PubMed Health⌕ Search

Biomedical subjects

P J Gaffney

Publications and source records attributed to P J Gaffney.

At least 19 recordsLinked to original sources
PubMed1992

Collaborative study of a proposed international standard for plasma fibrinogen measurement.

There is increased interest in the relationship between plasma fibrinogen levels and the incidence of coronary artery disease. The National Institute for Biological Standards and Control (UK) has completed a study to establish an International Standard for plasma fibrinogen. This study was conducted using a recommended assay procedure to measure the clottable material present in the proposed lyophilised Standard (coded 89/644). Twenty-two laboratories from nine countries took part in the study and analysis of the data allowed the calibration of 89/644 at 2.4 mg/ml clottable protein. Agreement with this figure was established in two laboratories using three or more different assays for plasma fibrinogen. Degradation studies of the proposed plasma fibrinogen Standard suggested that no loss of clottable protein was observed when the lyophilised material was stored at 20 degrees C for 1 year. The Fibrinogen Sub-Committee of the ISTH (Amsterdam, The Netherlands, June 1991) supported the establishment of 89/644 as an International Standard. This collaborative study will be presented to the Expert Committee on Biological Standardisation of the World Health Organisation at their 1992 session. In the meantime 89/644 will be distributed as the proposed International Standard for plasma fibrinogen measurement containing 2.4 mg/ml clottable protein.

Fibrinogen↗
PubMed1992

Screening for fibrin specific monoclonal antibodies: the development of a new procedure.

The acquisition of monoclonal antibodies specific for human fibrin has been impaired by the similarity in chemical composition between fibrinogen and fibrin and the conformational difference between immobilised and soluble fibrinogen. Five monoclonal antibodies (mabs) with a known affinity for fibrin have been subjected to screening procedures which involved the presentation of different forms of both fibrinogen and fibrin to the test mabs. It was observed by scanning electron microscopy that dried fibrin (denoted fibrin D), immobilised on the wells of PVC plates was morphologically similar to the fibrin found in human clots whereas PVC-immobilised fibrin monolayers (fibrin M) and a homogenised form of fibrin (fibrin FF) presented two very different morphological appearances. It was shown that lack of cross reactivity of a mab with an antigen (e.g. fibrinogen) was validly demonstrated only when both mab and antigen were present in the soluble state. These findings have allowed the generation of a screening procedure which involves the use of fibrin D on PVC plates in conjunction with whole human plasma incubated with the test antibody. This screening procedure has been validated using two mabs, one of which has an exclusive fibrin affinity while the other has a broad spectrum crossreactivity with both fibrinogen and fibrin. This procedure would ensure the acquisition of all the five fibrin-specific mabs used in this study while other less reliable screening procedures might not.

Antibodies, Monoclonal↗
PubMed1992

A collaborative study to establish an international standard for alpha-thrombin.

Since 1975 an International Standard for Thrombin of low purity has been used. While this standard was stable and of value for calibrating thrombins of unknown potency the need for a pure alpha-thrombin standard arose both for accurate calibration and for precise measurement of thrombin inhibitors, notably hirudin. An international collaborative study was undertaken to establish the potency and stability of an ampouled pure alpha-thrombin preparation. A potency of 97.5 international units (95% confidence limits 86.5-98.5) was established for the new alpha-thrombin standard (89/588) using a clotting-assay procedure. Stability data at various elevated temperatures indicated that the standard could be transported and stored with no significant loss of potency. Ampoules of lyophilised alpha-thrombin (coded 89/588) have been recommended as an International Standard for alpha-thrombin with an assigned potency of 100 international units per ampoule by the International Society for Thrombosis and Haemostasis (Thrombin and its Inhibitors Sub-Committee) in Barcelona, Spain in July 1990 while the Expert Committee on Biological Standardisation and Control of the World Health Organisation will consider its status at its next meeting in Geneva in 1991.

Drug Stability↗
PubMed1992

Kinetics of plasmin activation of single chain urinary-type plasminogen activator (scu-PA) and demonstration of a high affinity interaction between scu-PA and plasminogen.

