Heterologous expression of nattokinase in E. coli: Biochemical characterization and functional analysis of fibrin binding residues.

Heterologous expression of nattokinase, a potent fibrinolytic enzyme, has been successfully carried out in various microorganisms. However, the successful expression of this enzyme as a soluble protein was not achieved in E. coli. This study delves into the expression of nattokinase in E. coli as a soluble protein followed by its biochemical characterization and functional analysis for fibrinolytic activity. E. coli BL21C41 and pET32a vector host strain with pGro7 protein chaperone induced with IPTG at 16 °C 180 rpm for 16 h enabled the production of recombinant nattokinase in soluble fraction. Enzymatic assays demonstrated its protease activity, while characterization revealed optimal catalytic conditions at 37 °C and pH 8.0, with remarkable stability over a broad pH range (6.0-10.0) and up to 50 °C. The kinetic constants were determined as follows: Km = 25.83 ± 3.43 µM, Vmax = 62.91 ± 1.68 µM/s, kcat = 38.45 ± 1.06 s-1, and kcat/Km = 1.49 × 106 M-1 s-1. In addition, the fibrinolytic activity of NK, quantified by the fibrin plate hydrolysis assay was 1038 ± 156 U/ml, with a corresponding specific activity of 1730 ± 260 U/mg and the assessment of clot lysis time on an artificial clot (1 mg) was found to be 51.5 ± 2.5 min unveiling nattokinase's fibrinolytic potential. Through molecular docking, a substantial binding energy of -6.46 kcal/mol was observed between nattokinase and fibrin, indicative of a high binding affinity. Key fibrin binding residues, including Ser300, Leu302, and Asp303, were identified and confirmed. These mutants affected specifically the fibrin binding and not the proteolytic activity of NK. This comprehensive study provides crucial conditions for the expression of protein in soluble form in E. coli and biochemical properties paving the way for future research and potential applications in medicine and biotechnology.

Protracted fibrinolysis resistance following cardiac surgery with cardiopulmonary bypass: A prospective observational study of clinical associations and patient outcomes.

BACKGROUND: Surgery on cardiopulmonary bypass (CPB) elicits a pleiomorphic systemic host response which, when severe, requires prolonged intensive care support. Given the substantial cross-talk between inflammation, coagulation, and fibrinolysis, the aim of this hypothesis-generating observational study was to document the kinetics of fibrinolysis recovery post-CPB using ClotPro® point-of-care viscoelastometry. Tissue plasminogen activator-induced clot lysis time (TPA LT, s) was correlated with surgical risk, disease severity, organ dysfunction and intensive care length of stay (ICU LOS). RESULTS: In 52 patients following CPB, TPA LT measured on the first post-operative day (D1) correlated with surgical risk (EuroScore II, Spearman's rho .39, p < .01), time on CPB (rho = .35, p = .04), disease severity (APACHE II, rho = .52, p < .001) and organ dysfunction (SOFA, rho = .51, p < .001) scores, duration of invasive ventilation (rho = .46, p < .01), and renal function (eGFR, rho = -.65, p < .001). In a generalized linear regression model containing TPA LT, CPB run time and markers of organ function, only TPA LT was independently associated with the ICU LOS (odds ratio 1.03 [95% CI 1.01-1.05], p = .01). In a latent variables analysis, the association between TPA LT and the ICU LOS was not mediated by renal function and thus, by inference, variation in the clearance of intraoperative tranexamic acid. CONCLUSIONS: This observational hypothesis-generating study in patients undergoing cardiac surgery with cardiopulmonary bypass demonstrated an association between the severity of fibrinolysis resistance, measured on the first post-operative day, and the need for extended postoperative ICU level support. Further examination of the role of persistent fibrinolysis resistance on the clinical outcomes in this patient cohort is warranted through large-scale, well-designed clinical studies.

Association of the metabolic syndrome with PAI 1act and clot lysis time over a 10-year follow up in an African population.

