The role of thrombomodulin lectin-like domain in inflammation

Thrombomodulin (TM) is a cell surface glycoprotein which is widely expressed in a variety of cell types. It is a cofactor for thrombin binding that mediates protein C activation and inhibits thrombin activity. In addition to its anticoagulant activity, recent evidence has revealed that TM, especially its lectin-like domain, has potent anti-inflammatory function through a variety of molecular mechanisms. The lectin-like domain of TM plays an important role in suppressing inflammation independent of the TM anticoagulant activity. This article makes an extensive review of the role of TM in inflammation. The molecular targets of TM lectin-like domain have also been elucidated. Recombinant TM protein, especially the TM lectin-like domain may play a promising role in the management of sepsis, glomerulonephritis and arthritis. These data demonstrated the potential therapeutic role of TM in the treatment of inflammatory diseases

Role of thrombin in coronary artery bypass grafting

Thrombin is a multifunctional protease, which has a central role in the development and progression of coronary atherosclerotic lesions and it is a possible mediator of myocardial ischemia-reperfusion injury. Its generation and procoagulant activity are greatly upregulated during cardiopulmonary bypass (CPB). On the other hand, activated protein C, a physiologic anticoagulant that is activated by thrombomodulin-bound thrombin, has been beneficial in various models of ischemia-reperfusion. Therefore, our aim in this study was to test whether thrombin generation or protein C activation during coronary artery bypass grafting (CABG) associate with postoperative myocardial damage or hemodynamic changes. To further investigate the regulation of thrombin during CABG, we tested whether preoperative thrombophilic factors associate with increased CPB-related generation of thrombin or its procoagulant activity. We also measured the anticoagulant effects of heparin during CPB with a novel coagulation test, prothrombinase-induced clotting time (PiCT), and compared the performance of this test with the present standard of laboratory-based anticoagulation monitoring. One hundred patients undergoing elective on-pump CABG were studied prospectively. A progressive increase in markers of thrombin generation (F1+2), fibrinolysis (D-dimer), and fibrin formation (soluble fibrin monomer complexes) was observed during CPB, which was further distinctly propagated by reperfusion after myocardial ischemia, and continued to peak after the neutralization of heparin with protamine. Thrombin generation during reperfusion after CABG associated with postoperative myocardial damage and increased pulmonary vascular resistance. Activated protein C levels increased only slightly during CPB before the release of the aortic clamp, but reperfusion and more significantly heparin neutralization caused a massive increase in activated protein C levels. Protein C activation was clearly delayed in relation to both thrombin generation and fibrin formation. Even though activated protein C associated dynamically with postoperative hemodynamic performance, it did not associate with postoperative myocardial damage. Preoperative thrombophilic variables did not associate with perioperative thrombin generation or its procoagulant activity. Therefore, our results do not favor routine thrombophilia screening before CABG. There was poor agreement between PiCT and other measurements of heparin effects in the setting of CPB. However, lower heparin levels during CPB associated with inferior thrombin control and high heparin levels during CPB associated with fewer perioperative transfusions of blood products. Overall, our results suggest that hypercoagulation after CABG, especially during reperfusion, might be clinically important.Trombiini on hyytymisjärjestelmän keskeinen entsyymi, jolla on tärkeä osuus sepelvaltimotaudin synnyssä ja etenemisessä. Lisäksi trombiini on mahdollinen välittäjäaine hapen puutteeseen ja sen jälkeiseen verenkierron palautumiseen liittyvässä sydänlihasvauriossa. Trombiinin tuotanto ja veren hyytymistä aiheuttava vaikutus lisääntyvät voimakkaasti kehon ulkoisen verenkierron aikana. Aktivoitu proteiini C puolestaan on elimistön luonnollinen hyytymistä estävä aine, eli antikoagulantti, joka aktivoituu trombiinin vaikutuksesta. Tutkimuksen tarkoituksena oli selvittää, liittyvätkö trombiinin tuotanto tai proteiini C:n aktivoituminen sepelvaltimo-ohitusleikkauksen jälkeiseen sydänlihasvaurioon tai verenkierron muutoksiin. Trombiinin säätelyn selvittämiseksi tutkimme lisäksi, lisääntyykö trombiinin tuotanto tai sen vaikutukset potilailla, joilla on veren hyytymistä lisääviä tekijöitä, sekä vertasimme uutta hyytymistutkimusta [prothrombinase-induced clotting time (PiCT)] vakiintuneisiin laboratoriomenetelmiin kliinisesti sydänleikkauksessa käytetyn antikoagulantin, hepariinin, vaikutusten mittaamisessa. Tutkimusaineistona oli sata elektiivistä potilasta, joille tehtiin sepelvaltimo-ohitusleikkaus sydämen pysäytystä ja kehon ulkoista verenkiertoa käyttäen. Trombiinin tuotannon, trombiinin hyytymistä aiheuttavan vaikutuksen ja fibrinolyysin merkkiaineiden (protombiinin fragmenttien F1+2, liukoisten fibriinimonomeerikompleksien ja D-dimeerin) pitoisuudet lisääntyivät voimakkaasti leikkauksen aikana erityisesti sydämen verenkierron palauttamisen jälkeen. Tämän ns. reperfuusiovaiheen trombiinin tuotanto liittyi leikkauksen jälkeiseen keuhkoverenkierron vastuksen kohoamiseen ja leikkauksen jälkeen todettuun sydänlihasvaurioon. Proteiini C aktivoitui viiveellä trombiinin tuotantoon nähden ja aktivoitu proteiini C liittyi leikkauksen jälkeiseen verenkierrolliseen toipumiseen eri tavoin leikkausta edeltävästi ja sen eri vaiheissa. Vaikka perinnöllinen veren hyytymisalttius lisää yleisesti veritulppariskiä, leikkausta edeltävissä tutkimuksissa todettuun hyytymisalttiuteen ei liittynyt lisääntynyttä leikkauksen aikaista hyytymisaktivaatiota. Siitä huolimatta, että erilaisten hepariinin vaikutusten mittaamisessa käytettyjen laboratoriomenetelmien tulosten vastaavuus oli huono, osoitettiin heparinisaation asteen liittyvän kääntäen verensiirtojen tarpeeseen ja hyytymisaktivaatioon. Kaikkiaan tutkimuksessa osoitettiin hyytymisaktivaatiolla olevan haitallisia vaikutuksia sydänleikkauksen yhteydessä

