Fibrinolytic Activity Research Articles

Fibrinolytic Enzyme from Green Microalgae: A New Potential Drug for Thrombolytic Therapy?

Thrombosis is characterized by the pathological formation of fibrin clots within a blood vessel, leading to the obstruction of blood flow. Fibrinolytic enzymes from microorganisms have been shown to be more efficient and safer in dissolving clots. Then, this study aimed to evaluate the cell growth and fibrinolytic enzyme production of Tetradesmus obliquus under different cultivation conditions. T. obliquus grew under autotrophic and mixotrophic conditions using different concentrations of corn steep liquor (0.25 ≤ CSL ≤ 4.00%). The cells were concentrated and lysed via two different methods (sonication or homogenization) to trigger the release of the enzyme. It was precipitated via acetone or ammonium sulfate additions and purified using ion exchange chromatography. The highest biomass productivity (Px = 130 ± 12.8 mg∙L−1day−1), specific growth rate (µmax = 0.17 ± 0.00 day−1), and fibrinolytic activity (391 ± 40.0 U∙mg−1) was achieved on a mixotrophic cultivation at a 0.25% CSL concentration. The results showed that the homogenizing method had better performance in the release of enzyme, and the precipitation with acetone obtained the highest fibrinolytic activity (567 ± 49.3 U∙mg−1). The purified enzyme showed a specific activity of 1221 ± 31 U∙mg−1 and a molecular mass of 97 kDa. So, the fibrinolytic enzyme from T. obliquus had higher activity when compared to the other fibrinolytic enzymes, being a potential source for the development of therapeutic agents in thrombosis treatment. Additional studies are needed to investigate the biochemical properties and biological profile of this enzyme.

RATIONALE FOR PRESCRIBING ANTICOAGULANT THERAPY IN THE COMPLEX TREATMENT OF PURULENT-NECROTIC COMPLICATIONS OF DIABETIC FOOT SYNDROME

Abstract. This article is based on the study of hemostasis indicators in 25 patients with diabetic foot syndrome. Diabetic foot syndrome (DFS) develops against the background of diabetic neuropathy, micro- and macroaniopathy, osteoarthropathy, which lead to frequent traumatization of the soft tissues of the foot, infection with the subsequent development of purulent-necrotic processes on the foot, and in the course of progression leads to high limb amputations. According to the international federation, which deals with the problem of diabetes mellitus (DM), the 21st century is considered a pandemic of DM. It should be noted that the number of patients with diabetes is increasing every year. Patients with diabetes are characterized by an atherothrombotic state caused by multiple disorders in the hemostasis system. Hemostatic disorders in patients with diabetes are complex and include activation of natural mechanisms of the coagulation system, inhibition of fibrinolytic activity, and various platelet dysfunctions.
 The purpose of the study is to substantiate the appointment of anticoagulation therapy in patients with SDS.
 Materials and methods. On the basis of KNP KMKL No. 6 in Kyiv in 2023, for a period of 6 months, 69 patients with SDS were treated. The state of indicators of the coagulation link of the hemostasis system in 49 people was studied. At the same time, 26 patients had purulent-necrotic complications of the neuroischemic form of SDS, 23 people were healthy (comparison group). The patients were divided according to the article and age, according to the duration of diabetes mellitus, and according to the stages according to Wagner. The following indicators were determined for patients: prothrombin index (PTI), plasma recalcification time, fibrinogen, B-naphthol test, ethanol test, Willebrand factor, endothelin-1, coagulation factor (VIII), fibronectin.
 The results of the study and their discussion: Based on the study, 61.5 % of patients with SDS are of working age. Of them, 57.7 % are men, 42.3 % are women. The largest number of patients with SDS is observed in the group of patients who have had diabetes for more than 10 years - 57.7 %, and the largest number of patients with stage III-IV according to Wagner - 88.5 %. In patients with diabetes with neuroischemic form of SDS, the PTI level is increased by 28.8 % and the prothrombin time is shortened by 21 %, which indicates an increase in the activity of external blood clotting mechanisms. An increase in fibrinogen by 29.7 % indicates an increase in the activity of coagulation hemostasis. Willebrand factor increased by 34.5 %, coagulation factor (VIII) increased by 20.3 %, fibronectin increased by 83.01 %, which indicates an increase in aggregation and procoagulant processes, which in turn increases thrombus formation.
 Conclusion. Thus, with diabetic foot syndrome, hypercoagulation is observed, which leads to an increase in the level of thrombus formation, which justifies the appointment of anticoagulant therapy in the complex treatment of diabetic foot syndrome.