BACKGROUND AND AIMS: The association between the metabolic syndrome (MetS) and plasminogen activator inhibitor-1 (PAI-1) has been well established in cross-sectional studies. It is less clear whether this translates into decreased clot lysis rates and very little information is available on non-European populations. Little is known regarding prospective associations and whether clot lysis progressively worsens in MetS individuals over time. We determined the prospective association of MetS with PAI-1 activity (PAI-1act) and clot lysis time (CLT) over a 10-year period. METHODS AND RESULTS: As many as 2010 African men and women aged ≥30 years were stratified according to MetS status and number of MetS criteria (0-5). We also determined the contribution of the PAI-1 4G/5G polymorphism to these associations and identified which MetS criteria had the strongest associations with PAI-1act and CLT. Both PAI-1act and CLT remained consistently elevated in individuals with MetS throughout the 10-year period. PAI-1act and CLT did not increase more over time in MetS individuals than in controls. The 4G/5G genotype did not influence the association of PAI-1act or clot lysis with MetS. Increased waist circumference, increased triglycerides and decreased HDL-C were the main predictors of PAI-1act and CLT. CONCLUSIONS: Black South Africans with MetS had increased PAI-1act and longer CLTs than individuals without MetS. The inhibited clot lysis in MetS did, however, not deteriorate over time compared to controls. Of the MetS criteria, obesity and altered lipids were the main predictors of PAI-1act and CLT and are thus potential targets for prevention strategies to decrease thrombotic risk.

Plasminogen activator inhibitor-2 and impaired fibrinolysis in pregnancy and sickle cell anemia.

PURPOSE: This is the first study that aimed to determine antigen levels in plasma and genotypes of PAI-2 in pregnant and non-pregnant homozygous sickle cell anemia (SCA) patients. METHODS: The study subjects were all Bahraini females in the reproductive age group. The study population included 31 pregnant homozygous SS (SCA) patients. Three control groups were also studied to evaluate the effect of pregnancy and SCA on PAI-2 levels and fibrinolysis: (1) 31 healthy non-pregnant volunteers; (2) 31 cases of normal pregnancy; and (3) 20 non-pregnant SCA patients. Pregnancies were screened in the second (TM2) and third (TM3) trimesters. Global coagulation, fibrinolysis rate (euglobulin clot lysis time, ECLT), PAI-2 antigen (ELISA), and PAI-2 Ser(413)/Cys polymorphism (restriction fragment length polymorphism analysis) were determined. RESULTS: Feto-maternal complications were documented in both pregnancy groups. PAI-2 antigen levels were undetectable in the non-pregnant groups, but was quantifiable in both pregnant groups. Impaired fibrinolysis rate and rising PAI-2 levels with progression of pregnancy were observed in both healthy and SCA subjects. These changes were more prominent in SCA, although the rise in ECLT was less steep and PAI-2 antigen levels were not significantly different compared to normal pregnancy in the third trimester. No correlation was observed between PAI-2 genotypes and plasma antigen levels. Also, no significant difference in feto-maternal complications was found in normal (n = 25) versus SCA pregnant patients (n = 30). CONCLUSIONS: These observations suggest that with progression of pregnancy, increasing PAI-2 levels contribute to the hypercoagulable state, particularly in SCA patients.

Novel characteristics of soluble fibrin: hypercoagulability and acceleration of blood sedimentation rate mediated by its generation of erythrocyte-linked fibers.

Soluble fibrin (SF) in blood consists of monomers lacking both fibrinopeptides A with a minor population in multimeric clusters. It is a substantial component of isolated fibrinogen (fg), which spontaneously self-assembles into protofibrils progressing to fibers at sub-physiologic temperatures, a process enhanced by adsorption to hydrophobic and some metal surfaces. Comparisons of SF-rich (FR) and SF-depleted (FD) fg isolates disclosed distinct molecular imprints of each via an adsorption/desorption procedure using gold surfaced silica microplates. Accelerated plasminogen activator-induced lysis and decreased stiffness (G') of thrombin-induced FR fg clots were revealed by thomboelastography. Erythrocyte sedimentation (ESR) in afibrinogenemic plasma (Hematocrit 25-33%) was accelerated by FR fg nearly threefold that of FD fg. Stained smears disclosed frequent rouleaux formations and fibers linking stacked erythrocytes in contrast to no rouleaux by FD fg. Rouleaux formations were more pronounced at 4 °C than at ambient temperatures and at fiber-membrane contacts displayed irregular, knobby membrane contours. One of several FR fg isolates also displayed incomplete fiber networks in cell-free areas. What is more, pre-mixing FR fg with each of three monoclonal IgG anti-fg antibodies at 1.5 mol/mol fg, that inhibited fibrin polymerization, prevented rouleaux formation save occasional 2-4 erythrocyte aggregates. We conclude that spontaneously generated SF fibers bound to erythrocytes forming intercellular links culminating in rouleaux formation and ensuing ESR acceleration which in clinical settings reflects hypercoagulability. Also, the results can explain the reported fg binding to erythrocytes via ligands such as CD47, stable in vivo RBC aggregates in capillaries, and red areas of pathologic thrombi.

