bluuebunny/crossref_metadata_2025 huggingface dataset:
GENERATION OF REACTIVE OXYGEN METABOLITES BY PHAGOCYTOSING ENDOTHELIAL CELLS: REGULATORY ROLE OF THE GLYCOCALYX
Authors: Görög P , Pearson J D , Kakkar V V
Published: August 2018

We have studied the ability of particular stimuli to induce the release of reactive oxygen metabolites from sub-cultured monolayers of human umbilical vein endothelial cells. Superoxide production was measured as the superoxide dismutase-inhibitable reduction of exogenous cytochrome C, and H2O2 production was measured by the horseradish peroxidase catalysed oxidation of scopoletin. Basal release of either metabolite from undisturbed monolayers was low (80pmol O2 and 65pmol H2O2 in 3 h from dishes of 3×108 cells). Addition of 1μM diameter polystyrene microspheres (4.5×108 particles/dish) which were phagocytosed by the cells progressively over 3h, caused a dramatic increase in release of both metabolites (2nmol O2 and 490pmol H2O2 at 3h). Release of O2 was linear, whereas release of H2O2 was more rapid in the first hour. Addition of formaldehyde-fixed human platelets (2.5×l06/dish) significantly enhanced O2 production (490pmol at 2h) and raised, thought not significantly, H2O2 release (130pmol at 2h); addition of a stabilized lipid emulsion (lipofundin-S ; particle diameter 1μm, 3.6×108 particles/dish) caused a significant rise in O2 production (1.3nmol at 2h) but measurement of its effect on H2O2 release was prevented by interference in the assay. Similar rises in H2O2 and O2 production were induced by treatment with 10™7M phorbol myristate acetate. Pretreatment of endothelial cells with 12.5mU /ml neuraminidase or 0.5U/ml heparinase for 60 min to alter their glycocalyx composition substantially enhanced the effect of microspheres on H2O2 and O2 generation, particularly at early times (lh). We conclude that the interactions of particulates, including platelets and lipids, with endothelial cells can lead to the generation of significant pericellular levels of reactive oxygen species. These metabolites may oxidise a wide variety of nearby molecules, leading to cell damage and altered uptake characteristics for lipoproteins containing peroxidised lipids. These effects are exacerbated when endothelial cell glycocalyx composition is disrupted.
REGULATION OF INTERLEUKIN-2 (IL-2) PRODUCTION BY ENDOTHELIAL CELL (EC) DERIVED SUBSTANCES
Authors: Teitel J M , Shore A , McBarron J
Published: August 2018

We recently reported that vascular EC support the staphylococcal protein A (SPA)-induced production of IL-2 by T cells, and furthermore do so synergistically with monocytes. We have now assessed the contributions of EC membrane-associated and secreted factors to these functions. IL-2 was measured by either human tonsil blast of CTLL cell assay. Separation of EC from T cells by a permeable membrane (.45μ pore size) prevented IL-2 production. Consistent with this, supernatant from resting EC (ECs) was also unable to induce IL-2 generation. In addition, neither a crude EC plasma membrane preparation nor paraformaldehyde-fixed EC supported IL-2 production. The combination of fixed EC + ECs likewise did not reconstitute the accessory cell role of intact EC. This was not due to the absence of class II MHC antigens, since EC which were fixed after incubation with interferon (to induce HLA-DR expression) were also unable to promote IL-2. However, the addition of ECs to cultures of T + live EC enhanced IL-2 production by 42% (mean, n=7). This effect did not require the presence of detectable IL-1 in the ECs, and was not reproduced by purified IL-1. When added to unfractionated peripheral blood mononuclear cells (PBM), both fixed EC and ECs enhanced IL-2 production (increments of 120 ± 37% and 115 ± 44% respectively). This effect was dose related, and peaked at 20% ECs by volume. Incubation of EC with calcium ionophore (10™6 M) yielded a supernatant (ECs-i) with greatly enhanced IL-2 promoting activity despite the fact that ionophore alone suppressed IL-2: lllu for T + EC + ECs-i vs. 7u for T + EC + ECs + ionophore. Therefore, substances secreted by EC and presented on the EC surface are able to augment mitogen induced IL-2 production by T cells in the presence of accessory cells. The EC-derived mediator is apparently distinct from IL-1. EC may function in concert with monocytes as important immune regulatory cells at the blood/tissue interface.
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.
EFFECTS OF STANDARD HEPARIN AND A LOW MOLECULAR WEIGHT HEPARIN (KABI 2165) ON PLASMA LEVEL OF t-PA AND PA INHIBITOR
Authors: Agnelli G , Born J , Cosmi I , Levi M , ten Cate J W
Published: August 2018

