bluuebunny/crossref_metadata_2025_split huggingface dataset:
IMPROVEMENT OF T-PA PROPERTIES BY MEANS OF SITE DIRECTED MUTAGENESIS
Authors: Haigwood N , Pâques E-P , Mullenbach G , Moore G , DesJardin L , Tabrizi A 
Published: August 2018

The clinical relevance of tissue-plasminogen-activator (t-PA) as a potent thrombolytic agent has recently been established. It has however been recognized that t-PA does not fulfill all conditions required for an ideal thrombolytic pharmaceutical agent; for example, its physiological stability and its short half life in vivo necessitate the use of very large clinical doses. We have therefore attempted to develop novel mutant t-PA proteins with improved properties by creating mutants by site-directed mutagenesis in M13 bacteriophage. Seventeen mutants were designed, cloned, and expressed in CHO cells. Modifications were of three types: alterations to glycosylation sites, truncations of the N- or C-termini, and amino acids changes at the cleavage site utilized to generate the two chain form of t-PA. The mutant proteins were analyzed in vitro for specific activity, fibrin dependence of the plasminogen activation, fibrin affinity, and susceptibility to inhibition by PAI.In brief, the results are: 1) some unglycosylated and partially glycosylated molecules obtained by mutagenesis are characterized by several-fold higher specific activity than wild type t-PA; 2) truncation at the C-terminus by three amino acids yields a molecule with increased fibrin specificity; 3) mutations at the cleavage site lead zo a decreased inhibition by PAI; and 4) recombinants of these genes have been constructed and the proteins were shown to possess multiple improved properties. The use of site directed mutagenesis has proved to be a powerful instrument to modulate the biological properties of t-PA.
CATALYTIC ACTIVITIES OF ONE-CHAIN tPA AS REVEALED BY AN ANALOGUE RESISTANT TO PLASMIN CLEAVAGE
Authors: Petersen L C , Johannessen M , Foster D , Kumar A , Mulvihill E 
Published: August 2018

Substitution of Arg275 with Gly in the activation site of tPA provides a one-chain recombinant analogue, tPA-Gly275, which is very resistant to cleavage by plasmin. The amidolytic activity of tPA-Gly275 with simple synthetic substrates was investigated and compared to the kinetics obtained with authentic one- and two-chain tPA. Both one-chain (zymogen) forms possess enzymatic activity, however, in the absence of fibrin it is much lower than that of two-chain tPA. Fibrin enhances the activity of the one-chain tPA forms, but not of two-chain tPA.A chromogenic assay was developed for measurement of plasminogen activation. Due to the presence of a high affinity plasmin substrate (D-Val-Phe-Lys-pNA) and aprotinin in the reaction mixture, the assay ensures a low steady-state concentration of free plasmin during the measurement. With this assay and with tPA-Gly275 it is possible to measure plasminogen activation kinetics of one-chain tPA without any significant two-chain tPA formation even in the presence of fibrin. The intactness of tPA-Gly275 was confirmed by direct measurement of the one-/two-chain tPA content by means of reduced SDS-PAGE combined with Western blotting after exposure to plasmin digestion in the presence and absence of fibrin. The results suggest that one-chain tPA possesses enzymatic activity also with plasminogen as the substrate, however, the activity is much lower than that of two-chain tPA. Addition of fibrin profoundly enhances the plasminogen activation rate of both tPA-Gly275, one-chain, and two-chain authentic tPA to approx. the same maximal level. Taken together these observations indicate that fibrin binding can induce an activated state of the intact tPA ‘zymogen’
BOW CAN WE INHIBIT 5HT-INDUCED PLATELET AGGREGATION AND WHY SHOULD WE BOTHER?
Authors: Bevan Jane , Heptinstall S 
Published: August 2018