Impaired fibrin clot lysis is associated with increased mortality in patients after coronary artery bypass grafting: A long-term follow-up study.

BACKGROUND: Mortality after coronary artery bypass grafting (CABG) is primarily thromboembolic by nature. We investigated whether impaired fibrinolysis observed in cardiovascular diseases is associated with long-term mortality following CABG. METHODS: The study population comprised 292 consecutive patients (aged 64.6 ± 8.1 years) who underwent scheduled CABG. We measured plasma clot lysis time (CLT) preoperatively as a measure of fibrinolysis capacity. Cardiovascular and all-cause deaths were recorded during a median follow-up of 13.8 years. RESULT: CLT positively correlated with age (r = .56, p < .001), fibrinogen (r = .25, p = .002) and EuroSCORE I (r = .32, p < .001). The cardiovascular and overall mortality rates were 3.0 and 4.9 per 100 patient-years (32.4% vs 52.8%) respectively. In patients who died from cardiovascular and all causes, CLT was prolonged compared with survivors (both p < .050). Multivariable Cox regression analysis adjusted for potential confounders showed that long-term cardiovascular and all-cause deaths were associated with CLT (HR per 10 min 1.206; 95% CI 1.037-1.402, p = .015 and HR 1.164; 96% CI 1.032-1.309, p = .012), low-density lipoprotein cholesterol (HR per 1 mmol/L 1.556; 95% CI 1.205-2.010, p < .001 and HR 1.388; 96% CI 1.125-1.703, p = .002), C-reactive protein (HR per 10 mg/L 1.171; 95% CI 1.046-1.312, p = .006 and HR 1.127; 95% CI 1.005-1.237, p = .022) and EuroSCORE I (HR 1.173; 95% CI 1.016-1.355, p = .030 and HR 1.183; 95% CI 1.059-1.317, p = .003 respectively). Type 2 diabetes was solely associated with overall mortality (HR 1.594; 96% CI 1.088-2.334, p = .017). CONCLUSIONS: In this study, we showed that reduced fibrin clot susceptibility to fibrinolysis is weekly associated with long-term mortality in advanced CAD.

Arterial and venous blood sampling is equally applicable for coagulation and fibrinolysis analyses.

OBJECTIVES: No consensus exists upon whether arterial and venous blood samples are equivalent when it comes to coagulation analyses. We therefore conducted a comparative cohort study to clarify if arteriovenous differences affect analyses of primary and secondary hemostasis as well as fibrinolysis. METHODS: Simultaneous paired blood samplings were obtained from a cannula in the radial artery and an antecubital venipuncture in 100 patients immediately before or one day after thoracic surgery. Analyses of platelet count and aggregation, International Normalized Ratio (INR), activated partial thromboplastin time (APTT), antithrombin, thrombin time, fibrinogen, D-dimer, rotational thromboelastometry (ROTEM), thrombin generation, prothrombin fragment 1 + 2, and an in-house dynamic fibrin clot formation and lysis assay were performed. RESULTS: No differences were found between arterial and venous samples for the far majority of parameters. The only differences were found in INR, median (IQR): venous, 1.1 (0.2) vs. arterial, 1.1 (0.2) (p<0.002) and in prothrombin fragment 1 + 2: venous, 289 (209) pmol/L vs. arterial, 279 (191) pmol/L (p<0.002). CONCLUSIONS: The sampling site does not affect the majority of coagulation analyses. Small differences were found for two parameters. Due to numerically very discrete differences, they are of no clinical relevance. In conclusion, the present data suggest that both samples obtained from arterial and venous blood may be applied for analyses of coagulation and fibrinolysis.

