Abnormalities of Coagulation and Fibrinolysis Assessed by Thromboelastometry in an Endotoxic Shock Model in Piglets Treated with Nitric Oxide and Hydrocortisone.

This is an animal model study to investigate changes in hemostasis during endotoxemic shock and to determine whether the combination of inhaled nitric oxide (iNO) + intravenous hydrocortisone had an effect on clot formation and fibrinolysis. iNO selectively decreases pulmonary artery pressure, without affecting cardiac index or systemic vascular resistance; however, the results of studies on the possible consequences of iNO administration on coagulation are inconsistent and require further research. Thirty-four piglets were included. Administering endotoxin caused severe hypodynamic shock. Half of the animals received iNO (30 ppm) + hydrocortisone, starting 3 h after endotoxin infusion and continuing to the end of the study. All animals developed coagulation disorders, manifested by a tendency to hypocoagulation; at the same time, fibrinolysis was impaired. Coagulation and fibrinolysis disorders persisted after endotoxin infusion was discontinued, with worse severity in the animals that died before the study was terminated. Administering iNO + hydrocortisone did not cause further changes in coagulation and fibrinolysis parameters, either during or after the endotoxin challenge, suggesting that potential therapeutic interventions with iNO to lower pulmonary arterial pressure will not affect hemostasis.

Ultrasound combined with urokinase under key-shaped bone window enhances blood clot lysis in an in vitro model of spontaneous intracerebral hemorrhage.

OBJECTIVE: Minimally invasive surgery for spontaneous intracerebral hemorrhage is impeded by inadequate lysis of the target blood clot. Ultrasound is thought to expedite intravascular thrombolysis, thereby facilitating vascular recanalization. However, the impact of ultrasound on intracerebral blood clot lysis remains uncertain. This study aimed to explore the feasibility of combining ultrasound with urokinase to enhance blood clot lysis in an in vitro model of spontaneous intracerebral hemorrhage. METHODS: The blood clots were divided into four groups: control group, ultrasound group, urokinase group, and ultrasound + urokinase group. Using our experimental setup, which included a key-shaped bone window, we simulated a minimally invasive puncture and drainage procedure for spontaneous intracerebral hemorrhage. The blood clot was then irradiated using ultrasound. Blood clot lysis was assessed by weighing the blood clot before and after the experiment. Potential adverse effects were evaluated by measuring the temperature variation around the blood clot in the ultrasound + urokinase group. RESULTS: A total of 40 blood clots were observed, with 10 in each experimental group. The blood clot lysis rate in the ultrasound group, urokinase group, and ultrasound + urokinase group (24.83 ± 4.67%, 47.85 ± 7.09%, 61.13 ± 4.06%) was significantly higher than that in the control group (16.11 ± 3.42%) (p = 0.02, p < 0.001, p < 0.001). The blood clot lysis rate in the ultrasound + urokinase group (61.13 ± 4.06%) was significantly higher than that in the ultrasound group (24.83 ± 4.67%) (p < 0.001) or urokinase group (47.85 ± 7.09%) (p < 0.001). In the ultrasound + urokinase group, the mean increase in temperature around the blood clot was 0.26 ± 0.15°C, with a maximum increase of 0.38 ± 0.09°C. There was no significant difference in the increase in temperature regarding the main effect of time interval (F = 0.705, p = 0.620), the main effect of distance (F = 0.788, p = 0.563), or the multiplication interaction between time interval and distance (F = 1.100, p = 0.342). CONCLUSIONS: Our study provides evidence supporting the enhancement of blood clot lysis in an in vitro model of spontaneous intracerebral hemorrhage through the combined use of ultrasound and urokinase. Further animal experiments are necessary to validate the experimental methods and results.

Injectable polyamide-amine dendrimer-crosslinked meloxicam-containing poly-γ-glutamic acid hydrogel for prevention of postoperative tissue adhesion through inhibiting inflammatory responses and balancing the fibrinolytic system.

