DOCSLIB.ORG

Activities of Thrombin (Allostery/Fibrinogen/Protein C/Thrombomodulin) QUOC D

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Activities of Thrombin (Allostery/Fibrinogen/Protein C/Thrombomodulin) QUOC D Proc. Natl. Acad. Sci. USA Vol. 92, pp. 5977-5981, June 1995 Biophysics An allosteric switch controls the procoagulant and anticoagulant activities of thrombin (allostery/fibrinogen/protein C/thrombomodulin) QUOC D. DANG, ALESSANDRO VINDIGNI, AND ENRICO DI CERA* Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, MO 63110 Communicated by Carl Frieden, Washington University School ofMedicine, St. Louis, MO, March Z 1995 (received for review December 6, 1994) ABSTRACT Thrombin is an allosteric enzyme existing in We recently demonstrated that thrombin is an allosteric two forms, slow and fast, that differwidely in their specificities enzyme, present in two distinct forms in equilibrium: the slow toward synthetic and natural amide substrates. The two forms and fast forms (9, 10). These forms were so defined originally are significantly populated in vivo, and the allosteric equilib- from their ability to interact with and cleave small chromo- rium can be affected by the binding of effectors and natural genic amide substrates; the slow form shows a significantly substrates. The fast form is procoagulant because it cleaves reduced catalytic efficiency. Because the two forms are sig- fibrinogen with higher specificity; the slow form is anticoag- nificantly populated under physiological conditions (9, 11), ulant because it cleaves protein C with higher specificity. assessing their role in the procoagulant and anticoagulant Binding ofthrombomodulin inhibits cleavage offibrinogen by activities of the enzyme is important. the fast form and promotes cleavage of protein C by the slow form. The allosteric properties of thrombin, which has tar- MATERIALS AND METHODS geted two distinct conformational states toward its two fun- damental and competing roles in hemostasis, are paradig- Human a-thrombin was purified and tested for activity as matic of a molecular strategy that is likely to be exploited by described (9,12). Human fibrinogen, PC, APC, and rabbit lung other proteases in the blood coagulation cascade. TM of the highest purity and activity were from Hematologic Technologies (Essex Junction, VT). These reagents yielded The physiologic response to a vascular lesion entails a number results consistent with published data (see Results). The chro- of sequential enzymatic steps catalyzed by distinct serine mogenic substrate S2266 (H-D-Val-Leu-Arg-p-nitroanilide), proteases (1, 2). The cascade of events culminates in the specific for APC, was from Chromogenix (Molndal, Sweden). generation of thrombin, which then catalyzes the conversion of The slow and fast forms were studied under experimental fibrinogen into fibrin monomers, leading to clot formation. conditions of 5 mM Tris/0.1% PEG (pH 8.0) at 25°C. Ionic Efficiency of the coagulation system requires that the response strength was kept constant at 200 mM with NaCl, when be rapid, localized at the injury site, and then readily termi- studying the fast form, or with choline (Ch) chloride, when nated. This result is accomplished through a complex network studying the slow form (9, 10). In studies involving PC, the slow of regulatory interactions. The components of this network form of thrombin was studied under conditions of 195 mM share a surprisingly high degree of structural homology (3). ChCl/5 mM NaCl (see below). Hence, a crucial question arises as to the molecular origin of Release of fibrinopeptides A and B (FpA and FpB) by the specificity in the cascade. Is there an underlying molecular slow and fast forms was quantified by reverse-phase HPLC, strategy shared by different members of the cascade or is using a Waters 717plus Autosampler for 1-ml injections into specificity accomplished differently by each protease? Under- the HPLC system equipped with a Waters C18 column (4.6 x standing the physicochemical and structural basis of protease 250 mm). Elution was done at a flow rate of 1 ml/min, with a specificity in the coagulation cascade would provide new tools gradient containing 80 mM sodium phosphate buffer, pH 3.1 for engineering proteases of desired specificity and eventually (solvent A) and acetonitrile (solvent B). Optimal separation lead to more efficient pharmacological strategies for control- was obtained by using 17% of solvent B from 0 to 15 min, ling hemostasis. followed by a 10-min linear gradient to 40% of solvent B. The Thrombin function conveys most of the complexity of the effluent was monitored at 205 nm. Extinction coefficients for entire coagulation cascade. This enzyme accomplishes two FpA and FpB were derived by calibration and quantitative opposite roles in hemostasis. The procoagulant role entails amino acid analysis of highly pure standards. The sequential conversion of fibrinogen into the fibrin clot and enhancement release of FpA and FpB was analyzed according to the of its own conversion from prothrombin through feed-back mechanism of Shafer and coworkers (13) with the expressions activation of proteases upstream in the cascade. The antico- agulant role encompasses activation of protein C (PC) with [FpA] = [FpA].