Previous and recent reports have suggested a fibrinolysis-enhancing property of standard heparin (SH) and low molecular weight heparin (LMWH) but these observations have never been confirmed in a clinical study fulfilling appropriate methodological criteria.Aim of this study was to evaluate in a cross-over double blind placebo-controlled study the effect of SH and LMWH (K 2165) on fibrinolysis. Six healthy volunteers (age 22-30) received a bolus dose of the following treatments: l)Placebo, 2)Kabi SH,5000 I.U., 3) Kabi 2165,5000 anti Xa U, 4)Kabi 2165, 10000 anti Xa U. A 4 day wash-out period was adopted.The volunteers had been fasting for at least 12 hours before the infusion and were sitting during the experiment.Before starting the infusion and at 1,2,5,10,15,30,60,120 minutes thereafter, blood samples were collected for the assay of t-PA activity, PA inhibitor activity, plasminogen, alfa2-antiplasmin, fibrinplate lysis area (FPLA), fibrinogen, aPTT, antithrombin III and anti-Xa activity. Placebo and Kabi 2165,5000 U had no effect on t-PA plasma level, however both SH and Kabi 2165,10000 U produced a statistically significant increase of t-PA level at 1 hour after the infusion. The t-PA increase lasted for at least 2 hours after the infusion; at that time the average t-PA activity was 0.14 U/ml for SH and 0.09 U/ml for Kabi 2165,10000 U (p<0.05, AN0VA,average pre-infusion level and placebo at 2 hours:0.01 U/ml).At that time plasma anti-Xa was 0.11 U/ml for SH and 1.05 U/ml for Kabi 2165,10000 U. SH and Kabi,10000 produced no effect on PA inhibitor.No effect of any treatment on fibrinogen,alfa2-antiplasmin,plasminogen, FPLA and antithrombin III was observed. We conclude that in this study both SH and Kabi 2165 produced a delayed and sustained increase of t-PA. The relevance of this effect remains to be established.
LOW MOLECULAR WEIGHT HEPARIN (Enoxaparin) COMPARED WITH UNFRACTIONATED HEPARIN THRICE DAILY IN PREVENTION OF POSTOPERATIVE THROMBOSIS. A RANDOMIZED MULTICENTRE TRIAL
Authors: Samama M , Bernard P , Bonnardot J P , Tissot E , Lanson Y , Combe-Tamzali S
Published: August 2018

Three consecutive randomized open studies have been carried out in 892 patients undergoing abdominal, gynecological, thoracic or urological surgery. They were over 40 years old and presented at least one of the following risk factors for thrombosis: previous thromboembolism, obesity, varicose veins, malignancy (30% in these studies), pre-operative hospitalization over 5 days, estrogen therapy, chronic cardiac disease or bronchitis. The two groups of each trial were well matched with regard to population characteristics. The third trial included higher rate of patients undergoing urologic surgery. Isotopic venous thromboses and bleeding complications were assessed after subcutaneous administration of a low molecular weight (LMW) heparin fragment (Enoxaparin, 1 mg = 100 Anti-Xa I.U.) or unfractionated heparin (UH). The 3 studies compared 3 × 5,000 IU UH daily with 1 × 60 mg, 1 × 40 mg, or 1 × 20 mg LMW heparin daily. Thromboembolic event rates were not significantly different among the groups (UH : 3.6, 2.8, 7.6% respectively compared to LMWH : 3, 2.8, 3.7%). Significant decrease of hematocrit and hemoglobin were only observed in patients receiving 60 mg Enoxaparin (as compared to UH) whilst in the 2 other trials no difference could be evidenced between the 2 populations. The metaanalysis of the three studies on the “intention to treat” patients gave results consistent with those observed in good compliers. The three consecutive studies showed homogeneous results (p = 0.20), the Mantel Haenszel test did not evidence a global difference between Enoxaparin and unfractionated Heparin (p = 0.54). These results suggest that an optimal dosage of 20 mg per day of Enoxaparin is safe and as efficient as UH 5,000 IU × 3 in the prevention of post-operative thrombosis in this population.
EFFECT OF COLLAGEN AND CITRATE ON HEPARIN-HEDIATED PLATELET ANTIAGGREGATORY ACTIVITY
Authors: Saba S R , Saba H I , Morelli G A
Published: August 2018