Establishing a physical barrier between the peritoneum and the cecum is an effective method to reduce the risk of postoperative abdominal adhesions. Meloxicam (MX), a nonsteroidal anti-inflammatory drug has also been applied to prevent postoperative adhesions. However, its poor water solubility has led to low bioavailability. Herein, we developed an injectable hydrogel as a barrier and drug carrier for simultaneous postoperative adhesion prevention and treatment. A third-generation polyamide-amine dendrimer (G3) was exploited to dynamically combine with MX to increase the solubility and the bioavailability. The formed G3@MX was further used to crosslink with poly-γ-glutamic acid (γ-PGA) to prepare a hydrogel (GP@MX hydrogel) through the amide bonding. In vitro and in vivo experiments evidenced that the hydrogel had good biosafety and biodegradability. More importantly, the prepared hydrogel could control the release of MX, and the released MX is able to inhibit inflammatory responses and balance the fibrinolytic system in the injury tissues in vivo. The tunable rheological and mechanical properties (compressive moduli: from âˆ¼ 57.31 kPa to âˆ¼ 98.68 kPa;) and high anti-oxidant capacity (total free radical scavenging rate of âˆ¼ 94.56 %), in conjunction with their syringeability and biocompatibility, indicate possible opportunities for the development of advanced hydrogels for postoperative tissue adhesions management.

Comparison of coagulation and fibrinolysis in Irish Wolfhounds and age-matched control dogs using tissue plasminogen activator-augmented viscoelastic testing.

OBJECTIVE: To determine if Irish Wolfhounds (IWs), like other sighthounds, are hyperfibrinolytic compared with nonsighthound dogs using 2 native and tissue plasminogen activator (tPA)-enhanced viscoelastic assays, one that is whole blood-based (viscoelastic coagulation monitor [VCM]) and the other that is plasma-based thromboelastography (TEG). DESIGN: Cohort study. SETTING: University teaching hospital. ANIMALS: A convenience sample of 27 IWs recruited from the Irish Wolfhound Association of New England Specialty and the local community, and 27 healthy, age-matched, large-breed control dogs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood samples including CBC, biochemistry, traditional coagulation, and viscoelastic testing were collected from IWs and control dogs. Twelve IWs had viscoelastic testing. IWs had lower fibrinogen concentrations (215.5 ± 57.8 vs 251.4 ± 64.5 mg/dL, P = 0.034) and formed weaker clots on both whole-blood VCM and plasma TEG assays (maximum clot firmness [VCM-MCF] = 39.4 [25.1-48.8] vs 48.5 [34.6-57.3], P = 0.0042; maximum amplitude [TEG-MA] = 22.7 [14.7-33.6] vs 32.2 [26.9-42.0], P < 0.0001). IWs were hyperfibrinolytic compared with control dogs on VCM whole-blood assays, with 25 U/mL tPA (lysis at 30 min [VCM-LI30] = 68.1 [0-100] vs\ 99.9 [63.3-100], P = 0.0009; lysis at 45 min [VCM-LI45] = 31.0 [0-100] vs 98.1 [38.4-100], P = 0.0002) but hypofibrinolytic compared with controls on TEG plasma assays with 50 U/mL tPA (lysis at 30 min [TEG-LY30] = 45.7 [4.6-94.6] vs 93.7 [12.3-96.5], P = 0.0004; lysis at 60 min [TEG-LY60] = 68.7 [29.7-96.8] vs 95.7 [34.4-97.6], P = 0.0003). Minimal fibrinolysis was measured on whole-blood VCM or plasma TEG assays without the addition of tPA, and there were no differences between the 2 groups. CONCLUSIONS: Weaker clots were found in IWs than control dogs. With the addition of tPA, IWs had evidence of hyperfibrinolysis on whole-blood VCM assays and hypofibrinolysis on plasma TEG assays compared with control dogs. Without the addition of tPA, however, both groups of dogs showed minimal fibrinolysis on viscoelastic testing.

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model.

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates. The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.

Point-of-care testing for tranexamic acid efficacy: a proof-of-concept study in cardiac surgical patients.

