The effect of tranexamic acid on synovium of patients undergoing arthroplasty and anterior cruciate ligament reconstruction surgery.

Preoperative hemorrhage can be reduced using anti-fibrinolytic medicine tranexamic acid (TXA). During surgical procedures, local administration is being used more and more frequently, either as an intra-articular infusion or as a perioperative rinse. Serious harm to adult soft tissues can be detrimental to the individual since they possess a weak ability for regeneration. Synovial tissues and primary fibroblast-like synoviocytes (FLS) isolated from patients were examined using TXA treatment in this investigation. FLS is obtained from rheumatoid arthritis (RA), osteoarthritis (OA), and anterior cruciate ligament (ACL)-ruptured patients. The in vitro effect of TXA on primary FLS was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays for cell death, annexin V/propidium iodide (PI) staining for apoptotic rate, real-time PCR for p65 and MMP-3 expression, and enzyme-linked immunosorbent assay (ELISA) for IL-6 measurement. MTT assays revealed a significant decrease in cell viability in FLS of all groups of patients following treatment with 0.8-60 mg/ml of TXA within 24 h. There was a significant increase in cell apoptosis after 24 h of exposure to TXA (15 mg/ml) in all groups, especially in RA-FLS. TXA increases the expression of MMP-3 and p65 expression. There was no significant change in IL-6 production after TXA treatment. An increase in receptor activator of nuclear factor kappa-Β ligand (RANK-L) production was seen only in RA-FLS. This study demonstrates that TXA caused significant synovial tissue toxicity via the increase in cell death and elevation of inflammatory and invasive gene expression in FLS cells.

Tranexamic acid may promote melanocores clustering in keratinocytes through upregulation of Rab5b.

Tranexamic acid (TXA) is a promising therapeutic agent in melasma that can act on multiple pathophysiologic mechanisms of melasma. However, it is unclear whether TXA affects melanin in keratinocytes. To explore the effect of TXA on melanocores in keratinocytes. The melanocore-incorporated keratinocytes were constructed by co-incubating normal human epidermal keratinocytes (NHEK) with melanocores. After being treated with TXA, autophagy- and melanin-related protein expressions were detected. Then, transcriptome sequencing was used to compare the genetic changes in melanocore-incorporated keratinocytes before and after TXA treatment and further verified the differentially expressed genes. At the same time, the distribution of melanocores in human keratinocytes was observed by transmission electron microscopy. We found that TXA does not promote melanin degradation in primary keratinocytes by inducing autophagy. Protein transport and intracellular protein transport-related genes were enriched after TXA treatment, and Rab5b was significantly upregulated. Transmission electron microscopy showed that the percentage of melanocores distributed in clusters increased after treatment with TXA, which was reduced after Rab5b silencing. In addition, results suggested that melanocores could colocalize with Rab5b and lysosome-associated membrane protein1 (LAMP1). Our study found that Rab5b may be involved in the melanocore distribution in keratinocytes. TXA may promote the clustering distribution of endocytic melanocores through upregulation of Rab5b, representing a potential mechanism of TXA treatment against melasma.

Multiplexed Organelles Portrait Barcodes for Subcellular MicroRNA Array Detection in Living Cells.

Multiplexed profiling of microRNAs' subcellular expression and distribution is essential to understand their spatiotemporal function information, but it remains a crucial challenge. Herein, we report an encoding approach that leverages combinational fluorescent dye barcodes, organelle targeting elements, and an independent quantification signal, termed Multiplexed Organelles Portrait Barcodes (MOPB), for high-throughput profiling of miRNAs from organelles. The MOPB barcodes consist of heterochromatic fluorescent dye-loaded shell-core mesoporous silica nanoparticles modified with organelle targeting peptides and molecular beacon detection probes. Using mitochondria and endoplasmic reticulum as models, we encoded four Cy3/AMCA ER-MOPB and four Cy5/AMCA Mito-MOPB by varying the Cy3 and Cy5 intensity for distinguishing eight organelles' miRNAs. Significantly, the MOPB strategy successfully and accurately profiled eight subcellular organelle miRNAs' alterations in the drug-induced Ca2+ homeostasis breakdown. The approach should allow more widespread application of subcellular miRNAs and multiplexed subcellular protein biomarkers' monitoring for drug discovery, cellular metabolism, signaling transduction, and gene expression regulation readout.

