bluuebunny/crossref_metadata_2025_split huggingface dataset:
PLASMINOGEN ACTIVATOR INHIBITOR (PAI) ACTIVITY AS WELL AS PAI-1 AND PAI-2 ANTIGEN IN HEALTHY INDIVIDUALS AND HOSPITALIZED PATIENTS
Authors: Kruithof E K O , Nicoloso G , Gudinchet A , Bachmann F 
Published: August 2018

The activity of plasminogen activators (PA) is regulated in part by PA-inhibitors. Two distinct PAIs have been identified: PAI-1 and PAI-2. To be able to measure these proteins specifically we have purified PAI-1 and PAI-2, raised antisera and developed specific radioimmunoassays (RIAs), with detection limits in plasma of 10 and 15 ng/ml, respectively.In a population of over 350 individuals, PAI activity correlated well with PAI-1 :Ag (r=0.699), but not with PAI-2:Ag (r=0.070). In healthy individuals PAI-1 concentrations varied considerably (between 10 and 85 ng/ml, median 29 ng/ml). During venous occlusion (VO) applied in 40 healthy individuals, average PAI-1 in plasma increased fran 27 before, to 43 and 59 ng/ml after 10 and 20 min VO (p <0.001, Wilcoxon test). Overall fibrinolytic activity increased 15 fold, and t-PA:Ag levels eightfold. Increased PAI activity and PAI-1 antigen levels with respect to controls were found in patients with cardiovascular or thromboembolic disease, malignancies, hepatic insufficiency, after major trauma and in the postoperative period. This wide spectrum of pathologies with increased PAI-1 levels supports previous suggestions that PAI-1 behaves as an acute phase reactant. In patients undergoing extracorporeal circulation PAI activity and PAI-1 antigen were measured before and 1 h, 1 d and 7 d after the operation. Highest values of activity and antigen were observed 1 h postoperatively. These values remained elevated the day after the operation, but returned to preoperative levels within 7 days.PAI-2 antigen concentrations were at or below the detection limit in all controls and the majority of patients. Very high levels of PAI-2 (above 30 ng/ml) were only observed in pregnant women and in patients with hepatocellular carcinoma.The specific measurement of PAIs thus show increased levels in a variety of clinical conditions. The availability of specific RIAs permits to conduct prospective studies and to evaluate to what degree elevated PAI-1 and PAI-2 concentrations are correlated with the stage of the disease, its prognosis and the risk to develop thromboembolic complications.
THE PLATELET ALLOANTIGEN Zwa (P1A1) IS EXPRESSED BY CULTURED ENDOTHELIAL CELLS
Authors: Giltay J C , Leeksma O C , Borne A E G Kr v d , van Mourik J A 
Published: August 2018

Zwa (P1A1) is a platelet specific alloantigen, located on glycoprotein (GP) IIIa, and is of pathogenic importance in alloimmunologic disorders such as neonatal alloimmune thrombocytopenia and post transfusion purpura. As endothelial cells synthesize a plasma membrane protein complex which is structurally closely related to the platelet membrane GP IIb/IIIa complex, we examined whether these cells also express Zwa. Employing a variety of immunochemical techniques, our studies show that endothelial cells indeed can express this antigen at the plasma membrane surface.We also compared the expression of Zwa on platelets, isolated from umbilical cord blood, with the expression of Zwa on cultured endothelial cells, isolated from the same umbilical cord, of a number of neonates. Both platelets and endothelial cells obtained from the same individual, either expressed Zwa (Zwa positive individuals) or lacked expression of Zwa (Zwa negative individuals). These findings strongly suggest that endothelial-and platelet Zwa are encoded by the same genes.Thus, Zwa is not exclusively expressed by platelets, also endothelial cells express this alloantigen.An important consequence could be, that in alloimmunologic disorders in which the Zwa antigen is implicated, not only the platelets, but also the vessel wall is involved.
REGULATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ACTIVITY IN PLATELETS BY PHOSPHORYLATION
Authors: Grant P G , Mannarino A F , Colman R W 
Published: August 2018

