Fibrinolytic Research Articles

Evaluating the thrombolytic property of Juniperus Sabina tincture using spectrophotometry

Objectives: We aimed to explore the thrombolytic property of Juniperus Sabina by measuring the absorbance level of released haemoglobin (Hb) during clot lysis. Materials and Methods: Approximately 0.3 mL of blood was added to four 15 mL centrifuge tubes and blood clots were allowed to form naturally. The clots were incubated for 45 min and washed with normal saline. Using a visible spectrophotometer, the absorbance of Hb was recorded at five wavelengths: 405, 416, 541, 576 and 580 nm. Lyophilised recombinant streptokinase (SK) was used as standard control. J. Sabina mother tincture (83.28% v/v) of 1:10 (T1) and 1:20 (T2) dilutions were studied. Distilled water and ethyl alcohol mixed in a ratio of 1:1 (hydro alcohol) were used as a negative control. Results: Thrombolytic activity was compared by recording the absorbance of Hb at five wavelengths (405, 416, 541, 576 and 580 nm) every 10 min from 0 to 60 min. Absorption of light by the released Hb during thrombolysis was highest at 416 nm for SK, T1 at 405 and 416 nm and HA and T2 at 405 nm. Comparatively, T1 showed higher absorption than HA. In general, the peak absorbance of Sabina concentrations was more when compared to negative controls in the higher wavelengths of 541, 576 and 580 nm. However, this difference was not statistically significant. The mean and standard deviation from the data were calculated to find the amount of dispersion for all the samples at different wavelengths. The within- and between-group significance was determined using analysis of variance. Conclusion: This study has shown that Sabina in the dilutions of 1:10 and 1:20 does not induce thrombolysis in vitro. By and large, further studies with different concentrations of Sabina Q with a sophisticated spectrophotometer need to be tested for thrombolytic activity.

Computational modeling of blood clot lysis considering the effect of vessel wall and pulsatile blood flow.

Stroke is one of the major causes of global death, which can occur due to blockage in a blood vessel by a clot. The immediate dissolving of the clot is essential to restore the blood flow and prevent tissue necrosis. Clot dissolution can be achieved via thrombolytic therapy using plasminogen activators. In this study, a clot dissolution model is developed for a three-dimensional patient-specific carotid artery that investigates the effect of different vessel wall models on clot dissolution. The lysis pattern of the clot and hemodynamics of blood flow are evaluated using three different models of the vessel wall, namely, rigid, linear elastic, and Mooney-Rivlin hyperelastic. The effect of flow condition is considered by solving the Navier-Stokes equationsfor the free flow domain and the Brinkman equationfor the clot domain with the same pressure and velocity fields. This will result in continuous pressure and velocity over the interfaces of the free flow and clot domains. The blood inflow is assumed to be pulsatile. In addition, the species transport driven by diffusion and convection is considered to be different in the porous medium and plasma. The obtained results show that in all models, the starting time of clot volume decrease is almost the same and the clot starts dissolving from the inner curvature of the artery. However, in the hyperelastic model, dissolving the clot takes longer compared to the other two models. By monitoring the vessel wall deformation, the exact time of vessel recanalization is determined.

Association of Fibrinolytic Potential and Risk of Mortality in Cancer Patients.

Cancer is a leading cause of death, and the fibrinolytic system shows cooperative effects that facilitate the growth of tumors and the appearance of metastases. This prospective study aimed to evaluate the fibrinolytic potential in cancer patients and its association with mortality outcomes using the fluorometric method of simultaneous thrombin and plasmin generation. The study included 323 cancer patients and 148 healthy individuals. During the 12-month follow-up, 68 patients died. Compared to the control group, cancer patients showed alterations in thrombin production consistent with a hypercoagulability profile, and an increase in plasmin generation. Mortality risk was associated with two parameters of thrombin in both univariate and multivariable analysis: maximum amplitude (Wald 11.78, p < 0.001) and area under the curve (Wald 8.0, p < 0.005), while such associations were not observed for plasmin. In conclusion, this was the first study able to demonstrate the simultaneous evaluation of thrombin and plasmin generation in newly diagnosed untreated cancer patients. Patients with cancer have been observed to exhibit a hypercoagulable profile. During the study, two parameters linked to thrombin generation, MA and AUC, were identified and found to have a potential association with mortality risk. However, no associations were found with parameters related to plasmin generation.

