Lysine Analogues Research Articles

Abstract 131: Plasminogen Oxidized Phospholipid Adducts Drive Macrophage Activation In Through TLR2

Oxidized phospholipids (OxPL) are potent proinflammatory mediators that can trigger toll-like receptor 2 (TLR2) signaling and inflammasome activation in macrophages. Plasminogen (PLG), a key enzyme in the fibrinolytic pathway, has been shown to harbor OxPL adducts. We hypothesized that PLG can drive inflammasome activity in macrophages through OxPL activation of TLR2 in a fibrin-independent mechanism. To assess PLG-induced macrophage activation, total RNA sequencing was used to quantify gene changes in response to human PLG treatments, and we observed robust induction of pro-inflammatory genes, including Il1b, Il6, Nos2, Il12 , and Nlrp3 in primary mouse macrophages. In addition, a 400-fold increase in IL-6 protein was observed in the media. To determine if this activation is specifically via TLR2, a HEK293-NF-kB reporter system was used to screen PLG-induced activation of human TLRs, and we found that human PLG significantly-induced NF-kB signaling and transactivation in reporter cells expressing TLR2, TL1/2, TLR2/6, but failed to activate TLRs1/3/4/5/6/9. PLG activation of TLR2 was found to be blocked by pre-treatment of C29, a small molecule inhibitor of TLR2. In addition, when Tlr2-/- primary macrophages were treated with human PLG a 30% reduction in Il1b, Il6 and Tnf gene (mRNA) expression was observed compared to wild-type controls. Furthermore, we found PLG-TLR2 activation to be fibrin-independent, as there was no reduction in PLG activation by the lysine analogs tranexamic acid and e-aminocaproic acid or the plasmin activity inhibitor - VPLCK in our HEK293-NF-kB reporter system. To characterize the effect of PLG on inflammation in vivo , wild-type and Plg-/- mice were injected with AAV8-PCSK9 and treated with an atherogenic diet for 16 weeks. Remarkably, the Plg-/- mice demonstrated reduced plaque mass and aortic lipid content compared to wild-type control mice, with a 40% reduction in en face Sudan IV staining and a 10% reduction in lesion area by Oil-Red-O staining of neutral lipids. Collectively these results support that PLG-OxPL likely activate inflammasomes in macrophages through TLR2 and suggest this is likely to be involved in atherogenesis in vivo .

DnaK Functions as a Moonlighting Protein on the Surface of Mycoplasma hyorhinis Cells.

Mycoplasma hyorhinis is a common pathogen of swine and is also associated with various human tumors. It causes systemic inflammation, typically polyserositis and polyarthritis, in some infected pigs. However, the pathogenic mechanism of M. hyorhinis remains unclear. DnaK is a highly conserved protein belonging to the heat-shock protein 70 family of molecular chaperones, which plays important roles as a moonlighting protein in various bacteria. In the present study, we identified the surface exposure of M. hyorhinis DnaK. Two virulent strains expressed more DnaK on their surface than the avirulent strain. Thereafter, the potential moonlighting functions of DnaK were investigated. Recombinant M. hyorhinis DnaK (rMhr-DnaK) was found to be able to adhere to swine PK-15 cells and human NCI-H292 cells. It also bound to four extracellular matrix components—fibronectin, laminin, type IV collagen, and vitronectin—in a dose-dependent manner. ELISA demonstrated an interaction between rMhr-DnaK and plasminogen, which was significantly inhibited by a lysine analog, ε-aminocaproic acid. rMhr-DnaK-bound plasminogen was activated by tissue-type plasminogen activator (tPA), and the addition of rMhr-DnaK significantly enhanced the activation. Finally, a DnaK-specific antibody was detected in the serum of pigs immunized with inactivated vaccines, which indicated good immunogenicity of it. In summary, our findings imply that DnaK is an important multifunctional moonlighting protein in M. hyorhinis and likely participates extensively in the infection and pathogenesis processes of M. hyorhinis.

