrinolysis biomarkers such as (DD) and S100A10. However, other biomarkers such as t-PA, plasmin-anti plasmin (PAP), PAI-1, and PF 1+2 remain normal or not elevated. Furthermore, there is evidence of thrombin generation given the high F1+2 fibrinopeptide [46]. Globally, there is a balance between fibrinolysis, thrombin activation (PF 1&2), fibrin formation (FP A & FPB), and degradation (DD) such that coagulation tests (fibrinogen, PT, and aPTT) are normal despite elevated DD and low ML on ROTEM [42]. The discrepancy in test results is plausibly due to the high level of S100A10, which is active on cellular membrane surfaces in vivo, but without activity ex vivo under testing conditions. The membrane-bound S100A10, plasminogen, t-PA, and Annexin-2 bind onto cell surfaces in close proximity form a tetramer. It is upregulation under hypoxic conditions in trauma leads to localized fibrin degradation despite low systemic t-PA-plasmin levels.

3.7.4 Lowfibrinolysis

3.7.4.1 Hypofibrinolysis

This condition occurs due to lowfibrinolytic state without evidence of prior activation due to impaired t-PA release in the presence of elevated baseline fibrinolytic inhibitors [56]. The terminology deployed in this case is contentious given that it is also considered to correspond to a failure of fibrinolysis activation following clotting, especially when ELT is the test [29]. Traditionally, these hypofibrinolysis patients were taken to be those lacking fibrinolytic activity on venous occlusion test. However, this test is now largely obsolete and impractical in trauma victims, though the test has fallen out of favor and may not be practical in trauma victims. The laboratory features include normal routine coagulation tests, lowfibrinolytic activity on VHA, but DD and PAP levels are proportional to injury burden. A unique feature of this phenotype is that patients are responsive to recombinant t-PA challenge unlike in fibrinolysis [56].

3.7.4.2 Fibrinolysis resistance

In fibrinolysis resistance, which potentially may also include hypofibrinolysis and fibrinolysis shutdown on ECLT test [29], formed blood clots or thrombi are resistant to lysis by plasminogen activators [57, 58], and thus persists in vascular lumen [59]. In plasma, resistance is quantified by VHA (TEG/ROTEM) modified assays spiked with t-PA [60] in which resistant samples are unresponsive. Fibrinolysis resistance, putatively due to an imbalance between endogenous t-PA and PAI-1, shares some similarities with fibrinolysis shutdown, although other factors such as fibrin architecture, vWF, NETs, and microparticles are thought to offer in vivo contribution [58].

3.7.4.3 Fibrinolysis shutdown

In this condition, it is postulated that fibrinolysis, activated at some time point, is inactivated by the time of blood sampling. This phenotype was decoded over 60 years ago [61, 62] after observing that dilute blood lysis time and ELT were shorter 45 minutes into surgical operations but became prolonged during the postoperative period consistent with decreased fibrinolysis. The long lysis time on ELT correlated with reduced t-PA activity with concurrent elevated peak t-PAI-1 inhibitor on the first postoperative day patterns, which were similar to myocardial infarction and trauma at diagnosis and during recovery phases [63, 64].

Prior to adoption of VHA in surgery and trauma, ELT showed lack of response on subsequent testing of the same patient after some time interval. The adoption of VHA helped to explain the discrepancy in the test findings due to rebound PAI-1 inhibitors after triggering of coagulation and release of t-PA [29]. Currently, fibrinolysis shutdown is demonstrated by elevated DD and PAP but decreased functional lysis on VHA (either TEG or ROTEM) [52]. Two tests, t-PA hypersensitivity rapid TEG (r-TEG) or r-ROTEM, are used to delineate lowfibrinolysis due to hypofibrilysis from shutdown [65]. Fibrinolysis shutdown is associated with high rates of organ failure and death [34].

The postulated mechanisms of fibrinolysis shutdown include (a) PAI-1 inhibition, (b) neutrophil elastase impairment of fibrinolysis [66] through degradation of plasminogen [67] and plasmin [68], (c) and degradation of alpha-chain of fibrin that blocks activation of plasminogen [69].

There are variants of fibrinolysis shutdown that include:

1. Acute: There is evidence of fibrinolysis activation such as elevated DD and PAP, and low t-PA due to rapid hepatic clearance within 1 hour of trauma. In addition, VHA displays lowfibrinolytic activity unrelated to inhibitors since they have been depleted through consumption but retain increased sensitivity to t-PA challenge [56].