(1 - e-at) [1] participation of thrombomodulin (TM), a cofactor on the and membrane of endothelial cells (4-6). Activated protein C (APC) rapidly inactivates factors Va and VIIIa, both of which are involved in thrombin generation. Thrombin, therefore, [FpB] = [FpB](1 - ae-b-b [2] finely tunes its own production in the blood through the balance between positive and negative feed-back loops, and where [FpA]o. and [FpB]. are the asymptotic values of the much interest now exists in understanding the molecular concentrations of FpA and FpB, while a and b are equal to the mechanism that allows this enzyme to accomplish its dual role kcat/Km ratio for FpA and FpB, respectively, times the enzyme in hemostasis (7, 8). concentration. The publication costs of this article were defrayed in part by page charge Abbreviations: Ch, choline; FpA, fibrinopeptide A; FpB, fibrinopeptide payment. This article must therefore be hereby marked "advertisement" in B; PC, protein C; APC, activated protein C; TM, thrombomodulin. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 5977 Downloaded by guest on September 27, 2021 5978 Biophysics: Dang et al Proc. Natl. Acad. Sci. USA 92 (1995) Binding of TM to thrombin was quantified from the inhibition enhancement of the hydrolysis rate. For this reason, hydrolysis of hirudin binding. The equilibrium dissociation constant for of PC by the slow form of thrombin was studied under hirudin binding to thrombin, Ki, was measured as described (9) as conditions of 195 mM ChCl and 5 mM NaCl. At this Na+ a function of TM concentration. The value of Ki was found to concentration thrombin is =90% in the slow form, which increase linearly with the TM concentration, as expected for mimics the conditions of0.2 M NaCl, where thrombin is =90% competitive inhibition. The equilibrium dissociation constant for in the fast form (9). TM, Kd, was derived from the equation The Michaelis-Menten parameters in Eqs. 4-6 were deter- mined by analysis of progress curves of substrate hydrolysis K= OK(1 + [TM]) [3] using KINSIM and FITSIM. The values of TKm and Tkcat (see Eq. 6) for the hydrolysis of S2266 by thrombin in the various solution conditions were as follows: 210 ± 10 ,uM and 26 ± 1 where 0Ki is the value of Ki in the absence of TM. s-1 (0.2 M NaCl), 490 ± 20 AM and 29 ± 1 s-1 (195 mM ChCl, was The interaction of thrombin with PC studied by using 5 mM NaCl), 270 ± 20 AM and 29 ± 1 s-1 (195 mM ChCl/5 progress curves of substrate hydrolysis. This strategy is simple, mM NaCl/10 nM TM). No effect of Ca2+ at 5 mM was found accurate, and reliable. It has the advantage of monitoring PC under all conditions examined, and likewise 10 nM TM had no activation continuously and greatly minimizes the amounts of effect on the hydrolysis of S2266 by the fast form. The values expensive reagents needed for determination of the kinetic of CKm and ckcat (see Eq. 5) for the hydrolysis of S2266 by APC parameters. The general reaction scheme for catalytic conver- in the various solution conditions were as follows: 300 ± 10 ,uM sion of PC is and 11.3 ± 0.2 s-1 (0.2 M NaCl), 1.0 ± 0.1 mM and 2.0 ± 0.1 Km kat s-1 (195 mM ChCl/5 mM NaCl), 1.0 ± 0.1 mM and 6.0 ± 0.3 T+PC TPC - > T + APC, [4] s-1 (195 mM ChCl/5 mM NaCl/5 mM CaCl2). Thrombo- modulin at 10 nM had no effect on activity of APC, regardless CKm Ckcat APC+ S APCS APC+P, [5] of solution conditions, and 5 mM Ca2' had no effect under all conditions studied in 0.2 M NaCl. Progress curves of S2266 and hydrolysis were measured to derive the specificity (kcat/Km ratio for Eq. 4) of the slow and fast forms toward TKm Tkt PC. The T + S <'TS T + P. [6] S2266 concentration was 50 ,uM in all cases, and PC was used at 300 nM, well below estimated Km values (19). The thrombin The product P is the p-nitroaniline released from a chromo- concentration was 2 nM. Under these conditions release of genic substrate (S2266) specific for APC and provides the p-nitroaniline due to cleavage by thrombin and APC was signal to be followed at 405 nm as a function of time. The extremely sensitive to the kcat/Km ratio for PC activation, release of P depends on three reactions: cleavage of S2266 by thereby allowing accurate measurements of this parameter; APC (Eq. 5), activation of PC by thrombin (Eq. 4), and this was due to the constraints imposed by the parallel Eqs.
