Lysine Binding Sites Research Articles

Ixonnexin from Tick Saliva Promotes Fibrinolysis by Interacting with Plasminogen and Tissue-Type Plasminogen Activator, and Prevents Arterial Thrombosis

Tick saliva is a rich source of modulators of vascular biology. We have characterized Ixonnexin, a member of the “Basic-tail” family of salivary proteins from the tick Ixodes scapularis. Ixonnexin is a 104 residues (11.8 KDa), non-enzymatic basic protein which contains 3 disulfide bonds and a C-terminal rich in lysine. It is homologous to SALP14, a tick salivary FXa anticoagulant. Ixonnexin was produced by ligation of synthesized fragments (51–104) and (1–50) followed by folding. Ixonnexin, like SALP14, interacts with FXa. Notably, Ixonnexin also modulates fibrinolysis in vitro by a unique salivary mechanism. Accordingly, it accelerates plasminogen activation by tissue-type plasminogen activator (t-PA) with Km 100 nM; however, it does not affect urokinase-mediated fibrinolysis. Additionally, lysine analogue ε-aminocaproic acid inhibits Ixonnexin-mediated plasmin generation implying that lysine-binding sites of Kringle domain(s) of plasminogen or t-PA are involved in this process. Moreover, surface plasmon resonance experiments shows that Ixonnexin binds t-PA, and plasminogen (KD 10 nM), but not urokinase. These results imply that Ixonnexin promotes fibrinolysis by supporting the interaction of plasminogen with t-PA through formation of an enzymatically productive ternary complex. Finally, in vivo experiments demonstrates that Ixonnexin inhibits FeCl3-induced thrombosis in mice. Ixonnexin emerges as novel modulator of fibrinolysis which may also affect parasite-vector-host interactions.

Amino Acids: Riddles from A to Y.

The recent Food and Drug Administration approval of the use of L-glutamine for the reduction of pain episodes in patients with sickle cell disease (SCD)2 highlights the importance of familiar amino acids in new therapeutics. For example, lysine analogs are used as antifibrinolytics to stop bleeds, whereas an arginine analog is used as an antithrombotic agent. What is the mechanism of action of L-glutamine in SCD? L-glutamine has a favorable impact on the ratio of reduced to total NAD and redox potential in sickle red blood cells (RBCs) exposed to oxidative stress (1). RBCs from individuals with SCD who were treated with L-glutamine showed reduced sickling on the peripheral blood smear and were less sickled and less adherent to vascular cells than RBCs from untreated individuals (2). Oral L-glutamine also reduced endothelial adhesion of sickle RBCs to human umbilical vein endothelial cells (3). Improved redox biology might make sickle RBCs less adhesive. Both tranexamic acid and e-aminocaproic acid, which are analogs of lysine, competitively block the binding of fibrinogen and fibrin to their lysine binding sites on plasminogen, thus reducing the rate of fibrinolysis. Aprotinin, a serine protease inhibitor, has been removed from the market due …

Effect of Specific Cleavage of Immunoglobulin G by Plasmin on the Binding and Activation of Plasminogen