The activation kinetics of single chain urinary-type plasminogen activator (scu-PA) by plasmin have been studied in detail. Nonstandard Michaelis-Menten kinetics were observed. To explain our results, we propose a model in which plasmin can exist in two conformations of lower activity (kcat/Km = 1.4 x 10(6) M-1 s-1) or higher activity (kcat/Km = 16.7 x 10(6) M-1 s-1) depending on whether a lysine binding site is occupied or free, respectively. These kinetic studies demonstrate that scu-PA interacts at this binding site (KD approximately 30 nM) and so is able to act as both a substrate and effector in this reaction. Binding was also demonstrated between scu-PA and Glu- or Lys-plasminogen at a high affinity site (KD approximately 65 nM), sensitive to the presence of lysine analogs. This suggests that scu-PA may be almost completely bound to plasminogen in plasma under normal physiological conditions and provides a possible explanation for the fibrin specificity of this activator, as discussed.

Aminocaproic Acid↗
PubMed1992

Potency assays for Anistreplase: comparison of the fibrin plate assay and a 96-well plate assay.

Several production lots of Anistreplase (Eminase) were assayed for potency by either two fibrin plate assays or a clot lysis assay performed in 96-well microtiter plates. The 96-well plate assay yielded comparable data to the fibrin plate assays and had the advantage of greater efficiency with respect to both time and reagents. As a result the newer method appears to be a suitable alternative to the fibrin plate assays for lot release of Anistreplase.

Analysis of Variance↗
PubMed1992

Studies on the mechanism of binding of serpins and serine proteases.

The serpin family of inhibitors have an important role in the control of coagulation and fibrinolysis. For a full understanding of how these pathways operate in vivo and correct measurement of enzyme and inhibitor activity, in vitro knowledge of the mechanism of action of serpins is essential. Using alpha 2-antiplasmin as a model inhibitor we find, in contrast to most previous reports, a reversible mechanism: E + I in equilibrium with EI in equilibrium with EI', where complex formation is two stepped, but both steps are reversible. Our work with plasmin in the presence of 50 mM aminohexanoic acid shows that binding of alpha 2-antiplasmin is very tight (but reversible) with an overall Ki (Ki final) = 4.0 pM. With chymotrypsin (a model serine protease) Ki final = 100 pM, so as expected binding of alpha 2-antiplasmin is weaker with chymotrypsin. However, analysis of the individual rate constants shows that the difference in strength of binding is accounted for by the dissociation rate constant for the second step (k-2) = 1.9 x 10(-6) s-1 for plasmin and 1.1 x 10(-4) s-1 for chymotrypsin. Thus k-2, the rate constant previously ignored, explains the different affinities of alpha 2-antiplasmin for these two enzymes. Furthermore, this model of two (or more) step, reversible binding is accepted for protease inhibitors of other families. With one of these, aprotinin (a Kunitz inhibitor) with plasmin we also obtain a two-stage reversible mechanism with a Ki final = 200 pM and the strength of inhibition is also largely determined by k-2 = 3.5 x 10(-5) s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Increased release of tissue plasminogen activator and its inhibitor in human parotid saliva upon stimulation.

Parotid saliva from 12 healthy volunteers was collected prior to and after 5 and 25 min of stimulation at a constant flow rate of 0.25 or 1.0 ml min-1. In the salivary samples the concentrations of tPA (tissue-type plasminogen activator), PAI-1 (plasminogen activator inhibitor type-1), albumin and total protein were determined and the activity of amylase, tPA and PAI assessed. Presence of both tPA and PAI-1 antigen was demonstrated in all samples, and in unstimulated saliva the ratio between the activator and its inhibitor was 1:7. Upon stimulation we found a significantly increased concentration of PAI-1, a less pronounced increase in tPA concentration, unchanged amylase and total protein levels and significantly decreased albumin concentration. tPA activity was significantly reduced after prolonged stimulation which had no effect on PAI activity. In stimulated saliva a significant positive correlation between concentration of tPA and PAI-1 was demonstrated. Stimulation with citric acid had no effect on output of albumin which is passively filtered from blood, whereas the increase in flow rate corresponded to the significantly increased secretion rate of total protein and amylase which is secreted by gland cells. The secretion pattern of tPA and PAI-1 differed significantly from that of albumin in showing markedly increased output rate during the stimulation period, and the relative increase in output of PAI-1 was significantly higher than that of amylase and total protein. Thus, the results from this study suggest an active release of both tPA and its main inhibitor PAI-1 into saliva.

Adult↗
PubMed1992

A comparison of the plasminogen activator activity units of urinary-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA).