Pharmacological Investigation of In-vitro Anti-inflammatory, Antimicrobial, Thrombolytic, Cytotoxic and In vivo Analgesic Activities of Ethanolic Leaf Extract of Diospyros malabarica

Aims: This study was designed to investigate the effects of leaf extracts of Diospyros malabarica plant (EEDML) on a number of in vitro activities, including antibacterial, anti-inflammatory, cytotoxic, and thrombolytic activities, as well as in vivo analgesic activities.&#x0D; Place and Duration of Study: The Laboratory of Phytochemistry and Pharmacology at the Department of Pharmacy and the Laboratory of Microbiology at Stamford University Bangladesh and the Bangladesh Council of Scientific and Industrial Research, Dhaka, conducted this investigation from January 2022 to June 2023.&#x0D; Methodology: The study employed the Egg Albumin Protein Denaturation Assay to evaluate the anti-inflammatory effects of various doses. The Disk Diffusion Method was employed for conducting an antimicrobial assay. Different tests were conducted to evaluate the effects of thrombolytic and cytotoxic substances. These tests included the Clot Lysis assay and the Brine Shrimp Lethality Assay. In-vivo analgesic testing was conducted using the hot plate method and the acetic acid-induced writhing test.&#x0D; Results: The experimental tests showed significant pain-relieving effects, with inhibition percentages of 46.2% and 66.61% observed at doses of 200 and 400 mg/kg, respectively. The anti-inflammatory test showed a significant inhibition of EEDML at a concentration of 1000μg/mL, with an inhibition rate of 85.45%. The antimicrobial test showed a moderate antimicrobial effect, with a zone of inhibition ranging from 07-15, depending on the microorganism and dosage. The thrombolytic test showed a clot lysis rate of 85.37%, while the cytotoxic test indicated an LC50 value of 1.47.&#x0D; Conclusion: EEDML's phytochemical displays strong pharmacological properties, suggesting its potential for drug discovery in diverse areas.

Carbon monoxide (CO) derived from the CO-releasing molecule CORM-2 reduces peritoneal adhesion formation in a rat model.

Although low-dose carbon monoxide (CO) administration has been shown to have an anti-fibrotic effect in various fibrotic diseases, its effects on peritoneal adhesion (PA), one of the postoperative complications, are not elucidated. In this study, the effect of CO-releasing tricarbonyldichlororuthenium (II) dimer (CORM-2) administration on the formation of PA and the underlying factors of its potential effect were investigated. After the induction of PA, rats were divided into four groups with 8 rats in each group. The rats received either (i) dimethyl sulfoxide:saline solution (1:10) as a vehicle, (ii) 2.5mg/kg CORM-2, (iii) 5mg/kg CORM-2, or (iv) inactive (i) CORM (iCORM) intragastrically every day for a duration of 7days. PA was not induced in rats (n = 8) designated as sham controls. Gross, histological, immunohistochemical and quantitative real-time polymerase chain reaction analyses were performed to evaluate the effectiveness of CORM-2 administration. Gross analysis showed that CORM-2 administration reduced PA formation compared to rats treated with vehicle. Histological and immunohistochemical examinations showed that increased collagen deposition, myofibroblast accumulation, microvessel density, and M1 macrophage count in the peritoneal fibrosis area of vehicle-treated rats decreased following CORM-2 treatments. PCR analyses showed that CORM-2 treatments decreased hypoxia-induced Hif1a, profibrotic Tgfb1, ECM components Col1a1 and Col3a1, collagen degradation suppressor Timp1, fibrinolysis inhibitor Serpine1, and pro-inflammatory Tnf mRNA expressions, while increasing the M2 macrophage marker Arg1 mRNA expression. These results suggested that CORM-2 administration reduces PA formation by affecting adhesiogenic processes such as pro-inflammatory response, fibrinolytic system, angiogenesis and fibrogenesis.

