Early abnormal fibrinolysis and mortality in patients with thermal injury: a prospective cohort study.

INTRODUCTION: Abnormal fibrinolysis early after injury has been associated with increased mortality in trauma patients, but no studies have addressed patients with burn injury. This prospective cohort study aimed to characterize fibrinolytic phenotypes in burn patients and to see if they were associated with mortality. METHODS: Patients presenting to a regional burn centre within 4 h of thermal injury were included. Blood was collected for sequential viscoelastic measurements using thromboelastography (RapidTEG™) over 12 h. The percentage decrease in clot strength 30 min after the time of maximal clot strength (LY30) was used to categorize patients into hypofibrinolytic/fibrinolytic shutdown (SD), physiological (PHYS) and hyperfibrinolytic (HF) phenotypes. Injury characteristics, demographics and outcomes were compared. RESULTS: Of 115 included patients, just over two thirds were male. Overall median age was 40 (i.q.r. 28-57) years and median total body surface area (TBSA) burn was 13 (i.q.r. 6-30) per cent. Some 42 (36.5 per cent) patients had severe burns affecting over 20 per cent TBSA. Overall mortality was 18.3 per cent. At admission 60.0 per cent were PHYS, 30.4 per cent were SD and 9.6 per cent HF. HF was associated with increased risk of mortality on admission (odds ratio 12.61 (95 per cent c.i. 1.12 to 142.57); P = 0.041) but not later during the admission when its incidence also decreased. Admission SD was not associated with mortality, but incidence increased and by 4 h and beyond, SD was associated with increased mortality, compared with PHYS (odds ratio 8.27 (95 per cent c.i. 1.16 to 58.95); P = 0.034). DISCUSSION: Early abnormal fibrinolytic function is associated with mortality in burn patients.

Effects of an acidic environment on coagulation dynamics.

Essentials Acidosis, an outcome of traumatic injury, has been linked to impaired procoagulant efficiency. In vitro model systems were used to assess coagulation dynamics at pH 7.4 and 7.0. Clot formation dynamics are slightly enhanced at pH 7.0 in blood ex vivo. Acidosis induced decreases in antithrombin efficacy offset impairments in procoagulant activity. SUMMARY: Background Disruption of hydrogen ion homeostasis is a consequence of traumatic injury often associated with clinical coagulopathy. Mechanisms by which acidification of the blood leads to aberrant coagulation require further elucidation. Objective To examine the effects of acidified conditions on coagulation dynamics using in vitro models of increasing complexity. Methods Coagulation dynamics were assessed at pH 7.4 and 7.0 as follows: (i) tissue factor (TF)-initiated coagulation proteome mixtures (±factor [F]XI, ±fibrinogen/FXIII), with reaction progress monitored as thrombin generation or fibrin formation; (ii) enzyme/inhibitor reactions; and (iii) TF-dependent or independent clot dynamics in contact pathway-inhibited blood via viscoelastometry. Results Rate constants for antithrombin inhibition of FXa and thrombin were reduced by ~ 25-30% at pH 7.0. At pH 7.0 (+FXI), TF-initiated thrombin generation showed a 20% increase in maximum thrombin levels and diminished thrombin clearance rates. Viscoelastic analyses showed a 25% increase in clot time and a 25% reduction in maximum clot firmness (MCF). A similar MCF reduction was observed at pH 7.0 when fibrinogen/FXIII were reacted with thrombin. In contrast, in contact pathway-inhibited blood (n = 6) at pH 7.0, MCF values were elevated 6% (95% confidence interval [CI]: 1%-11%) in TF-initiated blood and 15% (95% CI: 1%- 29%) in the absence of TF. Clot times at pH 7.0 decreased 32% (95% CI: 15%-49%) in TF-initiated blood and 51% (95% CI: 35%-68%) in the absence of TF. Conclusions Despite reported decreased procoagulant catalysis at pH 7.0, clot formation dynamics are slightly enhanced in blood ex vivo and suppression of thrombin generation is not observed. A decrease in antithrombin reactivity is one potential mechanism contributing to these outcomes.

Anticoagulant effects of statins and their clinical implications.

