Lysine Binding Sites Research Articles

Grain Lysine enrichment and improved stress tolerance in rice through protein engineering.

Amino acids are a major source of nourishment for people living in regions where rice is a staple food. However, rice grain is deficient in essential amino acids, such as lysine. The activity of dihydrodipicolinate synthase (DHDPS) enzyme is crucial for lysine production in higher plants, but it is highly regulated through a feedback inhibition by its end product lysine, leading to its limited activity in the grain and resulting in low lysine accumulation. We identified lysine binding sites in the DHDPS enzyme and introduced key mutations to make it lysine feedback insensitive. Using in vivo analysis and functional complementation assays, we confirmed that protein engineering of the DHDPS renders it insensitive to lysine. Expression of mutated DHDPS resulted in 29 % higher lysine and 15 % higher protein accumulation in rice grains than the wild type. Importantly, the lysine content in transgenic grains was maintained in cooked rice. Further, the transgenic plants exhibited enhanced stress tolerance along with better antioxidant levels, improved photosynthesis, and higher grain yield compared to wild type plants. We have shown for the first time in rice that protein engineering of DHDPS can lead to accumulation of lysine in grains and impart abiotic stress tolerance. This approach could improve health in regions with nutrient deficiencies and environmental stressors that challenge food production and human health.

POSITIVE IMPACT OF TRANEXAMIC ACID IN VARIOUS MEDICAL SETTINGS

The role of successful hemostasis cannot be overemphasized. Bleeding can occur due to various reasons and its cause is not always evident. Any delay in restoring the proper hemostasis is associated with increased risk of complications and therefore results in increased mortality. Discovered in 1962 by Japanese researchers Shosuke and Utako Okamoto, tranexamic acid (TXA) is an anti-fibrinolytic agent that inhibits plasminogen activation by blocking the lysine binding sites on plasminogen. Thanks to its ability to inhibit fibrinolysis it stabilizes the preformed fibrin mesh-work and has a beneficial effect in reducing blood loss in wide range of clinical settings. TXA has been shown to reduce the risk of perioperative bleeding in patients undergoing noncardiac surgeries. CRASH trials provided the largest body of evidence confirming effectiveness of early administration of TXA, defined as administration within 3 hours after trauma, in reducing mortality in patients with severe injuries and traumatic brain injury. Recent evidence indicates that TXA administration in treatment of postpartum hemorrhage results in notable mortality reduction. Topical or locally injected tranexamic acid may reduce blood loss and improve visibility of the surgical field. More high-quality studies are needed to determine safety, efficacy and dosage. The risk of serious adverse events, especially vascular occlusive events, in patients receiving TXA compared to placebo group did not differ significantly.

Plasminogen degrades α-synuclein, Tau and TDP-43 and decreases dopaminergic neurodegeneration in mouse models of Parkinson’s disease

Parkinson's disease (PD) is the second most frequently diagnosed neurodegenerative disease, and it is characterized by the intracellular and extracellular accumulation of α-synuclein (α-syn) and Tau, which are major components of cytosolic protein inclusions called Lewy bodies, in the brain. Currently, there is a lack of effective methods that preventing PD progression. It has been suggested that the plasminogen activation system, which is a major extracellular proteolysis system, is involved in PD pathogenesis. We investigated the functional roles of plasminogen in vitro in an okadaic acid-induced Tau hyperphosphorylation NSC34 cell model, ex vivo using brains from normal controls and methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and in vivo in a widely used MPTP-induced PD mouse model and an α-syn overexpression mouse model. The in vitro, ex vivo and in vivo results showed that the administered plasminogen crossed the blood‒brain barrier (BBB), entered cells, and migrated to the nucleus, increased plasmin activity intracellularly, bound to α-syn through lysine binding sites, significantly promoted α-syn, Tau and TDP-43 clearance intracellularly and even intranuclearly in the brain, decreased dopaminergic neurodegeneration and increased the tyrosine hydroxylase levels in the substantia nigra and striatum, and improved motor function in PD mouse models. These findings indicate that plasminogen plays a wide range of pivotal protective roles in PD and therefore may be a promising drug candidate for PD treatment.