2. Acquired: Early descriptions acquired fibrinolysis was in association with the administration of exogenous thrombolytics that became resistant to subsequent exposure to the same agent due to the development of inhibitors. In trauma, the onset of acquired fibrinolysis commences after 2 hours but can last for up to 24 hours. It is characterized by low fibrinolytic activity on VHA, low t-PA activity with increased resistance to lysis upon t-PA challenge, and an upregulation of PAI-1 [56].

3. Persistent: This is exhibited by fibrinolysis activation followed by delayed upregulation of inhibitors whose onset is after 24 hours and may persist for 7 days. It is associated with platelet degranulation with persistent release of PAI-1 into systemic circulation signaling failure to regain hemostatic homeostasis [56]. The mechanism for persistent fibrinolysis shutdown is still unclear, but a strong immunological implicated. An alternative mechanism is the presence of neutrophil elastase (NE) released from leukocytes that not only degrades fibrin to DD but also digests plasminogen eventually contributing to fibrinolytic shutdown [67].

4. Fibrin resistant: An emerging phenomenon in cases of absence of fibrinolytic activity in trauma is the development of fibrin architecture resistant to plasmin lysis [70]. In this phenotype, viscoelastic hemostatic assays (TEG or ROTEM) demonstrate no lysis despite high circulating levels of t –PA released from injured sites and/or exogenous t-PA spiking during testing. The postulated mechanism is rapid and massive thrombin generation that leads to the development of denser and coarse fibrin networks impermeable by plasmin lytic head [71, 72]. In addition, it has been demonstrated that TAFI activation by thrombin inhibits plasminogen interaction with fibrin [73, 74], as well as FXIII cross-linking of fibrin confer lytic resistance [57].

3.8 Evolution of fibrinolysis phenotypes over time period

The incidence of the different fibrinolytic phenotypes varies and depends on the severity of injury, assays type employed and time period post-injury when blood was drawn for analysis. Notably, plasma fibrinolytic activity is a dynamic process in which transition from one phenotype to another frequently occurs.

In trauma patients, levels of molecular protein biomarkers levels change with time. It has been reported that while t-PA levels drastically decrease in the first hour and continue to decrease over time, PAI-1 levels incrementally increase for five hours post trauma in an inverse manner to t-PA [75]. However, a DD pattern is unrelated to either, since there was abrupt increase over first hour and then rapid drop in levels. The events after one hour probably reflect the fibrinolytic shutdown.

Serial determination of euglobulin lysis time (ELT) or fibrin platelet method in surgical patients has revealed that the period of peak enhanced fibrinolysis activity coincides with mid surgery, followed by progressive shutdown occurring over the next 24 hours post-operation, and thereafter leveling off by day third or fourth days [76]. This reflects the dynamic nature of fibrinolytic actors and measurements taken at single particular time points may be less useful as opposed to the trend in assay results.

By far VHA (TEG or ROTEM) has been used in trauma patients to stratify in fibrinolytic phenotypes in trauma patients. At admission, a majority (44–64%) of patients are in fibrinolytic shutdown, which persists for 120 hours [77]. The first 3 hours are critical since it is the time of transition from hyperfibrinolysis to physiologic state that remains stable for the next 5 days. The hyperfibrinolysis and physiological phenotypes may be described as transitory since they diminish within a few hours, while shutdown increases (from 27–60%) over three hours from the time of trauma [78]. As such, over time, shutdown is the predominant phenotype. Patients who are in initial hypofibrinolysis/shutdown and who remain in the same status or who transition from or to other phenotypes are prone to high mortality, especially due to multiple organ failure [79]. Transitions from one fibrinolysis phenotype to another may be due to resuscitation measures, rapid hepatic clearance of activators, and administration of antifibrinolytics [56]. In liver transplant recipients, initial hyperfibrinolysis eventually transition to either early or late shutdown over 120 minutes [80, 81].

4. Post-trauma fibrinolysis and development of vascular thrombosis

4.1 Epidemiology of vascular thrombosis post trauma

Cases of post-trauma vascular thrombosis are grossly under-reported owing to the difficulties in diagnosing the condition. Available evidence from clinical, radiological, histopathological, and autopsy studies that when present post-traumatic vascular thrombosis is preceded by fibrinolytic activity. Though varying depending on the vascular territory and patient population literature indicates that with surveillance, the rates of vascular could be up to 60% for DVT [82] and 25% for PTE [83], while microvascular thrombosis is variable [84, 85]. Although the macrovascular events are amenable to radiological visualization or imaging techniques, microvascular occlusions, especially at trauma sites are never included in the diagnostic reports. Furthermore, arterial thrombosis events including myocardial infarctions, ischemic stroke, or renal involvements are usually excluded from post-traumatic vascular thrombosis reporting, even though they occur frequently. Surprisingly, the incidence of post-trauma venous thrombosis is high despite pharmacological anticoagulant thromboprophylaxis [86]. The inadequate response to anticoagulants strongly suggests that fibrinolytic impairment superimposed on the coagulation cascade contributes to post-traumatic vascular.

