Cardiovascular disease – including heart disease and stroke – continues to be the leading cause of death worldwide. The World Health Organisation (WHO) says it accounts for 23.6% of total deaths and 37.7% of deaths in Europe. The financial cost of cardiovascular disease is projected to more than double by 2030.
In most cases, acute cardiovascular events arise from the development of an unwanted thrombus formed by rupture of an atherosclerotic plaque. Thrombolytic drugs or ‘clot busters’ are biological agents used to break down unwanted clots.
The work of the fibrinolysis group includes maintaining a range of WHO International Standards in the field of thrombolytic drugs. We work with several global collaborators to study fundamental questions around the regulation of enzymes used as plasminogen activators that promote fibrinolysis.
Common thrombolytic agents we deal with include:
Tissue plasminogen activator (tPA or alteplase) is widely used as a thrombolytic drug and is currently the only licensed drug available to treat ischaemic stroke. We have published several studies exploring how tPA enzyme activity is regulated through interactions with fibrin. We use engineered variants of tPA expressed in insect cells. Recent highlights include a study of the behaviour of variants of jellyfish green fluorescent protein-labelled tPA (GFP-tPA) in clots studied by confocal microscopy in collaboration with Dr Krasimir Kolev and his team at Semmelweis University, Budapest.
We identified critical interactions between regulatory domains in tPA (the finger and kringle 2 domains) and fibrin aggregates that develop during fibrinolysis. The videos below – made using confocal microscopy with GFP-tPA – show the different ways clots break down when tPA is added to fibrin.
Video 1: In fibrin made of fine fibres, lysis is slow. tPA binds to the surface, remains in a tight lytic zone and moves through the clot. (Longstaff et al, Blood 117, 661-8, 2011)
Video 2: With thicker fibrin fibres, tPA binds to the surface but aggregates of fibrin form which strongly bind GFP-tPA. Lysis proceeds more quickly.
Video 3: A different pattern of fibrin lysis is seen when a variant of GFP-tPA is used with finger domain deleted. This demonstrates the importance of the finger domain – fibrin binding.
Extracellular DNA and histones have been the focus of intense investigation recently as factors increasing the risk of arterial and venous thrombosis. DNA and histones are released from dying cells, especially following a particular form of cell death – NETosis – associated with neutrophils in blood clots. We have investigated the effects of DNA and histones on fibrinolysis and proposed that these highly charged molecules are able to hold the fibrin network together and retard clot lysis. The time-lapse video below shows how clot lysis is slowed in the presence of DNA and histones in combination and also how fibrinolysis can then be enhanced by DNase (Longstaff et al, J Biol Chem 288 6946-56, 2013):
More recent studies have addressed the effectiveness of histones from NETs in neutralising the anticoagulant activity of heparins (Longstaff et al, Thromb Haemost, 115, 591-99, 2016).
Streptokinase is a long established thrombolytic drug derived from Streptococci sp. It remains popular in the developing world.
No longer under patent, it is increasingly produced in E. coli as a biosimilar. We have studied versions of recombinant streptokinase produced in E. coli and found they do not behave like streptokinase produced by Streptococcus equisimilis,which is the source of streptokinase used in the current WHO 3rd International Standard for Streptokinase, 00/464.
Our work shows the current International Standard for Streptokinase is not a suitable for recombinant streptokinase. (Thelwell and Longstaff, J Thromb Haemost 12, 1229-33, 2014)We have also investigated the kinetics of plasminogen activation by therapeutic streptokinase and streptokinase from a pathogenic Group A strain of S. pyogenes (Huish et al, PLOS ONE DOI:10.1371/journal.pone.0170936, 2017). This particular strain binds fibrinogen which is also found to strongly stimulate plasminogen activation by S. pyogenes streptokinase as shown in panel A of the figure below. Fibrinogen stimulates tPA activation to a lesser extent (panel B) and has virtually no effect on therapeutic streptokinase (panel C). Understanding the details of streptokinase activity regulation may help identify new routes to combatting streptococcal disease.
The fibrinolysis group is accredited to EN ISO 17025 to carry out European batch release on fibrin sealants, including thrombin and fibrinogen components.
We also perform European batch release for alpha-1-proteinase inhibitor (alpha-1-antitrypsin) and have worked with the European Pharmacopoeia to develop monographs for potency determination on a variety of biological drugs, including streptokinase, alpha-1-proteinase inhibitor and C1-esterase inhibitor.
The fibrinolysis group has experience of performing contract work to support manufacturers worldwide for a number of biologicals including streptokinase, urokinase, reteplase and desmoteplase. Our services include advice, assay development, contract assays on thrombolytic products and contract fills to make secondary standards.
Much research in the haemostasis area includes global assays that rely on detailed analysis of absorbance over time. These data are exported and analysed to produce useful parameters such as time to clotting, time to 50% lysis, etc. Apps have been developed, written in the open source language R and the Shiny Package and may be used to analyse these time courses courses (Longstaff, J Thromb Haemost 15, 1044-46, 2017, and accompanying editorial).Graphical outputs and tables of summary results are generated for further processing:
Microtitre plate of clotting and lysis curves with time to 50% lysis indicated by the dashed line
Individual curves may be analysed in detail
The range of available Shiny apps and links are provided below:
Dr Colin Longstaff - Principal ScientistDr Matthew Locke
15/106: Ancrod00/464: Streptokinase 11/184: High Molecular Weight Urokinase 98/714: Tissue plasminogen activator (tPA)94/730: tPA Antigen in plasma01/580: Thrombin97/536: Plasmin92/654: PAI-110/180: NIBSC standard Reteplase08/230: Streptodornase05/162: Alpha-1-Antitrypsin08/262: C1 esterase Inhibitor - plasma08/256: C1 esterase Concentrate 02/168: Prekallikrein activator