PECAM-1/Thrombus Ratio Correlates with Blood Loss during Off-Pump Coronary Artery Bypass Grafting (OPCAB) Surgery: A Preliminary Study.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is considered an antiplatelet molecule. Previously, we introduced a new parameter called the PECAM-1/thrombus ratio, which indicates the proportion of PECAM-1 in the thrombus and provides a precise description of human platelet activity (in vitro). The aim of this study was to determine whether the PECAM-1/thrombus ratio could serve as a predictive factor for bleeding events during off-pump coronary artery bypass grafting (OPCAB). To achieve this, we collected blood samples from 20 patients scheduled to undergo OPCAB surgery. We assessed the PECAM-1/thrombus ratio by evaluating thrombus formation on collagen fibers under flow conditions. Subsequently, we compared the ability of the PECAM-1/thrombus ratio in predicting bleeding risk with other methods that evaluate hemostasis activity. These methods included assessing platelet P-selectin secretion, platelet exposure of phosphatidylserine, plasma coagulation and fibrinolysis system activity, and thrombus formation using the T-TAS assay. Our findings revealed a positive correlation between the PECAM-1/thrombus ratio and the amount of blood component units transfused (BCUT) during the OPCAB surgery. Furthermore, BCUT did not show any significant correlation with other measured hemostasis parameters. This preliminary study suggests that the PECAM-1/thrombus ratio might be a good predictor of bleeding risk during the OPCAB procedure.

The Role of Fibrinogen on Abortion Incidence in Pregnant Women with Toxoplasmosis

Toxoplasma gondii is the cause of toxoplasmosis. More than 60% of the world's population is infected with T. gondii. The prevalence of acute toxoplasmosis in pregnant women is 4.8 per 1000 women. The risk of fetal infection until 13 weeks of gestation is about 15% and after that the risk of infection increases and reaches 72% at 36 weeks of gestation. Fibrinogen is one of the most important acute phase proteins in pregnant women. Physiologically, there is an increase in blood procoagulant levels (include fibrinogen), but anticoagulant levels in the blood decrease during pregnancy and the performance of the fibrinolytic system weakens. In this condition, if the woman is infected with toxoplasmosis during pregnancy, the excessive elevation of coagulation factors may increase the risk of thromboembolism. T. gondii infection is known to increase IFN-γ secretion in sheep and rat experimental animals, then secrete fibrinogen-like protein 2 (fgl2) which plays a role in fibrin deposition and thrombosis. Fgl2 is a transmembrane prothrombinase that directly cleaves prothrombin to thrombin. The prothrombinase activity of Fgl2 was observed in abortion. Overall, the coagulation system gets stronger in pregnant women. However, the inflammatory process due to toxoplasmosis results in a prothrombic state situation associated with increased fibrinogen levels. Increased fibrinogen further increases inflammation. Excessive increases in fibrinogen increase the risk of thromboembolism that causes placental infarct and causes abortion.

Principal component analysis of a swine injury model identifies multiple phenotypes in trauma.

Trauma is the third leading cause of death in the United States and the primary cause of death for people between the ages of 1 year and 44 years. In addition to tissue damage, trauma may also activate an inflammatory state known as trauma-induced coagulopathy (TIC) that is associated with clotting malfunctions, acidemia, and end-organ dysfunction. Prior work has also demonstrated benefit to acknowledging the type and severity of endothelial injury, coagulation derangements, and systemic inflammation in the management of trauma patients. This study builds upon prior work by combining laboratory, metabolic, and clinical metrics into an analysis of trauma phenotypes, evolution of phenotypes over time after trauma, and significance of trauma phenotype on mortality. Seventy 3-month-old female Yorkshire crossbred swine were randomized to injury and resuscitation groups. Principal component analysis (PCA) of longitudinal swine TEG data (Reaction time, Alpha-Angle, Maximum Amplitude, and Clot Lysis at 30 minutes), pH, lactate, and MAP was completed in R at baseline, 1 hour postinjury, 3 hours postinjury, 6 hours postinjury, and 12 hours postinjury. Subjects were compared by principal component factor scores to assess differences in survival, injury severity, and treatment group. Among injured animals, three phenotypes were observed at each time point. Five phenotypes were associated with differences in survival, and of these, four were associated with differences in injury severity. Phenotype alignment was not significantly different by treatment group. This application of PCA to a set of coagulation, hemodynamic, and organ perfusion variables has identified multiple evolving phenotypes after trauma. Some of these phenotypes may correlate with injury severity and may have implications for survival. Next steps include validating these findings over greater numbers of subjects and exploring other machine-learning techniques for phenotype identification. Level IV, Therapeutic/Care Management.