There is evidence indicating that statins (3-hydroxy-methylglutaryl coenzyme A reductase inhibitors) may produce several cholesterol-independent antithrombotic effects. In this review, we provide an update on the current understanding of the interactions between statins and blood coagulation and their potential relevance to the prevention of venous thromboembolism (VTE). Anticoagulant properties of statins reported in experimental and clinical studies involve decreased tissue factor expression resulting in reduced thrombin generation and attenuation of pro-coagulant reactions catalysed by thrombin, such as fibrinogen cleavage, factor V and factor XIII activation, as well as enhanced endothelial thrombomodulin expression, resulting in increased protein C activation and factor Va inactivation. Observational studies and one randomized trial have shown reduced VTE risk in subjects receiving statins, although their findings still generate much controversy and suggest that the most potent statin rosuvastatin exerts the largest effect.

Measuring the mechanical properties of blood clots formed via the tissue factor pathway of coagulation.

Thrombelastography (TEG) is a method that is used to conduct global assays that monitor fibrin formation and fibrinolysis and platelet aggregation in whole blood. The purpose of this study was to use a well-characterized tissue factor (Tf) reagent and contact pathway inhibitor (corn trypsin inhibitor, CTI) to develop a reproducible thrombelastography assay. In this study, blood was collected from 5 male subjects (three times). Clot formation was initiated in whole blood with 5 pM Tf in the presence of CTI, and fibrinolysis was induced by adding tissue plasminogen activator (tPA). Changes in viscoelasticity were then monitored by TEG. In quality control assays, our Tf reagent, when used at 5 pM, induced coagulation in whole blood in 3.93 ± 0.23 min and in plasma in 5.12 ± 0.23 min (n=3). In TEG assays, tPA significantly decreased clot strength (maximum amplitude, MA) in all individuals but had no effect on clot time (R time). The intraassay variability (CVa<10%) for R time, angle, and MA suggests that these parameters reliably describe the dynamics of fibrin formation and degradation in whole blood. Our Tf reagent reproducibly induces coagulation, making it an ideal tool to quantify the processes that contribute to mechanical clot strength in whole blood.

Characteristics of fibrin formation and clot stability in individuals with congenital type IIb protein C deficiency.

Many studies have shown that unregulated or excessive thrombin formation is potentially a cause of thrombosis; however, studies examining processes that contribute to fibrin stabilization in individuals predisposed to thrombosis are limited. In this study, we investigate a family with familial thrombosis via type IIb protein C (PC) deficiency. Using contact pathway inhibited whole blood, thrombin generation, fibrin clot formation and factor (f)XIII activation were monitored over time in control (n=5) and PC deficient (n=4, 34 - 69% PC by activity) subjects. The dynamics of thrombin generation varied significantly with the time required to reach the maximal rate of thrombin-antithrombin formation being much shorter in PC deficiency (5.8 ± 0.4 minutes) than in controls (9.7 ± 0.4 minutes; p < 0.001). PC deficient clots were significantly heavier than control clots (p < 0.001) and this difference could not be contributed exclusively to differences in fibrinogen levels between groups. FXIII was consumed faster in PC deficient subjects (23.7 ± 2.0 nM/minute) than in controls (5.1 ± 1.5 nM/minute; p < 0.0001) suggesting increased fXIII activation and incorporation of fXIIIa substrates into the clot. In plasma, the clot lysis time was increased in PC deficiency by both TAFIa dependent and independent mechanisms. Since PC deficient clots are both denser and show a greater degree of resistance to fibrinolysis, these clots would likely resist fibrinolysis and potentiate fibrin deposition observed in thrombosis.

Thrombin activatable fibrinolysis inhibitor activation and bleeding in haemophilia A.

