bluuebunny/crossref_metadata_2025_split huggingface dataset:
EFFECTS OF PLASMA KALLIKREIN AND BRADYKININ ON FIBRINOLYSIS AND THROMBOXANE PROSTACYKLIN FORMATION STUDIED IN MINIPIGS
Authors: Egberg Nils , Gréen Krister , Jacobsson Jan , Vester-gvist Ole , Wiman Bjöm , Gallimore Michael 
Published: August 2018

The effect of plasma kallikrein and bradykinin infusions into pigs on hemodynamic and hemostatic variables have been investigated. Both substances caused a pronounced decrease of the systemic blood pressure. The leucocyte count in periferal blood fell markedly, reaching a minimun within one hour after infusion of either of the substances.Signs that could be interpreted as a progressive disseminated intravascular coagulation with decrease of fibrinogen and platelet count was observed after kallikrein as well as bradykinin infusions. A pronounced increase of the plasma tissue plasminogen activator concentration followed both plasma kallikrein and bradykinin infusions. However, the peak concentration was found 5 minutes after bradykinin infusion but 60-120 minutes after kallikrein infusion, suggesting different mechanisms leading to the t-PA release. Concomittant with the maximun t-PA concentration there was a marked reduction of the plasminogen activator inhibitor (PAI) concentration. Three hours after drug infusions the PAI concentration was at or above preinfusion level. Kallikrein infusions caused a 10-20 fold increase of the urinary excretion of 2,3-dinor thromboxane B2 (metabolite of TxA2) and a 3-42 fold increase of 2,3-dinor-6-keto-PGFlalpha (metabolite of PGI2) excretion respectively. Corresponding data for bradykinin infusions were, 1.6-5 fold and 2-10 fold increases respectively. Possible links between leucocyte aggregation, prostanoid formation and fibrinolytic variables will be discussed.
KINETICS OF HEPARIN CO-FACTOR II (HCII):THROMBIN AND ANTITHROMBIN III (ATIII):THROMBIN INTERACTION. MOLECULAR WEIGHT DEPENDENCY
Authors: Ellis V E , Scully M F , Kakkar V V 
Published: August 2018

The influence of increasing concentrations of heparin of different molecular mass (Mr) has been compared in potentiation of the rate of HCII:thrombin interaction and of ATIII:thrombin interaction under pseudo first order conditions. Unfractionated and fractionated heparin showed a concentration dependent ascending and descending limb of stimulation of the rate which was closely similar for both inhibitors. Unfractionated heparin and fractions of 16.5 KDa or less showed a peak acceleration of the rate of interaction of thrombin with both inhibitors at 0.3×10−6 heparin although the observed maximum rate at this peak decreased with fall in Mr. For both inhibitors two high Mr fractions (22KDa and 32KDa) showed peak stimulation at a lower heparin concentration (0.3×10−7M) and 1.5 to 2 fold greater increase in rate than that observed with unfractionated heparin. Under these conditions it could be calculated that the potency of a 32KDa fraction was 1200iu/mg with respect to UF heparin (150iu/mg). Three further pools were prepared and ATIII .high affinity fraction prepared by chromatography. Acceleration of rate of interaction was measured according to concentration and inverse plots gave values for apparent Kd amd maximal rate.These results suggest that differences in the profiles of stimulation by high Mr fractions to those of lower Mr are related to higher binding affinities for the inhibitor permitting maximal binding of heparin before the descending part of the slope due to saturation of thrombin (according to the template hypothesis). Although close similarity was found between heparin stimulation of HCII and ATIII, potentiation of HCII inhibitory activity differed in that it was reversed by lower ionic strength and was not reversed by a heparin pentasaccharide with high affinity for ATIII.
EFFECT OF VARIOUS HEPARIN(OID)S ON HEPARIN COFACTOR II MEDIATED ANTI-THROMBIN ACTIVITY AND INHIBITION OF THROMBIN GENERATION IN VITRO
Authors: van Dinther T G , Hol F , Meuleman D G 
Published: August 2018