Cyclic nucleotide phosphodiesterases (PDE) provide the only known pathway for the hydrolysis of cyclic nucleotides in cells and thus have the potential for modulating the effects of cAMP and cGMP on cells. In platelets a rise in intracellular cAMP levels inhibits platelet aggregation and secretion. Since cAMP exerts many of its effects through a cAMP-dependent kinase we questioned whether phosphorylation of cAMP PDE might be a mode for regulation of PDE activity in platelets. When platelets were incubated for 10 min with forskolin (100 μM) the level of cAMP rose at least 10-fold.When the low Km cyclic nucleotide PDE was isolated from freeze-thaw lysates of forskolin treated platelets by chromatography on blue dextran-Sepharose, the specific activity of this enzyme was increased 3 to 13-fold over similarly processed control platelets. The specific activity of a second PDE, the cGMP-stimulated cAMP PDE, was increased 1.5 to 3-fold by forskolin treatment of platelets. Forskolin had no direct effect on either purified PDE. The stimulation of the low Km cAMP PDE activity by exposure of platelets to forskolin was blocked when the platelets were simultaneously treated with the protein kinase inhibitor H-8 (100 μM) which is most potent toward cAMP dependent protein kinase indicating that this kinase may be responsible for the stimulation. When platelets which had been prelabeled with 32P inorganic phosphate were treated with forskolin and the low Km cAMP PDE isolated by blue dextran-Sepharose chromatography, a protein migrating in SDS gels at Mr=110,000, the molecular weight of the low Km cAMP PDE, was labeled indicating that phosphorylation of the PDE occurred coincident with stimulation of activity. These results suggest that phosphorylation of the low Km cAMP PDE by protein kinase may be an important regulatory mechanism for cAMP PDE activity and cyclic nucleotide levels in platelets.
IDENTIFICATION OF A NEW HUMAN KININ, ILE-SER-BRADYKININ, BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AND SEQUENCE ANALYSIS IN OVARIAN CARCINOMA ASCITES
Authors: Müller E , Henschen A , Wunderer G 
Published: August 2018

Human blood has been shown to contain two different kinin precursors, i.e0 the high and the low molecular mass kininogen0 These two kininogens release the same kinins, with the starting sequences Met-Lys-Arg-Pro-, Lys-Arg-Pro- or just Arg-Pro-depending on the releasing enzyme. The kinin starting with Arg-Pro- is denoted as bradykinin. In rats a different kininogen, called T-kininogen, is also present, especially as the major acute-phase protein in this species. The corresponding kinin, T-kinin, has the starting sequence Ile-Ser-Arg-Pro-. This type of kininogen or kinin has previously never been detected in human tissues. However, during the course of the present study evidence for existance of a third human kininogen, giving rise to human T-kinin, was obtained.Ascites from patients with metastatic ovarian carcinoma has been shown to contain high amounts of vascular permeability-increasing activity as determined by a rat skin-Evans blue test. When the ascites was fractionated by gel filtration followed by reversed-phase high-performance liquid chromatography (HPLC) a component could be isolated which by its total sequence and amino acid composition was identified as Ile-Ser-bradykinin. Several degradation products of this kinin were also detected as separate components in the chromatographies. The human Ile-Ser-bradykinin appeared on reversed-phase HPLC in the same position as synthetic T-kinin. It could be differentiated in this chromatography system from Met-Lys-bradykinin, Lys-bradykinin and bradykinin. It may be assumed that human Ile-Ser-bradykinin is released_ from a third, so far unidentified human kininogen which is only or predominantly expressed under certain pathophysiological conditions, and that therefore this new kinin might be employed as a tumor marker.
PHARMACOKINETICS OF A LOW MOLECULAR WEIGHT HEPARIN (KABI 2165, FRAGMIN) ATFER INTRAVENOUS AND SUBCUTANEOUS ADMINISTRATION IN HUMAN VOLUNTEERS AND ITS IN VIVO NEUTRALIZATION BY PROTAMINE SULFATE
Authors: Albada J , Nieuwenhuis K K , Sixma J J 
Published: August 2018