Tranexamic acid integrated into platelet-rich fibrin produces a robust and resilient antihemorrhagic biological agent: a human cohort study.

OBJECTIVE: To investigate the incorporation of the antifibrinolytic agent tranexamic acid (TA) during platelet-rich fibrin (PRF) formation to produce a robust fibrin agent with procoagulation properties. STUDY DESIGN: Blood from healthy volunteers was collected. Into 3 tubes, TA was immediately added in 1-mL, 0.4-mL, and 0.2-mL volumes, and the fourth tube was without additions. After PRF preparation, the clots were weighed in their raw (clot) and membrane forms. PRF physical properties were analyzed using a universal testing system (Instron). Protein and TA levels in the PRF were analyzed using a bicinchoninic acid assay and a ferric chloride assay, respectively. RESULTS: The addition of TA to PRF led to a robust weight compared with sham control. PRF weight was greater in females in all tested groups. The addition of TA also led to greater resilience to tears, especially at 1-mL TA addition to the blood. Furthermore, TA addition led to a greater value of total protein within the PRF and entrapment of TA in the PRF. CONCLUSIONS: Addition of TA to a PRF preparation leads to robust PRF with greater protein levels and the amalgamation of TA into the PRF. Such an agent may enhance the beneficial properties of PRF and attribute procoagulation properties to it.

Tranexamic acid inhibits melanogenesis partially via stimulation of TGF-ß1 expression in human epidermal keratinocytes.

Oral tranexamic acid (TA) has been an effective treatment for melasma with unclear mechanism. The present study aimed to demonstrate the effect of TA on melanogenesis via regulation of TGF-ß1 expression in keratinocytes. We firstly determined the expression level of TGF-ß1 in TA-treated keratinocyte-conditioned medium (KCM). Then, the mRNA and protein levels of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein 1 (TRP-1) of human epidermal melanocytes (NHEMs) in the presence of TA-treated KCM were evaluated via RT-PCR and western blot analysis. Moreover, melanin content and tyrosinase activity were quantified. TGF-ß1 gene was knocked down by small interfering RNA (siRNA) in keratinocytes. The mRNA and protein levels of TGF-ß1 in keratinocytes were significantly increased after TA treatment. Melanin contents, tyrosinase activity, protein and mRNA levels of TYR, MITF and TRP-1 were downregulated in NHEMs in the presence of TA-treated KCM. Knockdown of TGF-ß1 in keratinocytes could attenuate the inhibitory effect of TA-treated KCM on melanogenesis. TA could stimulate TGF-ß1 expression in keratinocytes, which further inhibits melanogenesis through the paracrine signalling.

Application of a Thermosensitive In Situ Gel of Chitosan-Based Nasal Spray Loaded with Tranexamic Acid for Localised Treatment of Nasal Wounds.

The integrity of the nasal epithelium plays a crucial role in the airway defence mechanism. The nasal epithelium may be injured as a result of a large number of factors leading to nose bleeds, also known as epistaxis. However, local measures commonly used to treat epistaxis and improve wound healing present several side effects and patient discomfort. Hence, this study aims to address some of these drawbacks by developing a new formulation for nasal epithelial wound healing. Chitosan, a biodegradable and biocompatible polymer, was used to develop a thermosensitive nasal formulation for the delivery of tranexamic acid (TXA), one of the most effective pharmacological options to control bleeding with cost and tolerability advantages. The in situ gelation properties of the formulation upon administration in the nasal cavity were investigated in terms of gelation time and temperature. It was found that the developed formulation can undergo rapid liquid-to-gel phase change within approximately 5 min at 32°C, which is well within the human nasal cavity temperature range. The spray pattern, deposition and droplet size generated by the nasal spray was also characterised and were found to be suitable for nasal drug delivery. It was also observed that the in situ gelation of the formulation prevent nasal runoff, while the majority of drug deposited mainly in the anterior part of the nose with no lung deposition. The developed formulation was shown to be safe on human nasal epithelium and demonstrated six times faster wound closure compared to the control TXA solution.

Comparison of Cytotoxic Effects of Intra-Articular Use of Tranexamic Acid versus Epinephrine on Rat Cartilage.