Protein C- und Antithrombin III-Aktivität: Stellenwert bei der Diagnose und Verlaufsbeurteilung unterschiedlicher systemischer Entzündungssyndrome bei kritisch kranken Patienten

In der vorliegenden Dissertation wurde der Stellenwert der Protein C- und AT III-Aktivität bei der Diagnose und Verlaufsbeurteilung unterschiedlicher systemischer Entzündungs-syndrome an 323 kritisch kranken Patienten evaluiert. Zusammenfassend kann festgestellt werden, dass bei einem Großteil kritisch kranker Patienten eine verminderte Protein C- und AT III- Aktivität vorliegt, wobei Patienten mit einer schweren Sepsis oder septischem Schock, mit einem erniedrigten Albuminspiegel, einer Gerinnungsstörung und zunehmendem Organversagen die niedrigste Aktivität aufweisen. Zur Diagnose und Verlaufsbeurteilung von kritisch kranken Patienten ist weder die Protein C-, noch die AT III-Aktivität geeignet. Weiterhin weisen die Ergebnisse darauf hin, dass sowohl eine Gerinnungsstörung, als auch Vorgänge, die den Albuminspiegel beeinflussen (Leberinsuffizienz und Kapillarleck) eine mögliche Rolle beim Abfall der Protein C- bzw. AT III- Aktivität spielen könnten