Heparin has been reported to inhibit platelet aggregation. Our studies show that this activity is easily demonstrable in washed platelet systems, but fails to occur in citrated platelet-rich plasma (PRP) in the presence of a variety of agonists, except collagen. Studies were performed to answer the following questions: (1) Why does heparin inhibit the aggregation of washed platelets but not of citrated PRP, which is the system commonly used for platelet aggregation studies? (2) What is the effect of heparin on platelet aggregation occurring in whole blood, where it can be examined both with and without the presence of sodium citrate? (3) Why does heparin consistently inhibit the collagen-induced aggregation even in citrated PRP, while it fails to inhibit aggregation caused by other agonists? Results of the studies clearly demonstrated that heparin has the ability to directly react with sodium citrate, causing loss of its inhibitory activity on platelets. The antiaggregatory activity of heparin in the presence of collagen as the agonists appears to be directly related to the blocking of collagen’s agonist activity by heparin. Small concentrations of heparin which were unable to inhibit aggregation per se, effectively blocked the collagen agonist activity on platelet aggregation when heparin was directly added to collagen. Further studies showed that heparin, in a native whole blood platelet aggregation system (in the absence of any anticoagulant), exhibited significant inhibitory activity. This activity was lost when citrate was present in the whole blood preparation. These studies, therefore, indicate that failure of heparin to inhibit platelet aggregation in citrated PRP does not negate the importance of this inhibitory activity. Reactivity of heparin with sodium citrate renders citrated systems unsuitable for studying heparin's effect upon platelets. The whole blood platelet aggregation system without the presence of anticoagulants appears to be a more suitable system for the study of heparin and platelet aggregation, and is closer to the physiological system. Heparin exhibits marked inhibitory activity on platelet aggregation in this system, and this suggests it may be an important activity which deserves further attention.
TWO-DIMENSIONAL MULTIMERIC ANALYSIS OF PLASMA AND PLATELET VON WILLEBRAND FACTOR (VWF) WITH IDENTIFICATION OF PLATELET VWF FRAGMENTS EXPRESSING A NEO-ANTIGEN
Authors: Dent J , Roberts J , Ruggeri Z M , Zimmerman T S
Published: August 2018

SDS-agarose electrophoresis of von Willebrand factor (vWF) was followed by reduction, second dimension SDS-polyacrylamide gel electrophoresis and immunoblotting with monoclonal anti-vWF antibodies. The multiple bands in each multimer of plasma vWF from normal and IIA von Willebrand disease (vWD) patients were shown to contain varying proportions of the intact 225 kDa vWF subunit and fragments of 189, 176, and 140 kDa. Only one relatively minor band in each multimer was composed entirely of the intact 225 kDa subunit. Repeating bands in successively larger multimers up to the thirteenth, exhibited similar compositions, whereas the largest multimers contained only the intact 225 kDa subunit. Thus the complex multimeric pattern of plasma vWF is the result, at least in part, of proteolytic degradation, and smaller multimers may derive from proteolytic degradation of larger species. In contrast, none of the fragments present in plasma vWF were seen in the vWF derived from platelets. Rather, fragments of 172 and 182 kDa were present in the smallest one or two multimers, whereas the larger multimers contained only the intact subunit. The fragments of platelet vWF reacted only with one monoclonal antibody (K14) of the 80 tested. This antibody did not react with unreduced plasma vWF nor with the unreduced fragments generated by Staphylococcus aureus V8 protease digestion of plasma vWF and reacted very poorly with reduced intact vWF subunit. Thus, the monoclonal antibody K14 recognized a neo-antigenic epitope expressed on at least two fragments of normal platelet, but not plasma, vWF.
A FACTOR VIII BINDING DOMAIN RESIDES WITHIN THE AMINO-TERMINAL 272 AMINO ACID RESIDUES OF VON WILLEBRAND FACTOR
Authors: Foster P A , Fulcher C A , Marti T , Titani K , Zimmerman T S
Published: August 2018