The effects of various heparin(oid)s, standard heparin VII (SH), dermatan sulphate (DS), a low molecular weight fraction of heparin (UMW-H), FragminR (FRA), Org 10172 = low molecular weight heparinoid, the fraction of Org 10172 with high affinity for AT-III (HA-10172) and the low affinity fraction (LA-10172) respectively were examined on in vitro thrombin generation and inactivation.Thrombin inactivation in the presence of either heparin cofactor II (HC-II) or anti-thrombin III (AT-III) was assessed with two newly developed assays using the purified cofactors, thrombin and chromogenic substrate S2238 on microtiterplates. Thrombin generation in the presence of HC-II and AT-III was studied using purified factor Xa, prothrombin and blood platelet lysate and the residual thrombin activity was assessed amidolytically.The inhibition of the compounds on thrombin activity are summarized in the tableThe following conclusions can be drawn:- SH, LMW-H, HA-10172 and FRA potentiate the AT-III mediated inactivation of Ha more strongly than the HC-II mediated inactivation.- DS and LA-10172 show the reverse pattern of inactivation, while Org 10172 potentiates both inactivaton pathways to a similar extent.Thrombin generation in the presence of HC-II is inhibited by mw-heparin(oid)s at approx. 2-5 times lower concentrations than the HC-II mediated thrombin inactivation, while the inhibiting effect of SH in both assays is comparable.AT-III mediated thrombin generation inhibition and AT-III mediated thrombin inactivation is comparable as well for SH, LMW-H and FRA. In contrast, Org 10172 and its subfractions are approx. 10 times more potent on AT-III mediated thrombin generation inhibition than on AT-III mediated thrombin inactivation.Org 10172 shows low anti-thrombin activity and this activity is mainly mediated via FC-II.
IN VITRO REACTION MODELS OF THROMBIN AND ITS PHYSIOLOGICAL INHIBITOR ANTITHROMBIN III IN THE PRESENCE OF HEPARIN
Authors: Niederst P N , Asbach M , Ott M , Zimmermann R E 
Published: August 2018

Antithrombin III (AT III) neutralizes thrombin and other serine proteases of plasma coagulation system by forming a stable 1:1 covalent complex. The inhibition rates are greatly increased by the potent catalyst heparin. The catalytic mechanism of heparin was studied in the presence of dextran sulfate (DS), a thrombin-binding sulfated Polysaccharid. DS did not influence the reaction of AT III with heparin and the amidolytic activity of thrombin, but preincubation with thrombin could inhibit the catalytic activity of heparin in the reaction of thrombin with AT III. We conclude that the reaction of heparin with enzyme and inhibitor, thus forming a ternary complex, is necessary for its catalytic activity.It is known that heparin also converts AT III from an inhibitor to a substrate for thrombin in a dose dependent manner. By cleavage of the reaction site bound Arg(385)-Ser(386) an AT III-fragment (MG 50000 d) occurs, which has a decreased affinity to heparin and does not inhibit F I la. At physiological ionic strength we have only measured a small percentage of AT 111-proteolysis (4%, 1 U/ml Hep). The extent of AT III-fragment formation could be enhanced by lowering the ionic strength (max 44%, 1 U/ml Hep., 1=0,02).
ANTITHROMBIN III AND HEPARIN COFACTOR II IN PATIENTS WITH CHRONIC RENAL FAILURE
Authors: Toulon P , Jacquot C , Frydman M O , Vignon D , Aiach M 
Published: August 2018

Anti thrombin III (AT III) and heparin cofactor II (HC II) were measured in 77 blood donors, 82 patients with chronic renal failure (CRF) undergoing regular hemodialysis and 36 undialyzed patients with CRF. AT III was measured as heparin cofactor and HC II as dermatan sulfate cofactor using amidolytic assays.The results (mean ± SD expressed in percentage of the in pooled normal plasma) are summurized in the table.Subnormal AT III levels were found in both dialyzed and undialyzed patients with CRF, while HC II was significantly (p < 0.001) reduced in dialyzed patients (12 of them were found to have HC II levels below to lowest value founded in our control group: 68 %).In order to explain this decrease of HC II level in dialyzed patients with CRF, we compared both AT III and HC II activities before and after a dialysis session in 24 patients (12 with a low and 12 with normal before dialysis HC II activity). AT III and HC II increased significantly (p < 0.01) in all patients after dialysis. When related to total plasma proteins in order to suppress the influence of hemoconcentration induced by dialysis, AT III decreased significantly (p < 0.01) in the 24 patients while HC II increased significantly (p < 0.01) only in patients with low before dialysis HC II levels (the increase in HC II activity was found significantly in the pooled 24 patients).
PROPHYLAXIS OF THROMBOEMBOLISM DURING PREGNANCY IN THREE SISTERS WITH CONGENITAL ANTITHROMBIN III (AT m) DEFICIENCY COMBINED WITH REDUCED INDUCIBLE FIBRINOLYTIC ACTIVITY
Authors: Schoch U , Zanetti E , von Felten A 
Published: August 2018