Prognostic value of D-dimers in patients with COVID-19: narrative synthesis

Introduction. Contemporary researchers have suggested and demonstrated the hypothesis that the elevated level of D-dimers, which is a valuable marker of coagulation and fibrinolysis activation, can predict the severity of COVID-19, pulmonary complications, and thromboembolic events before they occur. Material and methods. The bibliographic resources were analyzed and selected from databases such as PubMed, Hinari, SpringerLink, and Google Search using keywords such as “COVID-19,” “SARS-CoV-2,” “coronavirus,” “D-dimers,” “biomarkers,” and “severity prediction,” which were used in various combinations to maximize search efficiency. Therefore, the manuscript contains 51 representative articles for the purpose of this synthesis article. Results. The D-dimer levels are significantly higher in patients with severe forms of COVID-19 compared to those with non-severe forms, in patients with acute respiratory distress syndrome compared to those without acute respiratory distress syndrome, and in deceased patients compared to those who have survived. D-dimers positively correlate with the degree of severity and the increased risk of progression to severe disease, inversely proportional to the survival rate. They can predict prognosis, determine therapeutic strategies, prevent complications, positively influence the disease’s course, and monitor the prognosis. Conclusions. D-dimers should be used as a pre-radiographic screening tool as early as possible after admission and as an indicator for risk stratification of venous thromboembolism in hospitalized patients with COVID-19. Based on the increase in D-dimer levels, adjusting therapeutic doses of anticoagulants is more beneficial for patients compared to administering prophylactic doses.

The role of plant adaptogen in the correction of parameters of the hemostasis system during ­emotional-immobilization stress in the experiment

Background. Maladaptive changes on the part of various organs and systems, in particular on the part of the hemostasis system, are manifested in extreme forms of physical inactivity, one of which is immobilization stress. If the organism is also subjected to psycho-emotional effects at the same time, then the combined effect of stressors is accompanied by an increase in maladaptive disorders.
 Aim. To establish the influence of emotional-immobilization stress on the hemostasis system, as well as the possibility of correcting changes by taking a course of eleutherococcus.
 Material and methods. The experiment was conducted on male rats, which were divided into four groups: the first group (n=14) control, the second group (n=14) animals treated with eleutherococcus for 30 days, the third group (n=14) rats, subjected to a single 3-hour water-immersion dipping, the fourth group (n=14) animals that took eleutherococcus for 30 days before the water-immersion dipping. The state of the hemostasis system was assessed in all experimental groups. Statistical processing of the results was performed using the Statistica 10.0 package (Statsoft, USA). Significance of differences was assessed using the nonparametric MannWhitney test and Student's t-test.
 Results. A single immobilization effect was accompanied by hypercoagulable changes, which characterized by shortening of the silicone (p=0.001) and kaolin (p=0.002) time. Completion of the 30-day adaptogen intake followed by emotional immobilization stress was accompanied by an increase in silicone (p=0.001), kaolin (p=0.021), prothrombin (p=0.021) and thrombin (p=0.006) time, which indicated hypocoagulation changes.
 Conclusion. The immobilization effect is characterized by hypercoagulability against the background of a decrease in the anticoagulant and fibrinolytic activity of the blood plasma; the course intake of eleutherococcus prior to immobilization is accompanied by hypocoagulant changes and an increase in the anticoagulant activity of the blood plasma.