We have identified a Factor VIII (FVIII) binding domain residing within the amino-terminal 272 amino acid residues of the mature von Willebrand Factor (vWF) subunit. Two dimensional crossed immunoelectrophoresis showed direct binding of purified human FVIII to purified human vWF. After proteolytic digestion of vWF with Staphylococcus aureus V8 protease, FVIII binding was seen only with the amino-terminal SP fragment III and not with the carboxy-terminal SP fragment II. A monoclonal anti-vWF antibody (C3) partially blocked FVIII binding to vWF and SP fragment III. FVIII also bound to vWF which had been adsorbed to polystyrene beads. This binding was inhibited in a dose dependent manner by whole vWF, SP fragment III, and by monoclonal antibody C3. Binding could not be inhibited by SP fragment I, which contains the middle portion of the vWF molecule, or by reduced and alkylated whole vWF. SP fragment II caused only minor inhibition. Trypsin cleavage of SP fragment III produced a 35-kDa fragment containing the amino-terminal 272 amino acid residues of vWF. This fragment reacted with monoclonal antibody C3 and inhibited the binding of FVIII to vWF in a dose dependent manner. The other major fragment obtained from this digestion was a two chain hetero-dimer composed of amino acid residues 273-511 and 674-728. This fragment did not inhibit FVIII binding. These studies demonstrate that a major FVIII binding site resides within the first 272 amino acid residues of vWF.
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).
AN ALANINE INSERTION IN α2-ANTIPLASMIN ‘ENSCHEDE’ ABOLISHES ITS PLASM IN INHIBITORY ACTIVITY
Authors: Homes W E , Lijnen H R , Nelles L , Kluft C , Collen D
Published: August 2018

Congenital deficiencies of the fibrinolytic inhibitor α2antiplasmin (α2AP) may result in bleeding disorders. An abnormal a AP (α2AP‘Enschede’) is known. 2 siblings with 3% functional activity and normal antigen level have parents with 50% activity and normal antigen. The protein interacts normally with the lysine-binding site(s) of plasmin(ogen) but does not inhibit plasmin irreversibly. α2AP Enschede is a plasmin substrate that like the normal protein releases a M 8,000 peptide upon reaction with plasmin. In the present study, Southern blot analysis, using an α2AP cDNA probe showed a restriction fragment length polymorphism within a small genomic DNA fragment of the Enschede family members. Cloning and sequencing of these fragments revealed a GCG inframe insertion that results in an alanine addition between amino acids 353 and 357, 7-10 positions NH -terminal to the reactive site PI residue, Arg364. This area is homologous to the A4 B-sheet of reactive site cleaved a -antitrypsin. Clones from each individual confirm the parents as true heterozygotes and the children as true homozygotes. A cloned genomic DNA sequence containing the insertion (V ) was exchanged for the normal sequence in a eukaryotic a AP expression plasmid. Recombinant α2AP‘Enschede’ (ra AfVAla) purified from the conditioned media of transfected Chinese Hamster Ovary Cells is analogous to plasma a α2AP‘Enschede’ with respect to interactions with plasmin and plasminogen. Preliminary analysis of the released Mr 8,000 recombinant peptide shows that its NH -terminus is the same as the peptide cleaved from normal a AP. Although ra α2APVAla does not inhibit plasmin irreversibly it does, however, act as a competitive inhibitor of hydrolysis of the chromogenic substrate S-2251 by plasmin.The K for this interaction is 25 nM. Thus, α2APAla retains a high affinity for the active center of plasmin. In conclusion, an Ala insertion near the reactive site of α2AP must have resulted in a structural perturbation that has abolished the plasmin inhibitory activity of a α2AP‘Enschede’. This variant may provide a model for further investigation of structure-function relationships in the serpins which determine the relative inhibitor vs. substrate properties.
INTERACTIONS OF ACTIVATED HAGEMAN FACTOR (Factor Xlla, HFa) WITH NORMAL Cl-INHIBITOR
Authors: Donaldson V H , Mitchell B H , Ratnoff O D
Published: August 2018