Effect of age on the efficacy of tranexamic acid: An analysis of heterogeneity of treatment effect within the CRASH-2 dataset.

Trauma is a major cause of morbidity and mortality in older adults and will become more common as the population ages. Tranexamic acid (TXA) is a lysine analogue frequently used in the setting of significant trauma with hemorrhage. The aim of this study is to investigate the heterogeneity of treatment effect of TXA as it relates to patient age during trauma care. We included patients from the CRASH-2 trial who were randomized within 3h of injury. Patients were stratified into age groups <26years, 26 to 35years, 36 to 45years, 46 to 55years, and>55years. Multiple logistic regression models were utilized to evaluate adjusted odds ratios (OR) with 95% confidence intervals (CI) for mortality. Heterogeneity of treatment effect was evaluated using Akaike and Bayesian information criteria to determine the optimum logistic regression model after which a Wald Chi-square test was utilized to evaluate statistical significance. On univariate analysis, TXA administration decreased mortality within the <26years cohort (decrease of 2.1%, 95% CI 0.2 to 4.0), 46 to 55years cohort (decrease 6.7%, 95% CI 2.7 to 10.7), and>55years cohort (decrease of 5.3%, 95% CI 0.4 to 10.3). On adjusted analysis, when compared to the 36 to 45years cohort, the <26year cohort experienced a decreased mortality (OR 0.72, 95% CI 0.62 to 0.85) whereas the >55year cohort experienced increased mortality (OR 1.8, 95% CI 1.5 to 2.2). Assessment for heterogeneity of treatment effect of TXA administration between groups approached but did not reach statistical significance (p=0.11). Mortality related to trauma increases with age, however, there does not appear to be heterogeneity of treatment effect for TXA administration among different age groups.

Efficient production of lysine from genetically modified Corynebacterium glutamicum by feedback inhibition resistant strain

Lysine is an essential amino acid required for the synthesis of proteins. Lysine deficiency leads to numerous irreversible damages to the brain and other body organs. It plays a vital role in the treatment of osteoporosis and Herpes Simplex Virus (HSV). Lysine is commonly produced through fermentation. However, its efficient production is still a bottle neck. The wild strains of microorganisms are unable to produce sufficient amino acid. The current study was designed to enhance the lysine production through feedback inhibition resistant strains. N-methyl-N-nitro-N-nitrosoguanidine (NTG) was utilized to mutate the Corynebacterium glutamicum strain B391 to increase the production of Lysine. C. glutamicum was used as fermenting agent and cultivated in molasses-based media. S-β-aminoethyl-L-cysteine (AEC) lysine analogue resistant mutants were selected. It was observed that 30 g/L and 55 g/L of lysine were produced by the parent and mutant strains, respectively. The increase in lysine production was observed with 10% of sugar concentration in 100 mL of molasses media (6.5 pH, and temperature 30°C) with inoculum size of 8%. Furthermore, two different molasses media with different ingredients (termed M1 and M2) were used for the lysine production. The lysine production M-2 was observed more prominent.

Local Tranexamic Acid Reduces Surgical Blood Loss

Tranexamic acid (TXA) is a synthetic lysine analog that reduces perioperative blood loss by blocking lysine-binding sites on plasminogen molecules. It has been reported to be effective in limiting blood loss and transfusion needs in various orthopedic surgeries and for pediatric patients at high risk of blood loss. Randomized controlled trials (RCTs) supporting the capacity of TXA to reduce blood loss and improve outcomes associated with various surgical procedures have increased fourfold in the 10 years since Evidence Corner addressed this topic, which highlighted the capacity of locally delivered TXA to reduce blood loss during orthopedic knee surgery and intravenously delivered TXA to reduce blood lost during elective cesarean sections. Research continues to explore whether the ideal method of TXA delivery is local or systemic. One systemic preoperative 10 mg/kg dose of TXA did not improve blood loss, wound healing, or pain-related outcomes associated with ambulatory foot and ankle surgery. However, repeated topical doses of TXA as well as combined systemic and topical doses improved blood loss, inflammation, and nutritional condition following hip arthroplasty. This installment of Evidence Corner reviews 2 recent studies exploring the effects of adding locally delivered TXA to intravenous (IV) systemic TXA during spinal surgery8 and confirming efficacy of a single dose of topical TXA on hip arthroplasty.