BACKGROUND Adequate visualization is known to be essential to perform arthroscopic procedures effectively and efficiently. We hypothesized that tranexamic acid may be considered as an alternative agent to reduce intra-articular bleeding during arthroscopic procedures, after comparing its potential chondrotoxicity with that of epinephrine. MATERIAL AND METHODS Seventy-two rats were randomized into 3 groups with 24 rats each. The injections were performed in the right knees, as follows: Group 1: 0.25 mL of tranexamic acid solution, Group 2: 0.25 mL of epinephrine solution, and Group 3: 0.25 mL of 0.9% saline, serving as control. One week after the injections, the animals were euthanized. Samples were evaluated histologically based on the Osteoarthritis Research Society International (OARSI) Histopathology Grading and Staging System and the "live/dead" staining technique to determine chondrocyte viability. RESULTS Comparison of epinephrine and tranexamic acid revealed significantly higher OARSI scores in the epinephrine group (epinephrine: 3.42±1.31, TA: 0.92±0.90; P<0.001). The most significant difference between the 2 groups was in the number of joints diagnosed with OARSI grade III. The percentage of viability was significantly higher in the tranexamic acid group when compared with the epinephrine group (tranexamic acid: 79.74±3.343; epinephrine: 63.81±1.914; P<0.05). CONCLUSIONS Based on the histologic parameters and chondrocyte viability, tranexamic acid is less cytotoxic than epinephrine in rat chondrocytes at the doses typically used in irrigation fluid, and may be a good alternative to epinephrine in arthroscopic surgery.

Volatile Decay Products in Breath During Peritonitis Shock are Attenuated by Enteral Blockade of Pancreatic Digestive Proteases.

There is a need to develop markers for early detection of organ failure in shock that can be noninvasively measured at point of care. We explore here the use of volatile organic compounds (VOCs) in expired air in a rat peritonitis shock model. Expired breath samples were collected into Tedlar gas bags and analyzed by standardized gas chromatography. The gas chromatograms were digitally analyzed for presence of peak amounts over a range of Kovach indices. Following the induction of peritonitis, selected volatile compounds were detected within about 1 h, which remained at elevated amounts over a 6 h observation period. These VOCs were not present in control animals without peritonitis. Comparisons with know VOCs indicate that they include 1,4-diaminobutane and trimethylamine N-oxide. When pancreatic digestive proteases were blocked with tranexamic acid in the intestine and peritoneum, a procedure that serves to reduce organ failure in shock, the amounts of VOCs in the breath decreased spontaneously to control values without peritonitis. These results indicate that peritonitis shock is accompanied by development of volatile organic compounds that may be generated by digestive enzymes in the small intestine. VOCs may serve as indicators for detection of early forms of autodigestion by digestive proteases.

A Novel Chemical Enhancer Approach for Transdermal Drug Delivery with C17-Monoglycerol Ester Liquid Crystal-forming Lipid.

Transdermal administration of drugs represents an excellent alternative to conventional pharmaceutical dosage forms. However, insufficient penetration of the active pharmaceutical substance through the skin is a common problem. Thus, in the present study we evaluated the skin permeation enhancing ability of liquid crystal (LC) topical formulations. A recently developed LC-forming lipid, C17- monoglycerol ester (MGE), was evaluated and compared with glycerol monoolate (GMO), which is considered as the gold standard for LC formulations. We initially prepared LC formulations containing drugs with different physiochemical properties (tranexamic acid [TXA], 4-methoxy-salicylic acid [4-MS], catechin [CC], and calcein [Cal]), and confirmed the LC phase structures in the prepared formulations using a polarizing light microscope and a small-angle X-ray scattering (SAXS). The physicochemical properties of these formulations were also assessed using a viscometer and a zetasizer. The release rate of the drugs from the LC formulations was determined using a dialysis release method. The skin penetration-enhancing ability of LC formulations was also investigated in an in vitro skin permeation study. The results showed that both MGE- and GMO-LC-forming lipids shared the same behavior in terms of their birefringence indexes, LC phase structures, particle sizes, and zeta potentials. Both the MGE- and GMO-LC formulations managed to improve the skin permeation for various drugs with a range of physiochemical properties. However, MGE formulations showed lower viscosity, faster drug release rate, and better skin penetration-enhancing ability than GMO formulations, strongly suggesting that the low viscosity of MGELC-forming lipids might influence drug diffusivity and permeability through the skin. The present MGELC formulation might be utilized as a promising new topical formulation for therapeutic drugs and cosmetic ingredients.

