bluuebunny/crossref_metadata_2025 huggingface dataset:
Urokinase versus Recombinant Tissue Plasminogen Activator in Thrombosed Central Venous Catheters:
Authors: Haire William D , Atkinsonn James B , Stephens Lana C , Kotulak Gall D
Published: July 2018

Fifty dysfunctional central venous catheters proven radiographically to be occluded by thrombus were blindly randomized to be injected with either 2 mg recombinant tissue plasminogen activator (t-PA) or 10,000 units of urokinase (UK) and allowed to incubate for 2 h. A second dose was allowed if catheter function was not restored with the first injection. Repeat radiograph contrast injection was done when catheter function was restored or after 2 doses of study drug were administered, whichever occurred first. Thirteen of 22 catheters randomized to UK had full function restored compared to 25 of 28 randomized to t-PA (p = 0.013). Radiographic contrast injection showed 7 catheters randomized to UK had complete resolution of the thrombus compared to 17 randomized to t-PA (p = 0.042). Four catheters randomized to UK had complete resolution of the thrombus after a single dose compared to 13 randomized to t-PA (p = 0.036). A novel dose of 2 mg of t-PA restored catheter function more reliably and dissolved thrombi faster than twice the standard, FDA-approved dose of UK
Antithrombin III: Associations with Age, Race, Sex and Cardiovascular Disease Risk Factors
Authors: Conlan Maureen G , Folsom Aaron R , Finch Andrea , Davis C E , Marcucci Gretchen , Sorlie Paul , Wu Kenneth K
Published: July 2018

Antithrombin III (AT III) is a major inhibitor of blood coagulation, and hereditary deficiency is associated with venous thrombotic disease. The Atherosclerosis Risk in Communities (ARIC) Study, a prospective study of 15,800 middle-aged men and women, measured AT III in its baseline examination. AT III levels were significantly higher in women than men, and in blacks than whites. AT III decreased with age in men but increased with age in women. In age- and race-adjusted analyses, AT III was positively associated with smoking, HDL-cholesterol, triglycerides (men only), and in women, with diabetes and lipoprotein^). AT III was negatively associated with educational level, body mass index in men, and use of female hormones in women. Most of these associations were confirmed in multivariate analysis. These correlations between AT III and other risk factors must be considered when evaluating AT III as a risk factor for venous or arterial thrombosis.
The Platelet Adhesion Capacity to Subendothelial Matrix and Collagen in a Flow Model during Storage of Platelet Concentrates for 7 Days
Authors: Boomgaard M N , Gouwerok C W N , Homburg C H E , de Groot Ph G , IJsseldijk M J W , de Korte D
Published: July 2018

The influence of storage of platelet concentrates (PC) on the adhesion capacity of platelets was studied. Twenty-four PC, 12 prepared by the buffy coat (BC) method and 12 by the platelet-rich plasma (PRP) method, were stored for 7 days at room temperature. On days 1,3 and 7 of storage, the platelet adhesion capacity to subendothelial matrix (SEM) and collagen was studied in a rectangular perfusion system under flow conditions in conjunction with the platelet aggregation capacity after stimulation and the adenine nucleotide content. The platelet adhesion capacity to collagen was constant until day 3 of storage and decreased to about 80% of the starting value on day 7 of storage. The adhesion capacity to SEM, however, had already decreased on day 3 to about 75% of the value of day 1 and was even more decreased on day 7 to about 45% of the starting value. On day 1, platelets prepared by the BC method displayed a higher adhesion capacity to collagen and a higher aggregation capacity after stimulation by collagen alone or in combination with ADP, compared to platelets prepared by the PRP method. No other significant differences in adhesion or aggregation capacity were observed between the PC prepared by the two different methods. Both platelet adhesion and aggregation response decreased during storage, as did the total adenine nucleotide content. This study shows that platelet function, as measured by the aggregation and adhesion capacity, of platelets prepared by the PRP method is more severely impaired during the first 3 days of storage as compared to the function of platelets prepared by the BC method.
In Vivo Antithrombotic Effect of Triflavin, an Arg-Gly-Asp Containing Peptide on Platelet Plug Formation in Mesenteric Microvessels of Mice
Authors: Sheu Joen-Rong , Chao Seh-Huang , Yen Mao-Hsiung , Huang Tur-Fu
Published: July 2018