Load more

Recommended publications
  • MONONINE (“Difficulty ® Monoclonal Antibody Purified in Concentrating”; Subject Recovered)
    CSL Behring IU/kg (n=38), 0.98 ± 0.45 K at doses >95-115 IU/kg (n=21), 0.70 ± 0.38 K at doses >115-135 IU/kg (n=2), 0.67 K at doses >135-155 IU/kg (n=1), and 0.73 ± 0.34 K at doses >155 IU/kg (n=5). Among the 36 subjects who received these high doses, only one (2.8%) Coagulation Factor IX (Human) reported an adverse experience with a possible relationship to MONONINE (“difficulty ® Monoclonal Antibody Purified in concentrating”; subject recovered). In no subjects were thrombo genic complications MONONINE observed or reported.4 only The manufacturing procedure for MONONINE includes multiple processing steps that DESCRIPTION have been designed to reduce the risk of virus transmission. Validation studies of the Coagulation Factor IX (Human), MONONINE® is a sterile, stable, lyophilized concentrate monoclonal antibody (MAb) immunoaffinity chromatography/chemical treatment step and of Factor IX prepared from pooled human plasma and is intended for use in therapy nanofiltration step used in the production of MONONINE doc ument the virus reduction of Factor IX deficiency, known as Hemophilia B or Christmas disease. MONONINE is capacity of the processes employed. These studies were conducted using the rel evant purified of extraneous plasma-derived proteins, including Factors II, VII and X, by use of enveloped and non-enveloped viruses. The results of these virus validation studies utilizing immunoaffinity chromatography. A murine monoclonal antibody to Factor IX is used as an a wide range of viruses with different physicochemical properties are summarized in Table affinity ligand to isolate Factor IX from the source material.
    [Show full text]
  • Coagulation Factors Directly Cleave SARS-Cov-2 Spike and Enhance Viral Entry
    bioRxiv preprint doi: https://doi.org/10.1101/2021.03.31.437960; this version posted April 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry. Edward R. Kastenhuber1, Javier A. Jaimes2, Jared L. Johnson1, Marisa Mercadante1, Frauke Muecksch3, Yiska Weisblum3, Yaron Bram4, Robert E. Schwartz4,5, Gary R. Whittaker2 and Lewis C. Cantley1,* Affiliations 1. Meyer Cancer Center, Department of Medicine, Weill Cornell Medical College, New York, NY, USA. 2. Department of Microbiology and Immunology, Cornell University, Ithaca, New York, USA. 3. Laboratory of Retrovirology, The Rockefeller University, New York, NY, USA. 4. Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA. 5. Department of Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, New York, NY, USA. *Correspondence: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2021.03.31.437960; this version posted April 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Summary Coagulopathy is recognized as a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. Other host proteases, including TMPRSS2, are recognized to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing viral entry.