BACKGROUND: Low-dose tranexamic acid (TXA) has been recently recommended for cardiopulmonary bypass (CPB) to reduce associated complications. Although point-of-care laboratory tests for TXA concentrations are unavailable, a novel TPA-test on the ClotPro® system can measure TXA-induced inhibition of fibrinolysis. We evaluated the performance of the TPA-test in vitro and in patients undergoing surgery requiring CPB. METHODS: Blood samples were obtained from six volunteers for in vitro evaluation of tissue plasminogen activator (tPA)-triggered fibrinolysis and the effects of TXA. This was followed by an observational study in 20 cardiac surgery patients to assess clinical effects of TXA on the TPA-test. RESULTS: Hyperfibrinolysis induced by tPA was inhibited by TXA ≥2 mg L-1 in a concentration-dependent manner, and was completely inhibited at TXA ≥10 mg L-1. In patients undergoing CPB, antifibrinolytic effect was detectable on TPA-test parameters after a 0.1 g bolus of TXA at the end of CPB, and complete inhibition of fibrinolysis was obtained with TXA ≥0.5 g. The antifibrinolytic effects of 1 g TXA on TPA-test parameters were gradually attenuated over 18 h after surgery. However, the fibrinolytic inhibition continued in four patients with estimated glomerular filtration rate (eGFR) ≤30 ml min-1 1.73 m-2. The eGFR had strong correlations with TPA-test parameters at 18 h after surgery (r=0.86-0.92; P<0.0001). CONCLUSIONS: The TPA-test is sensitive to low concentrations of TXA and serves as a practical monitoring tool for postoperative fibrinolytic activity in cardiac surgery patients. This test might be particularly useful in patients with severe renal impairment.

Platelet activity, coagulation, and fibrinolysis in long-term users of anabolic-androgenic steroids compared to strength-trained athletes.

INTRODUCTION: Use of anabolic-androgenic steroids (AAS) is associated with adverse cardiovascular (CV) effects, including potential prothrombotic effects. This study aimed to assess platelet activation and aggregation, coagulation, and fibrinolysis, in long-term AAS users compared to non-using strength-trained athletes. MATERIALS AND METHODS: Thirty-seven strength-trained men using AAS were compared to seventeen non-using professional strength-trained athletes at similar age (median 33 years). AAS use was verified by blood and urine analyses. Platelet Function Analyzer 100 (PFA-100) and whole blood impedance aggregometry with thrombin, arachidonic acid, and ADP as agonists, were performed to evaluate platelet aggregation. ELISA methods were used for markers of platelet activation. Fibrinogen, D-dimer, the coagulation inhibitors protein S and C activity, and antithrombin were measured by routine. Fibrinolysis was evaluated by Plasminogen Activator Inhibitor-1 (PAI-1) activity. RESULTS: There were no significant differences in platelet aggregation between the two groups. Von Willebrand factor was lower among the AAS users (p < 0.01), and P-Selectin was slightly higher (p = 0.05), whereas CD40 Ligand, ß-thromboglobulin, and thrombospondin did not differ significantly. No differences were found in the assessed coagulation inhibitors. Higher D-dimer levels (p < 0.01) and lower PAI-1 activity (p < 0.01) were found among the AAS users. CONCLUSIONS: The investigated long-term users of AAS did not exhibit elevated platelet activity compared to strength-trained non-using athletes. However, AAS use was associated with higher D-dimer levels and lower PAI-1 activity. These findings suggest that any prothrombotic effect of long-term AAS use may predominantly involve other aspects of the hemostatic system than blood platelets.

The effects of emicizumab on in vitro coagulation and fibrinolysis parameters in patients with disseminated intravascular coagulation with and without addition of anti-FVIII antibody.