Cow haemostasis and resistance of calves under hypoxia conditions

Intensification of the livestock industry is impossible without effective animal reproduction. The solution to this problem is possible only if a viable offspring is obtained, and their body resistance and safety are increased, which determines the research relevance. In this regard, determining the influence of the haemostasis system and blood properties of cows on the growth and development of the foetus, and subsequently on the resistance of newborn calves, depending on the condition at birth, determined the research aim. The conditions of foetal growth and development were found to be related to the activity of haemostatic factors and blood properties of cows. Hemocoagulation factors were active in animals that gave birth to calves in a state of hypoxia. The prothrombin time was 1.63, 1.40, 1.23 and 1.40 times lower, and the prothrombin haemostasis index was 1.53, 1.52, 1.35 and 1.46 times lower, respectively than in cows that gave birth to functionally active calves (p&lt;0.01). The thrombin time of haemostasis, partially activated thrombin time and fibrinogen content in cows of the experimental groups were higher than in animals of the control group. The blood viscosity of cows in the control group was lower (p&lt;0.01), and the blood coagulation of cows in the experimental groups was faster (p&lt;0.05). The increase in coagulation properties of the blood of cows that gave birth to calves in a state of hypoxia occurred against the background of a decrease in the activity of the fibrinolytic system (p&lt;0.05) and retraction of the blood clot. All this reduced the growth and development of the embryo and foetus. The placental ligamentous connection of foetuses born with signs of hypoxia was significantly greater than that of functionally active calves. The intensity and size of embryo growth were higher in the group of functionally active calves (p&lt;0.05). The activity of leukocyte defence factors, and, accordingly, the resistance of the organism of functionally active calves at birth was higher. The percentage of activated leukocytes in the blood of calves was practically the same, and the microbial count was higher in calves of the control group (p&lt;0.05). The results obtained in the course of the research can be implemented in the process of obtaining and rearing calves, and offered to all livestock farms in Ukraine

Probing interactions of red blood cells and contracting fibrin platelet clots

Contraction of blood clots plays an important role in blood clotting, a natural process that restores hemostasis and regulates thrombosis in the body. Upon injury, a chain of events culminate in the formation of a soft plug of cells and fibrin fibers attaching to wound edges. Platelets become activated and apply contractile forces to shrink the overall clot size, modify clot structure, and mechanically stabilize the clot. Impaired blood clot contraction results in unhealthy volumetric, mechanical, and structural properties of blood clots associated with a range of severe medical conditions for patients with bleeding and thrombotic disorders. Due to the inherent mechanical complexity of blood clots and a confluence of multiple interdependent factors governing clot contraction, the mechanics and dynamics of clot contraction and the interactions with red blood cells (RBCs) remain elusive. Using an experimentally informed, physics-based mesoscale computational model, we probe the dynamic interactions among platelets, fibrin polymers, and RBCs, and examine the properties of contracted blood clots. Our simulations confirm that RBCs strongly affect clot contraction. We find that RBC retention and compaction in thrombi can be solely a result of mechanistic contraction of fibrin mesh due to platelet activity. Retention of RBCs hinders clot contraction and reduces clot contractility. Expulsion of RBCs located closer to clot outer surface results in the development of a dense fibrin shell in thrombus clots commonly observed in experiments. Our simulations identify the essential parameters and interactions that control blood clot contraction process, highlighting its dependence on platelet concentration and the initial clot size. Furthermore, our computational model can serve as a useful tool in clinically relevant studies of hemostasis and thrombosis disorders, and post thrombotic clot lysis, deformation, and breaking.