Laboratory evaluation of a new integrative assay to phenotype plasma fibrinolytic system.

BACKGROUND: There is currently no universal and standardized test available to phenotype plasma fibrinolytic system. AIMS: Our main aims were to evaluate the performances of the 'global fibrinolysis capacity' assay (GFC) performed with the Lysis Timer® instrument, and to study the influence of some preanalytical conditions. METHOD: Euglobulin clot lysis time (ECLT) and GFC were performed under several preanalytical conditions. RESULTS: GFC showed satisfactory intra- and inter-run precision. Frozen controls and reagents showed stability over the studied period. There was no statistically significant difference between GFC assessed in plasma samples processed at 4 °C or at 20 °C. GFC assessed with frozen-thawed plasma samples was prolonged when compared to fresh samples (p = 0.014). The centrifugation scheme had no influence on PAI-1 activity levels, GFC and ECLT. Reference interval for GFC ranges from 29.3 (C I90% = 26.9-31.9) to 49.5 (90% CI = 45.9-52.2) minutes. In addition, a preliminary study in 40 healthy volunteers and 43 adult patients referred for investigation of a bleeding disorder was conducted to compare GFC and ECLT assays in their ability to classify samples with shortened or prolonged clot lysis times. Disagreements between ECLT and GFC were observed for 23 samples (out of 83), most of them minor. CONCLUSION: GFC is suitable and convenient for a broad clinical use and can be performed with frozen-thawed plasma samples. Unlike ECLT, GFC is designed to take into account the balance between inhibitors and activators of the fibrinolytic system and could detect both hypo- and hyperfibrinolytic states. Whether it is as suitable as or even better than ECLT to detect a bleeding tendency due to a hyperactive fibrinolytic system deserves to be properly investigated.

Endogenous fibrinolysis-Relevance to clinical thrombosis risk assessment.

The development of an obstructive luminal thrombus is pathological and considered a failure of endogenous fibrinolysis. The consequences may be fatal, or result in lasting downstream organ damage. Therefore, assessment of endogenous fibrinolytic status in an individual may identify those at risk of occlusive thrombus formation and provide prognostic information. Arterial thrombi are more platelet rich and more resistant to fibrinolysis than venous thrombi. Several recent studies using global tests of fibrinolysis in patients with acute coronary syndromes (ACS) have shown that despite dual antiplatelet therapy, patients with impaired fibrinolytic status have an increased risk of adverse cardiovascular events, compared with those with effective fibrinolytic function. Such data add significantly to the predictive value of established cardiovascular risk factors and conventional biomarkers. Most data reported have been obtained with the Global Thrombosis Test and the turbidimetric plasma clot lysis assay. A few small studies in patients with ischaemic stroke suggest a similar predictive role of fibrinolytic status assessment in these patients. Studies reporting an association between impaired fibrinolysis and future venous thrombotic events are limited, and in the form of case-control studies. Viscoelastic assays may have a role in the prediction of venous thromboembolic risk. Assays of fibrinolytic function should be used to obtain a more accurate risk of future thrombotic events, particularly in the setting of ACS. The availability of point-of-care tests helps facilitate this and should encourage future studies to assess personalised antithrombotic treatment combinations to optimise fibrinolytic status and reduce thrombosis risk.

Integration of VEK-30 peptide enhances fibrinolytic properties of staphylokinase.