    [Show full text]
  • Thrombin-Jmi
    THROMBIN-JMI - thrombin, topical (bovine) THROMBIN-JMI; THROMBIN-JMI PUMP SPRAY KIT; THROMBIN-JMI SYRINGE SPRAY KIT - thrombin, topical (bovine) THROMBIN-JMI SYRINGE SPRAY KIT - thrombin, topical (bovine) THROMBIN-JMI EPISTAXIS KIT - thrombin, topical (bovine) King Pharmaceuticals, Inc. ---------- THROMBIN, TOPICAL U.S.P. (BOVINE ORIGIN) THROMBIN-JMI® Thrombin, Topical (Bovine) must not be injected! Apply on the surface of bleeding tissue. DESCRIPTION The thrombin in Thrombin, Topical (Bovine Origin) THROMBIN-JMI® is a protein substance produced through a conversion reaction in which prothrombin of bovine origin is activated by tissue thromboplastin of bovine origin in the presence of calcium chloride. It is supplied as a sterile powder that has been freeze-dried in the final container. Also contained in the preparation are mannitol and sodium chloride. Mannitol is included to make the dried product friable and more readily soluble. The material contains no preservative. THROMBIN-JMI® has been chromatographically purified and further processed by ultrafiltration. Analytical studies demonstrate the current manufacturing process’ capability to remove significant amounts of extraneous proteins, and result in a reduction of Factor Va light chain content to levels below the limit of detection of semi-quantitative Western Blot assay (<92 ng/mL, when reconstituted as directed). The clinical significance of these findings is unknown. CLINICAL PHARMACOLOGY THROMBIN-JMI® requires no intermediate physiological agent for its action. It clots the fibrinogen of the blood directly. Failure to clot blood occurs in the rare case where the primary clotting defect is the absence of fibrinogen itself. The speed with which thrombin clots blood is dependent upon the concentration of both thrombin and fibrinogen.
    [Show full text]
  • Protein C Product Monograph 1995 COAMATIC® Protein C Protein C
    Protein C Product Monograph 1995 COAMATIC® Protein C Protein C Protein C, Product Monograph 1995 Frank Axelsson, Product Information Manager Copyright © 1995 Chromogenix AB. Version 1.1 Taljegårdsgatan 3, S-431 53 Mölndal, Sweden. Tel: +46 31 706 20 00, Fax: +46 31 86 46 26, E-mail: [email protected], Internet: www.chromogenix.se COAMATIC® Protein C Protein C Contents Page Preface 2 Introduction 4 Determination of protein C activity with 4 snake venom and S-2366 Biochemistry 6 Protein C biochemistry 6 Clinical Aspects 10 Protein C deficiency 10 Assay Methods 13 Protein C assays 13 Laboratory aspects 16 Products 17 Diagnostic kits from Chromogenix 17 General assay procedure 18 COAMATIC® Protein C 19 References 20 Glossary 23 3 Protein C, version 1.1 Preface The blood coagulation system is carefully controlled in vivo by several anticoagulant mechanisms, which ensure that clot propagation does not lead to occlusion of the vasculature. The protein C pathway is one of these anticoagulant systems. During the last few years it has been found that inherited defects of the protein C system are underlying risk factors in a majority of cases with familial thrombophilia. The factor V gene mutation recently identified in conjunction with APC resistance is such a defect which, in combination with protein C deficiency, increases the thrombosis risk considerably. The Chromogenix Monographs [Protein C and APC-resistance] give a didactic and illustrated picture of the protein C environment by presenting a general view of medical as well as technical matters. They serve as an excellent introduction and survey to everyone who wishes to learn quickly about this field of medicine.
    [Show full text]
  • EXPERT COMMITTEE on BIOLOGICAL STANDARDIZATION Geneva, 17 to 21 October 2016
    WHO/BS/2016.2282 ENGLISH ONLY EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 17 to 21 October 2016 An international collaborative study to calibrate the WHO 2nd International Standard for Ancrod (15/106) and the WHO Reference Reagent for Batroxobin (15/140) Craig Thelwell1ᶲ, Colin Longstaff1ᶿ, Peter Rigsby2, Matthew Locke1 and Sally Bevan1 1Biotherapeutics Group, Haemostasis Section and 2Biostatistics Section, National Institute for Biological Standards and Control, South Mimms, Herts EN6 3QG, UK ᶲProject leader for Ancrod; ᶿProject leader for Batroxobin NOTE: This document has been prepared for the purpose of inviting comments and suggestions on the proposals contained therein, which will then be considered by the Expert Committee on Biological Standardization (ECBS). Comments MUST be received by 16 September 2016 and should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to the Responsible Officer: Dr C M Nübling at email: [email protected] © World Health Organization 2016 All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press through the WHO web site: (http://www.who.int/about/licensing/copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.