DNA and histones impair the mechanical stability and lytic susceptibility of fibrin formed by staphylocoagulase.

Staphylocoagulase (SCG) is a virulence factor of Staphylococcus aureus, one of the most lethal pathogens of our times. The complex of SCG with prothrombin (SCG/ProT) can clot fibrinogen, and SCG/ProT-induced fibrin and plasma clots have been described to show decreased mechanical and lytic resistance, which may contribute to septic emboli from infected cardiac vegetations. At infection sites, neutrophils can release DNA and histones, as parts of neutrophil extracellular traps (NETs), which in turn favor thrombosis, inhibit fibrinolysis and strengthen clot structure. To characterize the combined effects of major NET-components (DNA, histone H1 and H3) on SCG/ProT-induced clot structure, mechanical and lytic stability. Recombinant SCG was used to clot purified fibrinogen and plasma. The kinetics of formation and lysis of fibrin and plasma clots containing H1 or core histones+/-DNA were followed by turbidimetry. Fibrin structure and mechanical stability were characterized with scanning electron microscopy, pressure-driven permeation, and oscillation rheometry. Histones and DNA favored the formation of thicker fibrin fibers and a more heterogeneous clot structure including high porosity with H1 histone, whereas low porosity with core histones and DNA. As opposed to previous observations with thrombin-induced clots, SCG/ProT-induced fibrin was not mechanically stabilized by histones. Similarly to thrombin-induced clots, the DNA-histone complexes prolonged fibrinolysis with tissue-type plasminogen activator (up to 2-fold). The anti-fibrinolytic effect of the DNA and DNA-H3 complex was observed in plasma clots too. Heparin (low molecular weight) accelerated the lysis of SCG/ProT-clots from plasma, even if DNA and histones were also present. In the interplay of NETs and fibrin formed by SCG, DNA and histones promote structural heterogeneity in the clots, and fail to stabilize them against mechanical stress. The DNA-histone complexes render the SCG-fibrin more resistant to lysis and thereby less prone to embolization.

Enhanced Cerebroprotection of Xenon-Loaded Liposomes in Combination with rtPA Thrombolysis for Embolic Ischemic Stroke.

Xenon (Xe) has shown great potential as a stroke treatment due to its exceptional ability to protect brain tissue without inducing side effects. We have previously developed Xe-loaded liposomes for the ultrasound-activated delivery of Xe into the cerebral region and demonstrated their therapeutic efficacy. At present, the sole FDA-approved thrombolytic agent for stroke treatment is recombinant tissue plasminogen activator (rtPA). In this study, we aimed to investigate the potential of combining Xe-liposomes with an intravenous rtPA treatment in a clinically relevant embolic rat stroke model. We evaluated the combinational effect using an in vitro clot lysis model and an in vivo embolic middle cerebral artery occlusion (eMCAO) rat model. The treatment groups received intravenous administration of Xe-liposomes (20 mg/kg) at 2 h post-stroke onset, followed by the administration of rtPA (10 mg/kg) at either 2 or 4 h after the onset. Three days after the stroke, behavioral tests were conducted, and brain sections were collected for triphenyltetrazolium chloride (TTC) and TUNEL staining. Infarct size was determined as normalized infarct volume (%). Both in vitro and in vivo clot lysis experiments demonstrated that Xe-liposomes in combination with rtPA resulted in effective clot lysis comparable to the treatment with free rtPA alone. Animals treated with Xe-liposomes in combination with rtPA showed reduced TUNEL-positive cells and demonstrated improved neurological recovery. Importantly, Xe-liposomes in combination with late rtPA treatment reduced rtPA-induced hemorrhage, attributing to the reduction of MMP9 immunoreactivity. This study demonstrates that the combined therapy of Xe-liposomes and rtPA provides enhanced therapeutic efficacy, leading to decreased neuronal cell death and a potential to mitigate hemorrhagic side effects associated with late rtPA treatment.