Empleo de ácido tranexámico en el herido de combate, experiencia de la sanidad militar española. Serie de casos y revisión de la literatura / Use of tranexamic acid in combat casualties. Experience of the Spanish medical corps. Clinical series and literature review

Objetivo. Describir la experiencia obtenida con el ácido tranexámico (ATX) durante la atención a bajas de combate en el hospital militar español desplegado en Herat (Afganistán) y analizar la bibliografía relacionada en el ámbito militar. Material y métodos. Con la aprobación de las instituciones militares pertinentes, se analizó la administración de ATX en bajas de combate entre marzo y mayo de 2014. De los 745 pacientes atendidos, 10 fueron por arma de fuego/artefacto explosivo (bajas de combate). El método estadístico empleado fue el descriptivo. Para variables categóricas se emplearon frecuencias absolutas y relativas en tanto por ciento (%). Como índices de la tendencia central, la media aritmética y la desviación estándar o la mediana y el rango intercuartílico. Los datos se obtuvieron del registro militar de pacientes atendidos en el hospital militar español de Herat. Resultados. En nuestra serie de datos, todos los pacientes recibieron ATX antes de las 3 primeras horas tras el ataque. La dosis empleada más prevalente fue un gramo iv (intravenoso). La hemorragia fue controlada en el 100% de los casos. Todos los pacientes sobrevivieron y en ninguno se objetivaron efectos secundarios. Estos datos coinciden con lo recomendado en las guías de atención a la baja de combate seguidas por sanidades militares de otros países de nuestro entorno. Conclusión. Todas las bajas en combate fueron tratadas con ATX durante las 3 primeras horas. La dosis más prevalente fue de un gramo iv. La hemorragia fue controlada en la totalidad de los casos. Todos los pacientes sobrevivieron sin efectos secundarios (AU)

Objective. To describe the experience with tranexamic acid (TXA) during the care of combat causalities treated in the Spanish military hospital based in Herat (Afghanistan) and to perform an analysis of the literature related to the military setting. Material and methods. With the approval of the appropriate military institutions, an analysis was performed on the use of TXA in combat casualties treated between March and May 2014. Of the 745 patients seen, 10 were due to a firearm/explosive device (combat casualties). A descriptive analysis was performed on the data collected. Absolute and relative frequencies (%) were used for the categorical variables. For central tendency measurements, the arithmetic mean and standard deviation or the median and interquartile range was calculated. The data were obtained from the military records of patients treated in the Herat military hospital. Results. All the patients in this series received TXA within the first 3 hours after the attack. The most frequent dose used was one gram i.v, with bleeding was controlled in 100% of cases. All the patients survived and none of them had secondary effects. These data agree with that recommended in the combat casualties treatment guide followed by military health in other countries in this setting. Conclusion. All combat casualties were treated with TXA within the first 3 hours. The most frequent dose used was one gram iv and bleeding was controlled in all cases. All the patients survived with no adverse effects being observed (AU)

Dosis única intravenosa de ácido tranexámico como medida de ahorro transfusional en prótesis total primaria de rodilla / Single intravenous tranexamic acid dose to reduce blood loss in primary total knee replacement