Purified Hageman factor activated with a mixture of brain sulfatides and bovine serum albumin (BSA) developed clot-promoting activity which was inhibited by purified normal human Cl-inhibitor, but during extended incubation there was an apparent loss of inhibition in these mixtures. When a preparation of Hageman factor fragments (HFf), which had a much lower specific Hageman factor coagulant activity, was incubated with BSA-sulfatides, its_coagulant activity was also enhanced and could be inhibited by normal Cl-inhibitor. When the HFa having a molecular weight of approximately 75,000 was mixed with the HFf preparation before Cl-inhibitor was added, there was a progressive shortening of the Hageman factor specific clotting times with increasing amounts of HFf in the mixtures, despite the addition of Cl-inhibitor. Moreover, as the amounts of HFf in the mixtures were increase a high molecular weight complex of about 190,000 containing HFa and Cl-inhibitor could no longer be identified in SDS-gel electrophoresis but a low molecular weight band (127,000) appeared, which probably contained HFf of about 22,000 molecular weight and Cl-inhibitor. Therefore, HFf appeared to compete effectively with the HFa (75,000) for Cl-inhibitor in these mixtures of purified reagents. It is also possible that HFf may have interacted with the HFa or the Cl-inhibitor changing the steric configuration of either one so that an interaction between HFa and Cl-inhibitor did not occur.
PLASMINOGEN ACTIVATION BY SINGLE-CHAIN UROKINASE (scu-PA) IN FUNCTIONAL ISOLATION - DEMONSTRATION OF A NOVEL MECHANISM
Authors: Ellis V , Scully M F , Kakkar V V
Published: August 2018

The kinetics of the activation of Glu- and Lys- plasminogen by single-chain urokinase (pro-urokinase) derived from the transformed human kidney cell line, TCL-598, has been studied and compared with two-chain urokinase (UK). Plasminogen activation was determined by the change in fluorescence polarization of fluorescein-labelled aprotinin (Trasylol), an essentially irreversible inhibitor of plasmin. This methodology allows plasmin production by scu-PA to be measured in functional isolation, with no interfering generation of two-chain UK. scu-PA was found to activate plasminogen to plasmin with Michaelis-Menten type kinetics. The Km for this reaction was determined as 70µM, with a catalytic constant of 2.25 min-l. The generation of two-chain plasmin was confirmed by reduced SDS-PAGE. Plasminogen activation by UK was found to have a similar Km but the kcat was 16-fold higher, at 36.0 min-l. This is in contrast to the amidolytic activity of scu-PA which was less than 0.2% that of UK. The activation of scu-PA to UK by plasmin was also characterized. Using these data it is possible to calculate the theoretical rate of plasminogen activation by scu-PA, in the absence of aprotinin when UK will be generated by plasmin action. The calculated rate was in good agreement with that determined experimentally when using the chromogenic substrate, S-2251. These data demonstrate that scu-PA has properties which distinguish it from conventional serine protease zymogens. There is a lack of activity against peptide substrates (and also DFP) demonstrating the inaccessibility of the substrate binding pocket. However, there is moderate activity against plasminogen suggesting that plasminogen may be acting as both an effector and a substrate for scu-PA.
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.
THE EFFECT OF PHORBOL DIESTERS AND INTERLEUKINS ON THE SECRETION OF VON WILLEBRAND FACTOR BY HUMAN ENDOTHELIAL CELLS IN CULTURE
Authors: Giddings J C , Shall L
Published: August 2018

