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.

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.

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.

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.

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.

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.

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.

Beneficial Properties of Bromelain.

Bromelain is a major sulfhydryl proteolytic enzyme found in pineapple plants, having multiple activities in many areas of medicine. Due to its low toxicity, high efficiency, high availability, and relative simplicity of acquisition, it is the object of inexhaustible interest of scientists. This review summarizes scientific reports concerning the possible application of bromelain in treating cardiovascular diseases, blood coagulation and fibrinolysis disorders, infectious diseases, inflammation-associated diseases, and many types of cancer. However, for the proper application of such multi-action activities of bromelain, further exploration of the mechanism of its action is needed. It is supposed that the anti-viral, anti-inflammatory, cardioprotective and anti-coagulatory activity of bromelain may become a complementary therapy for COVID-19 and post-COVID-19 patients. During the irrepressible spread of novel variants of the SARS-CoV-2 virus, such beneficial properties of this biomolecule might help prevent escalation and the progression of the COVID-19 disease.

Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo.

Thrombolytic and fibrinolytic therapies are effective treatments to dissolve blood clots in stroke therapy. Thrombolytic drugs activate plasminogen to its cleaved form plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The FDA-approved human tissue plasminogen activator Reteplase (rPA) is a non-glycosylated protein produced in E. coli. rPA is a deletion mutant of the wild-type Alteplase that benefits from an extended plasma half-life, reduced fibrin specificity and the ability to better penetrate into blood clots. Different methods have been proposed to improve the production of rPA. Here we show for the first time the transient expression in Nicotiana benthamiana of rPA fused to the immunoglobulin fragment crystallizable (Fc) domain on an IgG1, a strategy commonly used to improve the stability of therapeutic proteins. Despite our success on the expression and purification of dimeric rPA-Fc fusions, protein instability results in high amounts of Fc-derived degradation products. We hypothesize that the "Y"- shape of dimeric Fc fusions cause steric hindrance between protein domains and leads to physical instability. Indeed, mutations of critical residues in the Fc dimerization interface allowed the expression of fully stable rPA monomeric Fc-fusions. The ability of rPA-Fc to convert plasminogen into plasmin was demonstrated by plasminogen zymography and clot lysis assay shows that rPA-Fc is able to dissolve blood clots ex vivo. Finally, we addressed concerns with the plant-specific glycosylation by modulating rPA-Fc glycosylation towards serum-like structures including α2,6-sialylated and α1,6-core fucosylated N-glycans completely devoid of plant core fucose and xylose residues.

Andrographolide sulfonate attenuates alveolar hypercoagulation and fibrinolytic inhibition partly via NF-κB pathway in LPS-induced acute respiratory distress syndrome in mice.

BACKGROUND: Alveolar hypercoagulation and fibrinolytic inhibition are important characteristics during acute respiratory distress syndrome (ARDS), and NF-κB p65 signaling pathway is involved to regulate these pathophysiologies. We hypothesize that targeting NF-κB signal pathway could ameliorate alveolar hypercoagulation and fibrinolyitc inhibition, thus attenuating lung injury in ARDS. PURPOSE: We explore the efficacy and the potential mechanism of andrographolide sulfonate (Andro-S) on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS in mice. METHODS: ARDS was made by lipopolysaccharide (LPS) inhalation in C57BLmice. Andrographolide sulfonate (2.5, 5 and 10 mg/kg) was intraperitoneally given to the mice (once a day for three consecutive days) before LPS administration. NEMO binding domain peptide (NBD), an inhibitor of NF-κB, was used as the positive control and it replaced Andro-S in mice of NBD group. Mice in normal control received saline instead of LPS. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis of alveolar coagulation, fibrinolytic inhibition as well as of pulmonary inflammatory response after 8 h of LPS inhalation. NF-κB signal pathway in lung tissue was simultaneously determined. RESULTS: Andro-S dose-dependently inhibited tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 expressions either in mRNA or in protein in lung tissue of ARDS mice, and it also decreased the concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) while promoting the production of activated protein C (APC) in BALF. Meanwhile, Andro-S effectively inhibited inflammatory response (interleukin 1ß and myeloperoxidase) induced by LPS. LPS stimulation dramatically activated NF-κB signal pathway, indicated by increased expressions of phosphorylation of p65 (p-p65), p-IKKα/ß and p-IκBα and the higher p65-DNA binding activity, which were all dose-dependently reversed by Andro-S. Andro-S and NBD presented similar efficacies. CONCLUSIONS: Andro-S treatment improves alveolar hypercoagulation and fibrinolytic inhibition and attenuates pulmonary inflammation in LPS-induced ARDS in mice partly through NF-κB pathway inactivation. The drug is expected to be an effective choice for ARDS.

Hypofibrinolysis in type 2 diabetes and its clinical implications: from mechanisms to pharmacological modulation.