Objetivo. Evaluar la eficacia y seguridad de la administración de una dosis única intravenosa de ácido tranexámico como medida de ahorro transfusional en prótesis total primaria de rodilla. Material y métodos. Estudio observacional prospectivo de la administración de ácido tranexámico en pacientes intervenidos de prótesis total primaria de rodilla desde noviembre de 2013 a febrero de 2015, en los que se utilizó un sistema de recuperación de sangre autóloga. Se incluyeron en el estudio 98 pacientes distribuidos en dos grupos de 49 pacientes según la exposición a la administración de ácido tranexámico. La variable principal del estudio fue el número de pacientes que precisaron autotransfusión del sistema de recuperación de sangre autológa. Resultados. No se registraron pérdidas durante el seguimiento. No hubo diferencias significativas entre ambos grupos con respecto a las variables preoperatorias y hospitalarias. Los valores medios de hemoglobina y hematocrito preoperatorios, a las 24 y 48 h postoperatorias eran similares en ambos grupos. El volumen medio de sangrado en el sistema de recuperación de sangre autóloga y la pérdida media estimada de sangre fue menor en los pacientes a los que se había administrado ácido tranexámico, siendo las diferencias significativas. Ningún paciente del grupo en el que se administró ácido tranexámico precisó autotransfusión sanguínea. No se precisó alotransfusión sanguínea en los pacientes de la cohorte. No se registraron eventos adversos relacionados con la administración del ácido tranexámico. Conclusiones. El uso de una dosis única 15 mg/kg de ATX intravenoso en PTR primaria ha presentado una tasa de no autotransfusión ni alotranfusión sanguínea del 100%, sin aumento en la incidencia de eventos trombóticos. Por ello recomendamos su utilización en este grupo de pacientes, con una indicación que debe ser individualizada, justificar su uso en la historia clínica y precisar del consentimiento informado del paciente. Nivel de evidencia III (AU)

Objective. To evaluate the effectiveness and safety of a single intravenous dose of tranexamic acid in order to reduce blood loss in total knee replacement. Materials and methods. Prospective observational study of the administration of tranexamic acid in patients undergoing primary total knee arthroplasty from November 2013 to February 2015, in which an autologous blood recovery system was used. The study included 98 patients, distributed into two groups of 49 patients according to whether or not they received intravenous tranexamic acid. The primary endpoint was the number of patients requiring autologous transfusion from the recovery system autologous blood recovery system. Results. No drop-outs were recorded during follow-up. There were no significant differences between groups as regards the preoperative and hospital variables. The mean preoperative haemoglobin and haematocrit at 24 and 48 hours postoperatively were similar in both groups. The average volume of bleeding in the autologous blood recovery system and estimated average blood loss was lower in patients who had been administered tranexamic acid, with significant differences. No patients in the group that was administered tranexamic acid required blood autotransfusion. The transfusion rate was zero in the two groups. No adverse events related to the administration of tranexamic acid were recorded. Conclusions. Intravenous administration of tranexamic acid, according to the described protocol, has presented a non-autotransfusion or allo-transfusion rate of 100%, with no increased incidence of thrombotic events. Thus, its use in this group of patients is recommended. The indication should be individualized, its use justified in the patient medical records, and informed consent is mandatory (AU)

α-Enolase Causes Proinflammatory Activation of Pulmonary Microvascular Endothelial Cells and Primes Neutrophils Through Plasmin Activation of Protease-Activated Receptor 2.

UNLABELLED: Proinflammatory activation of vascular endothelium leading to increased surface expression of adhesion molecules and neutrophil (PMN) sequestration and subsequent activation is paramount in the development of acute lung injury and organ injury in injured patients. We hypothesize that α-enolase, which accumulates in injured patients, primes PMNs and causes proinflammatory activation of endothelial cells leading to PMN-mediated cytotoxicity. METHODS: Proteomic analyses of field plasma samples from injured versus healthy patients were used for protein identification. Human pulmonary microvascular endothelial cells (HMVECs) were incubated with α-enolase or thrombin, and intercellular adhesion molecule-1 surface expression was measured by flow cytometry. A two-event in vitro model of PMN cytotoxicity HMVECs activated with α-enolase, thrombin, or buffer was used as targets for lysophosphatidylcholine-primed or buffer-treated PMNs. The PMN priming activity of α-enolase was completed, and lysates from both PMNs and HMVECs were immunoblotted for protease-activated receptor 1 (PAR-1) and PAR-2 and coprecipitation of α-enolase with PAR-2 and plasminogen/plasmin. RESULTS: α-Enolase increased 10.8-fold in injured patients (P < 0.05). Thrombin and α-enolase significantly increased intercellular adhesion molecule-1 surface expression on HMVECs, which was inhibited by antiproteases, induced PMN adherence, and served as the first event in the two-event model of PMN cytotoxicity. α-Enolase coprecipitated with PAR-2 and plasminogen/plasmin on HMVECs and PMNs and induced PMN priming, which was inhibited by tranexamic acid, and enzymatic activity was not required. CONCLUSIONS: α-Enolase increases after injury and may activate pulmonary endothelial cells and prime PMNs through plasmin activity and PAR-2 activation. Such proinflammatory endothelial activation may predispose to PMN-mediated organ injury.