BACKGROUND: Emicizumab (Emi) is used as haemostatic prophylaxis for patients with haemophilia A (PwHA). Disseminated intravascular coagulation (DIC) is a condition characterized by persistent systemic activation of coagulation, but there is yet no information on coagulation and fibrinolysis potentials in Emi-treated PwHA with DIC. AIM: To examine the effect of Emi on coagulation and fibrinolysis potentials in HA-model DIC plasmas. METHODS: Plasma from a patient with sepsis-DIC (seven patients) was treated with anti-factor (F)VIII monoclonal antibody (HA-model DIC plasma) and incubated with Emi (50 µg/mL). The plasma was then assessed using clot-fibrinolysis waveform analysis (CFWA). Coagulation and fibrinolysis parameters were expressed as ratios relative to normal plasma (|min1|-ratio and |FL-min1|-ratio, respectively). PATIENTS AND RESULTS: In case 1, coagulant potential was slightly high and fibrinolytic potential was extremely low, presenting a coagulant-dominant state (|min1|-ratio/|FL-min1|-ratio: 1.1/.38). In cases 2-5, fibrinolytic potential was not suppressed, but there were marked hypercoagulant potentials, indicating relative coagulant-dominant states. In case 6, coagulant and fibrinolytic potentials were increased but well balanced (|min1|-ratio/|FL-min1|-ratio: 1.38/1.28). In case 7, both potentials were severely deteriorated in not only CFWA but also the thrombin/plasmin generation assay. The addition of Emi into the HA-model DIC plasmas increased |min1|-ratio values in all cases, but the coagulant potentials did not exceed the initial ones (DIC plasma before treatment with anti-FVIII antibody). CONCLUSIONS: The presence of Emi in the HA-model DIC plasma improved coagulation potentials, but did not increase coagulation potentials beyond those of DIC plasma in non-HA states.

The effect of ultrasound-assisted thrombolysis studied in blood-on-a-chip.

BACKGROUND: Thromboembolism, which leads to pulmonary embolism and ischemic stroke, remains one of the main causes of death. Ultrasound-assisted thrombolysis (UAT) is an effective thrombolytic method. However, further studies are required to elucidate the mechanism of ultrasound on arterial and venous thrombi. METHODS: We employed the blood-on-a-chip technology to simulate thrombus formation in coronary stenosis and deep vein valves. Subsequently, UAT was conducted on the chip to assess the impact of ultrasound on thrombolysis under varying flow conditions. Real-time fluorescence was used to assess thrombolysis and drug penetration. Finally, scanning electron microscopy and immunofluorescence were used to determine the effect of ultrasound on fibrinolysis. RESULTS: The study revealed that UAT enhanced the thrombolytic rate by 40% in the coronary stenosis chip and by 10% in the deep venous valves chip. This enhancement is attributed to the disruption of crosslinked fibrin fibers by ultrasound, leading to increased urokinase diffusion within the thrombus and accumulation of plasminogen on the fibrinogen α chain. Moreover, the acceleration of the dissolution rate of thrombi in the venous valve chip by ultrasound was not as significant as that in the coronary stenosis chip. CONCLUSION: These findings highlight the differential impact of ultrasound on thrombolysis under various flow conditions and emphasize the valuable role of the blood-on-a-chip technology in exploring thrombolysis mechanisms.

Histone content, and thus DNA content, is associated with differential in vitro lysis of acute ischemic stroke clots.

BACKGROUND: Fibrin, von Willebrand factor, and extracellular DNA from neutrophil extracellular traps all contribute to acute ischemic stroke thrombus integrity. OBJECTIVES: In this study, we explored how the proteomic composition of retrieved thromboemboli relates to susceptibility to lysis with distinct thrombolytics. METHODS: Twenty-six retrieved stroke thromboemboli were portioned into 4 segments, with each subjected to 1 hour of in vitro lysis at 37 °C in 1 of 4 solutions: tissue plasminogen activator (tPA), tPA + von Willebrand factor-cleaving ADAMTS-13, tPA + DNA-cleaving deoxyribonuclease (DNase) I, and all 3 enzymes. Lysis, characterized by the percent change in prelysis and postlysis weight, was compared across the solutions and related to the corresponding abundance of proteins identified on mass spectrometry for each of the thromboemboli used in lysis. RESULTS: Solutions containing DNase resulted in approximately 3-fold greater thrombolysis than that with the standard-of-care tPA solution (post hoc Tukey, P < .01 for all). DNA content was directly related to lysis in solutions containing DNase (Spearman's ρ > 0.39 and P < .05 for all significant histones) and inversely related to lysis in solutions without DNase (Spearman's ρ < -0.40 and P < .05 for all significant histones). Functional analysis suggests distinct pathways associated with susceptibility to thrombolysis with tPA (platelet-mediated) or DNase (innate immune system-mediated). CONCLUSION: This study demonstrates synergy of DNase and tPA in thrombolysis of stroke emboli and points to DNase as a potential adjunct to our currently limited selection of thrombolytics in treating acute ischemic stroke.

Therapeutic Strategies for Disseminated Intravascular Coagulation Associated with Aortic Aneurysm.