Tranexamic Acid in the Treatment of Melasma: A Comprehensive Review of Topical, Intradermal, and Oral Administration

A malfunction in human melanogenesis called melasma causes the epidermis to become hyperpigmented in certain regions of the body gradually. It significantly affects physical appearance, results in psychological and physiological distress, and diminishes those impacted individuals' quality of living. In order to restrict blood loss, tranexamic acid (TA), a plasmin inhibitor, is used for stopping unusual fibrinolysis. It works by permanently inhibiting lysine binding sites on plasminogen molecules, which prohibits plasminogen activator (PA) from converting plasminogen to plasmin. It seems sense that tranexamic acid might have an impact on keratinocyte association and function because plasminogen is additionally found in human epidermis basal cells and owing to the fact that PA has been shown to be produced by cultivated human keratinocytes. A comprehensive review of the literature demonstrates that although TA is administered by topical, oral, and intradermal injection as well as utilized as an adjuvant therapy Added to the laser therapy to treat melasma, its effectiveness has not been sufficiently proved. To fully understand the function of TA in the management of melasma, additional investigation is required.

Drug Utilization of Tranexamic Acid in the Department of Surgery

Synthetic lysine derivative tranexamic acid reversibly blocks plasminogen lysine binding sites, hence inhibiting fibrinolysis. In order to halt bleeding, antifibrinolytics encourage blood coagulation. During surgery, tranexamic acid dramatically lowers blood loss. The study's objective was to ascertain how often tranexamic acid was used in the surgery department. The study used observational methods. The study was carried out at Rajajinagar, Bengaluru's ESIC MC-PGIMSR. 28 participants (88%) in the trial received TXA after surgery, 3 (9%), during surgery, and 1 (3%), both during and after surgery. seventeen (53%) belonged to the 25–34 age range, seven (22%) to the 35–44 age range, and eight (25%) to the 18–24 age range. There were 13 (41%) females and 19 (59%) males. 23 individuals (72%) received 500 mg, 9 subjects received 1g, 29 subjects (91%) received BD (bis in die), 2 subjects (6%) received STAT, and 1 subject (3%) received SOS in this study. Of the 32 participants, 22 (69%) had an infection of the gastrointestinal system, 3 (9%) had endocrine disease, 3 (9%) had cancer, 3 (9%) underwent surgery for diabetic foot problems, and 1 (3%) had soft tissue damage. Of the thirty-two individuals, twenty-four (75%) spent six to ten days in the hospital, four (13%) stayed one to five days, three (9%) stayed eleven to fifteen days, and one (3%) stayed for the maximum duration of sixteen to twenty days. Concluding that TXA was given to most patients after surgery, it may be assumed that most blood loss happens after surgery. TXA was not given to any of the research participants during surgery. This study demonstrates the requirement for an antifibrinolytic TXA in cases where post-operative bleeding continues.

Tranexamic Acid and its Role in Total Knee Replacement

Abstract: Tranexamic acid is a synthetic lysine derivative that inhibits the lysine binding sites on plasminogen molecules in order to have an antifibrinolytic action. In patients with upper gastrointestinal bleeding, tranexamic acid was related to mortality decreases of 5 to 54% when compared to placebo. A 40% reduction was found by meta-analysis. Tranexamic acid therapy reduced mean menstrual blood loss in menstruating women with menorrhagia by 34 to 57.9% in comparison to placebo or control; the medication has also been successfully used to treat placental bleeding, postpartum hemorrhage, and cervix conization. In hemophiliac patients undergoing oral surgery, tranexamic acid considerably decreased mean blood losses, and it worked well as a mouthwash for dental patients taking oral anticoagulants. With the administration of the medication, blood loss was also decreased in patients having orthotopic liver transplantation or transurethral prostate surgery, and rebleeding rates were decreased in patients with traumatic hyphaema. Patients with hereditary angioneurotic edema have also claimed clinical improvement from tranexamic acid. The most frequent adverse reactions to tranexamic acid are nausea and diarrhea. Clinical trials have not shown that the medication increases the risk of thrombosis

A High-Throughput Small-Angle X-ray Scattering Assay to Determine the Conformational Change of Plasminogen.