Vortex-ultrasound for microbubble-mediated thrombolysis of retracted clots.

Endovascular sonothrombolysis has gained significant attention due to its benefits, including direct targeting of the thrombus with sonication and reduced side effects. However, the small aperture of endovascular transducers restricts the improvement of their potential clinical efficiency due to inefficient acoustic radiation. Hence, in an earlier study, we used vortex ultrasound with an endovascular ultrasound transducer to induce shear stress and enhance the clot lysis. In this study, the vortex acoustic transduction mechanism was investigated using numerical simulations and hydrophone tests. Following this characterization, we demonstrated the performance of the vortex ultrasound transducer in thrombolysis of retracted clots in in vitro tests. The test results indicated that the maximum lysis rates were 79.0% and 32.2% with the vortex ultrasound for unretracted and retracted clots, respectively. The vortex ultrasound enhanced the efficiency of the thrombolysis by approximately 49%, both for retracted and unretracted clots, compared with the typical non-vortex ultrasound technique. Therefore, the use of endovascular vortex ultrasound holds promise as a potential clinical option for the thrombolysis of retracted clots.

Increased thromboinflammatory load in hereditary angioedema.

C1 inhibitor (C1Inh) is a serine protease inhibitor involved in the kallikrein-kinin system, the complement system, the coagulation system, and the fibrinolytic system. In addition to the plasma leakage observed in hereditary angioedema (HAE), C1Inh deficiency may also affect these systems, which are important for thrombosis and inflammation. The aim of this study was to investigate the thromboinflammatory load in C1Inh deficiency. We measured 27 cytokines including interleukins, chemokines, interferons, growth factors, and regulators using multiplex technology. Complement activation (C4d, C3bc, and sC5b-C9/TCC), haemostatic markers (β-thromboglobulin (β-TG), thrombin-antithrombin complexes (TAT), prothrombin fragment 1 + 2 (F1 + 2), active plasminogen activator inhibitor-1 (PAI-1), and the neutrophil activation marker myeloperoxidase (MPO) were measured by enzyme immunoassays. Plasma and serum samples were collected from 20 patients with HAE type 1 or 2 in clinical remission and compared with 20 healthy age- and sex-matched controls. Compared to healthy controls, HAE patients had significantly higher levels of tumour necrosis factor (TNF), interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-7, IL-9, IL-12, and IL-17A, chemokine ligand (CXCL) 8, chemokine ligand (CCL) 3, CCL4, IL-1 receptor antagonist (IL-1RA), granulocyte-macrophage colony-stimulating factor (GM-CSF), fibroblast growth factor (FGF) 2 and platelet-derived growth factor (PDGF)-BB. HAE patients also had higher levels of TAT and F1 + 2. Although granulocyte colony-stimulating factor (G-CSF), β-TG and PAI-1 were higher in HAE patients, the differences did not reach statistical significance after correction for multiple testing. In conclusion, C1Inh deficiency is associated with an increased baseline thromboinflammatory load. These findings may reflect that HAE patients are in a subclinical attack state outside of clinically apparent oedema attacks.

Changes in the Fibrinolytic System of Patients Infected with Severe Acute Respiratory Syndrome Coronavirus 2.

In this study, coagulation and fibrinolysis parameters and their association with disease severity were investigated in coronavirus disease (COVID-19) patients. COVID-19 patients (n = 446) admitted to our institute between 21 February 2021 and 17 March 2022, were recruited. Clinical data and staging were collected from all patients. Blood samples were collected and analyzed for several parameters of fibrinolysis and coagulation, including alpha-2-antiplasmin(α2AP) and plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), D-dimer, and fibrinogen levels. The TAFI, fibrinogen, and tPA levels were significantly higher in participants who died compared to that of patients who recovered (p < 0.001). However, PAI-1, tPA, and TAFI were significantly higher in patients admitted to the ICU than those of the healthy controls (p < 0.001 for PAI-1 and tPA; p = 0.0331 for TAFI). Our results showed that stage C and D COVID-19 patients had significantly higher levels of PAI-1 (p = 0.003). Furthermore, stage D COVID-19 patients had significantly higher tPA and TAFI values (p = 0.003). Hypofibrinolysis was the most prevalent condition among patients with severe COVID-19. In this study, several coagulation markers were elevated, making them suitable prognostic markers for hypofibrinolysis.