A mechanism for hereditary angioedema caused by a lysine 311–to–glutamic acid substitution in plasminogen

AbstractPatients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.

Abstract TP139: Therapeutic Value Of Antifibrinolytic Medication In Acute Ischemic Stroke Patients With Alteplase-associated Intracerebral Hemorrhage

Background and Purpose: Intracerebral hemorrhage can occur in acute ischemic stroke patients receiving alteplase (recombinant tissue plasminogen activator). Antifibrinolytic medications such as the synthetic lysine analogs tranexamic acid and ε;-aminocaproic acid that inhibit fibrinolysis by attaching to the lysine-binding site of the plasminogen molecule have been utilized. However, the efficacy of antifibrinolytic medication in prevention of hematoma expansion is not known. Methods: We analyzed the effect of antifibrinolytic medication in acute ischemic stroke patients with intracerebral hemorrhage associated with Alteplase from 2012 to 2017. The Region-of-Interest method on a Horos Open Source Medical Image Viewer (Version 3.3.6) was utilized for volume measurement. We compared the change in hemorrhage volume in patients who did and did not receive antifibrinolytic medication. Results: A total of 36 patients (mean age 72.7±11.5, 56% were men) who received intravenous alteplase with subsequent intracerebral hemorrhage were identified. Fourteen patients received antifibrinolytic treatment; five patients had reduction or stabilization in hematoma volume (overall pre-treatment volume 13.87±15.74 cm 3 and post-treatment volume 16.74±29.67 cm 3 ). Mean fibrinogen level in the antifibrinolytic medication group was 297 mg/dL; two patients had levels &lt; 200 mg/dL. In the cohort of patients did not receive antifibrinolytics, 15 out of 22 patients had reduction or stabilization in hematoma volume (overall pre-treatment volume 19.25±33.33 cm 3 and post-treatment volume 17.41±26.56 cm 3 ). Mean volume difference in the group that received antifibrinolytic medication was 36% and 9% in the group that did not receive antifibrinolytic medication. [Figure 1] Conclusion: We did not identify any advantage of antifibrinolytic treatment in reduction or stabilization in hematoma volume in patients with Alteplase-associated intracerebral hemorrhages.

Model for surface-dependent factor XII activation: the roles of factor XII heavy chain domains

AbstractFactor XII (FXII) is the zymogen of a plasma protease (FXIIa) that contributes to bradykinin generation by converting prekallikrein to the protease plasma kallikrein (PKa). FXII conversion to FXIIa by autocatalysis or PKa-mediated cleavage is enhanced when the protein binds to negatively charged surfaces such as polymeric orthophosphate. FXII is composed of noncatalytic (heavy chain) and catalytic (light chain) regions. The heavy chain promotes FXII surface-binding and surface-dependent activation but restricts activation when FXII is not surface bound. From the N terminus, the heavy chain contains fibronectin type 2 (FN2), epidermal growth factor-1 (EGF1), fibronectin type 1 (FN1), EGF2, and kringle (KNG) domains and a proline-rich region. It shares this organization with its homolog, pro–hepatocyte growth factor activator (Pro-HGFA). To study the importance of heavy chain domains in FXII function, we prepared FXII with replacements of each domain with corresponding Pro-HGFA domains and tested them in activation and activity assays. EGF1 is required for surface-dependent FXII autoactivation and surface-dependent prekallikrein activation by FXIIa. KNG and FN2 are important for limiting FXII activation in the absence of a surface by a process that may require interactions between a lysine/arginine binding site on KNG and basic residues elsewhere on FXII. This interaction is disrupted by the lysine analog ε-aminocaproic acid. A model is proposed in which an ε-aminocaproic acid–sensitive interaction between the KNG and FN2 domains maintains FXII in a conformation that restricts activation. Upon binding to a surface through EGF1, the KNG/FN2-dependent mechanism is inactivated, exposing the FXII activation cleavage site.