Plasminogen (Plg) is the inactive form of plasmin (Plm) that exists in two major glycoforms, referred to as glycoforms I and II (GI and GII). In the circulation, Plg assumes an activation-resistant "closed" conformation via interdomain interactions and is mediated by the lysine binding site (LBS) on the kringle (KR) domains. These inter-domain interactions can be readily disrupted when Plg binds to lysine/arginine residues on protein targets or free L-lysine and analogues. This causes Plg to convert into an "open" form, which is crucial for activation by host activators. In this study, we investigated how various ligands affect the kinetics of Plg conformational change using small-angle X-ray scattering (SAXS). We began by examining the open and closed conformations of Plg using size-exclusion chromatography (SEC) coupled with SAXS. Next, we developed a high-throughput (HTP) 96-well SAXS assay to study the conformational change of Plg. This method enables us to determine the Kopen value, which is used to directly compare the effect of different ligands on Plg conformation. Based on our analysis using Plg GII, we have found that the Kopen of ε-aminocaproic acid (EACA) is approximately three times greater than that of tranexamic acid (TXA), which is widely recognized as a highly effective ligand. We demonstrated further that Plg undergoes a conformational change when it binds to the C-terminal peptides of the inhibitor α2-antiplasmin (α2AP) and receptor Plg-RKT. Our findings suggest that in addition to the C-terminal lysine, internal lysine(s) are also necessary for the formation of open Plg. Finally, we compared the conformational changes of Plg GI and GII directly and found that the closed form of GI, which has an N-linked glycosylation, is less stable. To summarize, we have successfully determined the response of Plg to various ligand/receptor peptides by directly measuring the kinetics of its conformational changes.

Intracellular tPA-PAI-1 interaction determines VLDL assembly in hepatocytes.

Apolipoprotein B (apoB)-lipoproteins initiate and promote atherosclerotic cardiovascular disease. Plasma tissue plasminogen activator (tPA) activity is negatively associated with atherogenic apoB-lipoprotein cholesterol levels in humans, but the mechanisms are unknown. We found that tPA, partially through the lysine-binding site on its Kringle 2 domain, binds to the N terminus of apoB, blocking the interaction between apoB and microsomal triglyceride transfer protein (MTP) in hepatocytes, thereby reducing very-low-density lipoprotein (VLDL) assembly and plasma apoB-lipoprotein cholesterol levels. Plasminogen activator inhibitor 1 (PAI-1) sequesters tPA away from apoB and increases VLDL assembly. Humans with PAI-1 deficiency have smaller VLDL particles and lower plasma levels of apoB-lipoprotein cholesterol. These results suggest a mechanism that fine-tunes VLDL assembly by intracellular interactions among tPA, PAI-1, and apoB in hepatocytes.

Screening, characterization and specific binding mechanism of aptamers against human plasminogen Kringle 5

Plasminogen Kringle 5 is one of the most potent cytokines identified to inhibit the proliferation and migration of vascular endothelial cells. Herein, six aptamer candidates that specifically bind to Kringle 5 were generated by the systematic evolution of ligands by exponential enrichment (SELEX). After 10 rounds of screening against Kringle 5, a highly enriched ssDNA pool was sequenced and the representative aptamers were subjected to binding assays to evaluate their affinity and specificity. The preferred aptamer KG-4, which demonstrated a low dissociation constant (Kd) of ∼ 432 nM and excellent selectivity for Kringle 5. A conserved “motif” of eight bases located at the stem-loop intersection, common to the aptamer, was further confirmed as the recognition element for binding with Kringle 5. The bulge formed by the motif and depression on the lysine binding site of Kringle 5 were both located at the binding interface, and the “induced fit” between their structures played a central role in the recognition process. Kringle 5 interacts KG-4 primarily through enthalpy-driven van der Waals forces and hydrogen bond. The key nucleotides A34 and C35 at motif on KG-4 and the positively charged amino acids in the loop 1 and loop 4 regions on Kringle 5 play a major role in the interaction. Furthermore, KG-4 dose-dependently reduced the proliferation inhibition of vascular endothelial cells by Kringle 5 and had a blocking effect on the function of Kringle 5 in inhibiting migration and promoting apoptosis of vascular endothelial cells in vitro. This study put a new light on protein-aptamer binding mechanism and may provide insight into the treatment of ischemic diseases by target depletion of Kringle 5.