Coagulofibrinolytic effects of recombinant soluble thrombomodulin in prolonged porcine cardiac arrest

AimTo evaluate coagulofibrinolytic abnormalities and the effects of ART-123 (recombinant human thrombomodulin alpha) in a porcine model of cardiac arrest and prolonged cardiopulmonary resuscitation (CA/CPR). MethodsFifteen pigs (n = 5 per group) underwent 8 minutes of no-flow CA followed by 50 minutes of mechanical CPR, while 2 pigs underwent sham arrest. CA/CPR animals were randomized to receive saline or 1 mg/kg ART-123 pre-arrest (5 minutes prior to ventricular fibrillation) or post-arrest (2 minutes after initiation of CPR). Arterial and venous blood samples were drawn at multiple time points for blood gas analysis and measurement of plasma and whole blood markers of coagulation and fibrinolysis. ResultsIn saline-treated CA/CPR, but not sham animals, robust and persistent activation of coagulation and fibrinolysis was observed throughout resuscitation. After 50 minutes of CPR, plasma tests and thromboelastography indicated a mix of hypercoagulability and consumptive coagulopathy. ART-123 had a robust anticoagulant effect, reducing both thrombin-antithrombin (TAT) complexes and d-dimer (p < 0.05 for each). The duration of anticoagulant effect varied depending on the timing of ART-123 administration. Similarly, ART-123 when given prior to cardiac arrest was found to have pro-fibrinolytic effects, increasing free tissue plasminogen activator (tPA, p = 0.02) and decreasing free plasminogen activator inhibitor-1 (PAI-1, p = 0.04). ConclusionA porcine model of prolonged CA/CPR reproduces many of the coagulofibrinolytic abnormalities observed in human cardiac arrest patients. ART-123 demonstrates a combination of anticoagulant and profibrinolytic effects, depending on the timing of its administration relative to cardiac arrest.

Purification of fibrinolytic enzyme from Bacillus amyloliquefaciens GUTU06 and properties of the enzyme

A producing-fibrinolytic enzyme strain was isolated with high yield. The strain was identified as Bacillus amyloliquefaciens. B. amyloliquefaciens GUTU06 fibrinolytic enzyme was purified by acetone precipitation and reverse micelle. Acetone precipitation condition and reverse micelle condition were examined. Results showed that the total reverse micelle extraction efficiency was 64.49 % ± 1.6 %. The purification fold of the entire process reached 13.38. The optimum pH of purified enzyme is 5, and the optimum temperature is 45 °C. Fe3+ and K+ can enhance the fibrinolytic activity of the enzyme. Compared to commercial fibrinolytic enzymes such as urokinase and lumbrukinase, GUTU06 fibrinolytic enzymes have a lower pH optimal range and higher temperature stability. The molecular weight of the enzyme was approximately 28 kDa. Reverse micelle extraction with cetyl trimethylammonium bromide as a surfactant combined with acetone precipitation is suitable for separating and purifying fibrinolytic enzymes and a promising technique for obtaining active proteins.