Amide-derived lysine analogues as substrates and inhibitors of histone lysine methyltransferases and acetyltransferases.

Histone lysine methyltransferases and acetyltransferases are two classes of epigenetic enzymes that play pivotal roles in human gene regulation. Although they both recognise and posttranslationally modify lysine residues in histone proteins, their difference in histone peptide-based substrates and inhibitors remains to be firmly established. Here, we have synthesised lysine mimics that posses an amide bond linker in the side chain, incorporated them into histone H3 tail peptides, and examined synthetic histone peptides as substrates and inhibitors for human lysine methyltransferases and acetyltransferases. This work demonstrates that histone lysine methyltransferases G9a and GLP do catalyse methylation of the most similar lysine mimic, whereas they typically do not tolerate more sterically demanding side chains. In contrast, histone lysine acetyltransferases GCN5 and PCAF do not catalyse acetylation of the same panel of lysine analogues. Our results also identify potent H3-based inhibitors of GLP methyltransferase, providing a basis for development of peptidomimetics for targeting KMT enzymes.

Arabidopsis N-acetyltransferase activity 2 preferentially acetylates 1,3-diaminopropane and thialysine

Polyamine acetylation has an important regulatory role in polyamine metabolism. It is catalysed by GCN5-related N-acetyltransferases, which transfer acetyl groups from acetyl-coenzyme A to the primary amino groups of spermidine, spermine (Spm), or other polyamines and diamines, as was shown for the human Spermidine/Spermine N1-acetyltransferase 1 (HsSSAT1). SSAT homologues specific for thialysine, a cysteine-derived lysine analogue, were also identified (e.g., HsSSAT2). Two HsSSAT1 homologues are present in Arabidopsis, namely N-acetyltransferase activity (AtNATA) 1 and 2. AtNATA1 was previously shown to be specific for 1,3-diaminopropane, ornithine, putrescine and thialysine, rather than Spm and spermidine. In the present study, in an attempt to find a plant Spm-specific SSAT, AtNATA2 was expressed in a heterologous bacterial system and catalytic properties of the recombinant protein were determined. Data indicate that recombinant AtNATA2 preferentially acetylates 1,3-diaminopropane and thialysine, throwing further light on AtNATA1 substrate specificity. Structural analyses evidenced that the preference of AtNATA1, AtNATA2 and HsSSAT2 for short amine substrates can be ascribed to different main-chain conformation or substitution of HsSSAT1 residues interacting with Spm distal regions. Moreover, gene expression studies evidenced that AtNATA1 gene, but not AtNATA2, is up-regulated by cytokinins, thermospermine and Spm, suggesting the existence of a link between AtNATAs and N1-acetyl-Spm metabolism. This study provides insights into polyamine metabolism and structural determinants of substrate specificity of non Spm-specific SSAT homologues.

Efficacy and safety of aprotinin in paediatric cardiac surgery: A systematic review and meta-analysis.