Aortic aneurysms are sometimes associated with enhanced-fibrinolytic-type disseminated intravascular coagulation (DIC). In enhanced-fibrinolytic-type DIC, both coagulation and fibrinolysis are markedly activated. Typical cases show decreased platelet counts and fibrinogen levels, increased concentrations of fibrin/fibrinogen degradation products (FDP) and D-dimer, and increased FDP/D-dimer ratios. Thrombin-antithrombin complex or prothrombin fragment 1 + 2, as markers of coagulation activation, and plasmin-α2 plasmin inhibitor complex, a marker of fibrinolytic activation, are all markedly increased. Prolongation of prothrombin time (PT) is not so obvious, and the activated partial thromboplastin time (APTT) is rather shortened in some cases. As a result, DIC can be neither diagnosed nor excluded based on PT and APTT alone. Many of the factors involved in coagulation and fibrinolysis activation are serine proteases. Treatment of enhanced-fibrinolytic-type DIC requires consideration of how to control the function of these serine proteases. The cornerstone of DIC treatment is treatment of the underlying pathology. However, in some cases surgery is either not possible or exacerbates the DIC associated with aortic aneurysm. In such cases, pharmacotherapy becomes even more important. Unfractionated heparin, other heparins, synthetic protease inhibitors, recombinant thrombomodulin, and direct oral anticoagulants (DOACs) are agents that inhibit serine proteases, and all are effective against DIC. Inhibition of activated coagulation factors by anticoagulants is key to the treatment of DIC. Among them, DOACs can be taken orally and is useful for outpatient treatment. Combination therapy of heparin and nafamostat allows fine-adjustment of anticoagulant and antifibrinolytic effects. While warfarin is an anticoagulant, this agent is ineffective in the treatment of DIC because it inhibits the production of coagulation factors as substrates without inhibiting activated coagulation factors. In addition, monotherapy using tranexamic acid in cases of enhanced-fibrinolytic-type DIC may induce fatal thrombosis. If tranexamic acid is needed for DIC, combination with anticoagulant therapy is of critical importance.

Marine Microbial Fibrinolytic Enzymes: An Overview of Source, Production, Biochemical Properties and Thrombolytic Activity.

Cardiovascular diseases (CVDs) have emerged as a major threat to global health resulting in a decrease in life expectancy with respect to humans. Thrombosis is one of the foremost causes of CVDs, and it is characterized by the unwanted formation of fibrin clots. Recently, microbial fibrinolytic enzymes due to their specific features have gained much more attention than conventional thrombolytic agents for the treatment of thrombosis. Marine microorganisms including bacteria and microalgae have the significant ability to produce fibrinolytic enzymes with improved pharmacological properties and lesser side effects and, hence, are considered as prospective candidates for large scale production of these enzymes. There are no studies that have evaluated the fibrinolytic potential of marine fungal-derived enzymes. The current review presents an outline regarding isolation sources, production, features, and thrombolytic potential of fibrinolytic biocatalysts from marine microorganisms identified so far.

Fibroblast growth factor-21 as a novel metabolic factor for regulating thrombotic homeostasis.

Fibroblast growth factor-21 (FGF-21) performs a wide range of biological functions in organisms. Here, we report for the first time that FGF-21 suppresses thrombus formation with no notable risk of bleeding. Prophylactic and therapeutic administration of FGF-21 significantly improved the degree of vascular stenosis and reduced the thrombus area, volume and burden. We determined the antithrombotic mechanism of FGF-21, demonstrating that FGF-21 exhibits an anticoagulant effect by inhibiting the expression and activity of factor VII (FVII). FGF-21 exerts an antiplatelet effect by inhibiting platelet activation. FGF-21 enhances fibrinolysis by promoting tissue plasminogen activator (tPA) expression and activation, while inhibiting plasminogen activator inhibitor 1 (PAI-1) expression and activation. We further found that FGF-21 mediated the expression and activation of tPA and PAI-1 by regulating the ERK1/2 and TGF-ß/Smad2 pathways, respectively. In addition, we found that FGF-21 inhibits the expression of inflammatory factors in thrombosis by regulating the NF-κB pathway.