Ácido tranexámico en cirugía ortopédica / Tranexamic acid in orthopedic surgery

El sangrado perioperatorio en ocasiones conduce a transfusiones sanguíneas no exentas de complicaciones y riesgos, con un alto gasto sanitario. Entre otros métodos de prevención, el tratamiento con ácido tranexámico (TXA) ha mostrado ser efectivo en la disminución de las pérdidas sanguíneas quirúrgicas y especialmente en el postoperatorio inmediato. Al respecto, los estudios que lo han evaluado en cirugía ortopédica muestran su eficacia y seguridad, administrado por vía tanto intravenosa como intraarticular. Las dosis habituales por vía intravenosa evaluadas oscilan entre 10 y 20 mg/kg, o en dosis fijas de 1 a 2 g, mientras por vía intraarticular varía entre 250 mg y 3 g. El TXA como antifibrinolítico tiene un potencial efecto trombótico y está contraindicado en aquellos pacientes con riesgo o antecedentes de trombosis. Su administración por vía tópica podría ser más segura aunque se precisan estudios que lo confirmen (AU)

Perioperative bleeding may require blood transfusions, which are sometimes not without complications and risks, with the subsequent increase in health care costs. Among other prevention methods, treatment with tranexamic acid (ATX) has shown to be effective in reducing surgical blood loss, especially in the immediate postoperative period. In this regard, studies evaluating ATX in orthopedic surgery show that it is effective and safe when administered intravenously or intra-articularly. The usual evaluated intravenous doses range between 10 mg/Kg and 20 mg/kg or a fixed dose of 1 g to 2 g; while intra-articularly, it varies between 250 mg and 3 g. ATX, as an anti-fibrinolytic has a potential thrombotic effect, thus it is contraindicated in those patients at risk or with a history of thrombosis. Its topical administration may be safer, but studies are needed to confirm this (AU)

Tranexamic acid overdosage-induced generalized seizure in renal failure.

We report a 45-year-old lady with chronic kidney disease stage 4 due to chronic tubulointerstial disease. She was admitted to our center for severe anemia due to menorrhagia and deterioration of renal function. She was infused three units of packed cells during a session of hemodialysis. Tranexamic acid (TNA) 1 g 8-hourly was administered to her to control bleeding per vaginum. Two hours after the sixth dose of TNA, she had an episode of generalized tonic clonic convulsions. TNA was discontinued. Investigations of the patient revealed no biochemical or structural central nervous system abnormalities that could have provoked the convulsions. She did not require any further dialytic support. She had no further episodes of convulsion till dis-charge and during the two months of follow-up. Thus, the precipitating cause of convulsions was believed to be an overdose of TNA.

Protein hydrolysate-induced cholecystokinin secretion from enteroendocrine cells is indirectly mediated by the intestinal oligopeptide transporter PepT1.

Dietary protein is a major stimulant for cholecystokinin (CCK) secretion by the intestinal I cell, however, the mechanism by which protein is detected is unknown. Indirect functional evidence suggests that PepT1 may play a role in CCK-mediated changes in gastric motor function. However, it is unclear whether this oligopeptide transporter directly or indirectly activates the I cell. Using both the CCK-expressing enteroendocrine STC-1 cell and acutely isolated native I cells from CCK-enhanced green fluorescent protein (eGFP) mice, we aimed to determine whether PepT1 directly activates the enteroendocrine cell to elicit CCK secretion in response to oligopeptides. Both STC-1 cells and isolated CCK-eGFP cells expressed PepT1 transcripts. STC-1 cells were activated, as measured by ERK(1/2) phosphorylation, by both peptone and the PepT1 substrate Cefaclor; however, the PepT1 inhibitor 4-aminomethyl benzoic acid (AMBA) had no effect on STC-1 cell activity. The PepT1-transportable substrate glycyl-sarcosine dose-dependently decreased gastric motility in anesthetized rats but had no affect on activation of STC-1 cells or on CCK secretion by CCK-eGFP cells. CCK secretion was significantly increased in response to peptone but not to Cefaclor, cephalexin, or Phe-Ala in CCK-eGFP cells. Taken together, the data suggest that PepT1 does not directly mediate CCK secretion in response to PepT1 specific substrates. PepT1, instead, may have an indirect role in protein sensing in the intestine.