The earliest tissue plasminogen activator (t-PA) preparations prepared from melanoma cells were expressed in urinary-type plasminogen activator (u-PA) units of activity using the u-PA International Standard (66/46). This report describes a comparison between u-PA and t-PA units by various types of fibrin plate procedure using both human and bovine fibrin. Within the biometric limits of this procedure it was found that the potency ratio of u-PA/t-PA was 3.5 (human fibrin), 5.29 (enriched bovine fibrin) and 4.6 (crude bovine fibrin). Specific activity figures of approximately 100,000 IU/mg for pure t-PA using the urokinase standard (66/46) would convert to approximately 350,000-530,000 IU/mg using the International Standards for t-PA (83/517 and 87/670). This latter figure is compatible with the reported specific activity for purified preparations of recombinant t-PA.

Animals↗
PubMed1991

Serpin-serine protease binding kinetics: alpha 2-antiplasmin as a model inhibitor.

We have examined in detail the kinetics of binding of the serpin alpha 2-antiplasmin to the serine proteases alpha-chymotrypsin and plasmin. These represent model systems for serpin binding. We find, in contrast to earlier published results with alpha 2-antiplasmin and plasmin, that binding is reversible, and slow binding kinetics can be observed, under appropriate conditions. Binding follows a two-step process with both enzymes, with the formation of an initial loose complex which then proceeds to a tightly bound complex. In the absence of lysine and analogues, equilibrium between alpha 2-antiplasmin and plasmin is achieved rapidly, with an overall inhibition constant (Ki') of 0.3 pM. In the presence of tranexamic acid or 6-aminohexanoic acid, lysine analogues that mimic the effects of fibrin, plasmin binding kinetics are changed such that equilibrium is reached slowly following a lag phase after mixing of enzyme and inhibitor. The Ki' is also affected, rising to 2 pM in the presence of 6-aminohexanoic acid concentrations above 15 mM. Thus extrapolation to the in vivo situation indicates that complex formation in the presence of fibrin will be delayed, allowing a burst of enzyme activity following plasmin generation, but a tight, pseudoirreversible complex will result eventually. Chymotrypsin is more weakly inhibited by alpha 2-antiplasmin, exhibiting an overall Ki' of 0.1 nM, after two-stage complex formation. The inhibition constant for the initial loose complex (Ki) is very similar for both enzymes. The difference in binding strength between the two enzymes is accounted for by the dissociation rate constant of the second step of complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminocaproic Acid↗
PubMed1990

A collaborative study to establish a standard for high molecular weight urinary-type plasminogen activator (HMW/u-PA).

An international collaborative study involving eleven laboratories located in eight countries was undertaken to establish an International Standard for high molecular weight urinary-type plasminogen activator (HMW/u-PA). The current International Reference Preparation (IRP code numbered 66/46) for urinary-type plasminogen activator (u-PA) or urokinase (see Nomenclature footnote) is a 66/34 molar ratio mixture of low molecular weight (LMW)--and high molecular weight (HMW)--u-PA's and is considered unsuitable as a standard for homogeneous preparations of HMW/u-PA. The putative standard for HMW/u-PA (code number, 87/594) was compared for potency in a clot lysis assay with the current IRP for u-PA (code number, 66/46) and a lyophilised preparation of single chain urinary-type plasminogen activator (SCuPA), the latter being used in the assay without prior activation by plasmin to its active two chain form (TCuPA). Both the proposed standard for HMW/u-PA (87/594) and the SCuPA compared in a statistically satisfactory manner in parallel line bioassays with the current IRP for u-PA (66/46), thus allowing potency estimates to be obtained for these two materials in relation to defined international units. Data from the eleven laboratories indicated that each ampoule of the proposed standard for HMW/u-PA contained 4,300 i.u. of activity and was stable for over 1 year at 4 degrees C. Most participants indicated that SCuPA expressed only a small amount of its activity without a prior activator step and this suggests that SCuPA assays need to be preceded by a plasmin activation step.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme Stability↗
PubMed1990

A collaborative study to establish the second international standard for streptokinase.

An International Standard for Streptokinase--Streptodornase (62/7) has been used to calibrate high purity clinical batches of SK since 1965. An international collaborative study, involving six laboratories, was undertaken to replace this standard with a high purity standard for SK. Two candidate preparations (88/826 and 88/824) were compared by a clot lysis assay with the current standard (62/7). Potencies of 671 i.u. and 461 i.u. were established for preparations A (88/826) and B (88/824), respectively. Either preparation appeared suitable to serve as a standard for SK. However, each ampoule of preparation A (88/826) contains a more appropriate amount of SK activity for potency testing, and is therefore preferred. Accelerated degradation tests indicate that preparation A (88/826) is very stable. The high purity streptokinase preparation, coded 88/826, has been established by the World Health Organisation as the 2nd International Standard for Streptokinase, with an assigned potency of 700 i.u. per ampoule.