Individuals with haemophilia A exhibit bleeding tendencies that are not always predicted by their factor (F)VIII level. It has been suggested that bleeding in haemophilia is due not only to defective prothrombin activation but also aberrant fibrinolysis. Thrombin activatable fibrinolysis inhibitor (TAFI) activation was measured in tissue factor (TF)-initiated blood coagulation in blood samples of 28 haemophiliacs and five controls. Reactions were quenched over time with FPRck and citrate and assayed for TAFIa and thrombin-antithrombin (TAT). The TAFIa potential (TP), TAFI activation rate and the TAFIa level at 20 min (TAFIa(20 min)) was extracted from the TAFI activation progress curve. In general, the time course of TAFI activation follows thrombin generation regardless of FVIII activity and as expected the rate of TAFI activation and TP decreases as FVIII decreases. The magnitude of TP was similar among the control subjects and subjects with <11% FVIII. In severe subjects with <1% FVIII at the time of blood collection, the TAFIa(20 min) was inversely and significantly correlated with haemarthrosis (-0.77, P = 0.03) and total bleeds (-0.75, P = 0.03). In all cases, TAFIa(20 min) was more strongly correlated with bleeding than TAT levels at 20 min. Overall, this study shows that TAFI activation in whole blood can be quantified and related to the clinical bleeding phenotype. Measuring TAFIa along with thrombin generation can potentially be useful to evaluate the differential bleeding phenotype in haemophilia A.

The influence of prophylactic factor VIII in severe haemophilia A.

Haemophilia A individuals displaying a similar genetic defect have heterogeneous clinical phenotypes. Our objective was to evaluate the underlying effect of exogenous factor (f)VIII on tissue factor (Tf)-initiated blood coagulation in severe haemophilia utilizing both empirical and computational models. We investigated twenty-five clinically severe haemophilia A patients. All individuals were on fVIII prophylaxis and had not received fVIII from 0.25 to 4 days prior to phlebotomy. Coagulation was initiated by the addition of Tf to contact-pathway inhibited whole blood ± an anti-fVIII antibody. Aliquots were quenched over 20 min and analyzed for thrombin generation and fibrin formation. Coagulation factor levels were obtained and used to computationally predict thrombin generation with fVIII set to either zero or its value at the time of the draw. As a result of prophylactic fVIII, at the time of the blood draw, the individuals had fVIII levels that ranged from <1% to 22%. Thrombin generation (maximum level and rate) in both empirical and computational systems increased as the level of fVIII increased. FXIII activation rates also increased as the fVIII level increased. Upon suppression of fVIII, thrombin generation became comparable in both systems. Plasma composition analysis showed a negative correlation between bleeding history and computational thrombin generation in the absence of fVIII. Residual prophylactic fVIII directly causes an increase in thrombin generation and fibrin cross-linking in individuals with clinically severe haemophilia A. The combination of each individual's coagulation factors (outside of fVIII) determine each individual's baseline thrombin potential and may affect bleeding risk.

Activated protein C inhibitor for correction of thrombin generation in hemophilia A blood and plasma1.

BACKGROUND: Replacement therapy for hemophilic patient treatment is costly, because of the high price of pharmacologic products, and is not affordable for the majority of patients in developing countries. OBJECTIVE: To generate and evaluate low molecular weight agents that could be useful for hemophilia treatment. METHODS: Potential agents were generated by synthesizing specific inhibitors [6-(Lys-Lys-Thr-[homo]Arg)amino-2-(Lys[carbobenzoxy]-Lys[carbobenzoxy]-O-benzyl)naphthalenesulfonamide] (PNASN-1)] for activated protein C (APC) and tested in plasma and fresh blood from hemophilia A patients. RESULTS: In the activated partial thromboplastin time-based APC resistance assay, PNASN-1 partially neutralized the effect of APC. In calibrated automated thrombography, PNASN-1 neutralized the effect of APC on thrombin generation in normal and congenital factor VIII-deficient plasma (FVIII:C < 1%). The addition of PNASN-1 to tissue factor-triggered (5 pm) contact pathway-inhibited fresh blood from 15 hemophilia A patients with various degrees of FVIII deficiency (FVIII:C < 1-51%) increased the maximum level of thrombin generated from 78 to 162 nm, which approached that observed in blood from a healthy individual (201 nm). PNASN-1 also caused a 47% increase in clot weight in hemophilia A blood. CONCLUSIONS: Specific APC inhibitors compensate to a significant extent for FVIII deficiency, and could be used for hemophilia treatment.

Anticoagulation by factor Xa inhibitors.