Plasmin and plasminogen prevent sepsis severity by reducing neutrophil extracellular traps and systemic inflammation.

Sepsis is a lethal syndrome characterized by systemic inflammation and abnormal coagulation. Despite therapeutic advances, sepsis mortality remains substantially high. Herein, we investigated the role of the plasminogen/plasmin (Plg/Pla) system during sepsis. Plasma levels of Plg were significantly lower in mice subjected to severe compared with nonsevere sepsis, whereas systemic levels of IL-6, a marker of sepsis severity, were higher in severe sepsis. Plg levels correlated negatively with IL-6 in both septic mice and patients, whereas plasminogen activator inhibitor-1 levels correlated positively with IL-6. Plg deficiency render mice susceptible to nonsevere sepsis induced by cecal ligation and puncture (CLP), resulting in greater numbers of neutrophils and M1 macrophages, liver fibrin(ogen) deposition, lower efferocytosis, and increased IL-6 and neutrophil extracellular trap (NET) release associated with organ damage. Conversely, inflammatory features, fibrin(ogen), and organ damage were substantially reduced, and efferocytosis was increased by exogenous Pla given during CLP- and LPS-induced endotoxemia. Plg or Pla protected mice from sepsis-induced lethality and enhanced the protective effect of antibiotics. Mechanistically, Plg/Pla-afforded protection was associated with regulation of NET release, requiring Pla-protease activity and lysine binding sites. Plg/Pla are important host-protective players during sepsis, controlling local and systemic inflammation and collateral organ damage.

Inactivation of the lysine binding sites of human plasminogen (hPg) reveals novel structural requirements for the tight hPg conformation, M-protein binding, and rapid activation.

Accelerated activation of the human plasminogen zymogen (hPg) to two-chain active plasmin (hPm) is achieved following conformational changes induced by ligand-binding at the lysine-binding sites (LBSs) in four of the five hPg kringle domains. In this manner, pattern D skin-trophic strains of Group A streptococci (GAS), through the expression of surface plasminogen-binding M-protein (PAM), immobilize surface hPg, thereby enabling rapid hPg activation by GAS-secreted streptokinase (SK). Consequently, GAS enhances virulence by digesting extracellular and tight cellular junctional barriers using hPm activity. Many studies have demonstrated the singular importance of the kringle-2 domain of hPg (K2hPg) to PAM-binding using hPg fragments. Recently, we showed, using full-length hPg, that K2hPg is critical for PAM binding. However, these studies did not eliminate any modulatory effects of the non-K2hPg LBS on this interaction. Moreover, we sought to establish the significance of the intramolecular interaction between Asp219 of the LBS of K2hPg and its serine protease domain binding partner, Lys708, to conformational changes in hPg. In the current study, selective inactivation of the LBS of K1hPg, K4hPg, and K5hPg revealed that the LBS of these kringle domains are dispensable for hPg binding to PAM. However, the attendant conformational change upon inactivation of K4hPg LBS increased the affinity of hPg for PAM by an order of magnitude. This finding suggests that the native hPg conformation encloses PAM-binding exosites or sterically hinders access to K2hPg. While simultaneous inactivation of the LBS of K1hPg, K4hPg, and K5hPg inhibited hPg/SK association alongside hPg activation, the replacement of Lys708 generated a slight conformational change that optimally accelerated hPg activation. Thus, we accentuate disparate functions of hPg LBS and conclude, using intact proteins, that K2hPg plays a central role in regulating hPg activation.

Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells

Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.