    [Show full text]
  • Functions of the Complement Components C3 and C5 During Sepsis
    The FASEB Journal • Research Communication Functions of the complement components C3 and C5 during sepsis Michael A. Flierl,*,1 Daniel Rittirsch,*,1 Brian A. Nadeau,* Danielle E. Day,* Firas S. Zetoune,* J. Vidya Sarma,* Markus S. Huber-Lang,† and Peter A. Ward*,2 *Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA; and †Department of Trauma-, Hand- and Reconstructive Surgery, University of Ulm Medical School, Ulm, Germany ABSTRACT Activation of the complement system is a mia (2, 3). Thus, some clinicians preferably refer to this key event in the pathogenesis of sepsis. Nevertheless, complex of symptoms as “sepsis syndrome.” It is of the exact mechanisms remain inadequately understood. concern that doctors have seen a rapid increase in In the current study, we examined the role of comple- hospitalization and mortality rates for severe sepsis in ment C3 and C5 in sepsis in wild-type and C3- or the United States between 1993 and 2003 while mortal- C5-deficient mice induced by cecal ligation and punc- ity rates only slightly decreased (4). During this 11-year ؊/؊ ture. When compared to wild-type mice, C5 showed period, the hospitalization rate has almost doubled and ؊/؊ identical survival, and C3 presented significantly is considerably higher than it has been previously reduced survival. Interestingly, this was associated with predicted, making septicemia now the 10th leading significant decreases in plasma levels of proinflamma- .(؊/؊ cause of death in the United States. (5 tory mediators. Moreover, although septic C3 ani- Encroachment of pathogens prompts the comple- mals displayed a 10-fold increase of blood-borne bac- ؊/؊ ment cascade, which plays a decisive role in the host’s teria, C5 animals exhibited a 400-fold increase in immune response (1, 6).
    [Show full text]
  • Replacing the First Epidermal Growth Factor-Like Domain of Factor IX with That of Factor VII Enhances Activity in Vitro and in Canine Hemophilia B
    Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B. J Y Chang, … , K M Brinkhous, H R Roberts J Clin Invest. 1997;100(4):886-892. https://doi.org/10.1172/JCI119604. Research Article Using the techniques of molecular biology, we made a chimeric Factor IX by replacing the first epidermal growth factor- like domain with that of Factor VII. The resulting recombinant chimeric molecule, Factor IXVIIEGF1, had at least a twofold increase in functional activity in the one-stage clotting assay when compared to recombinant wild-type Factor IX. The increased activity was not due to contamination with activated Factor IX, nor was it due to an increased rate of activation by Factor VIIa-tissue factor or by Factor XIa. Rather, the increased activity was due to a higher affinity of Factor IXVIIEGF1 for Factor VIIIa with a Kd for Factor VIIIa about one order of magnitude lower than that of recombinant wild- type Factor IXa. In addition, results from animal studies show that this chimeric Factor IX, when infused into a dog with hemophilia B, exhibits a greater than threefold increase in clotting activity, and has a biological half-life equivalent to recombinant wild-type Factor IX. Find the latest version: https://jci.me/119604/pdf Replacing the First Epidermal Growth Factor-like Domain of Factor IX with That of Factor VII Enhances Activity In Vitro and in Canine Hemophilia B Jen-Yea Chang,* Dougald M. Monroe,* Darrel W. Stafford,*‡ Kenneth M.