A study of fibrinolytic system components in donor groups depending on various titers of circulating anti-SARS-CoV-2 IgG in the bloodstream.

The fibrinolytic system plays an important role in controlling blood coagulation at each stage, from thrombin generation to fibrin clot cleavage. Currently, long-term multiorgan dysfunction post-coronavirus disease 2019 (COVID-19) may include coagulation disorders. Little information is available about the potential causes of post-COVID-19 coagulopathy, but one of them may be subpopulation IgG produced by the immune system against SARS-CoV-2. This article describes the changes in the main parameters of the fibrinolytic system in donors with various titers of anti-SARS-CoV-2 IgG, which is part of a complex study of the hemostasis system in these donor groups. We determined the most significant parameters of the fibrinolytic system, such as potential activity and amount of plasminogen and tissue plasminogen activator (tPA), amount of plasminogen activator inhibitor-1 (PAI-1), inhibitory potentials of α-2-antiplasmin, α-1-antitrypsin, α-2-macroglobulin in the blood plasma of donor groups. The obtained results represent the maximum and minimum values of measurement parameters among donor groups with titers of anti-SARS-CoV-2 IgG at least 10 ± 3 Index (S/C), and their statistical differences from the reference point [donor group with titer of anti-SARS-CoV-2 IgG 0 Index (S/C)]. We established the changes in fibrinolytic parameters depending on the titers of anti-SARS-CoV-2 IgG. One conclusion can be drawn from this: anti-SARS-CoV-2 IgG population may influence coagulation in the post-COVID-19 period. Further research in-vitro and in-vivo experimental models using selected and purified IgG may confirm our previous findings.

SARS-CoV-2 Spike Proteins and Cell-Cell Communication Induce P-Selectin and Markers of Endothelial Injury, NETosis, and Inflammation in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for the Pathogenesis of COVID-19 Coagulopathy.

COVID-19 progression often involves severe lung injury, inflammation, coagulopathy, and leukocyte infiltration into pulmonary tissues. The pathogenesis of these complications is unknown. Because vascular endothelium and neutrophils express angiotensin-converting enzyme-2 and spike (S)-proteins, which are present in bodily fluids and tissues of SARS-CoV-2-infected patients, we investigated the effect of S-proteins and cell-cell communication on human lung microvascular endothelial cells and neutrophils expression of P-selectin, markers of coagulopathy, NETosis, and inflammation. Exposure of endothelial cells or neutrophils to S-proteins and endothelial-neutrophils co-culture induced P-selectin transcription and expression, significantly increased expression/secretion of IL-6, von Willebrand factor (vWF, pro-coagulant), and citrullinated histone H3 (cit-H3, NETosis marker). Compared to the SARS-CoV-2 Wuhan variant, Delta variant S-proteins induced 1.4-15-fold higher P-selectin and higher IL-6 and vWF. Recombinant tissue factor pathway inhibitor (rTFPI), 5,5'-dithio-bis-(2-nitrobenzoic acid) (thiol blocker), and thrombomodulin (anticoagulant) blocked S-protein-induced vWF, IL-6, and cit-H3. This suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial-neutrophil interactions, S-proteins increase adhesion molecules, induce endothelial injury, inflammation, NETosis and coagulopathy via the tissue factor pathway, mechanisms involving functional thiol groups, and/or the fibrinolysis system. Using rTFPI, effectors of the fibrinolysis system and/or thiol-based drugs could be viable therapeutic strategies against SARS-CoV-2-induced endothelial injury, inflammation, NETosis, and coagulopathy.

Quantitative increases of extracellular vesicles in prolonged cold storage of platelets increases the potential to enhance fibrin clot formation.