A rat model of stavudine-induced hyperalgesia

Stavudine, a nucleoside reverse transcriptase inhibitor (NRTI) used to treat infection by the human immunodeficiency virus (HIV), causes peripheral neuropathy and pain in HIV-positive patients. The mechanisms of this toxic neuropathy are poorly understood, partly because of a lack of animal models of the disease process. I investigated whether long-term daily oral administration of stavudine affects nociception in Sprague-Dawley rats, and whether changes in nociception are accompanied by a general deterioration in the rats’ conditions, as reflected in activity and appetite. Daily stavudine administration induced mechanical hyperalgesia in rats within three weeks without affecting appetite, growth or physical activity, and this hyperalgesia persisted throughout the six weeks of stavudine administration. I then investigated whether central changes underlie the hyperalgesia caused by stavudine in rats by examining inflammatory cytokine secretion and neuronal death in the spinal cord. Daily stavudine administration caused an increase in cytokine-induced neutrophil chemo-attractant (CINC)-1 concentration in the spinal cord after six weeks, but early development of stavudine-induced hyperalgesia did not depend on increases in spinal concentrations of CINC-1 and interleukin (IL)-6, nor on apoptosis or necrosis of spinal neurones. The neurotoxicity of stavudine is thought to derive from mitochondrial toxicity, which has been linked to increased plasma lactate concentration and decreased plasma adiponectin levels caused by lipodystrophy. Thus, I investigated whether a systemic inflammatory response or metabolic dysregulation accompanied stavudine-induced hypernociception by examining plasma adiponectin, lactate, CINC-1 and IL-6 concentrations in rats administered daily stavudine. Plasma adiponectin, lactate, CINC-1 and IL-6 concentrations were unchanged following three or six weeks of daily stavudine administration. Therefore, I have shown that stavudine-induced hyperalgesia is not dependent on spinal cord plasticity, nor on a systemic inflammatory response or extensive metabolic malfunction. Instead, the hyperalgesia I observed may be caused by the adverse effects of stavudine on peripheral neurone functioning. As stavudine administration to healthy rats had no adverse effects besides inducing hyperalgesia and causing a rise in CINC-1 concentration in the spinal cord after six weeks, my results indicate that many other side effects commonly associated with stavudine treatment in HIV-positive patients may arise through interaction with the underlying HIV infection

Characterizing Neutrophil Response Following Naturally-Occurring Spinal Cord Injury

In treating spinal cord injury (SCI), it is important to identify agents that could diminish the inflammatory response that follows primary injury. Human and rodent SCI research shows an increase in neutrophils following injury, yet, little is known about cellular activation post-injury. This study was implemented in dogs with naturallyoccurring SCI resulting from intervertebral disk herniation (IVDH) to characterize the postinjury inflammatory response. Canine SCI parallels human SCI with respect to identifiable contusion with sustained compression; treatment modalities; and histopathic, molecular and magnetic resonance lesion phenotype. However, unlike most human SCI, dogs with IVDH-SCI do not have poly-trauma masking inflammatory responses to the injured cord. Cerebrospinal fluid (CSF), blood, and spinal cord tissue were characterized in dogs with SCI and in healthy controls recruited from Texas A&M University. Metabolite concentrations from CSF were measured using enzyme-linked immunosorbent assays and tandem mass spectrometry. Isolated peripheral blood neutrophil activity and expression of L-selectin were evaluated with flow cytometric analysis. Neutrophils were identified in damaged spinal cords of dogs with severe IVDH. The inflammatory response in dogs with SCI is increased in CSF metabolite profiles. Neutrophil activity in circulation is prolonged, and there is evidence to suggest a canine specific expression of neutrophil L-selectin. This is the first observation of neutrophils in the damaged canine spinal cord. This study demonstrates changes in the peripheral immune cells in an animal model that closely resembles human pathogenesis of neuroinflammation after spinal cord injury

The flavonoid quercetin and its potential as neuroprotectant in the therapy of acute traumatic CNS Injury : an experimental study