Triflavin, an Arg-Gly-Asp-containing snake venom peptide, inhibits platelet aggregation through the blockade of fibrinogen binding to the activated platelets. In this study, platelet thrombus formation was induced by irradiation of the mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. Electron microscopy reveals moderately damaged endothelial cells, as well as aggregates consisting almost exclusively of platelets with pseudopod formation, and degranulated appearance. Triflavin (10-20 µg/g) significantly prolonged the lag period of inducing platelet plug formation in mesenteric venules when it was intravenously infused. Triflavin (20 µg/g) prolonged the occlusion time about 2-fold (from control 112 ± 23 to 240 ± 47 s). Furthermore, PGE, briefly prolonged the occlusion time about 1.5-fold (from 105 ± 21 to 168 ± 20 s) when it was given by continuous infusion (40 µg/kg/min). On the other hand, triflavin was also effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at dose of 2-4 µg/g. Heparin (1.5 U/g) and indomethacin (200 µg/g) had no significant effect in prolonging the occlusion time or in reducing ADP-induced pulmonary embolism in mice. Therefore, triflavin is an effective antithrombotic agent in preventing the thromboembolism in these two in vivo models.
Oral Anticoagulation in the Prevention of One-Year Vein Graft Occlusion after Aortocoronary Bypass Surgery: Optimal Therapeutic Range and Practical Limitations
Authors: van der Meer J , Hillege H L , Dunselman P H J M , Mulder B J M , Michels H R , Buser P T , van Gilst W H , Lie K I ,   the CABADAS Research Group of the Interuniversity Cardiology Institute of The Netherlands
Published: July 2018

To assess the optimal level of oral anticoagulation to prevent occlusion of vein coronary bypass grafts, 318 patients from a graft patency trial were analysed retrospectively. Oral anticoagulant therapy was started one day before surgery and continued for one year, after which graft occlusion was assessed by angiography. The aimed level of anticoagulation was 2.8-1.8 International Normalized Ratio (INR). Clinical outcome was assessed by the incidence of myocardial infarction, thrombosis and major bleeding.

The observed anticoagulation level was 2.8-4.8 INR for 54%, and 1.8-3.8 INR for 75% of time per patient. Occlusion rates in patients who spent <35, 35-70, and ≥70% of time within INR range 2.8-1.8 were 10.5%, 10.8% and 11.8%, respectively (differences not statistically significant). Patients who spent ≥70% of time within INR range 1.8-3.8 versus 2.8-4.8 showed comparable occlusion rates. The risk of graft occlusion was not related to quality of anticoagulation early (0-3 months) or late (3-12 months) after surgery. Myocardial infarction, thrombosis and major bleeding occurred in 1.3%, 2.0% and 2.9% of patients.

To maintain vein graft patency in the first postoperative year by oral anticoagulation, a level within INR range 1.8-3.8 for ≥70% of time seems to be sufficient.
Elevated Levels of Factor VII Activity in the Postprandial State: Effect of the Factor VII Arg-GIn Polymorphism
Authors: Silveira Angela , Green Fiona , Karpe Fredrik , Blombäck Margareta , Humphries Steve , Hamsten Anders
Published: July 2018

A genetic polymorphism (Arg/Gln353) of coagulation factor VII was recently identified and shown to be associated with differences in basal factor VII coagulant activity. Postprandial lipaemia seems to exert an acute but evanescent effect on the activity of factor VII, and the influence of the Arg/Gln353 polymorphism on factor VII activation during postprandial lipaemia was therefore studied in male post-infarction patients [age 48.8 ± 3.3 years (mean ± SD)] with Arg/Arg (n = 23) and Arg/Gln (n = 8) genotypes. Factor VII antigen (VIlag) and activity along with plasma lipoproteins were determined before and after intake of a mixed meal-type of oral fat load. Patients with the Arg/Gln genotype had basal VIlag and activated factor VII (Vila) levels 75% and 48%, respectively, of those of patients homozygous for the Arg allele. In absolute terms, Vila increased more in homozygotes for the Arg allele (AO-6 h Vila 1.76 ± 1.48 ng/ml) than in heterozygotes (0.60 ± 0.27 ng/ml) in response to fat intake, but the percentage increase in Vila molecules did not differ significantly between subjects with Arg/Arg and Arg/Gln genotypes (37 ± 32% versus 27 ± 15%). This suggests that the influence of the Arg/Gln polymorphism on factor VII activity is mainly accounted for by differences in the basal factor VII protein level between genotypes. Since most of our lives are spent in the postprandial state, possession of the factor VII-Gln353 allele is likely to confer protection against coronary heart disease by reducing the amount of Vila produced in response to fat intake.
Heparin Enhances Endothelial Cell von Willebrand Factor Content by Growth Factor Dependent Mechanisms
Authors: Tannenbaum Susan H , Chao Elizabeth S , Gralnick Harvey R
Published: July 2018