Platelet derived extracellular vesicles (EVs) display a pro-coagulant phenotype and are generated throughout platelet concentrate (PC) storage. Cold storage (CS) of PCs is thought to provide a superior haemostatic advantage over room temperature (RT) storage and could prolong the storage time. However, the effect of storage conditions on EV generation and PC function is unknown. We investigated EV production under CS and RT conditions and assessed whether these EVs exhibited a more pro-coagulant phenotype in model experiments. Buffy-coat-derived PCs in a platelet additive solution (PAS) to plasma ratio of approximately 65:35 were stored at RT (22 ± 2°C) or CS (4 ± 2°C) for a prolonged storage duration of 20 days. Impedance aggregometry assessed platelet function. EVs were isolated throughout storage and quantified using nanoparticle tracking analysis. EVs were applied to a coagulation assay to assess the impact on fibrin clot formation and lysis. CS produced significantly larger EVs from day 4 onwards. EV concentration was significantly increased in CS compared to RT from day 15. EVs, regardless of storage, significantly reduced time to clot formation and maximum optical density measured compared to the no EV control. Clot formation was proportionate to the number of EV applied but was not statistically different across storage conditions when corrected for EV number. EVs in CS and RT units showed similar clot formation capacity. However, the higher number of larger EVs generated in CS compared to RT suggests PC units derived from CS conditions may overall exhibit a haemostatically superior capacity compared to RT storage.

Potensi Ekstrak Enzim Daun Pepaya (Carica papaya L.) Sebagai Agen Fibrinolitik Dengan Metode Clot Lysis in Vitro

Stroke is a disease caused due to blockage of blood vessels. Health department in 2013 stated that 15,4% of people died from strokes. One of the treatment therapies that can be used is fibrinolytic. Fibrinolytic agents have a role in lysing blood clots, these agents can be obtained from microbes, plants and animals. This study aims to determine the protein content contained in papaya leaf enzyme extract (Carica papaya L.), and to determine the potential of the enzyme extract at certain concentrations as an alternative to natural fibrinolytic drugs. The study began with plant determination, sampling, enzyme extraction using a blender, purification of the extract by precipitation using ammonium sulphate, protein content determination using the Lowry method, and testing the fibrinolytic activity of the clot lysis method in vitro. Three series of test concentrations were made, namely 20, 40, and 80%. The results of the research on papaya leaf extract showed that the protein content in purification pellet 1 was 196,22 µg/ml and purification pellet 2 was 306,33 µg/ml. In papaya leaves, fibrinolytic activity was proven with the optimal percentage of blood clot lysis in purification pellet 2 with a concentration of 80% which was 79%.

PRODUCTION OPTIMIZATION, PARTIAL PURIFICATION, AND THROMBOLYTIC ACTIVITY EVALUATION OF PROTEASE OF Bacillus cereus HSFI-10

Cardiovascular disease is the primary cause of mortality in the world due to the formation of blood clots or thrombi in blood vessels. Bacterial proteases commonly function as thrombus dissolver agents in the pharmaceutical industry. Bacterial isolate HSFI-10 (Holothuria scabra Fermented Intestine-10) previously isolated from Rusip fermented sea cucumber had demonstrated thrombolytic activity. This study aimed to produce crude protease of HSFI-10 strain at an optimized incubation time and determine the thrombolytic activity of crude and dialysate proteases on A, B, AB, and O blood types. Isolate HSFI-10 was first molecularly identified and found to be Bacillus cereus with a homology level of 99.80% with Bacillus cereus strain ST06. The optimum crude enzyme was obtained after 48-h incubation with an activity of 222.52 U/mL, which increased to 438.84 U/mL after ammonium sulfate precipitation and dialysis. Clot lysis activity of crude enzymes was measured based on the gravimetry method on blood in the ABO system, showing results that ranged from 68.99% to 69.76%, while the dialysate ranged from 81.16% to 82.52%. In conclusion, partial purification of bacterial protease could increase both its specific and thrombolytic activities on human blood in the ABO system, with only 1% activity variability between A, B, AB, and O blood types.