Staphylokinase (SAK), a 136 amino acid bacterial protein with profibrinolytic properties, has emerged as an important thrombolytic agent because of its fibrin specificity and reduced inhibition by α-2 antiplasmin. In an attempt to enhance the clot dissolution ability of SAK, a 30 amino acid peptide (VEK-30) derived from a plasminogen (Pg) binding protein (PAM), was fused at the C-terminal end of SAK with a RGD (Arg-Gly-Asp) linker. The chimeric protein, SAKVEK, was expressed in E. coli and purified as a soluble protein. Pg activation by equimolar complexes of SAKVEK and SAK with plasmin revealed that the fusion of VEK-30 peptide has significantly enhanced the catalytic activity of SAK. The kinetic constant, kcat /Km , of SAKVEK for the substrate Pg appeared 2.7 times higher than that of SAK and the time required for the fibrin and platelet rich clot lysis was shortened by 30% and 50%, respectively. The binary activator complex of SAKVEK with plasmin gets inhibited by α2- antiplasmin but remains protected in the presence of fibrin, very similar to SAK. Thus, the present study suggests that SAKVEK is more potent and effective as a thrombolytic agent due to its higher catalytic activity for Pg activation in a fibrin-specific manner and its ability to clear platelet-rich plasma clot faster than SAK.

Fibrin Network Formation and Lysis in Septic Shock Patients.

BACKGROUND: Septic shock patients are prone to altered fibrinolysis, which contributes to microthrombus formation, organ failure and mortality. However, characterisation of the individual patient's fibrinolytic capacity remains a challenge due to a lack of global fibrinolysis biomarkers. We aimed to assess fibrinolysis in septic shock patients using a plasma-based fibrin clot formation and lysis (clot-lysis) assay and investigate the association between clot-lysis parameters and other haemostatic markers, organ dysfunction and mortality. METHODS: This was a prospective cohort study including adult septic shock patients (n = 34). Clot-lysis was assessed using our plasma-based in-house assay. Platelet count, activated partial thromboplastin time (aPTT), international normalised ratio (INR), fibrinogen, fibrin D-dimer, antithrombin, thrombin generation, circulating fibrinolysis markers and organ dysfunction markers were analysed. Disseminated intravascular coagulation score, Sequential Organ Failure Assessment (SOFA) score and 30-day mortality were registered. RESULTS: Three distinct clot-lysis profiles emerged in the patients: (1) severely decreased fibrin formation (flat clot-lysis curve), (2) normal fibrin formation and lysis and (3) pronounced lysis resistance. Patients with abnormal curves had lower platelet counts (p = 0.05), more prolonged aPTT (p = 0.04), higher lactate (p < 0.01) and a tendency towards higher SOFA scores (p = 0.09) than patients with normal clot-lysis curves. Fibrinogen and fibrin D-dimer were not associated with clot-lysis profile (p ≥ 0.37). CONCLUSION: Septic shock patients showed distinct and abnormal clot-lysis profiles that were associated with markers of coagulation and organ dysfunction. Our results provide important new insights into sepsis-related fibrinolysis disturbances and support the importance of assessing fibrinolytic capacity in septic shock.

Rosuvastatin use increases plasma fibrinolytic potential: a randomised clinical trial.

We conducted a study to assess the effect of rosuvastatin use on fibrinolysis in patients with previous venous thromboembolism (VTE). This was a post hoc analysis within the STAtins Reduce Thrombophilia (START) study (NCT01613794). Plasma fibrinolytic potential, fibrinogen, plasmin inhibitor, plasminogen activator inhibitor-1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI) were measured before and after four weeks of rosuvastatin or no treatment in participants with prior confirmed VTE, after ending anticoagulant therapy. In the non-rosuvastatin group (n = 121), plasma fibrinolytic potential and individual fibrinolysis parameters did not change at the end of the study versus the baseline, whereas in the rosuvastatin group (n = 126), plasma fibrinolytic potential increased: the mean clot lysis time decreased by 8·75 min (95% CI -13·8 to -3·72), and plasmin inhibitor levels and TAFI activity were lower at the end of the study (-0·05 U/ml; 95% CI -0·07 to -0·02 and -4·77%; 95% CI -6·81 to -2·73, respectively). PAI-1 levels did not change and fibrinogen levels were 0·17 g/l (95% CI 0·04-0·29) higher. In participants with prior VTE, rosuvastatin use led to an increased fibrinolytic potential compared with non-statin use. Our findings support the need for further studies on the possible role for statins in the secondary prevention of VTE.

Preparation of Peptide and Recombinant Tissue Plasminogen Activator Conjugated Poly(Lactic-Co-Glycolic Acid) (PLGA) Magnetic Nanoparticles for Dual Targeted Thrombolytic Therapy.