AT III deficiency is associated with a high risk of venous thromboembolism, particularly in pregnancy. As prophylactic treatment it has been recommended to normalize plasma levels of AT in by use of AT III concentrates, besides giving heparin.We report on the prophylactic treatment of three sisters (age 21, 25, 32 years) with congenital AT in deficiency (38-53%, normal: 80-120%) as well as a reduced inducible fibrinolytic activity (1.2, 5.8, 7.9%, normal: >8.5%), who already had suffered from severe thromboembolism. During pregnancy prophylactic measures were taken individually, depending on the plasma level of 3-throm-boglobulin (BTG) determined every 2-3 weeks. At the time of the first increase of BTG (around 10th week of gestation) prophylaxis with s. c.heparin 2x7'500IU/d was started, leading to normalization of BTG. When BTG was again elevated, the dose of heparin was successively increased up to 2x15'000IU/d; thereby, functional AT IH levels remained in the range of 28-50%. Two patients received only heparin throughout the pregnancy. However, in one patient BTG levels could not be normalized by heparin alone (60-130ng/ml, normal: <43ng/ml). Injections of AT in concentrate, 11000IU, led to reduction of BTG within 2 hrs (60 → 42, 220 → 61 ng/ml). Therefore, AT m was given from the 25th week of gestation in increasing amounts up to 5'000IU/week (funct. AT m in plasma: 51 -72%) in addition to heparin (2x12'500IU/d), resulting in BTG levels of 33-51 ng/ml. From the onset of labour, all patients received AT IU concentrates (two patients l'OOOIU every second day, one patient 1'0001U daily) together with i. v. heparin 20'000-27'500IU/d, until the oral anticoagulant treatment started after delivery had reached therapeutic levels. The amount of AT m concentrate totally administered was 5'000IU in two patients and 66'000IU in one patient. None of the patients showed ever signs of venous thrombosis.Our observations demonstrate that instead of an AT in substitution with the aim to normalize its plasma level, an effective thrombosis prophylaxis - monitored by plasma levels of BTG - may be achieved with heparin alone or combined with low amounts of AT in concentrate.
RANDOMIZED TRIAL OF ANTITHROMBIN III VERSUS PLACEBO IN PATIENTS UNDERGOING PERITONEO-VENOUS SHUNT OPERATION
Authors: Korninger C , Klepetko W , Miholic J , Schwarz Ch , Lechner K 
Published: August 2018

A randomized trial of Antithrombin III (AT III) versus placebo was performed in patients undergoing peritoneo-venous shunt operation because of intractable ascites. 10 patients with alcoholic liver cirrhosis (Child's stages B and C) were enrolled. Randomization was performed according to the stage of liver disease and to preoperative AT III levels. AT III concentrate (kindly provided by Kabi) was infused in 5 patients, twice daily for 4 days, at a dose of 20 U/kg BW, starting 12 hours prior to operation. Coagulation studies were performed preoperatively, and on postoperative days 1, 3 and 7.In all patients, ethanol gelation test was positive on postoperative days 1 and 3, indicating the presence of fibrin monomers. No difference between the two groups was seen with respect to prothrombin times, coagulation factor levels, FPA concentrations or fibrinolysis parameters. It is concluded that the severity of disseminated intravascular coagulation can be tempered by AT III substitution, but, with the administered dosage, DIC cannot be prevented.
ANIONIC INHIBITION OF PLASMINOGEN ACTIVATION
Authors: Castellino F J , Urano T , Chibber B , de Serrano Sator V 
Published: August 2018