The relicensing of aprotinin in Europe and Canada has stimulated discussions on its usefulness in paediatric cardiac surgery. To systematically evaluate the available evidence on the efficacy and safety of aprotinin in paediatric cardiac surgery. Systematic review of all randomised and observational studies comparing aprotinin with tranexamic acid, epsilon aminocaproic acid, placebo or no drug in paediatric cardiac surgery. Meta-analyses were performed on efficacy and safety outcomes. PubMed, Cochrane Central Register of Controlled Trials, Web of Science and Embase were searched from January 2000 to March 2021. Studies that enrolled children under 18 years undergoing cardiac surgery with cardiopulmonary bypass. Thirty-two studies enrolling a total of 63 894 paediatric cardiac procedures were included. Aprotinin significantly reduced total blood loss [mean difference -4.70 ml kg-1, 95% confidence interval (CI), -7.88 to -1.53; P = 0.004], postoperative transfusion requirements and the incidence of surgical re-exploration for bleeding [odds ratio (OR) 0.74, 95% CI, 0.56 to 0.97; P = 0.03]. Aprotinin had no effects on 30-day mortality (OR 1.02, 95% CI, 0.93 to 1.11; P = 0.73) and on other safety outcomes, except for the incidence of renal replacement therapy (RRT), which was significantly increased in patients given aprotinin (OR 1.29, 95% CI, 1.08 to 1.54; P = 0.006). Findings from observational and randomised controlled trials did not largely differ. A sub-group analysis in neonates showed that aprotinin significantly reduced packed red blood cell transfusions and the incidence of postoperative surgical re-exploration for bleeding and/or tamponade. When compared with lysine analogues, aprotinin was more effective at reducing bleeding and transfusion without increasing the risk of side effects. This meta-analysis suggests that aprotinin is effective and well tolerated in paediatric cardiac surgery. Given the large heterogeneity of the results and the risk of selection bias in observational studies, large randomised controlled trials are warranted.

Utilization of Tranexamic Acid in Surgical Orthopaedic Practice: Indications and Current Considerations.

Tranexamic acid (TXA) is a lysine analog that exhibits an anti-fibrinolytic effect by directly preventing the activation of plasminogen as well as inhibiting activated plasmin from degrading fibrin clots, thereby promoting hemostasis and reducing the duration and quantity of blood loss. The aims of this study were to summarize the indications, routes of administration, safety, and clinical outcomes of TXA use throughout the different subspecialities in orthopedic surgery. Given that orthopedic procedures such as TKA, THA, fracture fixation, and various spine surgeries involve significant intraoperative blood loss, TXA is indicated in providing effective perioperative hemostasis. Additionally, use of TXA in orthopedic trauma has been indicated as a measure to reduce blood loss especially in a group with potential for hemodynamic compromise. TXA has been implicated in reducing the risk of blood transfusions in orthopedic trauma, joint surgery, and spine surgery, although this effect is not seen as prominently in sports medicine procedures. There remains disagreement in literature as to whether TXA via any route of administration can improve other clinically significant outcomes such as hospital length of stay and total operative time. Procedures that rely extensively on clarity on visualization of the surgical field such as knee and shoulder arthroscopies can greatly benefit from the use of TXA, thereby leading to less intraoperative bleeding, with better visual clarity of the surgical field. While most studies agree thrombosis due to TXA is unlikely, new research in cells and animal models are evaluating whether TXA can negatively impact other aspects of musculoskeletal physiology, however with conflicting results thus far. As of now, TXA remains a safe and effective means of promoting hemostasis and reducing intraoperative blood loss in orthopedic surgery.

Enhancement of lysine biosynthesis confers high-temperature stress tolerance to Escherichia coli cells

Lysine, a nutritionally important amino acid, is involved in adaptation and tolerance to environmental stresses in various organisms. Previous studies reported that lysine accumulation occurs in response to stress and that lysine supplementation enhances stress tolerance; however, the effect of lysine biosynthesis enhancement on stress tolerance has yet to be elucidated. In this study, we confirmed that lysine supplementation to the culture medium increased intracellular lysine content and improved cell growth of Escherichia coli at high temperature (42.5 °C). Lysine-overproducing strains were then isolated from the lysine analogue S-adenosylmethionine-resistant mutants by conventional mutagenesis and exhibited higher tolerance to high-temperature stress than the wild-type strain. We identified novel amino acid substitutions Gly474Asp and Cys554Tyr on ThrA, a bifunctional aspartate kinase/homoserine dehydrogenase (AK/HSDH), in the lysine-overproducing mutants. Interestingly, the Gly474Asp and Cys554Tyr variants of ThrA induced lysine accumulation and conferred high-temperature stress tolerance to E. coli cells. Enzymatic analysis revealed that the Gly474Asp substitution in ThrA reduced HSDH activity, suggesting that the intracellular level of aspartate semialdehyde, which is a substrate for HSDH and an intermediate for lysine biosynthesis, is elevated by the loss of HSDH activity and converted to lysine in E. coli. The present study demonstrated that both lysine supplementation and lysine biosynthesis enhancement improved the high-temperature stress tolerance of E. coli cells. Our findings suggest that lysine-overproducing strains have the potential as stress-tolerant microorganisms and can be applied to robust host cells for microbial production of useful compounds.Key points• Lysine supplementation improved the growth of E. coli cells at high temperature.• The G474D and C554Y variant ThrA increased lysine productivity in E. coli cells.• The G474D substitution in ThrA reduced homoserine dehydrogenase activity.• E. coli cells that overproduce lysine exhibited high-temperature stress tolerance.