Recombinant tissue plasminogen activator (rtPA) is the only thrombolytic agent that has been approved by the FDA for treatment of ischemic stroke. However, a high dose intravenous infusion is required to maintain effective drug concentration, owing to the short half-life of the thrombolytic drug, whereas a momentous limitation is the risk of bleeding. We envision a dual targeted strategy for rtPA delivery will be feasible to minimize the required dose of rtPA for treatment. For this purpose, rtPA and fibrin-avid peptide were co-immobilized to poly(lactic-co-glycolic acid) (PLGA) magnetic nanoparticles (PMNP) to prepare peptide/rtPA conjugated PMNPs (pPMNP-rtPA). During preparation, PMNP was first surface modified with avidin, which could interact with biotin. This is followed by binding PMNP-avidin with biotin-PEG-rtPA (or biotin-PEG-peptide), which was prepared beforehand by binding rtPA (or peptide) to biotin-PEG-maleimide while using click chemistry between maleimide and the single -SH group in rtPA (or peptide). The physicochemical property characterization indicated the successful preparation of the magnetic nanoparticles with full retention of rtPA fibrinolysis activity, while biological response studies underlined the high biocompatibility of all magnetic nanoparticles from cytotoxicity and hemolysis assays in vitro. The magnetic guidance and fibrin binding effects were also confirmed, which led to a higher thrombolysis rate in vitro using PMNP-rtPA or pPMNP-rtPA when compared to free rtPA after static or dynamic incubation with blood clots. Using pressure-dependent clot lysis model in a flow system, dual targeted pPMNP-rtPA could reduce the clot lysis time for reperfusion by 40% when compared to free rtPA at the same drug dosage. From in vivo targeted thrombolysis in a rat embolic model, pPMNP-rtPA was used at 20% of free rtPA dosage to restore the iliac blood flow in vascular thrombus that was created by injecting a blood clot to the hind limb area.

Fibrinolysis in trauma patients: wide variability demonstrated by the Lysis Timer.

Hyperfibrinolysis contributes to the pathophysiology of trauma-induced coagulopathy. At present, systematic administration of tranexamic acid (TXA) is recommended in all patients in the early phase of trauma. However, there is some debate regarding whether TXA is beneficial in all trauma patients. A rapid and accurate tool to diagnose hyperfibrinolysis may be useful for tailoring TXA treatment. We conducted a proof-of-concept study of consecutive adult trauma patients. A first blood sample was obtained at the time of pre-hospital care (T1). Patients received 1 g of TXA after T1. A second sample was obtained on arrival at the emergency unit (T2). We examined coagulation, fibrin and fibrinogen formation and degradation. Fibrinolysis was assessed by determining tissue plasminogen activator (t-PA) antigen and plasminogen activator inhibitor 1 (PAI-1) activity and global fibrinolysis capacity assay using a device developed by Hyphen BioMed: the Lysis Timer (GFC/LT). The study population consisted of 20 patients (42 ± 21 years, index of severity score 32 ± 21). Both coagulation and fibrinolysis were altered at T1. GFC/LT values exhibited hyperfibrinolysis only in five patients. Principal component analysis carried out at T1 showed two main axes of alteration. The major axis was related to coagulation, altered in all patients, while the second axis was related to fibrinolysis. GFC/LT was mainly influenced by PAI-1 activity while fibrin monomers were related to the severity of trauma. At T2, GFC/LT exhibited the marked effect of TXA on clot lysis time. In conclusion, GFC/LT demonstrated huge variation in the fibrinolytic response to trauma.

Advancements in Managing Intracerebral Hemorrhage: Transition from Nihilism to Optimism.