A method of ELISA for measuring the binding of different samples of immunoglobulin (IgG) and its fragments to human plasminogen (Pg) has been developed. Instead of plasminogen, the heavy chain of plasminogen (Pg-H) containing five ligand-binding kringle domains, immobilized on the surface of the plate, was used in this method as a detector. It was found that IgG treated with plasmin (IgGPm-t) binds to the immobilized Pg-H 2.84 times more strongly than intact IgG. Both IgG samples showed a weak nonspecific binding to the immobilized light chain of plasminogen (Pg-L). It was shown that 0.2 M L-lysine inhibits the binding of IgGPm-t and does not affect the nonspecific binding of intact IgG to the immobilized Pg-H, indicating the involvement of lysine-binding regions of Pg-H in binding to IgGPm-t. A preliminary treatment of IgG samples with carboxypeptidase В (CPB) inhibited the binding of IgGPm-t and did not affect the nonspecific binding of intact IgG to the immobilized Pg-H, which indicates a key role of the С-terminal lysine of IgGPm-t in the specific binding to the lysine-binding sites of Pg. The study of the effects of intact IgG and IgGPm-t on the rate of activation of Glu- and Lys-forms of Pg (Glu-Pg and Lys-Pg) by a tissue activator of Pg (tPA) and urokinase (uPA) in buffer showed that intact IgG completely inhibited the activation of Glu-Pg and Lys-Pg with both tPA and uPA. Presumably, the inhibitory effect of intact IgG is due to steric hindrances that it creates for protein–protein interactions of the activators with the zymogen. IgGPm-t accelerated the generation of plasmin from Pg. In this case, the stimulatory effect of IgGPm-t on the activation of Glu-Pg under the action of tPA was ∼25% higher than on the activation of Lys-Pg, which is explained by more significant conformational changes in the Glu-Pg molecule compared with the Lys-Pg molecule after their binding to IgGPm-t. The results suggest that the specific cleavage of IgG by plasmin may be one of the ways by which the plasminogen/plasmin system is involved in various physiological and pathological processes.

Conformationally organized lysine isosteres in Streptococcus pyogenes M protein mediate direct high-affinity binding to human plasminogen

The binding of human plasminogen (hPg) to the surface of the human pathogen group A Streptococcus pyogenes (GAS) and subsequent hPg activation to the protease plasmin generate a proteolytic surface that GAS employs to circumvent host innate immunity. Direct high-affinity binding of hPg/plasmin to pattern D GAS is fully recapitulated by the hPg kringle 2 domain (K2hPg) and a short internal peptide region (a1a2) of a specific subtype of bacterial surface M protein, present in all GAS pattern D strains. To better understand the nature of this binding, critical to the virulence of many GAS skin-tropic strains, we used high-resolution NMR to define the interaction of recombinant K2hPg with recombinant a1a2 (VKK38) of the M protein from GAS isolate NS455. We found a 2:1 (m/m) binding stoichiometry of K2hPg/VKK38, with the lysine-binding sites of two K2hPg domains anchored to two regions of monomeric VKK38. The K2hPg/VKK38 binding altered the VKK38 secondary structure from a helical apo-peptide with a flexible center to an end-to-end K2hPg-bound α-helix. The K2hPg residues occupied opposite faces of this helix, an arrangement that minimized steric clashing of K2hPg We conclude that VKK38 provides two conformational lysine isosteres that each interact with the lysine-binding sites in K2hPg Further, the adoption of an α-helix by VKK38 upon binding to K2hPg sterically optimizes the side chains of VKK38 for maximal binding to K2hPg and minimizes steric overlap between the K2hPg domains. The mechanism for hPg/M protein binding uncovered here may facilitate targeting of GAS virulence factors for disease management.

Abstract 14: Characterization of the Oxidized Phospholipid Modification of Apolipoprotein(a) Kringle KIV10: Insights Into the Site of OxPC Addition

Elevated plasma levels of lipoprotein(a) (Lp(a)) are an independent and causal risk factor for coronary heart disease and aortic valve stenosis. Lp(a) consists of a low density lipoprotein (LDL)-like particle covalently linked to the unique glycoprotein apolipoprotein(a) (apo(a)). We have shown that apo(a) contains a covalent oxidized phosphocholine (oxPC) adduct on the KIV 10 domain, and that perturbation of the strong lysine binding site (LBS) in this kringle results in a lack of covalent oxPC addition. We have implicated this modification in proinflammatory processes, such as the ability of apo(a) to induce interleukin-8 expression in macrophages. Apo(a) from Old World monkeys and apes lacks covalent oxPC modification and contains mutations in KIV 10 , some of which impact the LBS. To identify the amino acids in human apo(a) that are covalently modified by oxPC, we mutagenized a KIV 10 KV di-kringle apo(a) that contains covalent oxPC modification. We mutated to alanine all residues that are either substituted in primate apo(a) or that can act as acceptors for covalent oxPC addition. The resulting variants were then subjected to immunoblotting analysis with E06, an IgM antibody that binds to oxPC, and assessment of lysine-binding ability using affinity chromatography. Mutation of His33 to Ala abolishes oxPC modification of apo(a) while retaining the lysine binding ability of KIV 10 . Thus, we have identified the long-sought location of the oxPC modification of apo(a). Molecular dynamic simulations of the oxPC-deficient mutant show no gross changes in the structure of KIV 10 , in contrast to mutations that abolish both lysine binding ability and oxPC addition that result in a collapsed lysine binding pocket. Using mass spectrometry, we have also identified noncovalently-associated oxPC present on apo(a). Interestingly, the abundance of these species appears to be reduced in the apo(a) variant containing a mutation in the strong lysine binding site in KIV 10 . Our findings allow the ability to assess the contributions to pathogenesis of the strong lysine binding site and oxPC modification of apo(a) kringle IV type 10 independently, and thus will enhance our understanding of the mechanisms by which Lp(a) contributes to atherothrombotic diseases.

Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles.

The development of different cell culture models has greatly contributed to increased understanding of the hepatitis C virus (HCV) life cycle. However, it is still challenging to grow HCV clinical isolates in cell culture. If overcome, this would open new perspectives to study HCV biology, including drug‐resistant variants emerging with new antiviral therapies. In this study we hypothesized that this hurdle could be due to the presence of inhibitory factors in patient serum. Combining polyethylene glycol precipitation, iodixanol gradient, and size‐exclusion chromatography, we obtained from HCV‐seronegative sera a purified fraction enriched in inhibitory factors. Mass spectrometric analysis identified apolipoprotein(a) (apo[a]) as a potential inhibitor of HCV entry. Apo(a) consists of 10 kringle IV domains (KIVs), one kringle V domain, and an inactive protease domain. The 10 KIVs are present in a single copy with the exception of KIV type 2 (KIV2), which is encoded in a variable number of tandemly repeated copies, giving rise to numerous apo(a) size isoforms. In addition, apo(a) covalently links to the apolipoprotein B component of a low‐density lipoprotein through a disulfide bridge to form lipoprotein(a). Using a recombinant virus derived from the JFH1 strain, we confirmed that plasma‐derived and recombinant lipoprotein(a) as well as purified recombinant apo(a) variants were able to specifically inhibit HCV by interacting with infectious particles. Our results also suggest that small isoforms are less inhibitory than the large ones. Finally, we observed that the lipoprotein moiety of HCV lipoviroparticles was essential for inhibition, whereas functional lysine‐binding sites in KIV7, KIV8, and KIV10 were not required. Conclusions: Our results identify apo(a) as an additional component of the lipid metabolism modulating HCV infection. (Hepatology 2017;65:1851‐1864)

Tranexamic acid in treatment of melasma: A comprehensive review of clinical studies.

Melasma is a human melanogenesis dysfunction that results in localized, chronic acquired hyperpigmentation of the skin. It has a significant impact on appearance, causing psychosocial and emotional distress, and reducing the quality of life of the affected patients. Tranexamic acid (TA) is a plasmin inhibitor used to prevent abnormal fibrinolysis to reduce blood loss and exerts its effect by reversibly blocking lysine binding sites on plasminogen molecules, thus inhibiting plasminogen activator (PA) from converting plasminogen to plasmin. As plasminogen also exists in human epidermal basal cells and cultured human keratinocyte are known to produce PA, there is basic rationale that TA will affect keratinocyte function and interaction. A thorough literature review indicates that while TA is used through various route of administration including oral, topical, and intradermal injection and as adjutant therapy with laser to treat melasma, its efficacy is not established adequately. Further studies are needed to clarify the role of TA in treatment of melasma.