Analysis of fibrinolytic shutdown in trauma patients with traumatic brain injury

BackgroundCoagulation profiles following major trauma vary depending on injury pattern and degree of shock. The physiologic mechanisms involved in coagulation function at any given time are varied and remain poorly understood. Thromboelastography (TEG) has been used evaluate coagulation profiles in the trauma population with some reports demonstrating a spectrum of fibrinolysis to fibrinolytic shutdown on initial presentation. The objective of this study was to evaluate the fibrinolytic profile of patients with TBI using thromboelastography (TEG). We hypothesized that patients with TBI would demonstrate low fibrinolytic activity. MethodsAll trauma activations at an ACS-verified level 1 trauma center received a TEG analysis upon arrival from December 2019 to June 2021. A retrospective review of the results and outcomes was conducted, and TBI patients were compared to patients without TBI. Linear regression was used to evaluate the effect of patient and injury factors on fibrinolysis. Hyperfibrinolysis was defined as LY30 ​> ​7.7%, physiologic fibrinolysis as LY30 0.6–7.7%, and fibrinolytic shutdown as LY30 ​< ​0.6%. ResultsA total of 1369 patients received an admission TEG analysis. Patients with TBI had a significantly higher median ISS (16 vs. 8, p ​< ​0.001), lower median admission Glasgow Coma Scale (14 vs. 15, p ​< ​0.001), longer intensive care unit length of stay (3 vs. 2 days, p ​< ​0.0001), increased ventilator days (216 vs. 183, p ​< ​0.001), higher mortality (14.6% vs. 5.1%, p ​< ​0.001), but lower shock index (0.6 vs. 0.7, p ​< ​0.0001) compared to those without TBI. Median LY30 was found to be decreased in the TBI group (0.1 vs. 0.2, p ​= ​0.0006). Patients with TBI were found to have a higher rate of fibrinolytic shutdown compared those without TBI (68.7% vs. 63.5%, p ​= ​0.054). ISS, sex, and shock index were found to be predictive of LY30 on linear regression, but TBI was not (Β: 0.09, SE: 0.277, p ​= ​0.745). The rate of DVT/PE did not appear to be elevated in patients with TBI (0.8%) and without TBI (1.2%). ConclusionsTrauma patients with and without TBI were found to have high rates of fibrinolytic shutdown. Although there was a high incidence of fibrinolytic shutdown, it did not appear to have an impact on the rate of thrombotic complications. The clinical significance of these results is unclear and differs significantly from recent reports which demonstrated that TBI is associated with a 25% rate of fibrinolytic shutdown. Further investigation is needed to better define the fibrinolytic pathway in patients with trauma and TBI to develop optimal treatment algorithms.

D-dimer level differences in pneumonia patients than in COVID-19

Introduction: Pneumonia, also known as pneumonia, is caused by inflammation of the air sacs (alveoli) in one or both lungs. In severe pneumonia, intravascular and intra-alveolar coagulation occurs, which is a response to local and systemic inflammatory processes. The clinical consequence of these changes in coagulation is an increase in the level of D-Dimer which causes death. The purpose of this study was to evaluate D-Dimer in 29 pneumonia patients and 37 COVID-19 patients at Jemursari Hospital. Methods: This research is quantitative with a cross-sectional analytic observational method. The research sample was obtained from the Jemursari Islamic Hospital in Surabaya for pneumonia and Covid-19 patients in 2021 Results: The results of the study, a number of women more than men. In women 63,6% and in men 36,4%, while the age is 46-54 years in pneumonia patients 28% and in Covid-19 patients 30%. D-Dimer levels in pneumonia patients 1001-2000 ng/mL were 35% higher compared to COVID-19 patients 501-1000 ng/mL were 30%. Mann Whitney's non-parametric test results obtained p=0.041 (p&lt;0.05), meaning that there was a difference in D-Dimer levels between pneumonia patients and COVID-19. Conclusion: D-Dimer levels were more elevated in pneumonia patients than in Covid-19, so Covid-19 infection did not exacerbate coagulation and fibrinolysis activities.

Is there a potential role of inhibition of thrombin-activatable fibrinolysis inhibitor in regulation of local fibrinolytic activities on fibrin thrombi in patients with pulmonary embolism?

Is there a potential role of inhibition of thrombin-activatable fibrinolysis inhibitor in regulation of local fibrinolytic activities on fibrin thrombi in patients with pulmonary embolism?

