Thrombin | EPFL Graph Search

Thrombin (, fibrinogenase, thrombase, thrombofort, topical, thrombin-C, tropostasin, activated blood-coagulation factor II, blood-coagulation factor IIa, factor IIa, E thrombin, beta-thrombin, gamma-thrombin) is a serine protease, an enzyme that, in humans, is encoded by the F2 gene. Prothrombin (coagulation factor II) is proteolytically cleaved to form thrombin in the clotting process. Thrombin in turn acts as a serine protease that converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions. History After the description of fibrinogen and fibrin, Alexander Schmidt hypothesised the existence of an enzyme that converts fibrinogen into fibrin in 1872. Prothrombin was discovered by Pekelharing in 1894. Physiology Synthesis Thrombin is produced by the enzymatic cleavage of two sites on prothrombin by activated Factor X (Xa). The activity of factor Xa is greatly enhanced by binding to activate

Tissue Factor-Independent Coagulation Correlates with Clinical Phenotype in Factor XI Deficiency and Replacement Therapy

Alessandro Aliotta, Vanessa Carle, Christian Heinis

Background In factor XI (FXI) deficiency, bleeding cannot be predicted by routine analyses. Since FXI is involved in tissue factor (TF)-independent propagation loop of coagulation, we hypothesized that investigating the spatiotemporal separated phases of coagulation (TF-dependent and -independent) could improve diagnostics. Objectives This article investigates the correlation of parameters describing TF-dependent and -independent coagulation with the clinical phenotype of FXI deficiency and their ability to assess hemostasis after FXI replacement. Methods We analyzed: (1) plasma from healthy controls ( n =53); (2) normal plasma ( n =4) spiked with increasing concentrations of a specific FXI inhibitor (C7P); (3) plasma from FXI-deficient patients ( n =24) with different clinical phenotypes (13 bleeders, 8 non-bleeders, 3 prothrombotics); (4) FXI-deficient plasma spiked with FXI concentrate ( n =6); and (5) plasma from FXI-deficient patients after FXI replacement ( n =7). Thrombin generation was measured with the reference method calibrated automated thrombogram and with Thrombodynamics (TD), a novel global assay differentiating TF-dependent and -independent coagulation. Results C7P dose-dependently decreased FXI activity, prolonged activated partial thromboplastin time, and hampered TF-independent coagulation. In FXI-deficient bleeders, TD parameters describing TF-independent propagation of coagulation and fibrin clot formation were reduced compared with controls and FXI-deficient nonbleeders and increased in FXI-deficient patients with prothrombotic phenotype. Receiver operating characteristic analysis indicated that TF-independent parameters were useful for discriminating FXI-deficient bleeders from non-bleeders. In FXI-deficient plasma spiked with FXI concentrate and in patients receiving FXI replacement, TD parameters were shifted toward hypercoagulation already at plasma FXI levels around 20%. Conclusion TF-independent coagulation parameters assessed by TD have the potential to identify the clinical phenotype in FXI-deficient patients and to monitor FXI replacement therapy.

GEORG THIEME VERLAG KG2020

Thrombin-induced ischemic tolerance is prevented by inhibiting c-jun N-terminal kinase

Cristina Granziera, Pierre Magistretti, Jonathan Thevenet

We have studied ischemic tolerance induced by the serine protease thrombin in two different models of experimental ischemia. In organotypic hippocampal slice cultures, we demonstrate that incubation with low doses of thrombin protects neurons against a subsequent severe oxygen and glucose deprivation. L-JNKI1, a highly specific c-jun N-terminal kinase (JNK) inhibitor, and a second specific JNK inhibitor, SP600125, prevented thrombin preconditioning (TPC). We also show that the exposure to thrombin increases the level of phosphorylated c-jun, the major substrate of JNK. TPC, in vivo, leads to significantly smaller lesion sizes after a 30-min middle cerebral artery occlusion (MCAo), and the preconditioned mice were better off in the three tests used to evaluate functional recovery. In accordance with in vitro results, TPC in vivo was prevented by administration of L-JNKI1, supporting a role for JNK in TPC. These results, from two different TPC models and with two distinct JNK inhibitors, show that JNK is likely to be involved in TPC.

2007

The Split Luciferin Reaction

Aurélien Godinat

Studying biological processes on the level of live cells with the help of biocompatible reac-tions has tremendously advanced our understanding of basic biology. However, the great complexity of many human pathologies such as cancer, diabetes and neurodegenerative diseases requires new tools that would allow investigation of biological processes throughout the organism. The 2-cyanobenzothiazole (CBT)-based ligation reaction has received a recent interest in the chemical biology community. It has been reported in the literature for various applications, ranging from fluorescent labelling of proteins to nanostructures formation, and, most importantly, the reaction was shown to proceed in cells. This selective reaction between D-cysteine and hydroxy-CBT (HO-CBT) or amino-CBT (H2N-CBT), also named as split luciferin reaction, generates as product a D-luciferin analogue, one of the most commonly used substrates for bioluminescence imaging (BLI). Therefore, the split luciferin reaction has high potential for BLI applications. In this work, we have shown that production of a luciferin substrate via the split luciferin reaction can be visualized in live mice using BLI. Furthermore, the split luciferin approach allows interrogation of target tissues using a masking approach, where D-luciferin is formed only under certain conditions. This reaction was successfully applied to real-time non-invasive imaging of apoptosis, associated with caspase 3/7 activity. Caspase-dependent release of free D-cysteine from a caspase 3/7 specific peptide substrate allowed selective reaction with H2N-CBT in vivo to form 6-amino-D-luciferin with subsequent light emission in the presence of the firefly luciferase enzyme. Importantly, this strategy was found to be superior to the use of the commercially available DEVD-aminoluciferin substrate for imaging caspase 3/7 activity. The same methodology was extended to imaging activity of other caspases as well as thrombin enzyme in an in vitro set-up. Furthermore, the split luciferin approach enables dual imaging, where each reaction partner would be individually caged to report on separate biological events. This approach was used for simultaneous imaging of caspase 3 and Î²-galactosidase in vitro, validating the use of the split luciferin reaction for imaging multiple processes. Moreover, the split luciferin reaction was also successfully applied to both quantification of Neutrophil Elastase activity in vitro and real-time non-invasive imaging of Neutrophil Elastase in an in vivo inflammation model. Altogether, the present study suggests that the split luciferin approach is an efficient and versatile tool for in vivo applications.