Human umbilical vein endothelial cells (EC) were cultured in the presence of 4p-phorbol 12-myristate 13-acetate (PMA, 10ug/l), interleukin 1 (IL-1, 1 unit/ml) and interleukin 2 (IL-2, 1 unit/ml), and secretion of von Willebrand factor activity (vWF, Ristocetin co-factor) and von Willebrand factor antigen (vWFAG, ELISA Technique) measured at intervals. Confluent control EC were treated with PMA, IL-1 and IL-2, and the supernatant medium assayed for release of vWF and vWFAg. Treated cells were also examined for vWFAg by immuno-fluorescence. The levels of both vWF and vWFAg in cultures containing IL-1 were significantly higher than those in control cultures after 5-6 days growth. Moreover, vWF and vWFAg increased significantly in the supernatant of confluent control EC incubated further in the presence of IL-1. Furthermore, the characteristic fluorescence pattern of endothelial vWFAg was markedly reduced in EC treated with IL-1. The levels of vWF and vWFAg in cultures containing PMA were also significantly higher than those of control cultures. In these conditions, however, the growth of cells appeared to be enhanced, and confluence was observed after about 6 days in the presence of PMA compared to 9 - 10 days in control cultures. The mean levels of vWF and vWFAg in the supernatant of EC incubated with PMA were higher than the control values but the differences were not statistically significant. Immunofluorescence of PMA-treated cells suggested that vWFAg might be less granular than in control cells but the differences were not as marked as those seen with IL-1. The results of all assays in the presence of IL-2 were not significantly different from those of control cells. In all instances no morphological evidence of endothelial injury was observed and more than 90% of cells remained viable at the termination of cultures. The results indicated that the synthesis and release of vWF were increased in the presence of PMA, and secretion of vWF was stimulated by IL-1. The data suggest that secreted vWF might contribute to the previously reported enhanced procoagulant and adhesive properties of EC treated with these substances.
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.
PLATELET INTERACTION WITH ARTIFICIAL SURFACES UNDER DEFINED FLOW CONDITIONS: ROLE OF VON WILLEBRAND FACTOR
Authors: Badimon L , Badimon J J , Turitto V , Fuster V
Published: August 2018

Platelets deposit onto artificial surfaces to form mono or multilayers which under certain conditions lead to gross thrombus formation and eventual failure of graft material. Both hemodynamic and blood related factors determine the interaction of blood cells with prosthetic inplants. The objective of this study has been to ascertain the extent to which vWF plays a role in platelet deposition onto artificial surfaces over a range of shear conditions. Polytetrafluoroethylene (PTFE) vascular graft segments were exposed to blood flow in a tubular perfusion chamber with well characterized rheological conditions. The perfusion chamber with test material was placed within an extracorporeal circuit (carotid artery-jugular vein) in normal swine and swine with inherited homozygous vWD. Platelet deposition was measured by labelling autologous platelets with 111Indium and also by optical morphometry of epoxy embedded specimens. Electron microscopy was performed in selected samples. Studies were performed with heparinized blood (300u/Kg iv) for perfusion times of 1 to 10 minutes. Platelet deposition increased with wall shear rate and with exposure time in the presence of vWF (vWF: 89 ± 11%) but in its absence (vWF: < 3%) platelet deposition was significantly(*) inhibited at high (1690s 1, shown below) but not at low (212s x) shear rate. Results are Platelets x 106 /cm2±lSE.