Pharmacokinetics of a low molecular weight heparin (LMWH) were studied in healthy volunteers. After an intravenous bolus injection of 5000 anti-Xa U in 5 healthy volunteers anti Xa activity disappeared according to the combination of saturable and a linear mechanism, preceded by a rapid initial disappearance. The apparent half-life of the anti Xa activity is about twice as long as that of standard heparin. In another set of experiments 5000 anti Xa U of LMWH were immediately followed by 50 mgr of Protamine Sulphate (PS). The curve of the anti Xa-activity parallelled the original curve at a level of about 30-40%. No rebound phenomenon was observed. The same dose of the LMWH followed by 100 mg of PS resulted in an anti Xa disappearance curve at an obvious higher level of about 50%. Also at this dose no rebound phenomenon was noticed.A continuous infusion of 10.000 anti Xa U/24 h during 10 hours was followed by 15.000 anti Xa U/24 h for another 10 hours after which the dose was raised to 20.000 anti Xa U/24 h for another 10 hours. Only the first infusion period resulted in a plateau fase. At the end of these experiments anti Xa activity was neutralized by 50 mg P.S. i.v. resulting in the disappearance of less than 50% of anti Xa activity. After subcutaneous administration of 15.000 anti Xa U (corresponding to the dose for i.v. treatment per day with this LMWH) peak levels of 1,1-1,8 anti Xa were reached after 3-4 hours. Supra-optimal anti Xa levels (higher than 0.9) were observed in all volunteers during a period of 5 hours. After 24 hours in none of the volunteers any anti Xa-activity could be detected.Conclusions:In contrast to previous reports pharmacokinetics of this LMWH do not essentially differ from those of standard heparin apart from its longer half-life and its high bioavialability after subcutaneous injection.
EFFECTIVENESS AND TOLERABILITY OF CV-3988, A SELECTIVE PAF ANTAGONIST, AFTER INTRAVENOUS ADMINISTRATION TO MAN
Authors: Arnout J , Van Hecken A , Delepeleire I , Miyamoto Y , Holmes I , Deschepper P , Vermylen J 
Published: August 2018

Platelet activating factor (PAF) is a naturally occurring phospholipid with a wide spectrum of biological activities. Although PAF has been ascribed a potential role in various conditions including inflammation, asthma, glomerulonephritis and thrombosis, its precise function in physiologic/pathophysiologic processes remains unclear. The introduction of selective PAF receptor antagonists could represent a useful tool to extend our knowledge of the role of this mediator in health and disease.We have investigated the efficacy and tolerability of (RS)-2-methoxy-3-(octadecylcarbomoyloxy)propy1 2-(3-thiazolio)-ethyIphosphate (CV-3988, Takeda Chem. Ind), a selective PAF antagonist with structural analogies with PAF, after intravenous infusion in man in a double-blind, placebo-controlled study. The compound, in doses from 750 to 2,000 pg/kg, significantly reduced platelet sensitivity to PAF. The threshold aggregating concentration (TAC) of PAF was defined as the minimal concentration causing an irreversible aggregation with a maximal amplitude of at least 50% of the difference in light transmission between platelet rich plasma and platelet poor plasma. It increased in a dose-dependent manner reaching 3.6 times the basal TAC (p<0.0005) at the end and 2.60 times the basal TAC (p<0.0005) 4 hours after infusion of the highest dose. The TAC of PAF returned to the basal value within 24 hours after the end of the infusion.CV-3988 did not cause major side effects nor changes in blood pressure, pulse or respiratory rate. However, small but clinically insignificant changes in plasma haemoglobin and serum haptoglobin were seen at the end and four hours after the end of the infusion, indicating a slight haemolysis probably by high local concentrations at the infusion site.Our results indicate that, when adequate infusion volumes and infusion rates are used, CV-3988 can be safely administered to man and should be useful in elucidating the role of PAF in health and disease.
CHARACTERIZATION OFTHE 5’FLANKING REGION FOR THE HUMAN FIBRINOGEN β GENE
Authors: Huber P , Dalmon J , Laurent M , Courtois G , Thevenon D , Marguerie G 
Published: August 2018

Fibrinogen is coded by three separate genes located in a 50kb region of chromosome 4 and organized in a α - β - γ orientation with an inversion of the gene 3- A human genomic library was constructed using the EMBL4 phage and screened with cDNA probes coding for human fibrinogen Aα, Bβ and γ chains. Clones, covering the fibrinogen locus,were identified, and their organization was analyzed by means of hybridization and restriction mapping. Among these clones one recombinant phage containing the β gene and large 5’ and 3’ -flanking sequences was isolated.To identify the regulatory sequences Dpstream from the human β gene, a 1.5 kb fragment of the immediate 5’-flanking region was sequenced. The SI mapping experiments revealed three transcription initiation sites. PotentialTATA and CAAT sequences were identified upstream the initiation start points at the positions -21 and -58 from the first initiation start point.Comparison of this sequence with that previously reported for the same region upstream from the human γ gene revealed no significant homology which suggests that the potential promoting sequences of these genes are different. In contrast, comparison of the 5’flanking regions of human and rat β genes showed more than 80% homology for 142 bp upstream from the gene. This highly conserved region is a potential candidate for a regulatory sequence of the human β gene.To verify this activity, a β fibrinogen minigene was constructed by deletion of the internal part of the normal gene and including 3.4kb of the 5’flanking region and 1.4kb of the 3’flanking region. The minigene was transfected into HepG2, a human hepatoma cell line, to show whether the 5’flanking region of the human fibrinogen gene contains DNA sequences sufficient for efficient transcription in HepG2. Constructions of several parts of the sequenced 5’flanking region of the human β gene with the gene of the chloramphenical acetyl transferase have been also transfected in the HepG2 cells to determine the specificity of the gene expression and to localize the sequences controlling the transcription of the gene.
SILENT PULMONARY EMBOLISM IN PATIENTS WITH DEEP VEIN THROMBOSIS
Authors: Huisman M V , Buller H R , ten Cate J W , van Royen E A , Vreeken J 
Published: August 2018

