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Biomedical subjects

Colin Longstaff

Publications and source records attributed to Colin Longstaff.

9 recordsLinked to original sources
PubMed2005

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.

Animals↗
PubMed2005

A reunification of the US ("NIH") and International Unit into a single standard for Thrombin.

The existence of two different units for Thrombin in widespread international use has caused confusion for many years. The holders of the WHO International Standard (IS) for Alpha Thrombin and the US Standard (also known as the "NIH Standard") now report on a collaboration to reunite the International Unit (IU) and the US unit ("NIH unit"). A study was organised involving 25 laboratories in 15 countries to investigate the possibility of preparing a common Standard with a common unit and to investigate aspects of methodology that cause divergence of results using the IS and US Standard. Laboratories were asked to measure the potency of two candidate replacement standards (C, 01/578 and D, 01/580), and potencies were calculated relative to both the existing US Standard (lot J) and the IS (89/588). Data analysis of a total of 128 assays indicated that sample D would make an ideal replacement joint Standard with a potency of 110 IU/ampoule (equivalent to 110 US units per ampoule) based on data from clotting assays. No significant differences in results were observed using fibrinogen of human or bovine origin, or using human plasma. Comparisons of chromogenic and clotting assays indicated that sample D had a similar high proportion of alpha thrombin to the current IS for Alpha Thrombin (89/588). Sample D was adopted as the IS for Thrombin (01/580) and the US Standard (lot K) with a potency of 110 IU/ampoule.

Animals↗
PubMed2004

Phospholipid barrier to fibrinolysis: role for the anionic polar head charge and the gel phase crystalline structure.

The massive presence of phospholipids is demonstrated in frozen sections of human arterial thrombi. Purified platelet phospholipids and synthetic phospholipids retard in vitro tissue-type plasminogen activator (tPA)-induced fibrinolysis through effects on plasminogen activation and plasmin function. The inhibition of plasminogen activation on the surface of fibrin correlates with the fraction of anionic phospholipid. The phospholipids decrease the amount of tPA penetrating into the clot by 75% and the depth of the reactive surface layer occupied by the activator by up to 30%, whereas for plasmin both of these parameters decrease by approximately 50%. The phospholipids are not only a diffusion barrier, they also bind the components of the fibrinolytic system. Isothermal titration calorimetry shows binding characterized with dissociation constants in the range 0.35-7.64 microm for plasmin and tPA (lower values with more negative phospholipids). The interactions are endothermic and thermodynamically driven by an increase in entropy, probably caused by the rearrangements in the ordered gel structure of the phospholipids (in line with the stronger inhibition at gel phase temperatures compared with liquid crystalline phase temperatures). These findings show a phospholipid barrier, which should be overcome during lysis of arterial thrombi.

Anions↗
PubMed2003

Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells.

Adenocarcinoma↗
PubMed2002

Interactions of the type III secretion pathway proteins LcrV and LcrG from Yersinia pestis are mediated by coiled-coil domains.

The type III secretion system is used by pathogenic Yersinia to translocate virulence factors into the host cell. A key component is the multifunctional LcrV protein, which is present on the bacterial surface prior to host cell contact and up-regulates translocation by blocking the repressive action of the LcrG protein on the cytosolic side of the secretion apparatus. The functions of LcrV are proposed to involve self-interactions (multimerization) and interactions with other proteins including LcrG. Coiled-coil motifs predicted to be present are thought to play a role in mediating these protein-protein interactions. We have purified recombinant LcrV, LcrG, and site-directed mutants of LcrV and demonstrated the structural integrity of these proteins using circular dichroism spectroscopy. We show that LcrV interacts both with itself and with LcrG and have obtained micromolar and nanomolar affinities for these interactions, respectively. The effects of LcrV mutations upon LcrG binding suggest that coiled-coil interactions indeed play a significant role in complex formation. In addition, comparisons of secretion patterns of effector proteins in Yersinia, arising from wild type and mutants of LcrV, support the proposed role of LcrG in titration of LcrV in vivo but also suggest that other factors may be involved.

Amino Acid Sequence↗
PubMed2002

Differences between neonates and adults in carbohydrate sequences and reaction kinetics of plasmin and alpha(2)-antiplasmin.