BACKGROUND: Therapeutic agents that regulate blood coagulation are critical to the management of thrombotic disorders, with the selective targeting of factor (F) Xa emerging as a promising approach. OBJECTIVE: To assess anticoagulant strategies targeting FXa. METHODS: A deterministic computational model of tissue factor (Tf)-initiated thrombin generation and two empirical experimental systems (a synthetic coagulation proteome reconstruction using purified proteins and a whole blood model) were used to evaluate clinically relevant examples of the two available types of FXa-directed anticoagulants [an antithrombin (AT)-dependent agent, fondaparinux, and an AT-independent inhibitor, Rivaroxaban] in experimental regimens relevant to long-term (suppression of new Tf-initiated events) and acute (suppression of ongoing coagulation processes) clinical applications. RESULTS: Computational representations of each anticoagulant's efficacy in suppressing thrombin generation over a range of anticoagulant concentrations in both anticoagulation regimens were validated by results from corresponding empirical reconstructions and were consistent with those recommended for long-term and acute clinical applications, respectively. All three model systems suggested that Rivaroxaban would prove more effective in the suppression of an ongoing coagulation process than fondaparinux, reflecting its much higher reactivity toward the prothrombinase complex. CONCLUSION: The success of fondaparinux in acute settings in vivo is not explained solely by its properties as an FXa inhibitor. We have reported that FIXa contributes to the long-term capacity of clot-associated catalysts to restart a coagulation process, suggesting that the enhanced anti-FIXa activity of fondaparinux-AT may be critical to its success in acute settings in vivo.

Thrombin generation and bleeding in haemophilia A.

Haemophilia A displays phenotypic heterogeneity with respect to clinical severity. The aim of this study was to determine if tissue factor (TF)-initiated thrombin generation profiles in whole blood in the presence of corn trypsin inhibitor (CTI) are predictive of bleeding risk in haemophilia A. We studied factor(F) VIII deficient individuals (11 mild, 4 moderate and 12 severe) with a well-characterized 5-year bleeding history that included haemarthrosis, soft tissue haematoma and annual FVIII concentrate usage. This clinical information was used to generate a bleeding score. The bleeding scores (range 0-32) were separated into three groups (bleeding score groupings: 0, 0 and < or = 9.6, >9.6), with the higher bleeding tendency having a higher score. Whole blood collected by phlebotomy and contact pathway suppressed by 100 microg mL(-1) CTI was stimulated to react by the addition of 5 pM TF. Reactions were quenched at 20 min by inhibitors. Thrombin generation, determined by enzyme-linked immunosorbent assay for thrombin-antithrombin was evaluated in terms of clot time (CT), maximum level (MaxL) and maximum rate (MaxR) and compared to the bleeding score. Data are shown as the mean+/-SD. MaxL was significantly different (P < 0.001) between the groups: 504 +/- 114, 315 +/- 117 and 194 +/- 91 nM; with higher thrombin concentrations in the groups with lower bleeding scores. MaxR was higher in the groups with a lower bleeding score; 97 +/- 51, 86 +/- 60 and 39 +/- 16 nM min(-1) (P = 0.09). No significant difference was detected in CT among the groups, 5.6 +/- 1.3, 4.7 +/- 0.7 and 5.6 +/- 1.3 min. Our empirical study in CTI-inhibited whole blood shows that the MaxL of thrombin generation appears to correlate with the bleeding phenotype of haemophilia A.

Empirical and theoretical phenotypic discrimination.

We have developed an integrated approach that combines empirical and computational methodologies to define an individual's thrombin phenotype. We have evaluated the process of thrombin generation in healthy individuals and individuals with defined pathologies in order to develop general criteria relevant to assess an individual's propensity for hemorrhage or thrombosis. Three complementary hypotheses have emerged from our work: (i) compensation by the ensemble of other coagulation proteins in individuals with specific factor deficiencies can 'normalize' an individual's thrombin generation process and represents a rationale for their unexpected phenotype; (ii) individuals with clinically unremarkable factor levels may present thrombin generation profiles typical of individuals with hemostatic complications; and (iii) in some hemostatic disorders a specific pattern of expression of a small ensemble of coagulation factors may be sufficient to explain the overall phenotype.