    [Show full text]
  • Assessing Plasmin Generation in Health and Disease
    International Journal of Molecular Sciences Review Assessing Plasmin Generation in Health and Disease Adam Miszta 1,* , Dana Huskens 1, Demy Donkervoort 1, Molly J. M. Roberts 1, Alisa S. Wolberg 2 and Bas de Laat 1 1 Synapse Research Institute, 6217 KD Maastricht, The Netherlands; [email protected] (D.H.); [email protected] (D.D.); [email protected] (M.J.M.R.); [email protected] (B.d.L.) 2 Department of Pathology and Laboratory Medicine and UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; [email protected] * Correspondence: [email protected]; Tel.: +31-(0)-433030693 Abstract: Fibrinolysis is an important process in hemostasis responsible for dissolving the clot during wound healing. Plasmin is a central enzyme in this process via its capacity to cleave fibrin. The ki- netics of plasmin generation (PG) and inhibition during fibrinolysis have been poorly understood until the recent development of assays to quantify these metrics. The assessment of plasmin kinetics allows for the identification of fibrinolytic dysfunction and better understanding of the relationships between abnormal fibrin dissolution and disease pathogenesis. Additionally, direct measurement of the inhibition of PG by antifibrinolytic medications, such as tranexamic acid, can be a useful tool to assess the risks and effectiveness of antifibrinolytic therapy in hemorrhagic diseases. This review provides an overview of available PG assays to directly measure the kinetics of plasmin formation and inhibition in human and mouse plasmas and focuses on their applications in defining the role of plasmin in diseases, including angioedema, hemophilia, rare bleeding disorders, COVID- 19, or diet-induced obesity.
    [Show full text]
  • Thrombin, Topical (Human) • Potential Risk of Thrombosis If Absorbed Systemically (5.2, 11)
    HIGHLIGHTS OF PRESCRIBING INFORMATION WARNINGS AND PRECAUTIONS • May carry a risk of transmitting infectious agents such as viruses and These highlights do not include all the information needed to use theoretically, the Creutzfeldt-Jakob disease (CJD) agent, despite EVITHROM safely and effectively. See full prescribing information. manufacturing steps designed to reduce the risk of viral transmission (11). EVITHROM* Thrombin, Topical (Human) • Potential risk of thrombosis if absorbed systemically (5.2, 11). For Topical Use Only ADVERSE REACTIONS Initial U.S approval: 2007 • Anaphylactic reactions may occur (6). INDICATIONS AND USAGE • Adverse events were reported in the clinical trial with similar frequency • As an aid to hemostasis whenever oozing blood and minor bleeding in the two study groups (EVITHROM or bovine thrombin group). The from capillaries and small venules is accessible and control of bleeding most common adverse event reported was procedural complications by standard surgical techniques is ineffective or impractical (1). and pruritus (6). None of the adverse events reported was considered • May be used in conjunction with an Absorbable Gelatin Sponge, USP (1). causally related to EVITHROM administration. • Immunogenicity was evaluated by testing for the development of DOSAGE AND ADMINISTRATION antibodies to highly purified antigens: human thrombin, human Factor • For topical use only. DO NOT INJECT (2.2). V/Va, bovine thrombin and bovine Factor V/Va. None of the patients • The amount required depends upon the area of tissue to be treated treated with EVITHROM developed antibodies to human thrombin or and the method of application. In clinical studies, volumes up to 10 ml to human Factor V/Va.
    [Show full text]
  • Reelin Signals Through Apolipoprotein E Receptor 2 and Cdc42 to Increase Growth Cone Motility and Filopodia Formation
    The Journal of Neuroscience, November 3, 2010 • 30(44):14759–14772 • 14759 Cellular/Molecular Reelin Signals through Apolipoprotein E Receptor 2 and Cdc42 to Increase Growth Cone Motility and Filopodia Formation Jost Leemhuis,1,2 Elisabeth Bouche´,1,3 Michael Frotscher,1,4 Frank Henle,1,2 Lutz Hein,2 Joachim Herz,1,6 Dieter K. Meyer,1,2 Marina Pichler,1,2 Gu¨nter Roth,5 Carsten Schwan,1,2 and Hans H. Bock1,3 1Center for Neuroscience, 2Institute of Experimental and Clinical Pharmacology and Toxicology, 3Department of Medicine II, 4Institute of Anatomy and Cell Biology, and 5Department of Microsystems Engineering, Albert-Ludwigs-University, 79104 Freiburg, Germany, and 6Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046 Lipoprotein receptor signaling regulates the positioning and differentiation of postmitotic neurons during development and modulates neuronal plasticity in the mature brain. Depending on the contextual situation, the lipoprotein receptor ligand Reelin can have opposing effects on cortical neurons. We show that Reelin increases growth cone motility and filopodia formation, and identify the underlying signaling cascade. Reelin activates the Rho GTPase Cdc42, known for its role in neuronal morphogenesis and directed migration, in an apolipoprotein E receptor 2-, Disabled-1-, and phosphatidylinositol 3-kinase-dependent manner. We demonstrate that neuronal vesicle trafficking, a Cdc42-controlled process, is increased after Reelin treatment and further provide evidence that the peptidergic VIP/ PACAP38 system and Reelin can functionally interact to promote axonal branching. In conclusion, Reelin-induced activation of Cdc42 contributes to the regulation of the cytoskeleton of individual responsive neurons and converges with other signaling cascades to orchestrate Rho GTPase activity and promote neuronal development.