Platelets are induced to aggregate when 5-hydraxytryptamine (5HT) is added to citrated whole blood and the extent of aggregation can be measured using a Whole Blood Platelet Counter. We have used this method to study a) 5HT-induced platelet aggregation in normal human blood and the effects of 14 5HT receptor antagonists and 2 Ca++-channel blockers, and b) aggregation in blood from patients with peripheral vascular disease (PVD). Previous studies of platelet aggregation in platelet-rich plasma have indicated an increased platelet sensitivity to 5HT in PVD, and a multicentre study of ketanserin (a S2 antagonist) is in progress.5HT induces a transient reversible aggregation in human whole blood which can be prevented by 5HT receptor antagonists. The inhibitory effects of 7 relatively potent antagonists (IC50 7 -41nM, e.g. ketanserin and pizotifen) could not be surmounted by increasing the concentration of 5HT, but the inhibitory effects of 7 less potent antagonists (IC50 0.28 - 53uM, e.g. mepyramine and amitriptyline) could be surmounted by 5-HT. One Ca++-channel blocker (verapamil) inhibited platelet aggregation but another (amlodipine) had very little effect. Verapamil inhibited 5HT-induced aggregation at much lower concentrations (IC50 1.6μM) than those required for aggregation induced by PAF, adrenaline or ADP (IC50 values 32, 33 and >100uM respectively) and the inhibition was insurmountable.5HT-induced platelet aggregation in blood from patients with PVD does not differ qualitatively or quantitatively from aggregation in blood from healthy, age- and sex-matched controls: patients (n = 13), aggregation 30 seconds after adding 5uM 5HT = 55.1% ± 13.3(s.d.); controls (n = 13) aggregation = 48.3% ± 18.9(s.d.). Neither was the platelet aggregation induced by ADP, U46619 or PAF different in patients and controls.We conclude that different 5HT receptor antagonists inhibit 5HT-induced platelet aggregation with different potencies and, apparantly, different mechanisms of action, and that verapamil has a selective effect on 5HT-induced aggregation at relatively low concentrations. Results obtained in PVD do not encourage the use of a 5HT antagonist in this condition.
EFFECTS OF SULFINPYRAZONE AND ITS METABOLITE G25671 ON PLATELET ACTIVATION AND DESENSITIZATION AND ON BRONCHOCONSTRICTION INDUCED BY THE PROSTAGLANDIN ENDOPEROXIDE ANALOGUE U46619
Authors: Hatmi M , Del Maschio A , Lefort J , De Gaetano G , Varqaftiq B B , Cerletti C 
Published: August 2018

In previous studies we have found (Br. 3. Pharmac. 85, 849, 1985) that a) human platelets pre-exposed to arachidonic acid or to the endoperoxide analogue, U46619 and then washed and resuspended, fail to respond to a second challenge by both arachidonic acid and U46619; b) desensitization by arachidonic acid and U46619 occurs at a site sensitive to endoperoxides / thromboxane (Tx) receptor antagonists; c) the desensitizing effects of U46619 are direct, whereas those of arachidonic acid are mediated by a cyclooxygenase-dependent metabolite. Sulfinpyrazone (100 μM) and its thioether metabolite G25671 (50 μM) are known to suppress arachidonic acid-induced platelet aggregation and TxB2 formation (Eur. 3. Pharmac, 101, 209, 1984). We now demonstrate that the presence of sulfinpyrazone or G25671 during platelet exposure to arachidonic acid or U46619 prevents desensitization. Platelet activation by the endoperoxide analogue U46619 is also prevented by sulfinpyrazone or G25671 (0.3-1 mM). The threshold aggregating concentrations of arachidonic acid and U46619 in healthy subjects before and after oral treatment with sulfinpyrazone were elevated by 2-3 fold and a good correlation between ex vivo and in vitro findings was established. We finally examined the actions of sulfinpyrazone and G25671 on the bronchoconstriction in vivo and parenchymal lung strip contraction in vitro induced by U46619. Neither drug had any preventive effect.Our results demonstrate that sulfinpyrazone and its metabolite G25671 are not only cyclooxygenase inhibitors but can also act as endoperoxide/Tx antagonists and indicate clearly that antagonism of U46619 by both drugs is selective for platelets.
PRESYSTEMIC DEACETYLATION OF LOW DOSES OF ENTERIC COATED ASPIRIN IN A PIG MODEL
Authors: Lloyd J V , Rodgers S E , Siebert D M , Bochner F , McIntosh G H , James M 
Published: August 2018