A non-randomized prospective study of the blood sparing effect of tranexamic acid in total knee replacement

<p class="abstract"><strong>Background:</strong> Several techniques are available to minimize the likelihood of blood transfusion following total knee arthroplasty. Tranexamic acid, an inhibitor of fibrinolysis that blocks the lysine-binding site of plasminogen to fibrin has been reported to reduce intraoperative and postoperative blood loss in patients undergoing total hip and total knee arthroplasties with or without cement. The objective of this study was to assess the efficacy of antifibrinolytic treatment along with other measures like saline adrenaline infusion, no drain, no tourniquet and hypotensive anaesthesia in reducing perioperative blood loss during total knee replacement<span lang="EN-IN">.</span></p><p class="abstract"><strong>Methods:</strong> Between January 2011 to January 2016, seventy five consecutive patients who had given written informed consent, undergoing a TKR received tranexamic acid 15 mg/kg body weight intravenous 5 minutes before the skin incision and two doses afterwards (3 and 6 hours after the first dose respectively). TKR was performed in a routine fashion without tourniquet. The saline adrenaline (1:200000) was infiltrated into the skin subcutaneous tissue and capsule before skin incision. A routine closure was carried out without drain. Total blood loss including the hidden blood loss was calculated. All patients were monitored for anemia and postoperative thromboembolic complications.<strong></strong></p><p class="abstract"><strong>Results:</strong> The average total blood loss in study group is 433 ± 148 ml. This is much lesser than what other studies have reported. Mean reduction in hemoglobin levels (gm/dl) between preoperative and postoperative readings is 1.6 gm/dl. One patient had a postoperative DVT which was treated with rivaroxaban 20 mg OD for 6 weeks (oral anticoagulant)<span lang="EN-IN">. </span></p><p class="abstract"><strong>Conclusions:</strong> Antifibrinolytic agents like tranexamic acid used along with other measures reported in this study produces a significant decrease in blood loss in patients undergoing total knee replacement<span lang="EN-IN">.</span></p>

Plasminogen coating increases initial adhesion of oral bacteria in vitro

Plasminogen is a major plasma protein and the zymogen of the broad spectrum protease plasmin. Plasmin activity leads to tissue degradation, direct and through activation of metalloproteinases. Infected tooth root canals, as a consequence of the inflammatory response and eventual necrosis, contain tissue fluid and blood components. These will coat the root canal walls and act as conditioning films that allow bacterial biofilms to grow and be a potential source of hematogenously spreading bacteria. We investigated the effect of in vitro surface conditioning with human plasminogen on the initial adhesion of bacteria. Four bacterial species, L. salivarius, E. faecalis, A. naeslundii, and S. gordonii, isolated from dental root canals, and three other oral streptococci, S. oralis, S. anginosus, and S. sanguinis, were grown in albumin- or plasminogen-coated flow chambers and studied by confocal laser scanning microscopy using the cell viability staining LIVE/DEAD and 16S rRNA fluorescence in situ hybridization (FISH). A. naeslundii, L. salivarius and in particular S. gordonii showed a higher initial adhesion to the plasminogen-coated surfaces. E. faecalis did not show any preference for plasminogen. Four-species biofilms cultured for 96 h showed that streptococci increased their proportion with time. Further experiments aimed at studying different streptococcal strains. All these adhered more to plasminogen-coated surfaces than to albumin-coated control surfaces. The specificity of the binding to plasminogen was verified by blocking lysine-binding sites with epsilon-aminocaproic acid. Plasminogen is thus an important plasma component for the initial adhesion of oral bacteria, in particular streptococci. This binding may contribute to their spread locally as well as to distant organs or tissues.

Molecular docking and dynamics simulation study of flavonoids as BET bromodomain inhibitors

Bromodomains (BRDs) are the epigenetic proteins responsible for transcriptional regulation through its interaction with methylated or acetylated histone residues. The lysine residues of Bromodomain-1 (BD1) of Brd4 undergo ε-N-Acetylation posttranslational modifications to control transcription of genes. Due to its role in diverse cellular functions, Brd4 of bromodomain family, was considered as a prominent target for many diseases such as cancer, obesity, kidney disease, lung fibrosis, inflammatory diseases, etc. In this study, an attempt has been made to screen compounds from flavonoids and extended flavonoids libraries targeting acetylated lysine (KAc) binding site of BD1 of Brd4 using docking and molecular dynamics simulations. Two different docking programs AutoDock and Glide were used to compare their suitability for the receptor. Interestingly, in both the docking programs, the screened flavonoids have occupied the same binding pocket confirming the selection of active site. Further the MMGBSA binding free energy calculations and ADME analysis were carried out on screened compounds to establish their anti-cancerous properties. We have identified a flavonoid which shows docking and Glide e-model score comparatively much higher than those of already reported known inhibitors against Brd4. The protein-ligand complex with top-ranked flavonoid was used for dynamics simulation study for 50 ns in order to validate its stability inside the active site of Brd4 receptor. The results provide valuable information for structure-based drug design of Brd4 inhibitors.