This study investigates reaction kinetics by slow-binding kinetics methods of both adult and fetal plasmin (Types 1 and 2) with adult and fetal alpha(2)-antiplasmin. In addition, carbohydrate sequences of Fetal and Adult Plasminogen Types 1 and 2, as well as fetal and adult alpha(2)-antiplasmin, were determined by mass spectrometric analysis. All curves of plasmin-alpha(2)-antiplasmin interaction followed the same pattern, indicating reversible slow-binding inhibition with an initial loose complex and a following tight complex. Differences between fetal and adult plasmin reactions with alpha(2)-antiplasmin were predominantly due to the initial loose complex. Values for K(i initial) in the reaction with adult alpha(2)-antiplasmin were 1.5 and 1.6 nM for Fetal Plasmin Types 1 and 2, respectively; compared to 0.3 and 0.7 nM for the corresponding adult types. Increasing concentrations of tranexamic acid resulted in a continuous increase of K(i initial) until a plateau was reached which was similar for all plasmin types. Almost identical values could be obtained when fetal alpha(2)-antiplasmin was used instead of adult alpha(2)-antiplasmin. Mass spectrometric analyses of the glycans present on plasminogen revealed a higher level of truncated N-glycans on the fetal material compared to the adult. The O-glycans of fetal and adult plasminogen were closely similar and only minor differences were observed between N-glycans of fetal and adult alpha(2)-antiplasmin. In conclusion, both fetal plasmin isoforms are less inhibited by alpha(2)-antiplasmin compared to the adult plasmin variants. These findings are important for the understanding of the physiology of the fibrinolytic system in neonates and provide further evidence that differences in glycosylation could be associated with marked effects on protein function.

Adult↗
PubMed2002

Plasminogen activation on the cell surface.

The plasminogen activation system appears to be widely involved in many biological processes in health and disease, but the regulation of plasmin generation or the mechanisms of stimulation by cell surface receptors are not well understood. Cell surface plasminogen activation requires binding sites for plasminogen substrate and activator enzyme before enhancement of plasmin generation rate is observed. The cell surface moieties involved in binding these reactants appear to be a mixed group of proteins and other molecules, many of which have been extensively investigated. The binding of plasminogen in particular is characterized by heterogeneous receptor molecules, present in high number but generally with low affinity for plasminogen. The low affinity of the interaction, with Kd values around 10(-6) M, presents considerable technical difficulties when studying and quantitating plasminogen binding to cells or isolated receptors. Studying plasminogen activation kinetics in the presence of cells also presents technical difficulties and raises difficult questions on interpretation of results. However, approaches developed to study enzyme activation systems in other areas of hemostasis may also be applied to the problems associated with pericellular proteolysis. Models should be developed that match In vitro experimental data and help us understand the meaning of kinetic constants derived from these systems. In this way it should be possible to better understand the regulation of plasminogen activation around the cell under normal conditions and in a variety of disease states where cell-associated plasminogen activation is believed to be up-regulated. Ultimately, a sound understanding of theses regulatory mechanisms will enable us to devise strategies for modulating proteolytic activity, test these approaches in well designed In vitro systems and relate these results to the in vivo situation.

Animals↗
PubMed2002

A collaborative study to establish the 3rd International Standard for tissue plasminogen activator.

An international collaborative study was organised to replace the 2nd International Standard (IS) for tissue plasminogen activator (tPA). The 2nd IS for tPA (86/670) was used to calibrate the replacement Standard, which was selected from two candidate materials included in the collaborative study. Participants were provided with five sets of four samples (A, B, C, D) and asked to use sample A (2nd IS, 86/670, 850 IU/ml) to determine the activity of B (86/624, approximately 850 IU/ml), C and D (coded duplicates of the same material, 98/714 approximately 11,000 IU/ml). A total of 14 laboratories returned results from Europe, USA, Japan and Australia, providing data from 60 independent assays. Four laboratories used a reference method based on a published monograph from the European Pharmacopoeia for Alteplase for Injection, 1998, and the remaining 10 used their own method. Fibrin was used as promoter of tPA activity by 12 out of the 14 laboratories, the remaining two used kits where fibrinogen fragments were the promoter. Data from this collaborative study and the previous study to establish the 2nd IS for tPA show that tPA from melanoma cells and recombinant tPA from CHO cells are both suitable materials as International Standards. It was agreed that sample C, D, recombinant tPA, 98/714, be established as the 3rd International Standard for tPA with a potency of 10,000 IU per ampoule, calculated as the mean value from laboratories using fibrin as a promoter of tPA activity. The standard was established by WHO in November 2000.

Animals↗