Chinese expert consensus on D-dimer laboratory testing and clinical application

D-dimer is a degradation product deriving from the plasmin-mediated degradation of cross-linked fibrin. It serves as a biomarker for the activation of coagulation and fibrinolysis, and has been extensively utilized in the diagnosis and treatment of various diseases. However, in recent years, issues related to D-dimer laboratory testing and its clinical application have become increasingly prominent. The lack of standardization in reports has led to confusion in both laboratory testing and clinical application. To address this, the Chinese Society on Thrombosis and Hemostasis brought together experts in the field of thrombosis and hemostasis in China to discuss and develop the 'Chinese Expert Consensus on D-Dimer Laboratory Testing and Clinical Application'. This consensus focuses on laboratory testing methods, quality requirements, performance evaluation, sample processing, analysis interference, result reports, and clinical application of D-dimer. Its aim is to optimize the key issues related to D-dimer testing in laboratories and provide guidance for the appropriate use and accurate interpretation of D-dimer in clinical settings.

The problem of inpatient thrombosis of coronary arteries in patients with COVID-19 on the background of intensive antithrombotic therapy

Myocardial infarction claims million lives on the planet each year. Pharmacotherapy in combination with percutaneous coronary interventions have significantly reduced morbidity and mortality in this cohort. Despite optimal therapy and prophylaxis, there is a group of patients with recurrent, often fatal, coronary thromboses. During the COVID-19 pandemic, some patients developed episodes of thrombosis in blood vessels of various organs, including brain, heart or lungs, even on therapeutic anticoagulation. The patient was admitted to the department of cardiology and reperfusion therapy, with a confirmed diagnosis of COVID-19 resulted in acute ST-segment elevation myocardial infarction. Patient underwent urgent coronary angiography with thromboaspiration and stenting of the infarct-dependent right coronary artery followed by optimal medical treatment with the use of triple antithrombotic therapy. His condition became stable with the resolution of ST-segment elevation on electrocardiogram. A few days later, the condition of the patient deteriorated again with recurrent of chest pain and a new ST segment elevation. Repeated coronary angiography revealed recurrent thrombosis in the right coronary artery. The mechanisms of thrombosis in COVID-19 are not fully understood. Among possible, the direct effect of the virus on tissues, activation of pro-inflammatory system and coagulation system, impaired vasodilation, and decreased fibrinolytic activity are discussed. These mechanisms may lead to vascular thrombosis in different organs, particularly heart, lungs, and brain.

Fungi Fibrinolytic Compound 1 Plays a Core Role in Modulating Fibrinolysis, Altering Plasma Clot Structure, and Promoting Susceptibility to Lysis.

Fibrin clot structure and function are major determinants of venous and arterial thromboembolic diseases, as well as the key determinants of the efficiency of clot lysis. Studies have revealed that fungi fibrinolytic compound 1 (FGFC1) is a novel marine pyranisoindolone natural product with fibrinolytic activity. Here, we explore the impacts of FGFC1 on clot structure, lysis, and plasminogen activation in vitro using turbidimetric, enzyme-linked immunosorbent assay, confocal and electron microscopy, urokinase, or plasmin chromogenic substrate. Clots formed in the presence of FGFC1 expressed reduced fibrin polymerization rate and maximum turbidity; however, they did not influence the lag phase of fibrin polymerization. In the absence of scu-PA (single-chain urokinase plasminogen activator), microscopy revealed that FGFC1 increased the number of protofibrils within fibrin fiber and the pore diameter between protofibrils, inducing clots to form a region of thinner and looser networks separated by large pores. The effects of FGFC1 on scu-PA-mediated plasma clot structure were similar to those in the absence of scu-PA. In addition, FGFC1 promoted the lysis of clots and increased the D-dimer concentration in lysate. FGFC1 increased the generation rate of p-nitroaniline in plasma. These results show that FGFC1 has fibrinolytic activity in plasma, leading to interference with the release of fibrinopeptide B to affect lateral aggregation of protofibrils and increase clot susceptibility to fibrinolysis by altering its structure.