The antithrombotic effect of aspirin might be enhanced if platelet cyclooxygenase could be inhibited in the portal ciculation while sparing cyclooxygenase in the systemic vascular endothelium. This might be achieved by modifying the dose and formulation to maximise presystemic aspirin clearance by the liver. To test this hypothesis low dose enteric coated aspirin (Astrix, 50mg single dose, lOOmg single dose and lOOmg daily for 1 week) was orally administered to pigs with permanent indwelling arterial and portal vein catheters. Plasma aspirin concentrations were measured by high performance liquid chromatography in blood obtained simultaneously from the artery and portal vein for 6 hours after dosage. Platelet aggregation and thromboxane generation were measured in 4 pigs before and after the lOOmg chronic dosage regimen. Aortic prostacyclin production was measured in aspirin treated (lOOmg daily for 1 week) and untreated pigs after sacrifice. After the 50mg single dose the arterial:portal areas under the plasma concentration versus time curve (AUC) ratio was 0.63±0.09 (n=6). In 3 pigs which received all 3 dosage regimens the arterial:portal AUC ratios were 0.48±0.05 after 50mg single dose, 0.52±0.02 after lOOmg single dose and 0.47+0.03 after lOOmg daily for 1 week. Platelet aggregation in response to sodium arachidonate (1.65mM) was completely abolished after chronic aspirin administration. Thromboxane production (pg/106 platelets) by this stimulus decreased from 536±117 before aspirin to 57±14 after aspirin (n=4; p=0.017). Aortic prostacyclin synthesis (ng/disc after 10 min incubation) was 1.66±0.28 (n=4) in untreated pigs and 0.97±0.25 (n=4) in treated pigs (p=0.06).With this slow release aspirin formulation there was substantial but incomplete clearance of aspirin by the liver. This may not be sufficient to spare cyclooxygenase in the systemic vessels from the effect of aspirin.
THE MOLECULAR ORGANIZATION OF HUMAN ALPHA 2-MACROGLOBULIN. AN IMMUNO ELECTRON MICROSCOPIC STUDY WITH MONOCLONAL ANTIBODIES
Authors: Delain E , Barrav M , Tapon-Bretaudière J , Pochon F , Van Leuven F 
Published: August 2018

Electron microscopy is a very convenient method to localize the epitopes of monoclonal antibodies (mAbs) at the surface of macromolecules for studying their tree-dimensional organization.We applied this immuno-electron microscopic method to human ct2-macroglobulin (ct2M). 29 anti-α2M mAbs have been tested with the four different forms of a2M : native and chymotrypsin-transformed tetramers, and the corresponding dimers, obtained by dissociation with divalent cations. These mAbs can be classified in three types : those which are specific for 1) the H-like transformed molecules, 2) the native molecules, and 3) those which can react with both forms of α2M.1) Among the H-like α2M specific mAbs, several react with the 20 kD-domain which is recognized by the cellular receptor of transformed a2M. This domain is located at the carboxyterminal end of each monomer. One IgG binds to the end of two adjacent tips of the H-like form.The other mAbs of this type bind to the α2M tips at non-terminal positions. Intermolecular connections built polymers of alternating α2M and IgG molecules.2) Among the native a2M-specific mAbs some are able to inhibit the protease-induced transformation of the native α2M. The binding sites of these mAbs are demonstrated on the native half-molecules. One of these mAbs was also able to react with transformed dimers, in a region corresponding very likely to an inaccessible epitope in the tetrameric transformed α2M molecule.3) Among the mAbs of this type, only two were able to inhibit the protease-induced transformation of α2M. Obviously, their epitopes should be close to the bait region of α2M. The other mAbs reacting with both α2M forms did not inhibit the α2M transformation.All these mAbs can be distinguished by the structure of the immune complexes formed with all forms of α2M. The epitopes are more easily located on the dimers and on the H-like transformed α2M than on the native molecules.From these observations, we propose a new model of the tree-dimensional organization of the human α2M in its native and transformed configurations, and of its protease-induced transformation.
GENE DELETIONS IN THE FACTOR IX LOCUS
Authors: Matthews R J , Anson D S , Peake I R , Bloom A L 
Published: August 2018