(More than) doubling down: Effective fibrinolysis at a reduced rt-PA dose for catheter-directed thrombolysis combined with histotripsy.

Deep vein thrombosis is a major source of morbidity and mortality worldwide. For acute proximal deep vein thrombosis, catheter-directed thrombolytic therapy is an accepted method for vessel recanalization. Thrombolytic therapy is not without risk, including the potential for hemorrhagic bleeding that increases with lytic dose. Histotripsy is a focused ultrasound therapy that generates bubble clouds spontaneously in tissue at depth. The mechanical activity of histotripsy increases the efficacy of thrombolytic therapy at doses consistent with current pharmacomechanical treatments for venous thrombosis. The objective of this study was to determine the influence of lytic dose on histotripsy-enhanced fibrinolysis. Human whole blood clots formed in vitro were exposed to histotripsy and a thrombolytic agent (recombinant tissue plasminogen activator, rt-PA) in a venous flow model perfused with plasma. Lytic was administered into the clot via an infusion catheter at concentrations ranging from 0 (control) to 4.54 µg/mL (a common clinical dose for catheter-directed thrombolysis). Following treatment, perfusate samples were assayed for markers of fibrinolysis, hemolysis, and intact red blood cells and platelets. Fibrinolysis was equivalent between the common clinical dose of rt-PA (4.54 µg/mL) and rt-PA at a reduction to one-twentieth of the common clinical dose (0.23 µg/mL) when combined with histotripsy. Minimal changes were observed in hemolysis for treatment arms with or without histotripsy, potentially due to clot damage from insertion of the infusion catheter. Likewise, histotripsy did not increase the concentration of red blood cells or platelets in the perfusate following treatment compared to rt-PA alone. At the highest lytic dose, a refined histotripsy exposure scheme was implemented to cover larger areas of the clot. The updated exposure scheme improved clot mass loss and fibrinolysis relative to administration of lytic alone. Overall, the data collected in this study indicate the rt-PA dose can be reduced by more than a factor of ten and still promote fibrinolysis when combined with histotripsy.

The α-globin chain of hemoglobin potentiates tissue plasminogen activator induced hyperfibrinolysis in vitro.

BACKGROUND: Severe injury predisposes patients to trauma-induced coagulopathy, which may be subdivided by the state of fibrinolysis. Systemic hyperfibrinolysis (HF) occurs in approximately 25% of these patients with mortality as high as 70%. Severe injury also causes the release of numerous intracellular proteins, which may affect coagulation, one of which is hemoglobin, and hemoglobin substitutes induce HF in vitro. We hypothesize that the α-globin chain of hemoglobin potentiates HF in vitro by augmenting plasmin activity. METHODS: Proteomic analysis was completed on a pilot study of 30 injured patients before blood component resuscitation, stratified by their state of fibrinolysis, plus 10 healthy controls. Different concentrations of intact hemoglobin A, the α- and ß-globin chains, or normal saline (controls) were added to whole blood, and tissue plasminogen activator (tPA)-challenged thrombelastography was used to assess the degree of fibrinolysis. Interactions with plasminogen (PLG) were evaluated using surface plasmon resonance. Tissue plasminogen activator-induced plasmin activity was evaluated in the presence of the α-globin chain. RESULTS: Only the α- and ß-globin chains increased in HF patients (p < 0.01). The α-globin chain but not hemoglobin A or the ß-globin chain decreased the reaction time and significantly increased lysis time 30 on citrated native thrombelastographies (p < 0.05). The PLG and α-globin chain had interaction kinetics similar to tPA:PLG, and the α-globin chain increased tPA-induced plasmin activity. CONCLUSIONS: The α-globin chain caused HF in vitro by binding to PLG and augmenting plasmin activity and may represent a circulating "moonlighting" mediator released by the tissue damage and hemorrhagic shock inherent to severe injury. LEVEL OF EVIDENCE: Prognostic, level III.

Quantitation of thrombin-activatable fibrinolysis inhibitor in human plasma by isotope dilution mass spectrometry.