We have found that anions play a significant role in the activation rates of Gluj-plasminogen (GlujPg) by streptokinase (SK) and urokinase (UK), and that these properties of anions can be correlated with their effects on the structure of GlujPg. In the case of activation by SK, a species of activator complex consisting of equimolar SK and GlujPg (SK-Glu]Pg*), that is sensitive to the presence of Cl- and fibrinogen (Fg), exists which decays to another complex of SK and GlujPg (SK-GlujPg'), the activity of which is not influenced by these effector molecules. The overall activation of GlujPg by SK-GlujPg* is inhibited by Cl- and stimulated by Fg. Kinetic studies indicate that Cl- is a mixed type inhibitor of the process, and Fg functions as a mixed type activator.The activation of GlujPg by both high- and low-molecular weight forms of UK is also inhibited by CL-, but is stimulated by e-amino caproic acid (EACA). The inhibition by Cl- does not occur in the presence of concentrations of EACA that saturate its weak binding sites on GlujPg, and the stimulation by EACA is maximally exhibited in the presence of CL-. Based upon structural studies of GlujPg in the presence of a variety of anions, such as Cl-, F-, I-, CNS-, OTs-, IO3
- and OAc-, we find that GlujPg can adopt a conformation (4.8S form) that is optimal to activation by UK, and one that is much less susceptible to such activation (5.8S form). The former occurs in certain salts and appears to correlate inversely with their ability to bind to the protein, or in the presence of any salt, plus saturating levels of EACA. The latter conformation is adopted in direct proportion with the ability of anions to interact with the protein, according to the Hofmeister series.In conclusion, we find that anions serve as inhibitors of activation of GlujPg, effects that are reversed by Fg (for SK) and EACA (for UK). This inhibition, which occurs at physiological concentrations of Cl-, serves to inhibit significantly plasminogen activation in normal circulation. Activation of plasminogen is enhanced upon its binding to surfaces (e.g., fibrin), via its lysine binding sites, an effect which may well contain a contribution from sequestration of Cl- from the protein.
New high capacity lysine-agarose based on divinylsul-fone activated agarose
Authors: Lihme A , Nielsen C S , Christensen U 
Published: August 2018

It is generally acknowledged that plasminogen is best purified on some kind of immobilized lysine. We here present results on plasminogen purification using a new type of lysine-agarose affinity chromatography.Divinylsulfone activated agarose1 were used for alpha-N -immobilization of L-lysine ("Mini leak", KernEuTec, P.O. Box 613, DK-2200). The intrinsic features of this matrix are 2,3:a) High density of active groups, high coupling capacities ( 40- 50mM ) of -NH2,-SH,-OH-groups.b) Very high mechanical stabilityc) Practically no leaking of immobilized ligands.he matrix is useful for fast and effeicient protein-purification in automated systems. Using a FPLC-system and a variety of exp. conditions lysine-mini1eak, compared with lysine-Sepharose (Pharmacia) consistently yielded more than tvice the amount of plasminogen from plasma and serum.1) L. Sundberg and J. Porath, (1975) Portides Biol. Fluids, Proc. Colloq., 23, 49-62.2) A. Lihme et a 1.,( 1 986 ) J. Chromatogr., 376 , 299-305.3) H.J. Allen and E.A.Z. Johnson,( 1 975 )Carbohydr . Res. 58, 253-265.
HETEROGENOUS EXPRESSION OF PLASMINOGEN ACTIVATOR (PA) GENES IN THE HUMAN SARCOMA CELL LINE HT1080
Authors: Laug Walter E 
Published: August 2018

Tumor cell derived PA activities are of crucial importance for tissue invasion and destruction by tumor cells. Therefore, we studied the expression of the PA genes in HT1080 cells using immunoenzymatic methods and specific PA gene probes.Immunenzymatic methods allowed only for the detection of urokinase like PA (u-PA) activities in HT1080 cells which was suppressed by treatment of the cells with dexamethasone (10-7 m). Despite the lack of u-PA activities, the cells still degraded extracellular tissue glycoproteins. Northern blot analysis with specific PA gene probe showed that HT1080 cells express both tissue type PA (t-PA) and u-PA. The enzymatic activities of t-PA were most likely masked by the simultaneous production of inhibitors of PA (PAI). Treatment of HT1080 cells with dexamethasone resulted in increased transcription of t-PA and decreased expression of the u-PA gene, explaining the unchanged tissue destruction by dexamethasone treated HT1080 cells.Cell clones secreting either large or small amounts of enzymatic PA activities were isolated from the parental HT1080 cell line using a fibrin agarose overlay technique.The expression of the u-PA gene was enhanced in high secreting PA clones compared to low secreting PA clones when analyzed on Northern blots. This heterogenous expression of the u-PA gene within the HT1080 cell line was confirmed by in situ hybridization with a specific u-PA gene probe.These findings demonstrate that PA gene expression can be missed with immunenzymatic methods due to simultaneous production of inhibitors of PA. In addition our results show that the expression of a given PA gene may be heterogenous on the cellular level within an established tumor cell line. These findings, therefore, suggest cellular variation of PA gene expression in tumor which may be of fundamental importance for tissue invasion and metastasis by cancer cells.
PREDICTION OF THE THREE-DIMENSIONAL STRUCTURE OF THE ENZYMATIC PART OF T-PA
Authors: Heckel A , Hasselbach K M 
Published: August 2018