Previous studies have indicated that the majority of haemophilia B patients who produce anti-factor IX inhibitors (antibodies) have some kind of deletion of the factor IX gene. We have analysed DNA from nine haemophilia B inhibitor patients using the Southern blotting method and hybridisation with (i) factor IX cDNA and intragenomic probes (ii) probes originating from flanking sequences up to 60kb 5' and 170kb 3' to the factor IX gene that have been isolated by gene walking experiments (D.S.Anson and G.G.Brownlee, unpublished observations).Two patients who are brothers (haemophilia B (Chicago I)) were shown to have a presumably identical complex rearrangement of the factor IX gene involving two separate deletions. The first deletion is approx. 5.0kb and removes exon e. The second deletion is between 9 and 29kb and removes exons g and h but leaves exon f intact. An abnormal TaqI fragment at one end of the deletions junctions acted as a marker for the inheritance of haemophilia B in the patients' family. Furthermore, an abnormal llkb Bglll fragment (detected with an intragenomic probe containing exon f) in DNA from both patients and their mother acted as a marker for the presence of both deletions. Since the patients' grandmother only showed the normal 12kb Bgl II fragment then both deletions appear to have arisen at the same time. We believe that haemophilia B (Chicago 1) is the first observation of a natural gene rearrangement involving two separate deletions within the same gene.Patient haemophilia B (Jersey 1) was revealed to have a deletion of at least 170kb including the entire factor IX gene and > 60kb of 5' flanking sequence. The 3' breakpoint of this deletion was mapped to between 80 and 140kb 3' to the factor IX gene. One further patient, haemophilia B (Boston I) was shown to have a deletion of > 230kb including the factor IX gene, > 60kb of 5' flanking sequence and >140kb of 3' flanking sequence. Five other inhibitor patients had a structurally intact gene as detected by this method.Although all nine haemophilia B inhibitor patients studied did not have a detectable plasma factor IX only in four of them is this absence due to a large deletion of the factor IX gene.
MOLECULAR BASIS OF HEMOPHILIA B: IDENTIFICATION OF THE DEFECT IN FACTOR IX VANCOUVER
Authors: Geddes V A , Louie G V , Brayer G D , MacGillivray R T A 
Published: August 2018

Factor IX Vancouver (fIX-V) is the cause of a moderate form of hemophilia B. An individual presenting with this disorder had 2.6% of normal procoagulant activity in his plasma but had 62% of the normal factor IX antigen level. Specific antibodies showed that fIX-V contains epitopes for both the heavy and light chains of factor IXa. To identify the defect involved, DNA was isolated from the lymphocytes of the male hemophiliac. Southern blot analysis using a full-length factor IX cDNA as a hybridization probe showed no gross differences between the fIX-V gene and the normal factor IX gene. The DNA from the hemophiliac was then partially digested with Sau3A and the resulting fragments (10-20kbp in size) were ligated into the BamHI site of λEMBL3. The DNA was then packaged into phage particles in vitro, and the recombinant phage were screened with the factor IX cDNA as a probe. Eight phage were isolated that contained overlapping DNA covering the complete gene for fIX-V. DNA sequence analysis of the protein-encoding regions, the intron/exon junctions and 5'-and 3'-flanking sequences revealed a single nucleotide change from the normal factor IX gene. The codon for amino acid 397 was changed from ATA (lie) to ACA (Thr). This mutation is in the catalytic domain of factor IXa and is novel amongst those hemophilia B mutations reported to date. Based on the known three dimensional structures of the pancreatic serine proteases, trypsin, elastase and chymotrypsin, models have been constructed for the structures of the catalytic domains of both the normal and Thr-397 mutant of factor IXa. These results suggest that the Thr-397 mutation may alter the conformation of the substrate binding region in the active site of factor IXa Vancouver through the formation of a hydrogen bond between the hydroxyl group of the Thr-397 side chain and the main chain carbonyl group of Trp-385. The postulated conformational change would lead to reduced binding affinity for the factor IXa substrate resulting in a reduction in the catalytic activity of fIXa-Vancouver.Supported in part by grants from the Medical Research Council of Canada (to GDB and RTAM).
A PROSPECTIVE RANDOMISED CONTROLLED STUDY OF MINI-DOSE WARFARIN PROPHYLAXIS OF DEEP VEIN THROMBOSIS IN MAJOR SURGERY
Authors: McKernan A , Thomson J M , Poller L 
Published: August 2018

A prospective randomised study has been undertaken to assess the clinical effectiveness of oral anticoagulation using minidose warfarin (1 mg daily for 2-4 weeks before major gynaecological surgery) compared with conventional oral anticoagulant prophylaxis and an untreated randomised control group. The conventional oral anticoagulant prophylaxis was based on a therapeutic range of 1.5 - 2.0 INR at the time of operation and 2.0 - 3.0 INR post-operatively. Overall the mini-dose warfarin group showed no pre-operative prolongation of the prothrombin time with the Manchester Reagent although a minority of patients showed a 1-2 second prolongation of the prothrombin time before operation. Post operatively the mini-dose warfarin group showed an exaggerated prolongation of the prothrombin time which normally occurs after operation and was observed in the untreated controls. Factor VII assays paralleled these findings. Minidose warfarin, while not prolonging the prothrombin time before operation, resulted in delayed platelet aggregation with the Chandler's tube technique in almost all patients.The incidence of deep vein thrombosis has been reduced in both mini-dose and conventional dose oral anticoagulant series compared with the untreated- group. It appears that the minimal changes in the prothrombin time, factor VII and platelet aggregation tests, observed in the mini-dose warfarin group, may offer sufficient protection against post-operative thrombosis in a moderate risk group undergoing abdominal or pelvic surgery.
USE OF INR FOR THE EXPRESSION OF PT IN PATIENTS UNDER ORAL ANTICOAGULANTS : COMPARISON OF RESULTS OBTAINED WITH FIVE THROMBOPLASTINS AND THREE DEVICES
Authors: Roussi J H , Francois D , Delemme J , Goguel A F 
Published: August 2018