A prothrombotic state is a typical feature of type 2 diabetes mellitus (T2DM). Apart from increased platelet reactivity, endothelial dysfunction, hyperfibrinogenemia, and hypofibrinolysis are observed in T2DM. A variety of poorly elucidated mechanisms behind impaired fibrinolysis in this disease have been reported, indicating complex associations between platelet activation, fibrin formation and clot structure, and fibrinolysis inhibitors, in particular, elevated plasminogen antigen inhibitor-1 levels which are closely associated with obesity. Abnormal fibrin clot structure is of paramount importance for relative resistance to plasmin-mediated lysis in T2DM. Enhanced thrombin generation, a proinflammatory state, increased release of neutrophil extracellular traps, elevated complement C3, along with posttranslational modifications of fibrinogen and plasminogen have been regarded to contribute to altered clot structure and impaired fibrinolysis in T2DM. Antidiabetic agents such as metformin and insulin, as well as antithrombotic agents, including anticoagulants, have been reported to improve fibrin properties and accelerate fibrinolysis in T2DM. Notably, recent evidence shows that hypofibrinolysis, assessed in plasma-based assays, has a predictive value in terms of cardiovascular events and cardiovascular mortality in T2DM patients. This review presents the current data on the mechanisms underlying arterial and venous thrombotic complications in T2DM patients, with an emphasis on hypofibrinolysis and its impact on clinical outcomes. We also discuss potential modulators of fibrinolysis in the search for optimal therapy in diabetic patients.

Docosahexaenoic Acid Suppresses Expression of Adipogenic Tetranectin through Sterol Regulatory Element-Binding Protein and Forkhead Box O Protein in Pigs.

Tetranectin (TN), a plasminogen-binding protein originally involved in fibrinolysis and bone formation, was later identified as a secreted adipokine from human and rat adipocytes and positively correlated with adipogenesis and lipid metabolism in adipocytes. To elucidate the nutritional regulation of adipogenic TN from diets containing different sources of fatty acids (saturated, n-6, n-3) in adipocytes, we cloned the coding region of porcine TN from a cDNA library and analyzed tissue expressions in weaned piglets fed with 2% soybean oil (SB, enriched in n-6 fatty acids), docosahexaenoic acid oil (DHA, an n-3 fatty acid) or beef tallow (BT, enriched in saturated and n-9 fatty acids) for 30 d. Compared with tissues in the BT- or SB-fed group, expression of TN was reduced in the adipose, liver and lung tissues from the DHA-fed group, accompanied with lowered plasma levels of triglycerides and cholesterols. This in vivo reduction was also confirmed in porcine primary differentiated adipocytes supplemented with DHA in vitro. Then, promoter analysis was performed. A 1956-bp putative porcine TN promoter was cloned and transcription binding sites for sterol regulatory-element binding protein (SREBP)-1c or forkhead box O proteins (FoxO) were predicted on the TN promoter. Mutating binding sites on porcine TN promoters showed that transcriptional suppression of TN by DHA on promoter activity was dependent on specific response elements for SREBP-1c or FoxO. The inhibited luciferase promoter activity by DHA on the TN promoter coincides with reduced gene expression of TN, SREBP-1c, and FoxO1 in human embryonic kidney HEK293T cells supplemented with DHA. To conclude, our current study demonstrated that the adipogenic TN was negatively regulated by nutritional modulation of DHA both in pigs in vivo and in humans/pigs in vitro. The transcriptional suppression by DHA on TN expression was partly through SREBP-1c or FoxO. Therefore, down-regulation of adipogenic tetranectin associated with fibrinolysis and adipogenesis may contribute to the beneficial effects of DHA on ameliorating obesity-induced metabolic syndromes such as atherosclerosis and adipose dysfunctions.

Bioevaluation of Pheretima vulgaris Antithrombotic Extract, PvQ, and Isolation, Identification of Six Novel PvQ-Derived Fibrinolytic Proteases.

Thrombosis is a disease that seriously endangers human health, with a high rate of mortality and disability. However, current treatments with thrombolytic drugs (such as recombinant tissue-plasminogen activator) and the oral anticoagulants (such as dabigatran and rivaroxaban) are reported to have a tendency of major or life-threatening bleeding, such as intracranial hemorrhage or massive gastrointestinal bleed with non-specific antidotes. In contrast, lumbrokinase is very specific to fibrin as a substrate and does not cause excessive bleeding. It can dissolve the fibrin by itself or convert plasminogen to plasmin by inducing endogenous t-PA activity to dissolve fibrin clots. Therefore, searching for potentially new therapeutic molecules from earthworms is significant. In this study, we first collected a strong fibrinolytic extract (PvQ) from the total protein of the Pheretima vulgaris with AKTA pure protein purification systems; its fibrinolytic bioactivity was verified by the fibrin plate assay and zebrafish thrombotic model of vascular damage. Furthermore, according to the cell culture model of human umbilical vein endothelial cells (HUVECs), the PvQ was proven to exhibit the ability to promote the secretion of tissue-type plasminogen activator (t-PA), which further illustrated that it has an indirect thrombolytic effect. Subsequently, extensive chromatographic techniques were applied to reveal the material basis of the extract. Fortunately, six novel earthworm fibrinolytic enzymes were obtained from the PvQ, and the primary sequences of those functional proteins were determined by LC-MS/MStranscriptome cross-identification and the Edman degradation assay. The secondary structures of these six fibrinolytic enzymes were determined by circular dichroism spectroscopy and the three-dimensional structures of these proteases were predicted by MODELLER 9.23 based on multi-template modelling. In addition, those six genes encoding blood clot-dissolving proteins were cloned from P. vulgaris by RT-PCR amplification, which further determined the accuracy of proteins primary sequences identifications and laid the foundation for subsequent heterologous expression.