Solution structure and functional characterization of human plasminogen kringle 5.

The ligand binding properties of the kringle 5 (K5) domain of human plasminogen have been investigated via intrinsic tryptophan fluorescence. The oleic acid (OA) affinity for K5 was quantified, yielding an association constant K(a) approximately 2.08 x 10(4) mM(-1). Simultaneously, it was determined that OA and trans-4-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) (K(a) approximately 50 mM(-1)) compete for binding to K5. The solution structure of K5 in the presence of 11 mM AMCHA was solved via NMR spectroscopy (protein heavy atom RMSD approximately 0.93 +/- 0.12 A). The AMCHA binding site was localized via (1)H/(15)N chemical shift perturbation mapping assisted by in silico docking. We have found that AMCHA binds at the canonical kringle lysine binding site (LBS), structured by the Pro54-Gly60 segment plus the neighboring Phe36, Thr37, Trp62, Leu71, and Tyr72 residues. The segment 30-42, encompassing LBS residues, appears to be endowed with a higher degree of structural flexibility as suggested by the relatively lower value of S(2), the generalized order parameter, consistent with a higher backbone heavy atom RMSD of approximately 1.22 A (vs 0.84 A overall) between the two monomeric units in the crystal unit cell, of potential significance for ligand binding. OA was found to perturb the same area of the protein, namely, the LBS, as well as Tyr74. Combined with previous studies, the observation of OA binding expands the range of ligands that interact with kringle 5 while it widens the scope of potential biological functions for kringle domains.

Recombinant activated factor VII and tranexamic acid are haemostatically effective during major surgery in factor XI-deficient patients with inhibitor antibodies.

One-third of patients with severe factor XI (FXI) deficiency caused by homozygosity for null alleles develop inhibitor antibodies following exposure to plasma. Haemostasis during surgery is achievable in such patients by recombinant activated factor VII (rFVIIa) at doses used in haemophilia A patients with an inhibitor to FVIII. However, thrombosis has occurred in three of 12 such patients. In this study we discerned whether low-dose rFVIIa would secure haemostasis and cause no thrombosis in patients with severe FXI deficiency and an inhibitor during surgery. In vitro, a very low concentration of rFVIIa (0.24 microg/ml) induced thrombin generation in FXI-deficient plasma quite similarly to 1.9 microg/ml (a concentration that is achieved in patients with haemophilia A and inhibitor after infusion of 80 microg/kg). Based on this finding, a protocol was designed for four patients with severe FXI deficiency and an inhibitor or immunoglobulin A deficiency who underwent five major surgical procedures. This included administration of tranexamic acid from two hours before surgery until seven to 14 days after, and single infusion of low-dose rFVIIa. No excessive bleeding or thrombosis were observed. In conclusion, a single low dose of rFVIIa and tranexamic acid secure normal haemostasis in patients with severe FXI deficiency who can not receive blood products.

Two families of synthetic peptides that enhance fibrin turbidity and delay fibrinolysis by different mechanisms.

When fibrin clots are formed in vitro in the presence of certain positively charged peptides, the turbidity is enhanced and fibrinolysis is delayed. Here we show that these two phenomena are not always linked and that different families of peptides bring about the delay of lysis in different ways. In the case of intrinsically adhesive peptides corresponding to certain regions of the fibrinogen gammaC and betaC domains, even though these peptides bind to fibrin(ogen) and enhance turbidity, the delay in lysis is mainly due to direct inhibition of plasminogen activation. In contrast, for certain pentapeptides patterned on fibrin B knobs, the delay in lysis is a consequence of how fibrin units assemble. On their own, these B knob surrogates can induce the gelation of fibrinogen molecules. The likely cause of enhanced clot turbidity and delay in fibrinolysis was revealed by a crystal structure of the D-dimer from human fibrinogen cocrystallized with GHRPYam, the packing of which showed the direct involvement of the ligand tyrosines in antiparallel betaC-betaC interactions.