Abstract TP130: Effect Of Antifibrinolytic Medication On Hematoma Expansion In Acute Ischemic Stroke Patients With Post Thrombolytic Intracerebral Hemorrhages

Background and Purpose: Antifibrinolytic medications such as the synthetic lysine analogs tranexamic acid and ε-aminocaproic acid inhibit fibrinolysis by attaching to the lysine-binding site of the plasminogen molecule. However, the efficacy of intravenous (IV) antifibrinolytic medication in the prevention of hematoma expansion in acute ischemic stroke patients who develop post thrombolytic intracerebral hemorrhage (ICH) is unclear. Methods: We analyzed the effect of antifibrinolytic medication in acute ischemic stroke patients with post thrombolytic ICH on hematoma expansion over an 8-year period. The Region-of-Interest; method on a Horos Medical Image Viewer (Version 3.3.6) was utilized for hematoma volume measurement on serial computed tomographic scans. We compared changes in hemorrhage volume in patients who did and did not receive IV antifibrinolytic medication. Results: A total of 53 patients (mean age 69.7±14.3, 53% were men) with post thrombolytic ICH were analyzed. Twenty-six patients received antifibrinolytic treatment: 20 and 6 patients received a single IV dose of ε-aminocaproic acid (5 g) or tranexamic acid (1 g), respectively. Ten patients (38%) had a reduction or stabilization in hematoma volume (overall pre and post-treatment volumes were 17.43±16.23 cm3 and 25.14±26.57 cm3 respectively). Mean fibrinogen level in the antifibrinolytic medication group was 277 mg/dL; 3 patients had levels &lt;200 mg/dL. In patients that did not receive antifibrinolytics, 16 (59%) out of 27 patients had a reduction or stabilization in hematoma volume (overall pre and post-treatment volumes were 19.74±32.32 cm3 and 27.15±52.58 cm3 respectively). The mean percent change in hematoma volume with antifibrinolytic medication treatment was 48% and without was 27%. [Figure 1] Conclusion: We did not identify any reduction in any of the measures of hematoma expansion with antifibrinolytic treatment in acute ischemic stroke patients with post thrombolytic ICH.

The usefulness of tranexamic acid for bleeding symptoms of chronic consumptive coagulopathy complicated by aortic disease: a single-institute, retrospective study of 14 patients

BackgroundTranexamic acid (TXA) is an antifibrinolytic drug that blocks lysine-binding sites on the profibrinolytic enzyme plasminogen. Aortic diseases with chronic consumption coagulopathy may lead to disseminated intravascular coagulation (DIC) and cause fatal bleeding. Although the use of antifibrinolytic agents in DIC is generally not recommended due to enhanced fibrin deposition risking thrombotic symptoms, the efficacy of TXA has been reported in several cases of DIC with aortic diseases. However, the efficacy and safety of TXA for bleeding symptoms of chronic consumption coagulopathy with aortic diseases have not been studied in detail.MethodsWe evaluated the efficacy of TXA in 14 patients with chronic consumptive coagulopathy due to aortic disease complicated by bleeding symptoms. Changes in coagulation and fibrinolysis parameters from baseline were analyzed with Wilcoxon matched-pairs signed-rank tests, excluding missing values. Kaplan-Meier curves were used to analyze overall survival.ResultsMedian age was 78.5 years (range, 66–89 years) and median observation period was 448 days (range, 0–2282 days). Twelve patients had chronic renal failure and 1 patient had chronic liver failure. Before starting treatment, median Japanese Ministry of Health and Welfare DIC diagnostic criteria score was 8 (range, 4–11) and median platelet count was 64 × 109/L (range, 25–97 × 109/L). Twelve patients underwent evaluation of bleeding symptoms after introduction of TXA, and 10 of those 12 patients showed improved bleeding tendencies within 30 days (median, 5.0 days). One patient with chronic liver failure showed worsening of bleeding symptoms. Although only one patient was initiated TXA in combination with anticoagulants, no significant worsening of thrombotic events was observed within 30 days.ConclusionsTXA therapy appears effective against chronic consumptive coagulopathy with bleeding due to aortic disease, with few side effects.

Development of thermostable Streptokinase by recombinant DNA technology as fibrinolytic agent for different thromboembolic disorders

Edicine is a therapeutic agent in the management of thromboembolic blockade. Streptokinase is presently utilized in clinical mase and is an extracellular protein secreted by certain strains of beta-hemolytic streptococci. It activates plasminogen to produce plasmin, an enzyme that breaks down fibrin clots through specific lysine binding sites.