In patients presenting with clinically suspected deep vein thrombosis symptomatic pulmonary embolism is rarely apparent. To assess the prevalence of asymptomatic pulmonary embolism in outpatients with proven deep vein thrombosis, perfusion ventilation lungscans were performed in 101 consecutive patients at the first day of treatment and after one week of therapy. Fifty-one percent of these patients had a high probability lung-scan at the start of treatment. In control patients (n=44) without deep venous thrombosis but referred through the same filter, the prevalence of high-proba-bility scans was only 5%. After one week of anticoagulant treatment complete to partial improvement was observed in 55% of the patients while in another 24% of the patients the scan remained normal.It is concluded that lungscan detected asymptomatic pulmonary embolism occurs frequently in patients presenting with symptomatic deep venous thrombosis and that the majority of these emboli resolve within one week of anticoagulant treatment.
THROMBUS IMAGING WITH MURINE MONOCLONAL ANTIBODIES AGAINST FIBRIN FRAGMENT D-DIMER IN A RABBIT JUGULAR VEIN THROMBOSIS MODEL
Authors: Holvoet P , Stassen J M , Collen D 
Published: August 2018

Three murine monoclonal antibodies (MA-6C1, MA-8D3 and MA-15C5) reacting with fragment D-dimer from crosslinked fibrin but not with monomeric fragment D were obtained by immunization of Balb/c mice with the highly purified fragment, fusion of spleen cells with a myeloma cell line, production of. ascites fluid in mice and purification of the antibodies on Affigel Blue. Fab fragments were isolated from papain digests. The IgG and Fab fragments were labeled with 125I, 131I or 123I using lactoper-oxidase. The disposition rates (t1/2) and thrombus to blood ratios, measured in groups of 3 rabbits with a non-occlusive jugular vein thrombus composed of whole human plasma were :These results indicate that, after 1 to 3 half-lives of the IgG or Fab fragments, using combinations of 2 or 3 monoclonal antibodies, thrombus to blood ratios of isotope of 5 to 7 are obtained. Such signal/noise ratios are sufficient for in vivo detection by exteijil gamma scintigraphy. This was preliminarily confirmed using 123I-labeled Fab fragments of the three antibodies in rabbits.
SERPINS: ANTITHROMBIN AND OTHER INHIBITORS OF COAGULATION AND FIBRINOLYSIS. EVIDENCE FROM AMINO ACID SEQUENCES
Authors: Carrell R W , Christey P D , Boswell D R 
Published: August 2018

A number of the key inhibitors of coagulation and fibrinolysis have recently been shown to be members of the same superfamily of serine protease inhibitors, the serpins. The archetypes of the group are alpha-l-antitrypsin and antithrombin and it includes antiplasmin, C1-inhibitor, heparin cofactor II and the newly recognised inhibitors of plasminogen activators and activated Protein C. Alignment of their structures shows that they have the same skeletal three-dimensional conformation and, by inference, the same general function mechanisms.The serpins have a reactive centre, primarily dependent on a single amino acid, exteriorly placed on a stressed peptide loop. This functions by offering the cognate protease a high-affinity substrate that resists complete cleavage to form a tight 1:1 complex of inhibitor and protease that is subsequently removed from the circulation. The loop is vulnerable to cleavage with resulting loss of inhibitory activity. This irreversible switch is utilised: pathologically by venom and invasive bacterial proteases; and physiologically by the neutrophil leucocyte to modify local inflammatory responses. These mechanisms contribute to the changes seen in DIC and the shock syndromes.Modelling of antithrombin indicates the likely topological features involved in the binding of heparin, namely a sphere of positive charge centred on the A and D helices and involving Arg 47, Lys 125, Arg 129 and probably Arg 132 and Lys 133.Because the serpins are largely dependent for their specificityon a single amino acid it is now possible to precisely tailor inhibitory activity by site specific mutation. This has been used to produce recombinant antitrypsins that function as an improved inhibitor of neutrophil proteases (valine or leucine reactive centre), or as an analogue of antithrombin (arginine reactive centre). An elegant application of this approach is the engineered mutants of antiplasmin recently described by Holmes, Collen and colleagues (Leuven).
CHARACTERIZATION OF SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR MUTANTS PRODUCED BY SITE-SPECIFIC MUTAGENESIS OF LYS
Authors: Nelles L , Linjnen H R , Demarsin E , Collen D , Holmes W E 
Published: August 2018