Insight into the thrombolytic ability of an extracellular fibrinolytic enzyme from Bacillus amyloliquefaciens GXU-1 isolated from Sipunculus nudus

Bacterial fibrinolytic enzymes have an important role in thrombolytic therapy due to their ability to dissolve fibrin. Therefore, purification, characterization and activity determination are of prime importance for bacterial fibrinolytic enzymes. In the current study, marine Bacillus amyloliquefaciens was first screened from Sipunculus nudus living in the Beibu Gulf of China and denoted as Guangxi University-1 (GXU-1). Then, an extracellular fibrinolytic enzyme (FEB-1) was purified from GXU-1 using ammonium sulfate precipitation, hydrophobic chromatography and gel filtration chromatography. The specific activity of FEB-1 was determined to be as high as 6789.74 U/mg. The relative molecular weight of FEB-1 was measured as 30 kDa through SDS‒PAGE. The optimum in vitro fibrinolytic activity of FEB-1 was identified at 37 °C and pH = 8. Furthermore, the activity of FEB-1 can be well preserved at 20–45 °C and pH = 6.0 to 9.0. The combination analysis of SDS‒PAGE and the molecular docking calculation revealed that FEB-1 can cleave more Aα- and Bβ-chains of fibrinogen than γ-chain. It is noteworthy that FEB-1 is comparatively stable under human-body environmental conditions, indicating its potential application in thrombosis therapy.

The Hemostatic System in Newborns and the Risk of Neonatal Thrombosis.

Newborns are the most vulnerable patients for thrombosis development among all children, with critically ill and premature infants being in the highest risk group. The upward trend in the rate of neonatal thrombosis could be attributed to progress in the treatment of severe neonatal conditions and the increased survival in premature babies. There are physiological differences in the hemostatic system between neonates and adults. Neonates differ in concentrations and rate of synthesis of most coagulation factors, turnover rates, the ability to regulate thrombin and plasmin, and in greater variability compared to adults. Natural inhibitors of coagulation (protein C, protein S, antithrombin, heparin cofactor II) and vitamin K-dependent coagulation factors (factors II, VII, IX, X) are low, but factor VIII and von Willebrand factor are elevated. Newborns have decreased fibrinolytic activity. In the healthy neonate, the balance is maintained but appears more easily converted into thrombosis. Neonatal hemostasis has less buffer capacity, and almost 95% of thrombosis is provoked. Different triggering risk factors are responsible for thrombosis in neonates, but the most important risk factors for thrombosis are central catheters, fluid fluctuations, liver dysfunction, and septic and inflammatory conditions. Low-molecular-weight heparins are the agents of choice for anticoagulation.

A Multilayered Imaging and Microfluidics Approach for Evaluating the Effect of Fibrinolysis in Staphylococcus aureus Biofilm Formation.

The recognition of microbe and extracellular matrix (ECM) is a recurring theme in the humoral innate immune system. Fluid-phase molecules of innate immunity share regulatory roles in ECM. On the other hand, ECM elements have immunological functions. Innate immunity is evolutionary and functionally connected to hemostasis. Staphylococcus aureus (S. aureus) is a major cause of hospital-associated bloodstream infections and the most common cause of several life-threatening conditions such as endocarditis and sepsis through its ability to manipulate hemostasis. Biofilm-related infection and sepsis represent a medical need due to the lack of treatments and the high resistance to antibiotics. We designed a method combining imaging and microfluidics to dissect the role of elements of the ECM and hemostasis in triggering S. aureus biofilm by highlighting an essential role of fibrinogen (FG) in adhesion and formation. Furthermore, we ascertained an important role of the fluid-phase activation of fibrinolysis in inhibiting biofilm of S. aureus and facilitating an antibody-mediated response aimed at pathogen killing. The results define FG as an essential element of hemostasis in the S. aureus biofilm formation and a role of fibrinolysis in its inhibition, while promoting an antibody-mediated response. Understanding host molecular mechanisms influencing biofilm formation and degradation is instrumental for the development of new combined therapeutic approaches to prevent the risk of S. aureus biofilm-associated diseases.

