Publications by authors named "Craig Thelwell"

13 Publications

  • Page 1 of 1

Quantitation of thrombin-activatable fibrinolysis inhibitor in human plasma by isotope dilution mass spectrometry.

Anal Biochem 2021 Oct 10:114413. Epub 2021 Oct 10.

National Institute for Biological Standards and Control, South Mimms, Potters Bar, EN6 3QG, UK. Electronic address:

Measurement of Thrombin-activatable fibrinolysis inhibitor (TAFI) in human plasma is dependent on reproducible assays. To date, standards for measuring TAFI are frequently calibrated relative to pooled normal human plasma and arbitrarily assigned a potency of 100% TAFI, despite variation in TAFI concentrations between plasma pools. Alternatively, TAFI calibrators can be assigned a value in SI units but the approach used for value assignment is not consistent and furthermore, if purified TAFI is used to determine TAFI concentration in plasma, may be adversely affected by matrix effects. A TAFI plasma standard in mass units with traceability to the SI unit of mass is desirable. We report here the establishment of a quantitative mass spectrometry method for TAFI in plasma. Traceability is obtained by reference to calibrators that consist of blank plasma spiked with a defined amount of purified TAFI, value assigned by amino acid analysis. The calibrators are run alongside the samples, using the same preparation steps and conditions; an acetonitrile assisted tryptic digestion and multi-dimensional liquid chromatography (LC) separation followed by SRM-MS analysis. We measured the TAFI quantitatively in human plasma with reproducibility, reliability and precision, and demonstrated the applicability of this approach for value assigning a common reference standard.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ab.2021.114413DOI Listing
October 2021

Structure, Mechanical, and Lytic Stability of Fibrin and Plasma Coagulum Generated by Staphylocoagulase From .

Front Immunol 2019 20;10:2967. Epub 2019 Dec 20.

Haemostasis Section, Biotherapeutics Group, National Institute for Standards and Control, Potters Bar, United Kingdom.

causes localized infections or invasive diseases (abscesses or endocarditis). One of its virulence factors is staphylocoagulase (SCG), which binds prothrombin to form a complex with thrombin-like proteolytic activity and leads to uncontrolled fibrin generation at sites of bacterial inoculation. The aim of this study was to characterize the formation, structure, mechanical properties and lysis of SCG-generated clots. Recombinant SCG was expressed in , purified and the amidolytic activity of its complexes with human prothrombin (SCG-PT) and thrombin (SCG-T) was determined using human thrombin as a reference. Fibrin clots were prepared from purified fibrinogen and human plasma using thrombin, SCG-PT or SCG-T as a coagulase. The kinetics of clot formation and lysis by tissue-type plasminogen activator (tPA) were monitored with turbidimetric assays. Fibrin ultrastructure was examined with scanning electron microscopy and small-angle X-ray scattering (SAXS). Fibrin clot porosity was characterized with fluid permeation assays, whereas the viscoelastic properties and mechanical stability were evaluated with oscillation rheometry. Compared to thrombin, the amidolytic and clotting activity of SCG-PT was 1.6- to 2.5-fold lower on a molar basis. SCG-T had equivalent amidolytic, but reduced clotting activity both on pure fibrinogen (1.6-fold), and in plasma (1.3-fold). The SCG-PT and SCG-T generated fibrin with thicker fibers (10-60% increase in median diameter) than thrombin due to increased number of fibrin protofibrils per fiber cross-section. According to the fluid permeability of the clots SCG-PT and SCG-T promoted the formation of more porous structures. The shear stress resistance in the pure fibrin and plasma clots generated by SCG-PT was significantly lower than in the thrombin clots (243.8 ± 22.0 Pa shear stress was sufficient for disassembly of SCG-PT fibrin vs. 937.3 ± 65.6 Pa in thrombin clots). The tPA-mediated lysis of both pure fibrin and plasma clots produced by SCG-PT or SCG-T was accelerated compared to thrombin, resulting in up to a 2.1-fold increase in tPA potency. Our results indicate that SCG generates a thrombus scaffold with a structure characterized by impaired mechanical stability and increased lytic susceptibility. This proneness to clot disintegration could have implications in the septic embolism from endocardial bacterial vegetation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933771PMC
November 2020

A new WHO reference reagent for activated blood coagulation factor X (FXa), human (15/102).