The use of tranexamic acid in reducing bleeding complications

Abstract Tranexamic acid inhibits fibrinolysis by competitively blocking the lysine binding sites of plasminogen, inhibiting binding between fibrin and plasminogen, and activation of plasminogen. It also competitively inhibits tissue plasminogen activator and inhibits plasmininduced platelet activation. The synthesis of tranexamic acid was first reported in 1962. It is used to treat or prevent excessive blood loss from trauma, surgery, and in various medical conditions including haemophilia and heavy menstrual bleeding. As a medicine affecting coagulation, it is listed in the World Health Organization’s List of Essential Medicines. Tranexamic acid remains a versatile and inexpensive agent with potential benefit in health services from the lowest to highest income countries. This pharmacy review focuses on the evidence base to support the use of tranexamic acid in acquired and inherited bleeding disorders to reduce bleeding complications.

Abstract 262: Lipoprotein(a) Stimulates Pro-Inflammatory Phenotypes in Vascular Cells: Demonstration of a Role for the Lysine-Binding Properties of Apolipoprotein(a)

Elevated plasma Lp(a) concentrations are a causal risk factor for a variety of atherosclerotic disorders. Although the role of Lp(a) in vascular disease remains poorly understood, the atherogenicity of Lp(a) may stem from the direct atherosclerotic effects of its LDL-like component, or from the presence of the unique protein component apolipoprotein(a) (apo(a)). It has been hypothesized that Lp(a) contributes to early atherogenesis by initiating a program of endothelial cell (EC) dysfunction which results in increased EC permeability and the adoption of a pro-coagulant and pro-inflammatory phenotype. In support of the latter, studies have shown that Lp(a) stimulates expression of cell adhesion molecules (CAMs) on ECs, and induces monocyte recruitment to these cells. In the present study we demonstrate that a recombinant 17 kringle form of apo(a) (17K) stimulates expression of CAMs on primary human umbilical vein endothelial cells (HUVECs) in both a time- and dose-dependent manner, as determined by qRT-PCR analysis. We found that 17K was able to induce the expression of ICAM1, E-Selectin, VCAM1, but not PE-CAM at pathophysiologically-relevant concentrations of apo(a). However, 17KD57A, lacking the strong lysine-binding site (sLBS) in KIV10, lacked these effects. 17K was also able to induce a dose dependant increase in ICAM1 expression in the THP-1 monocyte cell line, an effect that was completely abolished by the NF-B inhibitor BAY-11-7082. In a fluorescent monocyte adhesion assay, both Lp(a) and 17K apo(a) were able to stimulate increased monocyte adhesion to ECs. Using 17KD57A, we were able to show that this effect is dependent on the sLBS in KIV10. The presence of the sLBS in KIV10 has also been found to be required for the covalent oxidized phospholipid modification on apo(a). We report that multiple KIV10 sLBS mutants lack the oxPL modification, further supporting a pro-inflammatory role for an intact KIV10 domain. Taken together, these studies contribute to a more complete understanding of the pathophysiological role that the apo(a) component of Lp(a) plays in the initiation and progression of atherosclerosis, and has the potential to aid in the development new therapeutics aimed at reducing the harmful effects of Lp(a) in the vasculature.