There have been significant advancements in the management of intracerebral hemorrhage (ICH) stemming from new knowledge on its pathogenesis. Major clinical trials, such as Surgical Trial in Lobar Intracerebral Hemorrhage (STICH I and II), have shown only a small, albeit clinically relevant, advantage of surgical interventions in specific subsets of patients suffering from ICH. Currently, the aim is to use a minimally invasive and safe trajectory in removing significant brain hematomas with the aid of neuro-endoscopy or precise guidance through neuro-navigation, thereby avoiding a collateral damage to the surrounding normal brain tissue. A fundamental rational to such approach is to safely remove hematoma, preventing the ongoing mass effect resulting in brain herniation, and to minimize deleterious effects of iron released from hematoma to brain cells. The clot lysis process is facilitated with the adjunctive use of recombinant tissue plasminogen activator and sonolysis. Revised recommendations for the management of ICH focus on a holistic approach, with special emphasis on early patient mobilization and graded rehabilitative process. There has been a paradigm shift in the management algorithm, putting emphasis on early and safe removal of brain hematoma and then focusing on the improvement of patients' quality of life. We have made significant progress in transition from nihilism toward optimism, based on evidence-based management of such a severe global health scourge as intracranial hemorrhage.

Erythrocyte compression index is impaired in patients with residual vein obstruction.

Defective clot contraction has been postulated to contribute to thrombosis. We aimed to evaluate the association of residual vein obstruction (RVO) with erythrocyte compression within the whole-blood clot. We studied 32 patients with venous thromboembolism (VTE) taking vitamin K antagonists (VKAs) for at least 3 months (median time in therapeutic range 60%), including 12 (37.5%) with RVO, and 32 age- and sex-matched controls. In all study participants we evaluated whole blood clot retraction, expressed as the erythrocyte compression index (ECI), defined as a ratio of mean polyhedrocyte area to mean native erythrocyte area, along with clot area covered by polyhedrocytes, plasma clot permeability (Ks), clot lysis time (CLT), and thrombin generation. In both groups higher ECI, indicating impaired clot contraction, increased with older age, higher body mass index, red blood cell distribution width, and lower platelet count (all p < 0.05), but not with red blood cell count. In VTE patients ECI was 15.8% higher than in controls (median 63.6 vs. 54.9%, p = 0.021). Subjects with RVO had 20% higher ECI and 155% lower clot area covered by polyhedrocytes. RVO patients had also prolonged CLT by 41%, but not Ks, and elevated peak thrombin generation by 33%, as compared to those without RVO (all p < 0.05). This study is the first to show impaired compression of erythrocytes in RVO patients despite VKA anticoagulation. Altered ECI coexisted with hypolysability and increased thrombin generation. ECI might be useful in the diagnostic process of RVO or post-thrombotic syndrome and can help optimize the anticoagulant therapy.

Asthma is associated with enhanced thrombin formation and impaired fibrinolysis.

BACKGROUND: There is evidence that altered blood coagulation and fibrinolysis are involved in the pathogenesis of asthma. Increased thromboembolic risk has been reported in asthmatics. OBJECTIVE: To investigate whether enhanced thrombin generation and impaired fibrinolysis occur in asthmatics. METHODS: Plasma thrombin generation profile together with a computational assessment of thrombin dynamics and fibrinolytic capacity expressed as clot lysis time (CLT) were determined in 164 consecutive patients with stable asthma and 72 controls matched for age, gender, weight and smoking. RESULTS: Asthma patients had 20.2% increased endogenous thrombin potential (ETP), 41.4% higher peak thrombin concentration, 61% higher maximal prothrombin conversion rate, 15.5% faster rate of thrombin formation (all, P < 0.0001) and 10% lower thrombin decay capacity (P = 0.0004) compared with controls. Asthmatics had also 14.4% longer CLT (P = 0.001) associated with 21.3% higher plasminogen activator inhibitor-1 (PAI-1) (P < 0.0001), and 13% higher plasma α2 -macroglobulin (P = 0.0002). Using ETP and CLT above 75th percentile of the control values as the cut-off levels, we found increased risks of enhanced thrombin generation and hypofibrinolysis in asthmatics, also after correction for potential confounders. ETP and CLT were associated inversely with forced expiratory volume in 1 s/vital capacity (FEV1 /VC) index, after adjustment for age and body mass index. Non-allergic asthma (n = 70, 42.6%) was characterized by 17.5% longer CLT (P = 0.02), which positively associated with PAI-1. Thrombin generation profile was not affected by allergy. CONCLUSION AND CLINICAL RELEVANCE: Asthma is associated with enhanced thrombin generation and impaired fibrinolysis, which might contribute to thromboembolic events in this disease.