Surgery in rare bleeding disorders: the prospective MARACHI study

BackgroundDespite the wide use of bleeding scores and the reliability of clotting factor level measurement, bleeding risk stratification before surgery remains challenging in patients with rare inherited bleeding disorders. ObjectivesThis multicenter observational prospective study assessed in patients with rare coagulation factor deficiency, the perioperative hemostatic management choices by hemostasis experts and the bleeding outcomes after surgery. MethodsOne hundred seventy-eight patients with low coagulation activity level (factor [F] II, FV, combined FV-FVIII, FVII, FX, or FXI <50%) underwent 207 surgical procedures. The bleeding outcome, Tosetto’s bleeding score, and perioperative hemostatic protocols were collected. ResultsAmong the 81 procedures performed in patients with severe factor deficiency (level ≤10%), 27 were done without factor replacement (including 6 in patients at high bleeding risk), without any bleeding event. Factor replacement therapy was used mainly for orthopedic procedures. In patients with mild deficiency, 100/126 surgical procedures were carried out without perioperative hemostatic treatment. In patients with FVII or FXI deficiency, factor replacement therapy was in function of the procedure, bleeding risk, and to a lesser extent previous bleeding history. Tranexamic acid was used in almost half of the procedures, particularly in case of surgery in tissues with high fibrinolytic activity (76.8%). ConclusionsThe current perioperative hemostatic management of patients with rare bleeding disorders appears to be adapted. Among the 207 procedures, only 6 were associated with excessive bleeding. Our findings suggest that rather than the bleeding score, factor level and surgery type are the most relevant criteria for perioperative factor replacement therapy.

Recovery and partial purification of fibrinolytic protease from Pleurotus ostreatus and P. eryngii and cytotoxic and antioxidant activity of their extracts

Mushrooms are a source of primary and secondary metabolites. Little is known about the most suitable conditions for production of mushrooms by submerged fermentation. This article reports antioxidant and cytotoxic assays, in addition to quantitatively evaluating the content of proteases with fibrinolytic action in the crude extracts of two species of edible mushrooms produced in different formulations, as well as evaluating the recovery of these enzymes by aqueous two-phase systems (ATPS). The mushrooms Pleurotus ostreatus and Pleurotus eryngii, at concentration of 100 µg/mL, displayed inhibition of DPPH and ABTS radicals below 50%. In the cytotoxicity test, the cells human fibroblast cell lines (MRC-5) showed cell viability greater than 80%. Concerning fibrinolytic activity, P. eryngii presented 226.47 ± 7.26 U/mL, therefore being more efficient than P. ostreatus (71.5 ± 0.56 U/mL). In the recovery of the P. eryngii extract by ATPS, the fibrinolytic protease was partitioned in the salt phase (30.25 U/mL). The molecular mass of the proteases was between 75 and 100 kDa. These results prove the low cytotoxicity of the extracts produced and that fermentation in supplemented malt broth favored the excretion of fibrinolytic proteases compared to the other evaluated media.

Fibrinolytic Protease Activity of Crude Enzyme from Fermented Sunflower (Helianthus annuus) and Common Bean (Phaseolus vulgaris) seeds by Rhizopus microsporus var. oligosporus FNCC 6010 in Solid State Fermentation