PO-17 - Identification of IgG bound to plasminogen in oncologic diseases

The binding of plasminogen (Pg) to cell receptors and extracellular ligands facilitates its activation to plasmin, which stimulates the extracellular matrix degradation, neoangiogenesis and tumor invasion. Plasmin can also degrade IgG thereby exposing C-terminal lysine residues. Previously, we have found IgG specifically bounded to Pg in the plasma of patients with malignant tumors. To identify IgG degraded by plasmin in the plasma of cancer patients. Methods of ELISA were used for comparative research of levels of IgG bound to Pg in plasma of patients with the prostate cancer (PC, n=25) and lung cancer (LC, n=17). Plasma of healthy donors (n=29) was used as control. All patients signed informed consent for participation in this study. Affinity chromatography on Pg-sepharose was used for the quantification of IgG. Carboxypeptidase was used for remove of C-terminal lysine residues of the IgG. The program ATTESTAT was used for nonparametric analysis. The frequency of occurence of elevated levels of IgG to Pg in plasma was detected in 68% of patients with PC, 59% of patients with LC and only 12% of healthy women and 10% of healthy men. The quantification of antibodies in plasma samples showed that the quantity of IgG to Pg in patients with PC was 27% from the total amount of IgG and in healthy men - 9%. Treatment of diluted plasma samples with carboxypeptidase B abolished the elevated levels of IgG to Pg, as well as the specific activity of the purified IgG to Pg-sepharose. C-terminal lysine residues which are formed as a result of degradation of native IgG with plasmin can bind to lysine binding sites on the kringle domains of Pg. Increased levels of these degraded IgG can be marker at cancer.

The "Gatekeeper" Residue Influences the Mode of Binding of Acetyl Indoles to Bromodomains.

Small-molecule hits for the bromodomains of CREBBP and BAZ2B have been identified by scaffold hopping followed by docking of a set of ∼200 compounds containing the acetyl indole scaffold. Chemical synthesis of nearly 30 derivatives has resulted in ligands of representatives of three subfamilies of human bromodomains with favorable ligand efficiency. The X-ray crystal structures of three different bromodomains (CREBBP, BAZ2B, and BRPF1b) in complex with acetyl indole derivatives reveal the influence of the gatekeeper residue on the orientation of small-molecule ligands in the acetyl lysine binding site.

The preparation of a t-PA/nanoparticle conjugate via bio-affinity ligation

Event Abstract Back to Event The preparation of a t-PA/nanoparticle conjugate via bio-affinity ligation Xiaoli Liu1, Zengchao Tang1, Dan Li1* and Hong Chen1* 1 Soochow University, China Introduction: Tissue plasminogen activator (t-PA) is commonly used as a thrombolytic agent in the treatment of thrombotic diseases such as acute myocardial infarction and ischemic stroke. A major issue in the therapeutic use of t-PA is its very short half-life in the circulation (2–6 min) due to the effects of inhibitors, enzymes and antibodies in blood. Efforts have been made to overcome this deficiency by conjugating t-PA to synthetic materials to limit access of inhibitors or to materials with thrombus-targeted ability.[1] However, the linkages between t-PA and synthetic materials are commonly based on covalent bonding or electrostatic interactions, which are “invasive” and generally accompanied by loss of protein activity and immunogenic responses. Bio-affinity ligation based on the specific interactions between ligands and receptors has many advantages over covalent bonding and other nonspecific interactions for the conjugation of proteins to synthetic materials since it is benign to protein, highly specific and efficient, proceeds under physiological conditions and has no unwanted side reactions. Most proteins possess specific domains that can be recognized by some affinity ligand, so the interaction between such a domain and its affinity ligand can be utilized to link protein to materials. For t-PA, there is a high affinity lysine binding site in one of its two kringle domains that mediates its binding to carboxy-terminal lysine residues (ε-lysines) exposed on the surface of fibrin. Therefore ε-lysine is a natural affinity ligand that can be utilized to link t-PA to a synthetic material. Moreover, t-PA conjugated through ε-lysine mimics the one bound on the surface of fibrin in physiological fibrinolytic process, which is advantageous for limiting access of inhibitors. In this study, a t-PA/gold nanoparticle (t-PA/AuNP) conjugate was prepared via bio-affinity ligation. Materials and Methods: Here AuNPs were used as the model nanoparticles and modified with ε-lysine-terminated polyvinyl pyrrolidone (PVP), referred to as AuNPs-PVP-Lys. PVP functions as a bio-inert moiety to prevent the conjugate from interacting nonspecifically with other components in blood. t-PA was conjugated to AuNPs-PVP-Lys under physiological conditions through affinity interactions between ε-lysine exposed on AuNP surface and the specific domain in t-PA. Results and Discussion: Activity of the conjugated t-PA was compared with free t-PA both in vitro and in vivo. It is found that the t-PA/AuNP conjugate can not only maintain almost all enzyme activity of t-PA but also reduce the inhibition effect, leading to a prolonged circulation time (20.5 min). Conclusions: In conclusion, a t-PA/AuNPs-PVP-Lys conjugate was prepared under physiological conditions via bio-affinity ligation based on the bio-affinity interactions under physiological conditions. Compared with free t-PA, the conjugate retained almost full enzyme activity and clot-dissolving efficiency, and the t-PA was protected from inhibition by PAI-1 to some extent. Moreover, the conjugate showed prolonged circulation time in vivo. It will be of interest to apply this bio-affinity ligation more generally to conjugate synthetic materials with other proteins, especially those with important biomedical functions. National Natural Science Foundation of China (21204055, 21304062, 21334004); National Natural Science Foundation Fund for Distinguished Young Scholars (21125418); The Natural Science Foundation of the Jiangsu Higher Education Institutions of China (13KJB430020); China Postdoctoral Science Foundation (2013M541714)