Drug Stability↗
PubMed1990

The relationship of plasminogen activators and oncogenes to tumour invasion.

Accruing evidence suggests an association between increased activity of plasminogen activators and transformed cells, and urokinase activity with tumour aggressiveness. The PIP2 pathway seems to provide a link between oncogene-associated transformation, cellular proliferation, plasminogen-activator expression and tumour invasion and metastasis.

Cell Division↗
PubMed1989

Imaging of human thrombi in the rabbit jugular vein: I: Comparison of two fibrin-specific monoclonal antibodies.

The development of monoclonal antibodies with a specificity for cross-linked fibrin may have a potential role in the detection and of thrombi and thrombolytic therapy. In this study, two monoclonal antibodies with a specificity for fibrin have been examined. In vitro studies have shown NIBn 123 (which has a high affinity for X-oligomer) and DD-3B6 to bind to immobilised fibrin on PVC plates as well as plasma clots which were incubated in the presence of plasma. The Km values for NIBn 123 and DD-3B6 wre 1.0 x 10(10)/7.7 x 10(8) M and 2.6 x 10(8) M respectively. No significant binding to fibrinogen either immobilised or in solution was found. The binding of these antibodies to a human thrombus in the jugular vein of the rabbit was monitored over a 24 hour period. Preferential binding of each antibody reached a ratio of approximately 1.0 (jugular/heart) at 24 hours and an image was detected.

Animals↗
PubMed1989

Influence of various structural domains of fibrinogen and fibrin on the potentiation of plasminogen activation by recombinant tissue plasminogen activator.

Fibrinogen, fibrin, and related fragments have varying stimulatory effects on the initial rate of the activation of human plasminogen ([ Glu1]Pg) by recombinant tissue plasminogen activator (rt-PA). A detailed analysis of this enhancement was undertaken using various purified and complexed forms of the known domains of fibrin(ogen) with a view to gaining additional knowledge regarding the substructures of fibrinogen and fibrin that are important for their stimulatory capacities. Both arvin-mediated fibrin, as well as fibrinogen fragments generated as a result of its cleavage with CNBr, stimulate the activation in a biphasic manner, most likely as a result of changes in the promoter molecule accompanying the denaturation processes that are normally employed to either solubilize or generate these particular promoters. Using purified fibrinogen and fibrin fragments, it was found that fragment E, which binds to [Glu1]Pg, does not enhance the activation reaction, while fragment D1 has a potentiating effect. This suggests that the binding of [Glu1]Pg to fibrin(ogen) alone is not, in itself, sufficient for stimulation of activation to occur, but that the rt-PA-fibrin(ogen) interaction is fundamental to this same process. All purified and mixtures of fragments containing the fragment D domain (e.g., D2E, X-oligomer, fragment X) stimulate the reaction to a greater degree than fibrinogen and fragment D1. It is concluded that the fibrinogen D domain is a sine qua non for the enhancement reaction, while structures containing the E domain had a symbiotic effect on enhancement.

Binding Sites↗
PubMed1989

Sensitive and direct assays for functionally active plasminogen activators (tissue-type and urokinase-type) in plasma.

A previously described bioimmunoassay for tissue-type plasminogen activator (t-PA) has been modified in terms of antibody concentration and conditions of incubation to achieve a sensitivity range of approximately 5-500 IU/L of t-PA. A similar assay has been developed for urinary-type plasminogen activator (U-PA), also known as urokinase (UK), achieving a sensitivity range of approximately 5-500 X 10(-1) IU/L. The direct sensitive t-PA assay has been demonstrated to be a quantitative alternative to the traditional semiquantitative euglobulin clot lysis time (ECLT) assay, with correlation coefficients of 0.967 and 0.914, depending on the laboratory where the ECLT was carried out. This assay has the added advantages of avoiding loss of t-PA or UK during the formation of the euglobulin fraction and of calibration in terms of International Standards for both t-PA and UK. With both these assays, it has been demonstrated that the enhanced fibrinolytic activity observed after exercise results from increased levels of t-PA while the level of UK in plasma remains unaltered. Free t-PA (10-60 IU/L) has been demonstrated in resting plasma, whereas the level of free UK activity was 50-100 IU/L. Venous occlusion of matched groups of subjects with and without deep venous thrombosis (DVT) showed a wide variation of response in t-PA levels in each group and no change in UK levels. There was no difference between the DVT and non-DVT groups, and this suggests that the notion of the "nonresponder" being more prone to thrombosis needs to be reexamined.

Adult↗