Plasma and platelet vWF play an important role in platelet-deposition and stabilization of thrombus growth on a synthetic biomaterial as we have previously observed on vascular surfaces
RELATIONSHIP BETWEEN HAEMORRHAGIC AND LIPASE-RET EASING PROPERTIES OF HEPARIN AND LMV HEPARIN
Authors: Maggi A , Barrowcliffe T W , Gray E , Donati M B , Merton R E , Pangrazzi I
Published: August 2018

In a preliminary study, a good correlation (r = 0.97) was noted between the relative abilities of an unfractionated heparin, a LMW heparin, pentosan poly sulphate and dermatan sulphate to prolong the template bleeding time in rabbits and their lipase-releasing potencies. In the present study, we have measured the prolongation of both the template and transection bleeding times in groups of 5 rats given i.v. injections of 0.75 mg/kg of two different unfractionated heparins (UEH), A and B, three different LMW heparins, X, Y and Z, and a heparan sulphate, HS. Lipase release was measured in plasma samples from different groups of 5 rats, using a tritiated triolein method.

UFH A had the most haemorrhagic effect, with an approximate doubling of both template and transection bleeding times and was also the most potent lipase-releaser, giving an average lipase level of 1126 mu/ml. UFH B had no significant effect on the template bleeding time, but did prolong the transection time; its lipase releasing potency was 70% of UFH A. IMW heparin X had no effect on template or transection bleeding time and released only 40% lipase compared with UEH A. LMW heparins Y and Z did not affect the template bleeding time but did prolong the transection time; they released more lipase (60%) than LMW heparin X. Correlation coefficients with lipase release were 0.97 for the template bleeding time and 0.69 for the transection bleeding time. HS released only 7% lipase but gave significant prolongations of both bleeding times.

These results confirm a strong positive correlation between the haemorrhagic and lipase releasing properties of heparin and LMW heparin, suggesting very similar structural requirements for the two biological activities. This correlation exists also for dermatan sulphate and pentosan polysulphate, but not for the heparan sulphate sample tested.
HEPARIN IS NOT AN EFFICIENT INHIBITOR OF THE FACTOR Xa-DEPENDENT ACTIVATION OF FACTOR V AND FACTOR VIII
Authors: Ofosu F A , Modi G J , Buchanan M R , Hirsh J , Blajchman M A
Published: August 2018

We have previously proposed that the steps in coagulation most sensitive to inhibition by heparin are the thrombin-dependent activation of factor V and factor VIII. This observation was based on the demonstration that therapeutic concentrations of heparin or 1μM of the thrombin specific inhibitor, phe-pro-arg CH2Cl (PPACK) completely inhibited the activation of prothrombin when contact-activated plasma (CAP) was recalcified for up to 1 min. Under similar conditions, heparin and PPACK only partially inhibited the activation of factor X. Moreover, the addition of thrombin (lOnM) to CAP 1 min before that of heparin or PPACK reversed their inhibitory effects. We now provide further support for our hypothesis by showing that when the activity of thrombin is suppressed by heparin or PPACK, efficient activation of radiolabelled prothrombin occurs only when the factor Xa then present activates factor V and factor VIII. We compared the effects of HEP of PPACK on the following four systems for initiating the activation of prothrombin: (1) CAP; (2) CAP + lOnM thrombin; (3) CAP + InM Xa and (4) unactivated plasma + InM Xa + InM Va + coagulant phospholipids. In each system, the enzymes were added 1 min before the heparin or PPACK. In the absence of heparin or PPACK, all four systems generated the same amount of thrombin activity in 45s. Complete inhibition of prothrombin activation by heparin and PPACK was observed only in system 1 which did not contain exogenous thrombin or factor Xa. No inhibition by heparin or PPACK was observed when thrombin or factor Xa was added to CAP in systems (2) and (3). Only partial inhibition was observed in system (4) which contained exogenous prothrombi-nase complex. Factor Xa thus provides an effective by-pass mechanism for the activation of factor VIII and factor V in plasma containing therapeutic concentrations of heparin. Our data provide further evidence that the heparin-antithrombin III system is not effective in inactivating factor Xa. These results support the hypothesis that in unactivated normal plasma, the primary anticoagulant effect of heparin is the inhibition of the thrombin-dependent activation of factor V and factor VIII.
A COMPARISON OF DERMATAN SULPHATE AND HEPARIN AS ANTITHROMBOTIC DRUGS
Authors: Merton R E , Barrowcliffe T W , Thomas D P
Published: August 2018

Dermatan sulphate (DS) has been shown to accelerate thrcmbin inhibition by its action on heparin cofactor II (HCII) but has no effect on anti thrombin III (Tollefson et al., 1983). In this study, we have examined the in vitro anticoagulant effect of a purified preparation of DS (free from heparin and heparan sulphate), m comparison with that of unfractionated heparin (UEH). We have also studied the effect of DS and UEH in preventing experimental venous thrombosis in rabbits and in inhibiting thrombin generation, both in vitro and in ex vivo plasma samples.