J Thromb Haemost 2020 01;18(1):255-257

Biostatistics Group, National Institute for Biological Standards and Control, South Mimms, UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.14623DOI Listing
January 2020

Establishment of the WHO 2nd International Standard Factor V, plasma (16/374): communication from the SSC of the ISTH.

J Thromb Haemost 2019 Apr 28;17(4):695-697. Epub 2019 Feb 28.

National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.14403DOI Listing
April 2019

Endothelial cell functions impaired by interferon in vitro: Insights into the molecular mechanism of thrombotic microangiopathy associated with interferon therapy.

Thromb Res 2018 03 6;163:105-116. Epub 2018 Feb 6.

Section of Cytokines and Growth Factors, Division of Biotherapeutics, National Institute for Biological Standards and Control, United Kingdom.

Introduction: Interferon (IFN)-α and IFN-β approved for treatment of chronic hepatitis C viral infection and multiple sclerosis respectively have been linked to thrombotic microangiopathy (TMA) affecting renal function. Since the molecular mechanisms underlying this severe complication remain largely unclear, we aimed to investigate whether IFN affects directly in vitro endothelial cell functions associated with angiogenesis and blood haemostasis, as well as endothelial cell-derived vasodilators of nitric oxide (NO) and prostacyclin.

Methods: Proliferation and survival of human umbilical vein endothelial cells (HUVECs) were measured by BrdU incorporation and alamarBlue assays. Angiogenesis was evaluated in co-cultures of HUVECs and human dermal fibroblasts. Fibrinolysis molecules were measured with ELISA. NO and prostacyclin were measured using a fluorescent NO-specific probe and a competitive enzyme immunoassay, respectively.

Results: HUVEC proliferation was dose-dependently inhibited by IFN-β1a and IFN-β1b, but not by IFN-α2a and IFN-α2b. Consistently, IFN-β1a and IFN-β1b also reduced survival of HUVECs, but this again was not observed with IFN-α. However, both IFN subtypes inhibited VEGF-induced development of capillary-like structures, but the effect of IFN-α was less potent than IFN-β. In addition, both IFN subtypes upregulated interferon inducible protein 10 production from treated co-cultures while suppressing angiogenesis. Furthermore, intracellular NO generation was reduced by IFN-α2a and IFN-β1a, whereas prostacyclin release from HUVECs was not affected by IFN. Importantly, both IFN-β1a- and IFN-β1b-treated HUVECs showed a marked reduction in urokinase-type plasminogen activator release and a much greater secretion of plasminogen activator inhibitor-1 than tissue-type plasminogen activator compared with untreated cells, suggesting decreased fibrinolytic activity. IFN-α, however was less effective in modulating the fibrinolysis system.

Conclusions: We demonstrate the detrimental effects of IFN on endothelial cell functions mediated with angiogenesis and fibrinolysis, which could potentially cause the loss of physiological endothelium thromboresistance and facilitate the development of vascular complications in a clinical setting. Mechanistically, our findings have implications for understanding how IFN therapy can foster the development of TMA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2018.01.039DOI Listing
March 2018

Mechanism of plasmin generation by S100A10.

Thromb Haemost 2017 06 6;117(6):1058-1071. Epub 2017 Apr 6.

David M. Waisman*, Departments of Biochemistry & Molecular Biology and Pathology, Sir Charles Tupper Medical Building, 5850 College Street, room 11-N2, PO Box 15000, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada, Tel.: +1 902 494 1803, Fax: +1 902 494 1355, E-mail:

Plasminogen (Pg) is cleaved to form plasmin by the action of specific plasminogen activators such as the tissue plasminogen activator (tPA). Although the interaction of tPA and Pg with the surface of the fibrin clot has been well characterised, their interaction with cell surface Pg receptors is poorly understood. S100A10 is a cell surface Pg receptor that plays a key role in cellular plasmin generation. In the present report, we have utilised domain-switched/deleted variants of tPA, truncated plasminogen variants and S100A10 site-directed mutant proteins to define the regions responsible for S100A10-dependent plasmin generation. In contrast to the established role of the finger domain of tPA in fibrin-stimulated plasmin generation, we show that the kringle-2 domain of tPA plays a key role in S100A10-dependent plasmin generation. The kringle-1 domain of plasminogen, indispensable for fibrin-binding, is also critical for S100A10-dependent plasmin generation. S100A10 retains activity after substitution or deletion of the carboxyl-terminal lysine suggesting that internal lysine residues contribute to its plasmin generating activity. These studies define a new paradigm for plasminogen activation by the plasminogen receptor, S100A10.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1160/TH16-12-0936DOI Listing
June 2017

Activity Regulation by Fibrinogen and Fibrin of Streptokinase from Streptococcus Pyogenes.