A cDNA encoding full length single chain urokinase-type plasminogen activator (scu-PA) was cloned and sequenced. The recombinant scu-PA (rscu-PA) cDNA as well as the cDNA of two mutants constructed by deoxyoligonucleotide directed mutagenesis of Lys158 in rscu-PA to Gly158 (rscu-PA-Gly158 ) or to Glu158 (rscu-PA-Glu158 ) were inserted into SV40 early promoter/enhancer based expression vectors, which were used to transfect Chinese Hamster Ovary (CHO) cells. The expression products were purified from serum-free conditioned media by immunoadsorption on an insolubilized monoclonal antibody raised against natural scu-PA (nscu-PA), followed by gel filtration.The amidolytic activity of the three rscu-PAs was low (< 500 IU/mg). The mutant rscu-PAs, in contrast to the rscu-PA and nscu-PA, could not be converted into an amidolytically active two-chain form (tcu-PA) by plasmin. The mutant scu-PAs had a very low specific activity (< 1,000 IU/mg) on fibrin plates, whereas wild type rscu-PA had a specific activity < 1000 IU/mg. The mutant scu-PAs did not cause lysis of a I-fibrin labeled plasma clot immersed in citrated human plasma. Serum-free medium from a control transfected CHO cell line showed no significant plasminogen activating activity.In a purified system, both rscu-PA-Gly and rscu-PA-Glu activate plasminogen following Michaelis-Menten kinetics, with a much lower affinity (K = 60-80 yM) but with a higher catalytic rate constant (k2 = B.01 s-1) as compared to the wild type rscu-PA (K =1.0 yM, k = 0.002 s-1).It is concluded thaz conversion of scu-PA to tcu-PA is prerequisite for the activation of plasminogen. However, Lys158 seems to be important for the stability of the Michaelis complex between scu-PA and plasminogen.
PERTURBATION OF CULTURED ENDOTHELIAL CELLS ALTERS CELLULAR VON WILLEBRAND FACTOR DISTRIBUTION
Authors: Reinders J H , Verweii C L , Mourlk J A V , de Groot Ph G 
Published: August 2018

Endothelial cells, cultured from human umbilical veins, synthesize von Willebrand Factor (vWF), that is stored by the cells in Weibel-Palade bodies, secreted into the medium and incorporated into the extracellular matrix underneath the cells. We have studied the influence of perturbation by phorbol esters and thrombin on the cellular distribution of vWF. Short-term (< 1 hour) treatment of endothelial cells with phorbol ester PMA or thrombin resulted in the release of cellular stored vWF. Long-term treatment with perturbants evoked a distinct change in the endothelial cell distribution of vWF, evident 24 to 48 hours after exposure. While the contents of the vWF storage vesicles were gradually restored within 48 hours, enhanced amounts of vWF were secreted into the medium. However, PMA did not increase the endothelial cell contents of mRNA encoding for vWF. The number as well as the size of vWF storage granules in the cells increased after exposure to perturbants. The perturbed cells responded to stimuli in releasing stored vWF, the amounts secreted were even greater than those in control cells. The extracellular matrix lost its vWF contents as the result of PMA or thrombin treatment, by blocking deposition of vWF in the matrix, not by enhancing degradation of matrix vWF. In perfusion experiments, the adhesion of washed platelets onto the isolated matrix of perturbed cells was considerable less than that in controls. Addition of vWF to the perfusate overcame this impairment. Thus, perturbation of endothelial cells changes the cellular distribution of vWF.Supported in part by ZWO grants 13-30-31 and 13-90-91 and Netherlands Heart Foundation grant 28.004.