Discordant fibrin formation in hemophilia.

BACKGROUND: The conversion of fibrinogen to fibrin and its crosslinking to form a stable clot are key events in providing effective hemostasis. OBJECTIVES: To evaluate the relationship of fibrinopeptide (FP) release and factor (F) XIII activation in whole blood from hemophiliacs. PATIENTS/METHODS: We investigated FPA and FPB release, FXIII activation and fibrin mass in tissue factor-initiated coagulation in whole blood from individuals with hemophilia and healthy subjects. RESULTS: In hemophiliacs, the rates of fibrin formation were delayed as compared to healthy individuals. FPA/FPB release and FXIII activation were decreased in hemophiliacs vs. healthy individuals: 5.4 +/- 0.7 microM min(-1) to 1.7 +/- 0.4 microM min(-1) (P = 0.003), 2.3 +/- 0.6 microM min(-1) to 0.5 +/- 0.1 microM min(-1) (P = 0.025), and 12.1 +/- 0.7 nM min(-1) to 3.1 +/- 0.7 nM min(-1) (P < 0.0005), respectively. More FPA was released in hemophiliacs (6.6 +/- 1.2 microM) prior to clot time (CT) than in healthy individuals (2.6 +/- 0.4 microM, P = 0.013), whereas FPB and activated FXIII levels remained comparable. FXIII activation, which normally coincides with FPA release, was delayed in hemophiliacs. At CT in normal blood, the FPA concentration was 2.6-fold higher than that of FPB (P = 0.003), whereas in hemophiliacs this ratio was increased to 6.6-fold (P = 0.001). CONCLUSIONS: These data suggest that essential dynamic correlations exist between the presentations of fibrin I, fibrin II, and FXIIIa. The 'discordance' of fibrin formation in hemophiliacs results in clots that are more soluble than normal (43% lower mass; P = 0.02). The resulting poor physical clot strength probably plays a crucial role in the pathology of hemophilia.

Atorvastatin and quinapril inhibit blood coagulation in patients with coronary artery disease following 28 days of therapy.

BACKGROUND: We evaluated the antithrombotic effects of statins and angiotensin-converting enzyme inhibitor (ACEI) drugs in patients with coronary artery disease (CAD). METHODS AND RESULTS: Blood coagulation at the site of microvascular injury was assessed in 26 males with CAD before and after treatment with quinapril (10 mg day-1; n=13) or atorvastatin (40 mg day-1; n=13) for 4 weeks and an additional 4 weeks of combined therapy (quinapril+atorvastatin). Rates of prothrombin and factor V activation (FVa), fibrinogen (Fbg) cleavage and FVa inactivation showed that both quinapril and atorvastatin decreased the rates of: formation of thrombin B-chain (by 30.6%, P=0.007; and by 34.3%, P=0.003), formation of thrombin-antithrombin complexes (by 30.4%, P=0.0002; and by 40%, P=0.001), FV activation (by 19.1%, P=0.03; and by 21.8%, P=0.005) and Fbg depletion (by 29.2%, P=0.004; and by 32.7%, P=0.001). Atorvastatin alone accelerated FVa inactivation (P=0.005). A further 4 weeks of combined therapy enhanced most anticoagulant effects only when atorvastatin was added to quinapril. CONCLUSIONS: In CAD patients, atorvastatin and quinapril slowed blood clotting at the site of microvascular injury after 28 days of therapy. Addition of atorvastatin to quinapril, but not quinapril to the statin, enhanced the anticoagulant effects. Our findings might help explain the reduced risk of myocardial infarction or stroke in patients treated with statins and/or ACEIs and the lack of clinical benefits from ACEI added to prior statin therapy in patients at cardiovascular risk.

Thrombin generation profiles in deep venous thrombosis.

