C. Thelwell

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

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.

The regulation by fibrinogen and fibrin of tissue plasminogen activator kinetics and inhibition by plasminogen activator inhibitor 1.

Summary.   Background:  Tissue plasminogen activator (tPA) is unusual in the coagulation and fibrinolysis cascades in that it is produced as an active single‐chain enzyme (sctPA) rather than a zymogen. Two chain tPA (tctPA) is produced by plasmin but there are conflicting reports in the literature on the behaviour of sc‐ and tctPA and little work on inhibition by the specific inhibitor plasminogen activator inhibitor‐1 (PAI‐1) under physiological conditions.

Regulation of fibrinolysis by C-terminal lysines operates through plasminogen and plasmin but not tissue-type plasminogen activator.

Summary.  Background:  Binding of tissue‐type plasminogen (Pgn) activator (t‐PA) and Pgn to fibrin regulates plasmin generation, but there is no consistent, quantitative understanding of the individual contribution of t‐PA finger and kringle 2 domains to the regulation of fibrinolysis. Kringle domains bind to lysines in fibrin, and this interaction can be studied by competition with lysine analogs and removal of C‐terminal lysines by carboxypeptidase B (CPB).

Understanding the enzymology of fibrinolysis and improving thrombolytic therapy

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.

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

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.

The poor quality of streptokinase products in use in developing countries

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

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.

Biosimilars: the process is the product. The example of recombinant streptokinase

Worldwide, streptokinase remains the most used thrombolytic agent for the treatment of myocardial infarction. Recombinant streptokinase, from E. coli, is increasingly used in developing countries as a biosimilar of native streptokinase; however, potency assignments relative to the WHO International Standard (IS) are highly variable with potentially dangerous consequences. A proportion of recombinant streptokinase appears to be incompletely processed, retaining the amino‐terminal methionine engineered for intracellular expression.

An international collaborative study to establish the WHO 1st international standards for C1-inhibitor, plasma and concentrate.

*Biotherapeutics Group, Haemostasis Section; and Biostatistics Group, National Institute for Biological Standards and Control, South Mimms,UKTo cite this article: Thelwell C, Rigsby P, Longstaff C, on behalf of the ISTH-SSC Subcommittee on Factor XI and the Contact System. J ThrombHaemost 2011; 9: 2097–9.

An international collaborative study to investigate a proposed reference method for the determination of potency measurements of fibrinolytics in absolute units.

Traditionally, WHO International Standards (IS) have been calibrated in International Units (IU) by consensus following an international collaborative study. In the area of coagulation and fibrinolysis standards it is also common for laboratories involved in such studies to perform their own in-house methods, although guidelines may be defined to include recommendations of replication and randomization of sample testing to improve the robustness of the study. The historic basis of this approach has been to develop a common reference standard to facilitate comparisons of results for the relative potency of standard and test preparations in laboratories using different methods [1]. However, this approach has been criticized and suggestions for improvements have been made which would bring the standardization of biologicals more in line with other calibrators used in medicinal chemistry. Guidelines for the introduction of a metrologically sound approach to standardization have been detailed elsewhere [2]. General goals include standardization of methods and the introduction of a hierarchy of reference materials and procedures, each with an assigned uncertainty, to provide a system of metrological traceability where testing of routine samples can ultimately be traced back to a primary calibrator and primary reference method that are defined in SI units [3]. We have previously published a proposal for a reference method developed to measure the potency of thrombolytic products (plasminogen activators) [4] that did allow the calculation of activity in enzyme units (moles of product per second in the defined method), which could theoretically be converted into katals. The katal (mole per second) is the coherent derived SI unit of measurement for enzyme activity and is at the top of the hierarchy for the catalytic concentration of an enzyme [5]. Thus it would be possible theoretically to define the concentration of an IS not only in IU but also by absolute SI units. With the encouragement of the Fibrinolysis SSC, an international collaborative study was organized in which laboratories expert in fibrinolysis methods were recruited to perform the defined method [4] using the current IS for urokinase (uPA, 87/594), tissue plasminogen activator, (tPA, 98/714) and streptokinase (SK, 00/464). The study was planned as far as possible to remove possible sources of variation. All necessary reagents were provided, including IS, plasminogen substrate (NIBSC reagent 97/534), and chromogenic substrate for plasmin CS-41(03) (Hyphen BioMed, France). In addition, thrombin (NIBSC reagent 01/578) and fibrinogen concentrate (1st IS 98/614) were provided in order to make clots as consistently as possible in all laboratories. Plasmin (3rd IS, 97/ 536) was also provided to all laboratories to perform a series of assays in which a range of concentrations of chromogenic substrate was hydrolysed completely in order to calculate an extinction value for p-nitroaniline for each laboratory that was specific to the equipment they used. This value was critical to calculate the molar concentration of p-nitroaniline released during the plasminogen activation reaction, which in turn allows the molar concentration and rate of plasmin generation to be calculated and thus express the activity of the plasminogen activator in SI units. The only materials provided by the laboratories in the study were Tris buffer and microtitre plates. A detailed collaborative study protocol was agreed in conjunction with participating laboratories and other outside interested parties over a series of months ahead of the practical phase of the study. Twelve participants contributed a total of 36 assays, each of which included the three activators, uPA, tPA and SK at four doses, in quadruplicate for each point. Raw data of absorbance vs. time were returned to NIBSC for analysis, where rates of plasminogen Correspondence: C. Longstaff, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, Herts, EN6 3QG, UK. Tel.: +44 1707 641253; fax: +44 1707 641050; e-mail: clongstaff@ nibsc.ac.uk