4.2 Relation to fibrinolytic activity

Despite clinical and laboratory evidence of post-trauma fibrinolysis [42, 75], vascular thrombosis, in which impaired fibrinolysis plays a role, fibrinolysis [87, 88] remains a silent killer. There are three competing theories linking dysregulated fibrinolysis with thrombosis in trauma: (a) activation followed by suppression of lytic pathways leading to reduction of fibrin degradation products (FDPs) and D-dimers, (b) coexistent of lytic activity with overwhelming fibrin deposition associated with increased fibrin split products, and (c) formation of lysis resistant fibrin.

Fibrinolysis shutdown has largely been demonstrated in association with surgery [63] and has been shown to lead to high incidence of thrombosis complications despite heparin administration [89]. Many reports of thromboembolism in trauma have implicated fibrinolytic shutdown [80, 90].

Proponents of overwhelming fibrin deposition in the presence of active fibrinolysis have used D-dimers as a marker of thrombosis. In a cohort of trauma patients, serial D-dimer levels remained elevated above normal cutoff threshold levels for 14 days without evidence of bleeding. Instead, some patients were found to have PE. The elevated D-dimers though predictive of thrombosis outside trauma are difficult to interpret post-injury [91]. Similarly in surgery, postoperative fibrinolysis shutdown ascertained by ELT and fibrin plate method coincided with decreased FDP linked with the development of DVT or PE [76].

In non-trauma patients, vascular thrombosis has been linked to lowfibrinolysis states [88]. In such patients, hypofibrinolysis coincides with elevated inhibitor levels. This scenario is complicated by the concurrent increased t-PA in shutdown states. In effect, the elevated t-PA antigens are more predictive of thrombosis than fibrinolysis owing to their utility as a marker of endothelial damage [92]. This is because t-PA fibrinolytic activity is limited to plasminogen bound to cross-linked fibrin and surfaces, and therefore free-floating levels are insignificant to clot lysis.

Trauma patients are procoagulant [93] and show enhanced thrombin generation [94]. Thrombin contributes to fibrinolysis resistance by reducing profibrinolytic effects of thrombomodulin (TM) and activated protein C (APC) [73]. There is activation of TAFI, which blocks the binding of plasminogen to fibrin and activation of coagulation factor XIII, which covalently ligates fibrin, thus confers lysis resistance. Fibrin formed in the presence of high levels of thrombin is compact and impervious to permeation by plasmin lytic head [95, 96]. Furthermore, the formed thrombus in trauma is resistant to lysis due to incorporated RBC [97, 98, 99], FXIII [57], platelets [100, 101], and abnormal dense fibrin structure [59, 70, 71, 72, 102]. Other contributors to lysis resistance are neutrophil extracellular traps (NETs) [103, 104, 105] and circulating microparticles [106].

In trauma, the reactions of cellular elements are dynamic, with platelet reactions playing special role on lytic resistance. Specifically, in trauma, platelets contribute to lytic resistance by (a) producing over 90% of PAI-1, (b)release of FXIII that cross-links fibrin to diminish permeability by plasmin, (c) provision of a surface for catalytic coagulation reactions leading to thrombin burst and TAFI activation and release, (d) undergo clot retraction and release microparticles that promote coagulation cascade leading to overwhelming fibrin formation and deposition [107].

Tissue injuries associated with surgery, and trauma are associated with fibrinolysis in the early phases and vascular thrombosis in the later recovery phases. Although the fibrinolytic phenotypes evolve from one type to another, the shutdown phenotype increases with time [78] and is associated with the development of vascular thrombosis [80, 90, 108]. Although no single laboratory test can predict transition from fibrinolysis to thrombosis, some small studies indicate that resistance to t-PA on rapid thromboelastography/elastometry could be a useful test [109]. However, large clinical trials are needed to validate the claim.