Up to now the three-dimensional structure of t-PA or parts of this enzyme is unknown. Using computer graphical methods the spatial structure of the enzymatic part of t-PA is predicted on the hypothesis, the three-dimensional backbone structure of t-PA being similar to that of other serine proteases. The t-PA model was built up in three steps:1) Alignment of the t-PA sequence with other serine proteases. Comparison of enzyme structures available from Brookhaven Protein Data Bank proved elastase as a basis for modeling.2) Exchange of amino acids of elastase differing from the t-PA sequence. The replacement of amino acids was performed such that backbone atoms overlapp completely and side chains superpose as far as possible.3) Modeling of insertions and deletions. To determine the spatial arrangement of insertions and deletions parts of related enzymes such as chymotrypsin or trypsin were used whenever possible. Otherwise additional amino acid sequences were folded to a B-turn at the surface of the proteine, where all insertions or deletions are located. Finally the side chain torsion angles of amino acids were optimised to prevent close contacts of neigh bouring atoms and to improve hydrogen bonds and salt bridges.The resulting model was used to explain binding of arginine 560 of plasminogen to the active site of t-PA. Arginine 560 interacts with Asp 189, Gly 19 3, Ser 19 5 and Ser 214 of t-PA (chymotrypsin numbering). Furthermore interaction of chromo-genic substrate S 2288 with the active site of t-PA was studied. The need for D-configuration of the hydrophobic amino acid at the N-terminus of this tripeptide derivative could be easily explained.
REC. t-PA GENETICALLY MODIFIED AT THE CLEAVAGE SITE OF ONE-CHAIN TO TWO-CHAIN CONVERSION: ENZYMOLOGY AND DIAGNOSTIC APPLICATIONS
Authors: Rånby M , Brändstrôm A , Hansen L , Henson K , Larsen G 
Published: August 2018

Native one-chain t-PA is cleaved by plasmin or by trypsin after the Arg in the sequence -Gln-Phe-Arg-Ile-Lys-. Variants of one-chain t-PA where the -Arg- was replaced by a His (Arg to His) or by a Lys (Arg to Lys) or by a Thr (Arg to Thr) were made through genetic modification. The three mutants and the wild type were expressed in animal cells and purified in the one-chain form by affinity chromatography as was t-PA from Bowes melanoma cells. In contrast to wild type and melanoma t-PA the mutants reacted poorly with polyclonal antibodies raised against the peptide -Gln-Pro-Gln-Phe-Arg-Ile-Lys--Gly-Gly- indicating mutation in the sequence. Of these proteins only the Arg to Thr mutant was resistant to plasmin cleavage as evidenced by SDS-PAGE. t-PA antigen values (ELISA) and fibrinolytic activity values (fibrin clot lysis assay) yielded the following specific activities expressed in IU/|μg: 810 (Arg to His), 640 (Arg to Lys), 290 (Arg to Thr), 810 (wild type) and 660 (melanoma t-PA). The amidolytic activities for the one-chain proteins against D-Ile-Pro-Arg-pNA at pH 9.0 and 37°C, expressed in mOD per minute at 1 M-g/mL of enzyme were: 15.8 (Arg to His), 13.6 (Arg to Lys), 8.3 (Arg to Thr), 10.0 (wild type), 9.6 (melanoma t-PA) as compared to 55.2 for two-chain melanoma t-PA.All mutants including the uncleavable Arg to Thr mutant could be used in determination of PAI activity in plasma samples. Only one-chain t-PA reacts selectively with PAI 1. Thus, use of the Arg to Thr mutant represents a theoretical advantage in PAI 1 activity determination since preparations of this mutant most likely is free of contaminating two-chain t-PA.The plasminogen activation rate as measured in a coupled assay in the presence and absence of fibrin at 0.5 jiM plasminogen and 37°C was measured and the stimulation factor calculated. This was about 950 fold for the Arg to Thr mutant wich was considerably higher than that of melanoma one chain t-PA and the other mutants wich all were about 550 fold. The stimulation factor for melanoma two-chain t-PA was in the same experiment about 120 fold. The extra fibrin sensitivity of the Arg to Thr mutant resulted in improved soluble fibrin assay according to Wiman and Renby Thromb. Haemostas, (1986) 55:189-193.In conclusion: the use of a plasmin insensitive protein-engineered mutant of t-PA gives advantages in assays for PAI 1 and soluble fibrin.