Oxidative deamination of lysine residues by polyphenols generates an equilibrium of aldehyde and 2-piperidinol products

Polyphenols, especially catechol-type polyphenols, exhibit lysyl oxidase–like activity and mediate oxidative deamination of lysine residues in proteins. Previous studies have shown that polyphenol-mediated oxidative deamination of lysine residues can be associated with altered electrical properties of proteins and increased crossreactivity with natural immunoglobulin M antibodies. This interaction suggested that oxidized proteins could act as innate antigens and elicit an innate immune response. However, the structural basis for oxidatively deaminated lysine residues remains unclear. In the present study, to establish the chemistry of lysine oxidation, we characterized oxidation products obtained via incubation of the lysine analog N-biotinyl-5-aminopentylamine with eggshell membranes containing lysyl oxidase and identified a unique six-membered ring 2-piperidinol derivative equilibrated with a ring-open product (aldehyde) as the major product. By monitoring these aldehyde–2-piperidinol products, we evaluated the lysyl oxidase–like activity of polyphenols. We also observed that this reaction was mediated by some polyphenols, especially o-diphenolic-type polyphenols, in the presence of copper ions. Interestingly, the natural immunoglobulin M monoclonal antibody recognized these aldehyde–2-piperidinol products as an innate epitope. These findings establish the existence of a dynamic equilibrium of oxidized lysine and provide important insights into the chemopreventive function of dietary polyphenols for chronic diseases.

Incorporating Substrate Features in Fmoc Amino Acids to Develop Potent and Specific Butyrylcholinesterase Inhibitors for the Potential Treatment of Alzheimer's Disease

Alzheimer's Disease (AD) is a chronic neurodegenerative disease leading to irreversible memory loss and is the most common form of dementia. While a cure for AD is not available, inhibitors of the two major classes of cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are under investigation to mitigate the effects of this disease. In individuals with AD, studies showed that BChE activity increased while AChE activity was similar. The increased BChE activity is suggested to deplete acetylcholine, a neurotransmitter, and contribute to dementia. We identified Fmoc-amino acids as BChE inhibitors, and a lysine-based compound that shares structural similarities with the substrate was one of the most potent inhibitors. Although the original inhibitor had the same positive charge as acetylcholine, the inhibitor lacked methyl groups found in the actual substrate. We hypothesized that methylation of the lysine side chain better mimics leading to better inhibitors. Due to challenges in synthesizing a trimethylated product, we first synthesized a dimethyl amino analog of lysine using 34% formaldehyde, acetic acid, NaBH4, and 1,4-dioxane. The product was purified by chromatography and characterized by NMR. Enzyme kinetics assays monitored by UV-Vis spectroscopy were then used to determine inhibition constants (KI values). The KI values for the dimethylated product Fmoc-Lys(CH3)2-O- was four-fold lower than Fmoc-Lys-O- (40 μM versus 150 μM, respectively), indicating the dimethylammonium compound is a better inhibitor. Studies comparing BChE and AChE inhibition indicated that the compound selectively targets BChE relative to AChE. Computational docking was then used to visualize possible binding sites of the inhibitor. The results suggested that the cationic amino group was binding in the BChE active site similar to the substrate and supports the hypothesis that adding substrate features lead to a more potent BChE inhibitor. Currently, we are testing different synthesis approaches to obtain the trimethyl ammonium compound to then further test the effect of introducing substrate features. Together, the results are aimed at systematically evaluating the contributions of introducing substrate features in enzyme inhibitors to develop more potent inhibitors.