Mechanistic insights into Lp(a)-induced IL-8 expression: a role for oxidized phospholipid modification of apo(a)

Elevated lipoprotein (a) [Lp(a)] levels are a causal risk factor for coronary heart disease. Accumulating evidence suggests that Lp(a) can stimulate cellular inflammatory responses through the kringle-containing apolipoprotein (a) [apo(a)] component. Here, we report that recombinant apo(a) containing 17 kringle (17K) IV domains elicits a dose-dependent increase in interleukin (IL)-8 mRNA and protein expression in THP-1 and U937 macrophages. This effect was blunted by mutation of the lysine binding site in apo(a) kringle IV type 10, which resulted in the loss of oxidized phospholipid (oxPL) on apo(a). Trypsin-digested 17K had the same stimulatory effect on IL-8 expression as intact apo(a), while enzymatic removal of oxPL from apo(a) significantly blunted this effect. Using siRNA to assess candidate receptors, we found that CD36 and TLR2 may play roles in apo(a)-mediated IL-8 stimulation. Downstream of these receptors, inhibitors of MAPKs, Jun N-terminal kinase and ERK1/2, abolished the effect of apo(a) on IL-8 gene expression. To assess the roles of downstream transcription factors, luciferase reporter gene experiments were conducted using an IL-8 promoter fragment. The apo(a)-induced expression of this reporter construct was eliminated by mutation of IL-8 promoter binding sites for either NF-κB or AP-1. Our results provide a mechanistic link between oxPL modification of apo(a) and stimulation of proinflammatory intracellular signaling pathways.

Tranexamic Acid in Trauma Management

Hemorrhage is the major cause of death in patients with severe trauma. The early coagulopathy that occurs in patients with severe trauma, known as acute traumatic coagulopathy (ATC), has a major effect on morbidity and mortality. About a quarter of all patients with severe injury are reported to have coagulopathy at the time of arrival to hospital. The important mechanism of ATC is hyperfibrinolysis by up-regulation of activated protein C. Therefore anti-fibrinolytics are needed to treat trauma patients with ATC. Tranexamic acid (TXA) is a common anti-fibrinolytic broadly used in clinical practice. TXA is a lysine analogue and binds to lysine-binding site on plasminogen, interfering with plasminogen binding to fibrin. By inactivating plasmin, TXA can prevent hyperfibrinolysis. Clinical randomization of an anti-fibrinolytic in significant hemorrhage-2 proved TXA effective for patients significantly bleeding from traumatic injuries, reducing mortality in bleeding trauma patients without increasing the risk of thrombosis. The results of this trial led many trauma centers to include TXA in their major hemorrhage protocols. Early use of TXA is the simplest and most effective treatment to improve patient outcomes in trauma patients with significant hemorrhage. Therefore use of TXA should be considered in patients with major trauma. (J Acute Care Surg 2015;5:47-51)