1,2,3-Triazole Derivatives as Novel Antifibrinolytic Drugs

Fibrinolysis is a natural process that ensures blood fluidity through the removal of fibrin deposits. However, excessive fibrinolytic activity can lead to complications in different circumstances, such as general surgery or severe trauma. The current antifibrinolytic drugs in the market, aminocaproic acid (EACA) and tranexamic acid (TXA), require high doses repetitively to maintain their therapeutic effect. These high doses are related to a number of side effects such as headaches, nasal symptoms, or gastrointestinal discomfort and severely limit their use in patients with renal impairment. Therefore, the discovery of novel antifibrinolytics with a higher specificity and lower dosage could vastly improve the applicability of these drugs. Herein, we synthesized a total of ten compounds consisting of a combination of three key moieties: an oxadiazolone, a triazole, and a terminal amine. The IC50 of each compound was calculated in our clot lysis assays, and the best candidate (1) provided approximately a 2.5-fold improvement over the current gold standard, TXA. Molecular docking and molecular dynamics were used to perform a structure-activity relationship (SAR) analysis with the lysine binding site in the Kringle 1 domain of plasminogen. This analysis revealed that 1,2,3-triazole was crucial for the activity, enhancing the binding affinity through pi-pi stacking and polar interactions with Tyr72. The results presented in this work open the door to further investigate this new family as potential antifibrinolytic drugs.

A REVIEW ON TRANEXAMIC ACID

The objective is to study the effectiveness of hemostatic therapy with tranexamic acid in pregnant women with miscarriage that started. Tranexamic acid works by slowing the breakdown of blood clots, which helps to prevent prolonged bleeding. It belongs to a class of drugs known as antibrinolytics. Tranexamic acid is a synthetic derivative of the amino acid lysine and binds the 5 lysine binding sites on plasminogen. This inhibits plasmin formation and displaces plasminogen from the brin surface. It may also directly inhibit plasmin and partially inhibit brinogenolysis at higher concentrations. The investigators hypothesize that tranexamic acid as an adjunct to misoprostol will be more effective than misoprostol alone in stopping postpartum bleeding without recourse to further treatment in signicantly more women. The use of tranexamic acid, as hemostatic therapy in pregnant women with a miscarriage, signicantly reduces the duration of bleeding, promotes the accelerating the organization and resorption of intrauterine hematomas, reduces the duration of inpatient treatment. The rst trimester is associated with the highest risk for miscarriage. Most miscarriages occur in the rst trimester before the 12th week of pregnancy. A miscarriage in the second trimester (between 13 and 19 weeks) happens in 1% to 5% of pregnancies. Tranexamic acid appeared safe and effective for the prevention and management of bleeding during pregnancy.

Mass-spectrometric identification of oxidative modifications in plasma-purified plasminogen: Association with hypofibrinolysis in patients with acute pulmonary embolism

ObjectivesMechanisms behind disturbed fibrinolysis in pulmonary embolism (PE) are poorly understood. We hypothesized that oxidative stress-induced changes in plasminogen contribute to impaired fibrinolysis in patients with acute PE. MethodsOxidative and other modifications were investigated using mass-spectrometry in plasminogen purified from pooled plasma of 5 acute PE patients on admission and after 3 months of anticoagulant treatment, along with plasma clot lysis time, a measure of global efficiency of fibrinolysis, and a stable oxidative stress marker, plasma 8-isoprostane. ResultsTwenty sites of oxidation, 3 sites of carbonylation and 4 sites of S-nitrosylation were identified in plasminogen. The intensity of peptides oxidized at cysteine residues with respect to unmodified peptides decreased after 3 months of anticoagulation (p = 0.018). This was not observed for oxidized methionine residues (p = 0.9). Oxidized tryptophan (n = 4) and proline (n = 2), as well as carbonylation at 3 threonine residues were selectively identified in acute PE episode, not after 3 months. This was accompanied by 12.8% decrease in clot lysis time (p = 0.043). Deamidation occurred at the arginine, previously identified to undergo the cleavage by plasminogen activator. Methylated were two lysine-binding sites important for an interaction of plasminogen with fibrin. Other identified modifications involved: glycation, acetylation, phosphorylation, homocysteinylation, carbamylation and dichlorination (88 modifications at 162 sites). ConclusionsData suggest that oxidative stress-induced changes in plasminogen molecules may contribute to less effective global fibrinolysis in patients with acute PE. The comprehensive library of posttranslational modifications in plasminogen molecules was provided, including modifications of sites reported to be involved in important biological functions.