Every year, several thousand individuals suffer spinal cord injury (SCI) in North America, while 1.5 million suffer traumatic brain injury in the U.S.A. alone. Primary mechanical trauma to the CNS is followed by a complex pathology, including vascular dysregulation, ischemia, edema and traumatic hemorrhage. Secondary damage is to a large extent caused by oxidative stress and inflammatory processes, resulting in necrosis and apoptosis of neural cells. If secondary tissue injury could be limited by interference with any of the pathomechanisms involved, preservation of structure and function would increase the potential for functional recovery. Experiments performed in other laboratories have shown that the polyphenolic flavonoid quercetin acts as an anti-oxidant and anti-inflammatory, reduces edema formation and apoptotic cell death. Quercetin is also an excellent iron chelator. This action profile suggested a high therapeutic potential for acute CNS trauma. Therefore, I used models of both spinal cord injury and head trauma in adult male rats to test the hypothesis that administration of quercetin is beneficial for the therapy of acute traumatic CNS injury. While the primary focus of my work was on therapy of acute traumatic spinal cord injury, quercetin was also evaluated in the settings of chronic SCI and acute head trauma. I found that, in a rat model of mid-thoracic spinal cord compression injury, 1) administration of quercetin, starting 1 hr after injury and continued every 12 hr, improved recovery of motor function in the hind limbs in more than half of the injured animals to a degree that allowed previously paraplegic animals to step or walk. The minimum quercetin dose that was efficacious was 5 µmol/kg. The minimum treatment duration for optimal outcome was determined to be 3 days. In control animals, some spontaneous recovery of motor function did occur, but never to an extent that allowed animals to step or walk. Quercetin administration was associated with more efficient iron clearance from the site of injury, decreased inflammatory response as reflected in decrease of myeloperoxidase activity and decreased apoptosis of neural cells at the site of injury. 2) Quercetin administered in the same injury model as late as 2 weeks after injury, given in a higher dose than that used for treatment in the acute phase, still resulted in significant recovery of motor function in 40% of the injured animals, although at a lower level of performance, when compared to early onset of treatment. 3) Quercetin administered after moderate fluid percussion brain injury resulted in decreased oxidative stress, as reflected in higher tissue glutathione levels at the site of injury. In animals receiving quercetin, the amplitude of compound action potentials was significantly better maintained at 24 hr and 72 hr after injury than in saline-treated control animals. My experiments have shown that the flavonoid quercetin is neuroprotective in a rat model of brain trauma and in a rat model of spinal cord injury. My data show that administration of quercetin after CNS trauma promotes iron clearance, decreases oxidative stress and inflammation. Quercetin also decreases apoptotic cell death following neurotrauma. These results suggest that quercetin may be a valuable adjunct in the therapy of acute CNS trauma. There is a possibility that administration of quercetin may be beneficial even in certain settings of chronic CNS trauma. These conclusions are based solely on the results from animal experiments. However, the fact that few adverse reactions have been noted to date in either animal experiments or human trials targeting other diseases is encouraging for the progression to human clinical trials for patients with spinal cord injury

Inflammatory Diseases

"Inflammatory Diseases - A Modern Perspective" represents an extended and thoroughly revised collection of papers on inflammation. This book explores a wide range of topics relevant to inflammation and inflammatory diseases while its main objective is to help in understanding the molecular mechanism and a concrete review of inflammation. One of the interesting things about this book is its diversity in topics which include pharmacology, medicine, rational drug design, microbiology and biochemistry. Each topic focuses on inflammation and its related disease thus giving a unique platform which integrates all the useful information regarding inflammation

Detección de nuevas mutaciones en componentes de la vía de la proteina C asociadas con un mayor riesgo trombótico.