In the entire world, cardiovascular diseases (CVDs) are the main cause of death. For the treatment of CVDs, microbial fibrinolytic enzymes are highly regarded as novel therapeutic candidates. This study was purposed to determine the fibrinolytic protease activity produced by fungus source, which is Rhizopus microsporus var. oligosporus FNCC 6010 in fermented sunflower (Helianthus annuus) seed and common bean (Phaseolus vulgaris) seed. Fermentation was carried out by solid-state fermentation method at an initial pH of 5, incubation temperature of 33±1°C, and incubation time of 24 hours. The fermented seed was extracted to obtain supernatant as the crude enzyme. The proteolytic activity assay was done by the skimmed milk agar (SMA) plate method to obtain the proteolytic index, and the fibrinolytic activity assay was conducted by the fibrin-agarose plate method to get the fibrinolytic index. The results show that crude enzymes from fermented H. annuus and P. vulgaris seeds by R. microsporus have fibrinolytic protease activity with proteolytic index 2.64 ± 0.01 and 2.23 ± 0.04, respectively. The fibrinolytic index is 2.40 ± 0.06 and 1.64 ± 0.06, respectively. Therefore, the crude enzyme has the potential to be further researched as a candidate for thrombolytic agents. The purification, characterization, and in-depth research are needed to develop enzymes into preparations for preventing and treating CVDs.

Elevated factor VIII level: An implication for thrombosis during pregnancy

Hemostasis is the process by which the body maintains the integrity of blood vessels and regulates blood circulation. It involves arresting bleeding, and possibly mending broken or severed blood vessels. It is achieved through a series of steps, including vascular spasm, platelet plug formation, coagulation cascade, and fibrinolysis. Skipping any of these steps causes severe bleeding and death. During pregnancy, the hemostatic system undergoes significant changes to adapt to the physiological demands. These changes aim to prevent excessive bleeding during and after childbirth. Hypercoagulability, characterized by elevated clotting factors and reduced fibrinolytic activity, is common during pregnancy. While these alterations in the coagulation system help prevent postpartum bleeding, they also increase the mother's risk of thrombosis by a factor of five. This hypercoagulable state is caused by pregnancy-related factors like venous stasis, endothelial damage, and hormonal fluctuations. However, studies have shown that an increase in Factor VIII levels poses as a risk factor for both venous and arterial thrombosis. The determination of plasma factor VIII levels is influenced by genetic factors, as well as the levels of von Willebrand factor (VWF) and the individual's blood group. The findings of familial investigations have revealed that the concentration of factor VIII is indeed hereditary, exhibiting a lesser degree of variability among twins when compared with individuals who lack a familial connection. Continuous monitoring should be performed on pregnant patients with thrombosis risk factors. This intervention is implemented with the aim of mitigating the potential aggravation of deep vein thrombosis (DVT) and the subsequent development of post-thrombotic syndrome.

Nemopilema nomurai jellyfish venom attenuates phenotypic modulation of PDGF BB-induced vascular smooth muscle cells and κ-carrageenan-induced rat tail thrombosis

Jellyfish venoms have long been recognized as a potentially rich source of natural bioactive compounds with pharmacological potential for the creation of innovative drugs. Our previous study demonstrated that Nemopilema nomurai jellyfish venom (NnV) has a chymotrypsin-like serine protease with fibrinolytic activity in vitro. Therefore, the present study aims to investigate the potential effect of NnV on cell migration, proliferation, and differentiation of vascular smooth muscle cells (VSMC; A7r5 cells) involved in the probable mechanism pathways. We also determined its anti-thrombotic effect through κ-carrageenan-induced Sprague–Dawley (SD) rat tail thrombus model. NnV inhibits on Platelet-derived growth factor (PDGF)-BB-stimulated A7r5 cells migration and proliferation by decreasing matrix metalloproteinase 2 (MMP-2) level and phosphorylation of ERK and Akt in a dose-dependent manner, but not p38. Furthermore, NnV regulates the phenotype transition of differentiation in PDGF-BB-stimulated A7r5 cells via ɑ-SMA and calponin in a dose-dependent manner. In an in vivo study, NnV treatment demonstrated clear anti-thrombotic activity in a dose-dependent manner, which was associated with decreased thrombus formation and length in κ-carrageenan-induced SD rat tail. These findings suggested that NnV has a novel fibrinolytic enzyme that can be used to prevent and/or treat thrombosis-related cardiovascular disorders.