Use of Tranexamic Acid in Liposculpture: A Double-Blind, Multicenter, Randomized Clinical Trial.

Intraoperative hemostasis should be performed with great caution because bleeding is a huge enemy of patient safety during surgery. Tranexamic acid is a lysine synthetic derivate that inhibits fibrinolysis and diminishes the bleeding by blocking the five lysine-binding sites for plasminogen. The authors compare the efficacy of tranexamic acid versus placebo as a hemostatic agent in liposculpture procedures. The authors conducted a multicenter, double-blind, randomized, controlled clinical trial in patients who were scheduled for liposculpture in three plastic surgery centers (Colombia and Mexico) between January of 2019 and February of 2020. One hundred forty-one patients were randomly assigned into three groups: intravenous (1 g of tranexamic acid), subcutaneous (1 g of tranexamic acid), and placebo (normal saline). Forty-seven patients were assigned to each group. There were 30 male patients and 111 female patients. The main outcome was to evaluate the amount of postoperative bleeding between groups. The primary outcome was measured by the hemoglobin point loss at day 1 (preoperative hemoglobin minus hemoglobin at day 1 postoperatively) and the hemoglobin (in milligrams per deciliter) point loss at day 5 (preoperative hemoglobin minus hemoglobin at day 5 postoperatively). The authors found the intravenous intervention group to have a greater hemoglobin level than the other two groups on both the first postoperative day ( p = 0.0001) and the fifth postoperative day ( p = 0.001). There were no statistical differences in hemoglobin values between the placebo and the subcutaneous intervention groups. Intravenous tranexamic acid is a good therapeutic choice to implement on liposculpture procedures to decrease postoperative bleeding. The preoperative use of intravenous tranexamic acid not only decreases the bleeding rate after liposuction procedures, but also allows greater lipoaspirate volumes when performing high-definition liposculpture. Further studies are required to support the effectiveness of tranexamic acid within the infiltration solution. Therapeutic, I.

Investigation of binding mechanism for human plasminogen Kringle 5 with its potential receptor vWA1 domain in Cochlin by bio-specific technologies and molecular dynamic simulation

Given the significant clinical potential of human plasminogen Kringle 5 on tumours, it is crucial to seek its receptors for a thorough comprehension of its physiological functions and mechanism. Eleven candidates have been screened out in our previous works. In the present work, we further inquired whether the candidate, von Willebrand factor type A domain 1 in coagulation factor C homology protein (abbr. vWA1), was a potential receptor of Kringle 5, and investigated their binding mechanism by bio-specific experiments, frontal affinity analysis (FA), and molecular dynamic simulation (MDS). After the potential was validated by bio-specific experiments, the FA results stated that vWA1 exhibited a strong interaction towards Kringle 5 in the proportion of 1:1 with the binding constant of 4.18 × 104 L/mol. The MDS results showed that the binding was mainly driven by electrostatic and Van der Waals forces and occurred spontaneously, during which vWA1 and Kringle 5 mutually fit each other by conformational changing into more flexible and suitable structures including fluctuations for five loops and partial transformation into a random coil for α6-helix in vWA1. Moreover, lysine binding site Leu71-Tyr74 was speculated responsible for Kringle 5 in binding and Tyr72 to be the key amino acid residue. In short, this work not only confirmed vWA1 as a potential Kringle 5 receptor but also provided valuable information on the detailed binding, facilitating the application development of Kringle 5 in regulating immune or inhibiting tumour migration through vWA1.