Impact of tranexamic acid on blood loss and transfusion rate in children with cerebral palsy undergoing hip reconstruction with two or more osteotomies.

Tranexamic acid (TXA), a synthetic lysine analogue, has been used in orthopedic procedures to limit blood loss and prevent allogeneic blood transfusions. However, data are scarce on its use in hip reconstruction of patients with cerebral palsy (CP). This study examines the effect of TXA on patients with CP undergoing hip reconstruction with at least two osteotomies. This is a single-center, retrospective study of patients with CP who underwent hip reconstruction with two or more osteotomies from January 2013 to April 2019. There were 43 patients, with a mean age of 9.9years. Age, procedure, preoperative and postoperative hemoglobin/hematocrit, estimated blood loss (EBL), transfusions and length of stay were recorded. The patients were split into the following two groups: 24 patients who had received intraoperative TXA and 19 who had not. Age, EBL, mean preoperative and postoperative hemoglobin or hematocrit, preoperative to postoperative hematocrit drop, and length of stay were similar for the two groups (p > 0.05). The risk for intraoperative transfusion (21 vs. 17%), postoperative transfusion (26 vs. 8%), and any transfusion (42 vs. 21%) appeared to be greater in the group that did not receive TXA, but this difference did not achieve statistical significance. This pilot study shows patients with CP undergoing hip reconstruction with two or more osteotomies; the use of TXA, while not statistically significant, shows a trend toward a decreased need for allogeneic blood transfusion.

Interaction between Borrelia miyamotoi variable major proteins Vlp15/16 and Vlp18 with plasminogen and complement

Borrelia miyamotoi, a relapsing fever spirochete transmitted by Ixodid ticks causes B. miyamotoi disease (BMD). To evade the human host´s immune response, relapsing fever borreliae, including B. miyamotoi, produce distinct variable major proteins. Here, we investigated Vsp1, Vlp15/16, and Vlp18 all of which are currently being evaluated as antigens for the serodiagnosis of BMD. Comparative analyses identified Vlp15/16 but not Vsp1 and Vlp18 as a plasminogen-interacting protein of B. miyamotoi. Furthermore, Vlp15/16 bound plasminogen in a dose-dependent fashion with high affinity. Binding of plasminogen to Vlp15/16 was significantly inhibited by the lysine analog tranexamic acid suggesting that the protein–protein interaction is mediated by lysine residues. By contrast, ionic strength did not have an effect on binding of plasminogen to Vlp15/16. Of relevance, plasminogen bound to the borrelial protein cleaved the chromogenic substrate S-2251 upon conversion by urokinase-type plasminogen activator (uPa), demonstrating it retained its physiological activity. Interestingly, further analyses revealed a complement inhibitory activity of Vlp15/16 and Vlp18 on the alternative pathway by a Factor H-independent mechanism. More importantly, both borrelial proteins protect serum sensitive Borrelia garinii cells from complement-mediated lysis suggesting multiple roles of these two variable major proteins in immune evasion of B. miyamotoi.

Substrate Scope for Human Histone Lysine Acetyltransferase KAT8

Biomedically important histone lysine acetyltransferase KAT8 catalyses the acetyl coenzyme A-dependent acetylation of lysine on histone and other proteins. Here, we explore the ability of human KAT8 to catalyse the acetylation of histone H4 peptides possessing lysine and its analogues at position 16 (H4K16). Our synthetic and enzymatic studies on chemically and structurally diverse lysine mimics demonstrate that KAT8 also has a capacity to acetylate selected lysine analogues that possess subtle changes on the side chain and main chain. Overall, this work highlights that KAT8 has a broader substrate scope beyond natural lysine, and contributes to the design of new chemical probes targeting KAT8 and other members of the histone lysine acetyltransferase (KAT) family.