    [Show full text]
  • Activation by the Thrombin-Thrombomodulin Complex DEBORAH J
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10212-10216, September 1996 Cell Biology The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex DEBORAH J. STEARNS-KUROSAWA*, SHINICHIRO KUROSAWA*, JEFFERY S. MOLLICA*, GARY L. FERRELLt, AND CHARLES T. ESMON*tt§ *Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, *Departments of Pathology and Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, and tHoward Hughes Medical Institute, Oklahoma City, OK 73104 Communicated by Kenneth M. Brinkhous, University of North Carolina, Chapel Hill, NC, July 8, 1996 (received for review May 28, 1996) ABSTRACT Protein C activation on the surface of the protein C and the derivative devoid of the protein C Gla endothelium is critical to the negative regulation of blood domain (10, 11). Taken together, these data supported the coagulation. We now demonstrate that monoclonal antibodies possibility that an additional endothelial cell protein might that block protein C binding to the endothelial cell protein C facilitate protein C activation. receptor (EPCR) reduce protein C activation rates by the In this study, we describe monoclonal antibodies (mAbs) thrombin-thrombomodulin complex on endothelium, but that that bind to EPCR and block both protein C and APC binding antibodies that bind to EPCR without blocking protein C to cells stably transfected with EPCR (E7 cells), to human binding have no effect. The kinetic result of blocking the umbilical endothelial cells (HUVECs), and to EA.hy926 cells EPCR-protein C interaction is an increased apparent Km for (a transformed endothelial cell line). On those cells that the activation without altering the affinity of thrombin for express both TM and EPCR, these anti-EPCR antibodies thrombomodulin.
    [Show full text]
  • Update on the 8Th Congress of the Asian - Pacific Society of Thrombosis and Hemostasis
    www.apsth.org January 2014 Volume 4 No. 1 Message from the Chairman Inside Dear Members of APSTH, Message from the Chairman 1 It is my privilege to address you in this newsletter From the Editor 2 once again. Presentations from the Symposium 3 on Thrombosis and Haemostasis in The council met in Amsterdam on 1st July 2013 the Asian-Pacific during the XXIV Congress of the ISTH in Amsterdam when we welcomed several new members to the council, namely Dr. Raymond Wong from Hong Research News 6 Kong, Dr. Soo-Mee Bang from Korea, Dr. Ming Lai Heng Lee ISTH Sponsored Education Programs Hou from China, Dr. Lugyanti Sukrisman from In- in Asia 9 donesia, Dr. Claire McLintock from New Zealand, Dr. Ponlapat Rojnuckarin, and Update on the 8th APSTH Congress Dr. Yingyong Chinthammitr from Thailand. We also welcomed Dr. Satoshi Fujii as in Vietnam 12 the new Secretary General of APSTH as Dr. Yukio Ozaki relinquished this posi- tion to assume the new position as Treasurer of APSTH. Notably, the Executive Upcoming Meetings 13 Committee for the council, comprising of Dr. Lee Lai Heng, Dr. Yukio Ozaki, Dr. Pantep Angchaisuksiri, Dr. Doyuen Oh, Dr. Christopher Ward, Dr. Ming Hou, and APSTH Officers Dr. Satoshi Fujii, was formed with unanimous support from the council. Honorary Chairmen The executive committee very quickly took on their first task as they work with Akikazu Takada Prof. Tri and his team on the scientific programme of the next APCTH to be held Yasuo Ikeda in Hanoi from 10th to 11th October 2014. We look forward to an informative and Hatem Salem interactive programme with ample opportunities for networking and future col- Chairman laborations within the Asia Pacific region.
    [Show full text]