5. Clinical experiences in surgery and trauma

5.1 Fibrinolysis and liver transplantation

Early in orthotopic liver transplantation hemorrhage due to fibrinolysis demonstrated by TEG was an issue and administration of tranexamic acid reduced requirements for blood transfusion [110]. With this evidence, empiric Epsilon amino caproic acid (EACA) antifibrinolytics was implemented. However, it was observed that over time that such practice increased vascular thrombosis [111]. Perhaps these early investigators did not appreciate transient time course of hyperfibrinolysis that occurs during anhepatic stage that soon resolves after re-establishment of circulation [112, 113]. Although all the fibrinolytic phenotypes have been observed during liver transplantation [81], but the shutdown phenotype is associated with the development of thrombohaemorrhagic complications that begins 30 minutes after re-establishment of reperfusion in allograft recipient [80] that is predicted by thromboelastography [114]. With this knowledge, it can be potentially concluded that indiscriminate administration of antifibrinolytics against hyperfibrinolysis if not followed by anticoagulants may increase risk of vascular thrombosis. In reviews of antifibrinolytics in liver transplantation over the last 4 decades, it is quite evident that the twin problems of hemorrhage secondary to hyperfibrinolysis and thrombosis still remain real challenges [115]. The clinical challenge is lack of a useful predictive test to guide timing of antifibrinolytics and when to start anticoagulants.

5.2 Fibrinolysis and cardiac surgery

Fibrinolysis is activated during cardiac surgery, especially so during cardiopulmonary bypass [116]. Antifibrinolytics are usually administered to decrease blood loss during cardiac surgery. However, the most potent agent aprotinin has been associated with significant thromboembolism and as a result, its indication has been discontinued in many countries [117]. Given that all fibrinolytic phenotypes have been described in cardiac surgery, and that the incidence of shutdown ranging from 30 to 45% [118], it is still controversial the contribution of aprotinin to fibrinolytic shutdown and vascular thrombosis. Furthermore, it is not yet known the proportion of patients who would transition from hyperfibrinolysis to shutdown and the relation of shutdown to thrombosis in such patients. What is clear is antifibrinolytics should be administered following a strict [119], even though simple laboratory guidelines are still lacking.

5.3 CRASH-2

The use of tranexamic acid (TXA) against fibrinolysis to forestall blood loss in surgery and trauma has been practiced since its introduction more than 60 years ago. Antifibrinolytic activity of TXA can only be monitored by VHA or DD changes but not by using molecular biomarkers such as t-PA, PAP, and PAI-1. In the largest randomized clinical trial of TXA in trauma (CRASH-2 trial), it was shown that, compared to placebo, TXA radically reduced hemorrhage and mortality. Furthermore, incidences of vascular thrombosis were not significant. In this study, TXA only had clear benefit if administered within 3 hours of trauma. However, other clinical trials post-CRASH-2 have shown that indiscriminate administration was associated with high proportions of fibrinolysis shutdown and increased risk of thrombosis by a factor almost twice than those that did not. This finding reinforced an earlier observation of increased thromboembolism in animals administered TXA to reverse hemorrhagic shock-induced hyperfibrinolysis. The current call for selective and rational usage in trauma is based on the recognition that it is associated with greater fibrinolytic shutdown but does not improve clot strength for those already in shutdown. A greater benefit is achieved if TXA is given within 3 hours post-injury (CRASH-2) and in those with proven hyperfibrinolysis phenotype who form less than 20% of cases. Furthermore, indiscriminate usage of tranexamic acid is associated with increased risk of development of post-traumatic venous thromboembolism. Although some studies have indicated that TEG Ly3% could be the critical cutoff to start TXA in trauma patients [120], systematic reviews of published results of TXA, and thrombotic events are still conflicting since some point to increased incidence [121], while others are unequivocal owing to heterogenous cohort of the patient population studied [122].

6. Conclusion

Traumatic injury is associated with concomitant activation of blood coagulation cascade and fibrinolysis. Pathological imbalance between the two could potentially lead to increased risk of hemorrhage or vascular thrombosis. No single laboratory test best describes the spectrum of fibrinolysis in trauma, and thus a combination is used to stratify fibrinolysis phenotypes. The phenotypes are increased of hyperfibrinolysis, normal or physiologic, and decreased fibrinolysis activity, which may be further categorized as hypofibrinolysis, fibrinolysis resistance, and fibrinolysis shutdown. The fibrinolytic phenotypes evolve over time and may transition from one type to another. Overall, the increased fibrinolysis activity is present in the early phases post trauma, and decreased activity or shutdown predominates in the later phases coinciding with recovery. Fibrinolysis shutdown has been associated with the development of occlusive vascular thrombosis. The evolution of fibrinolysis phenotypes is modified by resuscitation strategies in the management of hemodynamics associated with shocks such as intravenous fluids and blood product transfusions. Furthermore, therapeutic interventions such as antifibrinolytics may accelerate transition to fibrinolysis shutdown thereby increasing the risk of vascular thrombosis. The twin problems of post-traumatic fibrinolysis and thrombosis still remain a challenge, but unfortunately, laboratory guidelines in the management of therapeutic interventions to forestall hemorrhage from fibrinolysis and minimization of transition to thrombosis remain largely un described.