Measurement of Thrombin-activatable fibrinolysis inhibitor (TAFI) in human plasma is dependent on reproducible assays. To date, standards for measuring TAFI are frequently calibrated relative to pooled normal human plasma and arbitrarily assigned a potency of 100% TAFI, despite variation in TAFI concentrations between plasma pools. Alternatively, TAFI calibrators can be assigned a value in SI units but the approach used for value assignment is not consistent and furthermore, if purified TAFI is used to determine TAFI concentration in plasma, may be adversely affected by matrix effects. A TAFI plasma standard in mass units with traceability to the SI unit of mass is desirable. We report here the establishment of a quantitative mass spectrometry method for TAFI in plasma. Traceability is obtained by reference to calibrators that consist of blank plasma spiked with a defined amount of purified TAFI, value assigned by amino acid analysis. The calibrators are run alongside the samples, using the same preparation steps and conditions; an acetonitrile assisted tryptic digestion and multi-dimensional liquid chromatography (LC) separation followed by MRM-MS analysis. We measured the TAFI quantitatively in human plasma with reproducibility, reliability and precision, and demonstrated the applicability of this approach for value assigning a common reference standard.

Temporal Transitions in Fibrinolysis after Trauma: Adverse Outcome Is Principally Related to Late Hypofibrinolysis.

BACKGROUND: The relationship between late clinical outcomes after injury and early dynamic changes between fibrinolytic states is not fully understood. The authors hypothesized that temporal transitions in fibrinolysis states using rotational thromboelastometry (ROTEM) would aid stratification of adverse late clinical outcomes and improve understanding of how tranexamic acid modulates the fibrinolytic response and impacts mortality. METHODS: The authors conducted a secondary analysis of previously collected data from trauma patients enrolled into an ongoing prospective cohort study (International Standard Randomised Controlled Trial Number [ISRCTN] 12962642) at a major trauma center in the United Kingdom. ROTEM was performed on admission and at 24 h with patients retrospectively grouped into three fibrinolysis categories: tissue factor-activated ROTEM maximum lysis of less than 5% (low); tissue factor-activated ROTEM maximum lysis of 5 to 15% (normal); or tissue factor-activated ROTEM maximum lysis of more than 15% (high). Primary outcomes were multiorgan dysfunction syndrome and 28-day mortality. RESULTS: Seven-hundred thirty-one patients were included: 299 (41%) were treated with tranexamic acid and 432 (59%) were untreated. Two different cohorts with low-maximum lysis at 24 h were identified: (1) severe brain injury and (2) admission shock and hemorrhage. Multiple organ dysfunction syndrome was greatest in those with low-maximum lysis on admission and at 24 h, and late mortality was four times higher than in patients who remained normal during the first 24 h (7 of 42 [17%] vs. 9 of 223 [4%]; P = 0.029). Patients that transitioned to or remained in low-maximum lysis had increased odds of organ dysfunction (5.43 [95% CI, 1.43 to 20.61] and 4.85 [95% CI, 1.83 to 12.83], respectively). Tranexamic acid abolished ROTEM hyperfibrinolysis (high) on admission, increased the frequency of persistent low-maximum lysis (67 of 195 [34%]) vs. 8 of 79 [10%]; P = 0.002), and was associated with reduced early mortality (28 of 195 [14%] vs. 23 of 79 [29%]; P = 0.015). No increase in late deaths, regardless of fibrinolysis transition patterns, was observed. CONCLUSIONS: Adverse late outcomes are more closely related to 24-h maximum lysis, irrespective of admission levels. Tranexamic acid alters early fibrinolysis transition patterns, but late mortality in patients with low-maximum lysis at 24 h is not increased.

Enhanced clot lysis by a single point mutation in a reteplase variant.