BACKGROUND: Reliable markers and methods to predict risk for thrombosis are essential to clinical management. OBJECTIVE: Using an integrated approach that defines an individual's comprehensive coagulation phenotype might prove valuable in identifying individuals at risk for experiencing a thrombotic event. METHODS: Using a numerical simulation model, we generated tissue factor (TF) initiated thrombin curves using coagulation factor levels from the Leiden Thrombophilia Study population and evaluated thrombotic risk, by sex, age, smoking, alcohol consumption, body mass index (BMI) and oral contraceptive (OC) use. We quantitated the initiation, propagation and termination phases of each individuals' comprehensive TF-initiated thrombin generation curve by the parameters: time to 10 nm of thrombin, maximum time, level and rate (MaxR) of thrombin generated and total thrombin. RESULTS: The greatest risk association was obtained using MaxR; with a 2.6-fold increased risk at MaxR exceeding the 90th percentile. The odds ratio (OR) for MaxR was 3.9 in men, 2.1 in women, and 2.9 in women on OCs. The association of risk with thrombin generation did not differ by age (OR:2.8 OR:2.5), BMI (OR:2.9 OR:2.3) or alcohol use. In both numerical simulations and empirical synthetic plasma, OC use created extreme shifts in thrombin generation in both control women and women with a prior thrombosis, with a larger shift in thrombin generation in control women. This suggests an interaction of OC use with underlying prothrombotic abnormalities. CONCLUSIONS: Thrombin generation based upon the individual's blood composition is associated with the risk for thrombosis and may be useful as a predictive marker for evaluating thrombosis on an individual basis.

Evaluation of the profile of thrombin generation during the process of whole blood clotting as assessed by thrombelastography.

The objective of this study was to evaluate the possibility of linking the tracing of whole blood clotting in a thrombelastograph (TEG) hemostasis system with the generation of thrombin assessed by thrombin/antithrombin complex (TAT). Citrated whole blood containing corn trypsin inhibitor from volunteers was clotted in the presence of CaCl2 and tissue factor. Clotting was monitored with the eight channels of a TEG system. At different time points, the whole blood TEG reaction cups were kept in a cold quenching solution, centrifuged, and the supernatants were kept at -80 degrees C until assayed for TAT by ELISA. The total thrombus generation (TTG) was calculated from the first derivative of the TEG waveform and was compared with thrombin generation measured by TAT. The two vector values--the TAT thrombin generation data and the corresponding TEG TTG--were analyzed using Pearson correlation coefficients (r) and linear, non-linear and natural log (ln) transformation of TAT values for least-squares goodness-of-fit curves. The best least-squares fit is an exponential curve. Linearizing using the ln of the TAT thrombin generation variable produces the same r (0.94) as of the exponential curve. The prediction equation is y = 8.0465 + 0.0005x (P < or = 0.0001), where y is the TAT thrombin generation variable in the ln transformation and x is the TEG TTG variable. The high magnitude of r and the high significance of the prediction equation demonstrate the high efficacy of the prediction of TAT thrombin generation by the use of TEG TTG.

Thrombin generation: phenotypic quantitation.

An individual's ability to generate thrombin following tissue factor stimulus was evaluated in 13 healthy male donors in a 6-month study. Thrombin generation in whole blood collected by phlebotomy, contact pathway suppressed by the presence of 100 micro g mL-1 corn trypsin inhibitor, was initiated by the addition of 5 pm tissue factor/10 nm phospholipid. Reactions were quenched at 20 min by the addition of an ethylenediaminetetraacetic acid (EDTA), benzamidine, FPRck cocktail. Thrombin generation was determined by an ELISA for thrombin-antithrombin III (TAT) complex formation. Results showed that the levels of TAT observed varied from 245 to 775 nm. Thrombin production was consistent within each individual, CVi = 11.6%, but varied significantly within the group, CVg = 25.2%, and correlated inversely with an individual's clotting time (r = - 0.54, P = 0.07). No correlations were individually observed between TAT and C-reactive protein, antithrombin III, factors II, V, VII, VIII, IX and X, fibrinogen and prothrombin time. However, computer simulations, which integrated each individual's coagulation factor levels using the Speed Rx method (Hockin et al., J Biol Chem 2002; 277: 18322), predicted maximum active thrombin levels (ranging from calculated values of 220-500 nm) consistent with the empirically determined values. Overall, these data suggest that thrombin generated in whole blood exclusively by tissue factor stimulation can be used as an integrative phenotypic marker to determine an individual's response to a tissue factor challenge.