A Novel Interaction between Complement Inhibitor C4b-binding Protein and Plasminogen That Enhances Plasminogen Activation

The complement, coagulation, and fibrinolytic systems are crucial for the maintenance of tissue homeostasis. To date numerous interactions and cross-talks have been identified between these cascades. In line with this, here we propose a novel, hitherto unknown interaction between the complement inhibitor C4b-binding protein (C4BP) and plasminogen of the fibrinolytic pathway. Binding of C4BP to Streptococcus pneumoniae is a known virulence mechanism of this pathogen and it was increased in the presence of plasminogen. Interestingly, the acute phase variant of C4BP lacking the β-chain and protein S binds plasminogen much stronger than the main isoform containing the β-chain and protein S. Indeed, the complement control protein (CCP) 8 domain of C4BP, which would otherwise be sterically hindered by the β-chain, primarily mediates this interaction. Moreover, the lysine-binding sites in plasminogen kringle domains facilitate the C4BP-plasminogen interaction. Furthermore, C4BP readily forms complexes with plasminogen in fluid phase and such complexes are present in human serum and plasma. Importantly, whereas the presence of plasminogen did not affect the factor I cofactor activity of C4BP, the activation of plasminogen by urokinase-type plasminogen activator to active plasmin was significantly augmented in the presence of C4BP. Taken together, our data demonstrate a novel interaction between two proteins of the complement and fibrinolytic system. Most complexes might be formed during the acute phase of inflammation and have an effect on the homeostasis at the site of injury or acute inflammation.

Preferential Acquisition and Activation of Plasminogen Glycoform II by PAM Positive Group A Streptococcal Isolates.

Plasminogen (Plg) circulates in the host as two predominant glycoforms. Glycoform I Plg (GI-Plg) contains glycosylation sites at Asn289 and Thr346, whereas glycoform II Plg (GII-Plg) is exclusively glycosylated at Thr346. Surface plasmon resonance experiments demonstrated that Plg binding group A streptococcal M protein (PAM) exhibits comparative equal affinity for GI- and GII-Plg in the "closed" conformation (for GII-Plg, KD = 27.4 nM; for GI-Plg, KD = 37.0 nM). When Plg was in the "open" conformation, PAM exhibited an 11-fold increase in affinity for GII-Plg (KD = 2.8 nM) compared with that for GI-Plg (KD = 33.2 nM). The interaction of PAM with Plg is believed to be mediated by lysine binding sites within kringle (KR) 2 of Plg. PAM-GI-Plg interactions were fully inhibited with 100 mM lysine analogue ε-aminocaproic acid (εACA), whereas PAM-GII-Plg interactions were shown to be weakened but not inhibited in the presence of 400 mM εACA. In contrast, binding to the KR1-3 domains of GII-Plg (angiostatin) by PAM was completely inhibited in the presence 5 mM εACA. Along with PAM, emm pattern D GAS isolates express a phenotypically distinct SK variant (type 2b SK) that requires Plg ligands such as PAM to activate Plg. Type 2b SK was able to generate an active site and activate GII-Plg at a rate significantly higher than that of GI-Plg when bound to PAM. Taken together, these data suggest that GAS selectively recruits and activates GII-Plg. Furthermore, we propose that the interaction between PAM and Plg may be partially mediated by a secondary binding site outside of KR2, affected by glycosylation at Asn289.

The effectiveness of tranexamic acid at reducing postoperative blood loss following cesarean section: a systematic review of quantitative evidence protocol

The effectiveness of tranexamic acid at reducing postoperative blood loss following cesarean section: a systematic review of quantitative evidence protocol