Recombinant tissue-type plasminogen activator (rtPA) is the clot lysis drug approved for clinical use, and is characterised by a short half-life and substantial inactivation by plasminogen activator inhibitor-1 (PAI-1). We previously discovered that a tPA mutation (A419Y) at the protease domain led to enhanced fibrinolysis activity. In the present study, we studied the mechanism of such mutation in enhancing the proteolytic activity, and whether such enhancement persists in reteplase, an United States Food and Drug Administration-approved tPA truncated variant. We constructed and expressed a series of reteplase-based mutants, including rPAG (glycosylated rPA), rPAG -Y (with A419Y mutant at rPAG ), rPAG -A4 (tetra-alanine mutation at 37-loop of rPAG ), and rPAG -A4/Y (with both) and evaluated their plasminogen activation and PAI-1 resistance. Surface plasmon resonance analysis showed that the rPAG had fibrin affinity comparable to full-length tPA. Moreover, rPAG -Y had 8·5-fold higher plasminogen activation and stronger tolerance to PAI-1 compared to rPAG . We also found that the mutations containing tetra-alanine (rPAG -A4 and rPAG -A4/Y) had dramatically reduced plasminogen activation and impaired clot lysis. In a pulmonary embolism murine model, rPAG -Y displayed a more efficient thrombolytic effect than rPAG . These results identified a novel mutant reteplase variant of tPA with increased fibrinolytic activity, laying the foundation for the development of a new potent fibrinolytic agent.

A randomised controlled trial to assess the antithrombotic effects of aspirin in type 1 diabetes: role of dosing and glycaemic control.

BACKGROUND: The enhanced thrombotic milieu in diabetes contributes to increased risk of vascular events. Aspirin, a key antiplatelet agent, has inconsistent effects on outcomes in diabetes and the best dosing regimen remains unclear. This work investigated effects of aspirin dose and interaction with glycaemia on both the cellular and protein components of thrombosis. METHODS: A total of 48 participants with type 1 diabetes and 48 healthy controls were randomised to receive aspirin 75 or 300 mg once-daily (OD) in an open-label crossover study. Light transmittance aggregometry and fibrin clot studies were performed before and at the end of each treatment period. RESULTS: Aspirin demonstrated reduced inhibition of collagen-induced platelet aggregation (PA) in participants with diabetes compared with controls, although the higher dose showed better efficacy. Higher aspirin dose facilitated clot lysis in controls but not individuals with diabetes. Collagen-induced PA correlated with glycaemic control, those in the top HbA1c tertile having a lesser inhibitory effect of aspirin. Threshold analysis suggested HbA1c levels of > 65 mmol/mol and > 70 mmol/mol were associated with poor aspirin response to 75 and 300 mg daily doses, respectively. Higher HbA1c was also associated with longer fibrin clot lysis time. CONCLUSIONS: Patients with diabetes respond differently to the antiplatelet and profibrinolytic effects of aspirin compared with controls. In particular, those with elevated HbA1c have reduced inhibition of PA with aspirin. Our findings indicate that reducing glucose levels improves the anti-thrombotic action of aspirin in diabetes, which may have future clinical implications. TRIAL REGISTRATION: EudraCT, 2008-007875-26, https://www.clinicaltrialsregister.eu/ctr-search/search?query=2008-007875-26 .

Cost-effectiveness Analysis of AngioJet and CDT for Lower Extremity Deep Vein Thrombosis Among Chinese Population.

AngioJet has sufficient safety and efficacy in the treatment of acute and subacute lower extremity deep vein thrombosis (LEDVT). But the price of consumables used by AngioJet is relatively high and there is a lack of relevant research on health economics to measure the benefits to patients. Objective of this study is to estimate the cost effectiveness of AngioJet compared with catheter-directed thrombolysis (CDT) among Chinese population. Using a Markov decision model, we compared the 2 treatment strategies in patients with LEDVT. The model captured the development of post-thrombotic syndrome (PTS), recurrent venous thromboembolism, and treatment-related adverse events within a lifetime horizon and the perspective of a third-party payer. Model uncertainty was assessed with one-way and Monte Carl sensitivity analyses. The clinical inputs were obtained from the literature. Costs obtained from the hospital accounts and the literature are expressed in US dollars ($). Utilities were defined as quality adjusted life years (QALY). In cost-effectiveness analysis, AngioJet accumulated $1064.6445/QALY compared with $2080.1561/QALY after CDT treatment alone. AngioJet has higher long-term cost-effectiveness than CDT at a willingness to pay threshold of $11 233.52. One-way sensitivity analysis showed that the utilities of PTS and post-LEDVT state had significant influence on the results and the model maintained a strong stability under ± 10% fluctuation of utilities. Monte Carl sensitivity analysis shows that AngioJet model has strong stability and AngioJet has higher long-term cost-effectiveness than CDT. AngioJet is likely to be a cost-effective alternative to the CDT for patients with LEDVT.