PLoS One 2017 26;12(1):e0170936. Epub 2017 Jan 26.

Biotherapeutics Section, National Institute for Biological Standard and Control, South Mimms, Herts, United Kingdom.

Streptokinase is a virulence factor of streptococci and acts as a plasminogen activator to generate the serine protease plasmin which promotes bacterial metastasis. Streptokinase isolated from group C streptococci has been used therapeutically as a thrombolytic agent for many years and its mechanism of action has been extensively studied. However, group A streptococci are associated with invasive and potentially fatal infections, but less detail is available on the mechanism of action of streptokinase from these bacteria. We have expressed recombinant streptokinase from a group C strain to investigate the therapeutic molecule (here termed rSK-H46A) and a molecule isolated from a cluster 2a strain from group A (rSK-M1GAS) which is known to produce the fibrinogen binding, M1 protein, and is associated with life-threatening disease. Detailed enzyme kinetic models have been prepared which show how fibrinogen-streptokinase-plasminogen complexes regulate plasmin generation, and also the effect of fibrin interactions. As is the case with rSK-H46A our data with rSK-M1GAS support a "trigger and bullet" mechanism requiring the initial formation of SK•plasminogen complexes which are replaced by more active SK•plasmin as plasmin becomes available. This model includes the important fibrinogen interactions that stimulate plasmin generation. In a fibrin matrix rSK-M1GAS has a 24 fold higher specific activity than the fibrin-specific thrombolytic agent, tissue plasminogen activator, and 15 fold higher specific activity than rSK-H46A. However, in vivo fibrin specificity would be undermined by fibrinogen stimulation. Given the observed importance of M1 surface receptors or released M1 protein to virulence of cluster 2a strain streptococci, studies on streptokinase activity regulation by fibrin and fibrinogen may provide additional routes to addressing bacterial invasion and infectious diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170936PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5268773PMC
August 2017

Fractal kinetic behavior of plasmin on the surface of fibrin meshwork.

Biochemistry 2014 Oct 26;53(40):6348-56. Epub 2014 Sep 26.

Department of Medical Biochemistry, Semmelweis University , 1094 Budapest, Hungary.

Intravascular fibrin clots are resolved by plasmin acting at the interface of gel phasesubstrate and fluid-borne enzyme. The classic Michaelis.Menten kinetic scheme cannot describe satisfactorily this heterogeneous-phase proteolysis because it assumes homogeneous well-mixed conditions. A more suitable model for these spatial constraints,known as fractal kinetics, includes a time-dependence of the Michaelis coefficient Km(F) = Km0F (1+ t)h, where h is a fractal exponent of time, t. The aim of the present study was to build up and experimentally validate a mathematical model for surface-acting plasmin that can contribute to a better understanding of the factors that influence fibrinolytic rates. The kinetic model was fitted to turbidimetric data for fibrinolysis under various conditions. The model predicted Km0(F) = 1.98 μM and h = 0.25 for fibrin composed of thin fibers and Km0(F) = 5.01 μM and h = 0.16 for thick fibers in line with a slower macroscale lytic rate (due to a stronger clustering trend reflected in the h value) despite faster cleavage of individual thin fibers (seen as lower Km0(F) ). ε-Aminocaproic acid at 1 mM or 8 U/mL carboxypeptidase-B eliminated the time-dependence of Km F and increased the lysis rate suggesting a role of C-terminal lysines in the progressive clustering of plasmin. This fractal kinetic concept gained structural support from imaging techniques. Atomic force microscopy revealed significant changes in plasmin distribution on a patterned fibrinogen surface in line with the time-dependent clustering of fluorescent plasminogen in confocal laser microscopy. These data from complementary approaches support a mechanism for loss of plasmin activity resulting from C-terminal lysine-dependent redistribution of enzyme molecules on the fibrin surface.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi500661mDOI Listing
October 2014

Biological standards for potency assignment to fibrinolytic agents used in thrombolytic therapy.