Protective effect of composite earthworm powder against diabetic complications via increased fibrinolytic function and improvement of lipid metabolism in ZDF rats.

Thrombosis is the leading cause of mortality globally. It is not only a complication but also a risk factor for progression of diabetes. However, alternative oral therapies and prophylaxis with less adverse effect for thrombosis have not been well studied. In this study, composite powder containing earthworm (CEP) was used and its fibrinolytic activity was measured. CEP was found to have a high urokinase-type plasminogen activator like activity in an in vitro assay. It also had significantly shortened euglobulin clot lysis time (ECLT) at 4 and 24 h after ingestion in Sprague Dawley rats. Zucker Diabetic Fatty rats were used to assess the effect of CEP on diabetes and diabetic nephropathy. After 10 weeks of feeding, CEP significantly shortened ECLT and attenuated HbA1c, hepatic lipid accumulation, and urinary albumin excretion and improved glomerular mesangial matrix score. Therefore, CEP may have beneficial effects on diabetes and diabetic nephropathy.

Purification and characterization of a novel C-type hemolytic lectin for clot lysis from the fresh water clam Villorita cyprinoides: a possible natural thrombolytic agent against myocardial infarction.

Villorita cyprinoides (black clam) is a fresh water clam that belongs as a bivalve to the group of mollusc. The saline extracts from the muscle reveal high titers of agglutination potency on trypsin-treated rabbit erythrocytes. With the help of affinity chromatography a hemolytic protein with lectin activity which could all be inhibited by D-galactose were isolated. The lectins were separated on DEAE-cellulose and the main component was purified after an additional step of gel filtration on sephadex G-75. The main component is a non-glycosylated protein with a molecular weight of 96,560 Da determined by MALDI-ToF, consisting of a single protein chain and characterized by the lack of polymers and intermediate disulfide bonds. The pure main lectin with clot lytic feature shows two bands at molecular weights 36,360 and 26, 520 Da. Optimal inhibition of the pure lectin is achieved by D-galactose containing oligo- and polysaccharides. The lectin activity decreased above 40 °C and was lost at 62 °C, the stability over the pH range between 7.0 and 8.0 and requires divalent cations for their activity. The novel C-type hemolytic lectin for clot lysis from the clam Villorita cyprinoides was identified and evaluated, the purified hemolytic lectin (0.35 mg/ml and 0.175 mg/ml) enhanced clot lysis activity when compared to the different concentration (5 mg/ml and 1 mg/ml) of commercial streptokinase. In the present study identified hemolytic lectin was a rapid and effective clot lytic molecule and could be developed as new drug molecule in future.

Global fibrinolytic activity, PAI-1 level, and 4G/5G polymorphism in Thai children with arterial ischemic stroke.

BACKGROUND: Prolonged euglobulin clot lysis time (ECLT) and increased level of plasminogen activator inhibitor-1 (PAI-1) were reported to be risk factors of arterial ischemic stroke (AIS) by some studies; however, these findings were not supported by other studies. The objective of this study was to determine the association of ECLT, PAI-1 level, and polymorphisms of 4G and 5G of PAI-1 gene to the development of AIS in Thai children. METHODS: This study included patients aged 1-18 years old. Diagnosis of AIS was confirmed by imaging study. The control group was age- and sex-matched healthy subjects. Demographic data were recorded, and blood was tested for ECLT, PAI-1 level, lipid profiles, fasting blood sugar (FBS), and 4G and 5G polymorphisms of PAI-1 gene. RESULTS: There were 70 subjects participating in this study, consisting of 30 patients and 40 controls. Demographic data, lipid profiles, and FBS were similar between the 2 groups. Furthermore, ECLT and PAI-1 level did not differ between patient and control groups; however, both showed significant correlation (r = .352, P = .006). The 4G/5G polymorphism was the most common genotype in both patient and control groups (69.0% vs. 80.0%). However, 4G and 5G polymorphisms of PAI-1 gene did not correlate with PAI-1 level in this study (P = .797). CONCLUSIONS: The PAI-1 level and 4G/5G polymorphism may not be a risk factor of AIS in this population. It was also found that the 4G/5G polymorphism was the most common PAI-1 genotype in this study.