Exposure of plasminogen and a novel plasminogen receptor, Plg-RKT, on activated human and murine platelets

AbstractPlasminogen activation rates are enhanced by cell surface binding. We previously demonstrated that exogenous plasminogen binds to phosphatidylserine-exposing and spread platelets. Platelets contain plasminogen in their α-granules, but secretion of plasminogen from platelets has not been studied. Recently, a novel transmembrane lysine-dependent plasminogen receptor, Plg-RKT, has been described on macrophages. Here, we analyzed the pool of plasminogen in platelets and examined whether platelets express Plg-RKT. Plasminogen content of the supernatant of resting and collagen/thrombin-stimulated platelets was similar. Pretreatment with the lysine analog, ε-aminocaproic acid, significantly increased platelet-derived plasminogen (0.33 vs 0.08 nmol/108 platelets) in the stimulated supernatant, indicating a lysine-dependent mechanism of membrane retention. Lysine-dependent, platelet-derived plasminogen retention on thrombin and convulxin activated human platelets was confirmed by flow cytometry. Platelets initiated fibrinolytic activity in fluorescently labeled plasminogen-deficient clots and in turbidimetric clot lysis assays. A 17-kDa band, consistent with Plg-RKT, was detected in the platelet membrane fraction by western blotting. Confocal microscopy of stimulated platelets revealed Plg-RKT colocalized with platelet-derived plasminogen on the activated platelet membrane. Plasminogen exposure was significantly attenuated in thrombin- and convulxin-stimulated platelets from Plg-RKT−/− mice compared with Plg-RKT+/+ littermates. Membrane exposure of Plg-RKT was not dependent on plasminogen, as similar levels of the receptor were detected in plasminogen−/− platelets. These data highlight Plg-RKT as a novel plasminogen receptor in human and murine platelets. We show for the first time that platelet-derived plasminogen is retained on the activated platelet membrane and drives local fibrinolysis by enhancing cell surface–mediated plasminogen activation.

Tranexamic acid rapidly inhibits fibrinolysis, yet transiently enhances plasmin generation in vivo.

Tranexamic acid (TXA) is a lysine analogue that inhibits plasmin generation and has been used for decades as an antifibrinolytic agent to reduce bleeding. Recent reports have indicated that TXA can paradoxically promote plasmin generation. Blood was obtained from 41 cardiac surgical patients randomly assigned to TXA or placebo before start of surgery (preOP), at the end of surgery (EOS), then again on postoperative day 1 (POD-1) as well as POD-3. Plasma levels of tissue-type plasminogen activator (t-PA), urokinase (u-PA), the plasmin-antiplasmin (PAP) complex, as well as t-PA and u-PA-induced clot lysis assays were then determined. Clot lysis and PAP complex levels were also assessed in healthy volunteers before and at various time points after taking 1 g TXA orally. Surgery induced an increase in circulating t-PA, yet not u-PA at EOS. t-PA levels were unaffected by TXA; however, u-PA levels were significantly reduced in patients on POD-3. t-PA and u-PA-induced clot lysis were both inhibited in plasma from TXA-treated patients. In contrast, PAP complex formation, representing plasmin generation, was unexpectedly enhanced in the plasma of patients administered TXA at the EOS time point. In healthy volunteers, oral TXA effectively blocked fibrinolysis within 30 min and blockade was sustained for 8 h. However, TXA also increased PAP levels in volunteers 4 h after administration. Our findings demonstrate that TXA can actually augment PAP complex formation, consistent with an increase in plasmin generation in vivo despite the fact that it blocks fibrinolysis within 30 min. This may have unanticipated consequences in vivo.