Authors:
Craig Thelwell

Semin Thromb Hemost 2014 Mar 30;40(2):205-13. Epub 2014 Jan 30.

Biotherapeutics, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, United Kingdom.

Thrombolytic drugs are used for the treatment of thrombotic disorders such as acute myocardial infarction, acute ischemic stroke, and pulmonary embolism. Biological standards are used for potency assignment to the range of fibrinolytic proteins used in thrombolytic therapy. The World Health Organization (WHO) International Standards are primary reference materials, calibrated in arbitrary units (international unit), assigned by collaborative study using the range of assay methods available at the time. Provided the standard and test material are equivalent, adhering to the principle of measuring like versus like, the exact nature of the assay method is unimportant. This approach has been applied successfully for several decades since the advent of fibrinolytic treatment, ensuring consistency for potency labeling and the correct dosing of patients. The emergence of generic biosimilar products and new recombinant variants poses a challenge to this system, where functional differences impact on the relative biological activity in different assay systems. A more demanding system of standardization may therefore be required on the basis of international reference materials with associated reference methods. WHO recognizes this, and where possible and practical is seeking to incorporate concepts of traceability, uncertainty, and commutability to International Standards. However, some caution is needed because limitations on the characterization of many complex biologicals remain real, and a flexible approach is required on the basis of real-world needs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/s-0033-1364188DOI Listing
March 2014

Differential scanning fluorimetry: rapid screening of formulations that promote the stability of reference preparations.

J Pharm Biomed Anal 2013 Apr 8;77:163-6. Epub 2013 Jan 8.

Standardisation Science, National Institute for Biological Standards and Control, Health Protection Agency, South Mimms, Potters Bar Herts, UK.

When formulating a biopharmaceutical protein, its stability in the liquid state is critical. In addition, when preparing biological reference materials the stability, both when lyophilised and after reconstitution, needs to be determined. In order to optimise the stability in aqueous conditions (as indicated by Tmelt or denaturation point) the impact of different excipient choices should be evaluated. Micro differential scanning calorimetry is a well established method for these applications but can be time consuming even when an autosampler is used. Differential scanning fluorimetry (DSF) is a novel technique which measures the fluorescence of a dye when bound to the hydrophobic regions of a denatured protein. We have investigated these techniques for their suitability using alpha-1-protease inhibitor (A1PI) as a model system and found similar trends in terms of the impact of different excipients by both methods. DSF is a promising method and has advantages in terms of speed and quantities of biological material required and can be performed using a PCR instrument.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jpba.2013.01.006DOI Listing
April 2013

The interplay between tissue plasminogen activator domains and fibrin structures in the regulation of fibrinolysis: kinetic and microscopic studies.

Blood 2011 Jan 21;117(2):661-8. Epub 2010 Oct 21.

National Institute for Biological Standards and Control, South Mimms, UK.

Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domain-agglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2010-06-290338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3031486PMC
January 2011

Understanding the enzymology of fibrinolysis and improving thrombolytic therapy.

FEBS Lett 2005 Jun 7;579(15):3303-9. Epub 2005 Apr 7.

Division of Haematology, National Institute for Biological Standards and Control, South Mimms, Herts, EN6 3QG, UK.

Cardiovascular disease is responsible for 17 million deaths per year but acute myocardial infarction and stroke can be treated with thrombolytics ("clot busters"), which are plasminogen activators. However, despite many years of study and huge investment from the pharmaceutical industry, clinical trials of new drugs have often been disappointing. Part of the problem may be our incomplete understanding of the regulation of plasminogen activation in vivo. We have developed precise in vitro methods and with the application of computer simulations, we hope to improve our understanding of plasminogen activation to facilitate improvements in thrombolytic therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2005.03.058DOI Listing
June 2005

Regulatory frameworks in developing countries.

Nat Biotechnol 2005 Apr;23(4):413; author reply 413-4

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nbt0405-413aDOI Listing
April 2005
-->