RESUMEN La hemostasia es el conjunto de procesos que controlan la fluidez de la sangre y la integridad del sistema vascular a través de cuatro grandes mecanismos, la coagulación y anticoagulación controlan la formación del coágulo, mientras que la fibrinolisis y la antifibrinolisis controlan la eliminación del coágulo. La coagulación sanguínea requiere mecanismos de regulación que eviten su propagación y extensión de un modo incontrolado. De hecho, la trombosis es la principal causa de mortalidad y morbilidad en países desarrollados. El sistema de la proteína C constituye uno de los principales mecanismos de anticoagulación, puesto que presenta actividades anticoagulantes, antiinflamatorias, antiapoptóticas, neuroprotectoras y profibrinolíticas. Para la activación de la proteína C se requiere el preciso ensamblaje de, al menos, cuatro proteínas sobre la superficie de la célula endotelial la trombina, la trombomodulina, la proteína C y el receptor endotelial de la proteína C (EPCR). La proteína C así activada (APC) se une a su cofactor la proteína S e inactiva a los cofactores V activado y VIII activado frenando, por tanto, la cascada de la coagulación. Por ello, como hipótesis planteamos que cualquier cambio que altere la expresión o el preciso ensamblaje de las proteínas que forman el complejo de activación de la proteína C, podría dar lugar a una reducción de la generación de APC. Además, dada la función anticoagulante del sistema de la proteína C, una reducción de la generación de APC daría lugar a un incremento del riesgo trombótico. Hemos desarrollado un ensayo para la cuantificación de los niveles circulantes de APC que es rápido, fácil de llevar a cabo y con un límite de detección lo suficientemente bajo como para detectar niveles muy reducidos de APC circulante posiblemente presentes en algunas patologías trombóticas. Mediante este ensayo hemos determinado que un nivel reducido de APC es un factor de riesgo prevalente e independiente de tromboembolismo venoso, el cual parece estar genéticamente determinado. 1 La determinación de la proteína C activada nos ha permitido seleccionar pacientes con tromboembolismo venoso y con niveles de proteína C activada persistentemente bajos. En estos pacientes, hemos llevado a cabo la búsqueda de nuevas mutaciones en los genes que codifican para las proteínas del complejo de activación de la proteína C, identificando un número considerable de alteraciones en estos cuatro genes. Al secuenciar el gen de la trombomodulina identificamos el polimorfismo C1418T que produce un cambio de Alanina a Valina en el aminoácido 455. Este polimorfismo está asociado con niveles elevados de APC y con un menor riesgo trombótico. Al secuenciar el gen del EPCR identificamos diversas mutaciones. El polimorfismo A4600G que produce un cambio de Serina a Glicina en el aminoácido 219, determina los niveles plasmáticos de EPCR soluble. A su vez, identificamos el polimorfismo G4678C en la región 3´ no traducida del EPCR, el cual está asociado con niveles elevados de APC y con una disminución del riesgo trombótico, como hemos comprobado también ocurre en los individuos portadores de la mutación factor V Leiden, uno de los principales factores de riesgo trombótico. Al secuenciar el gen de la proteína C identificamos la mutación G1435A la cual produce un cambio de Glutámico a Lisina en el aminoácido 16, un residuo esencial para que la proteína C ejerza su función anticoagulante. Esta mutación es privativa de una familia en la que los individuos con historia de trombosis son portadores de la mutación. La presencia de esta mutación produce una deficiencia de proteína C tipo II. ____________________________________________________________________________________________________The protein C anticoagulant pathway plays a crucial role in the control of thrombus formation. Protein C circulates in plasma as an inactive zymogen that is converted to the active enzyme, activated protein C (APC) on the surface of endothelial cells by the thrombin-thrombomodulin complex. Another receptor, the endothelial protein C receptor (EPCR) binds protein C on the endothelial cell surface and further enhances the rate of protein C activation. APC is then released from the complex, binds protein S and inhibits thrombin formation by inactivating coagulation factors Va and VIIIa. The protein C pathway also plays a significant role in inflammatory processes, and displays anti-apoptotic and neuroprotective activities. Therefore, alterations in the expression or in the assembly of these four proteins could lead to a reduction in APC levels and to an increase in the thrombotic risk. We have developed a sensitive, rapid and reproducible assay for the determination of circulating APC levels. Using this assay, we have observed that reduced APC levels constitute an independent prevalent risk for venous thrombosis, which seems to be hereditary. We have identified several mutations in the proteins that form the protein C activation complex. The C1418T (Ala455Val) polymorphism in the thrombomodulin gene is associated with increased APC levels and decreased risk of venous thromboembolism. The A4600G (Ser219Gly) polymorphism in the EPCR gene determines the levels of soluble EPCR. And the G4678C polymorphism located in the 3´ untranslated region of EPCR is associated with increased levels of circulating APC and decreased risk of venous thrombosis, as we also observed in carriers of the factor V Leiden mutation, the most common genetic risk factor for familial venous thrombosis