The aim of our study is to know if INR (International Normalized Ratio) is really the best way to express PT (Prothrombin Time) in patients under oral anticoagulants (AVK). In this way, PT of 85 patients treated by AVK and 30 normal controls were determined with 5 different reagents and three devices : manual technic, KC10 (AHS Dade), Coag A Pet 200 (Gal Diagnostics, Organon). The reagents were : Thromborel S (Behring) extracted from human placenta, Thrombotest (Nyegaard) from ox brain, and 3 rabbit thromboplastins : Neo-plastine (Diagnostica Stago), Thromboplastine calcique (Bio-Merieux), Simplastin + (Gal Diagnostics). The results are expressed in seconds, percentage according to the dilution curve and INR with ISI given by the manufacturers.Analysis of results shows :- Influence of the technic : Whatever the technic is, mean results obtained with the same reagent, expressed in % are very close ; expressed in INR mean results are dispersed with all the reagents, ex :- Influence of the reagent : Results performed with the same technic whatever the reagent is expressed in INR are very close, whereas in % the dispersion is large.Analysis of variance and Student test show highly significant differences between all reagents for expression in %, whe reas expressed in INR (KC10) the difference is not statistical ly significant.INR leads an improvement in the dispersion of results but it seems necessary to have at least two ISI for each reagent, one determined by manual technic or device like KC10, the other one determined on photo-optical device.
FLUORESCENCE STUDIES OF THE BINDING OF FACTOR Va TO PHOSPHOLIPID VESICLES
Authors: Krishnaswamy S , Mann K G 
Published: August 2018

The free sulphydryl groups in factor Va were analyzed under native and denaturing conditions using dithio-bis-nitrobenzoic acid (DTNB). The heavy chain (D) and the light chain (E) of factor Va were found to contain one free sulphydryl each under native conditions and following denaturation. Intact factor Va contained two free sulphydryls after denaturation while only one of these groups was accessible to DTNB under native conditions. Analysis of the rates of modification of the accessible sulphydryl in factor Va, and direct visualization of factor Va reacted with fluorescent sulphydryl modifiers and analyzed by SDS-PAGE, indicated that the accessible group was present in component D. Factor Va was labelled with the sulphydryl-directed flurophore N-pyrene-l-malemide with no loss in functional activity. Fluorescently modified Va (PYR-Va) contained between 0.83 and 0.91 moles pyrene and showed a concomittant loss in the free sulphydryls accessible to DTNB. Fluorescence polarization studies indicated that the binding of PYR-Va to phospholipid vessicles composed of phosphatidylcholine (PC) and phosphatidylserine (PS) was accompanied by a significant increase in the fluorescence polarization of the pyrene moiety. Systematic analysis of the binding of PYR-Va to PCPS (75%PC, 25%PS) indicated that the binding interaction was characterized by a Kd=2.7x10−9 M and a stoichiometry of 42 monomeric phospholipids per lipid combining site. The binding of PYR-Va to PCPS was independent of added calcium ion and could be reversed by the addition of unlabelled factor Va. Analysis of the displacement curves indicated that native factor Va and PYR-Va mutually excluded each other with identical affinities (Kd=2.5×10−9 M). Isolated component D had no effect on the interaction between PYR-Va and PCPS, while isolated component E was capable of disrupting the PYR-Va-PCPS interaction (Kd=3.1×10−9 M%) indicating that this subunit entirely mediates the interaction between Va and PCPS. No detectable binding of PYR-Va was observed when 100% PC vesicles were used and systematic variation of the PS content of the vesicles indicated that this parameter linearly influenced the stoichiometry of the PYR-Va-PCPS interaction with a minimal effect on the dissociation constant for the reaction. The data indicate that the factor Va-binding site is determined by the PS content of the lipid bilayer.(Supported by NIH grants HL-35058 and HL-34575)