Dermatan had low activity in vitro by APTT and anti-Xa assays (< 5 iu/ mg). When thrombin generation was measured in vitro, 1 μg/ml UEH was sufficient to inhibit thrombin formation. Although 1 μg/ml DS reduced thrombin generation to 40% of control values, there was no further reduction when the concentration of DS was increased to 8 μg/ml.

When DS was injected into rabbits (n = 10), a dose of 150 μg/kg inpaired thrombogenesis m a Wessler stasis model. The mean thrombus score was reduced to 25% of the control values, although thrombosis could not be completely prevented, even after an eight-fold increase m dose (1250 μg/kg). When the duration of stasis was extended from 10 to 20 minutes, there was no impairment of thrombosis (mean thrombus score 100%) following 1250 μg/kg of DS. Thrombin generation measured in ex vivo plasma after 150 μg/kg of DS was 72% (s.e.m. 63-81) of that measured in pre-injection plasma. In contrast, 150 μg/kg of heparin prevented thrombosis after both 10 and 20 minutes stasis (mean score 0%) and thrombin generation was reduced to 17% (s.e.m. 12-23) of control values m ex vivo plasma samples.

Unlike heparin, DS does not completely abolish thrcmbin generation in vitro and is not as potent as UEH in inhibiting thrombin generation m ex vivo plasma. While DS has demonstrable antithrombotic activity under defined conditions, it is less effective than heparin and increasing doses of DS do not improve antithrombotic effectiveness in this model.
CONFORMATIONAL EQUILIBRIA IN THE γ CHAIN COOH-TERMINUS OF HUMAN FIBRINOGEN
Authors: Cierniewski C S , Budzynski A Z
Published: August 2018

Synthetic peptides and fragments cleaved from native fibrinogen are used in studies to localize binding sites for various ligands. We addressed the question how the native conformation of a selected γ chain segment is affected by scission of the original chain. The conformation of the γ chain COOH-terminus of intact fibrinogen and its various fragments containing this region has been compared by an immunochemical analysis. An antibody population specific for the native epitope within the γ391-405 segment was isolated by affinity chromatography on the corresponding synthetic peptide. Between 19.2 and 22.8% of antibodies were obtained from three different antisera indicating that this region represents one of the major epitopes of native fibrinogen. Anti-γ391-405 antibodies were used to determine the value of Kconf the equilibrium constant for the interconversion of the non-native and native conformations of this epitope. The measurements were done using native fibrinogen, fragments D1 and DD, γ chain and γ391-405 synthetic peptide. In addition, the effect of 5 M guanidine-HCl on the conformation of fragments D1 and DD, which is known to abolish their antipolymerizing activity, was studied. Radioiodinated fibrinogen was used in the determination of Kconf, and quantitative analytical parameters, CI50% and CIs, calculated from competition between 125I-fibrinogen and the fibrinogen derivatives under study for binding to the immunochemically purified antibody. The measurements indicated that the epitope is unperturbed by iodination of fibrinogen and that 38.3% of fragment D1, 8.9% of fragment DD, 3.6% of the γ chain and less than 0.008% of the γ391-405 molecules adopt in aqueous solution the native conformation within the epitope. Denaturation of fragment D1 with 5 M guanidine-HCl affected only slightly the conformation of this γ chain determinant. More significant changes in the conformation were observed when fragment DD was denatured. The results suggest that long-range interactions are necessary for the stabilization of the native structure in the region of fibrinogen that interacts with the antibody and which is in close vicinity to the polymerization site, crosslinking site, and platelet recognition site.