Von Willebrand factor (vWf), a multimeric adhesive glycoprotein synthesized, stored, and secreted in megakaryocytes and endothelial cells, is normally found in plasma, platelets and subendothelium. While many substances mediate the release of vWf from endothelial cells, factors that enhance vWf synthesis and partitioning to its regulated pathway are currently unknown. We studied the effect of pharmacologic doses of heparin on the vWf content of endothelial cells. After a lag of 8 h and in the presence of crude or purified growth factor, heparin at doses between 0.25 and 2 U (1.4-11 μg)/ml, increased the content of high moleculr weight vWf. The increased amounts of vWf were localized to Weibel-Palade bodies and extracellular matrix. Lower molecular weight highly sulfated heparin or heparin-like compounds were most active in growth factor dependent endothelial cell vWf expression. There was no clear importance of polysaccharide sequence or protein core.
Epidemiology of Haemophilia in Greece: An Overview
Authors: Koumbarelis Emmanuil , Rosendaal Frits R , Gialeraki Argyri , Karafoulidou Anastasia , Noteboom Willy M P , Loizou Calliopi , Panayotopoulou Catherine , Markakis Constantine , Mandalaki Titica
Published: July 2018

Demographic data of the Greek haemophilia A and B population for the period 1972-1993 were analyzed. Prevalence at birth including known not-registered patients was calculated at 23.1 per 100,000 male births. However, the observed prevalence in 1993 was only 61% of the expected. Since 1975 the proportion of mild cases had significantly increased. Adjusted by age, severity and HIV status reproductive fitness of haemophiliacs was 0.62. Overall mortality was 2.6 times higher than in the general population, but 7.9 times among patients with severe haemophilia and 16.4 among HIV(+) haemophiliacs. Fifty out of 78 deaths occurred among HIV(+) patients and 28 of these were caused by AIDS. Inhibitor patients did not show excess mortality due to bleeding. Cancer mortality was equal to normal, but the number of deaths from ischaemic heart disease was 0.25 of the expected. Risk of death due to cerebral haemorrhage was 3.8 times higher in HIV(+) haemophiliacs than in HIV(-).
The Venous Antithrombotic Profile of Naroparcil in the Rabbit
Authors: Millet Jean , Theveniaux Jocelyne , Brown Neil L
Published: July 2018

The venous antithrombotic profile of naroparcil or (4-[4-cyanoben-zoyl]-phenyl)-1.5-dithio-β-D-xylopyranoside was investigated in the rabbit following single i. v. and oral administration. Naroparcil attenuated thrombus development in a Wessler stasis model of venous thrombosis (jugular vein) employing bovine factor Xa as a thrombogenic stimulus giving ED50 values of 21.9 mg/kg and 36.0 mg/kg after respectively i. v. and oral administration. Venous antithrombotic activity was maximal 2-3 h after i. v. administration and 4-8 h after oral administration. Four hours after the oral administration of maximal antithrombotic (Wessler model, factor Xa) doses (100 and 400 mg/kg), naroparcil had no significant effect on bleeding time. In platelet poor plasma obtained from animals treated 4 h previously with various doses (25 to 400 mg/kg) of naroparcil, there was no detectable anti-factor Xa nor antithrombin activity. Similarly, naroparcil had no effect on APTT nor on thrombin time. A sensitized thrombin time (to about 35 s) was modestly but significantly increased following oral administration of the compound at 400 mg/kg. However, thrombin generation by the intrinsic pathway was reduced in a dose-related manner, maximal reduction being 65% at 400 mg/kg. The same doses of naroparcil enhanced the formation of thrombin/heparin cofactor II complexes at the expense of thrombin/antithrombin III complexes in plasma incubated with (125I)-human a-thrombin and induced the appearance of dermatan sulfate-like material in the plasma of treated rabbits, as measured by a heparin cofactor II-mediated thrombin inhibition assay. The results suggest that naroparcil could have a safe venous antithrombotic profile following oral administration (antithrombotic effect compared to bleeding risk). It is probable that part of the mechanism of action of the β-D-xyloside, naroparcil, is due to the induction of chondroitin sulfate-like glycosaminoglycan biosynthesis, this material being detectable in the plasma.
Laboratory Diagnosis of APC-Resistance: A Critical Evaluation of the Test and the Development of Diagnostic Criteria
Authors: de Ronde Hans , Bertina Rogier M
Published: July 2018

The APC-resistance test consists of two APTT’s, one in the presence and one in the absence of a fixed amount of Activated Protein C (APC), and is a simple and reliable method to detect a reduced sensitivity to the anticoagulant action of APC (APC-resistance). At a fixed concentration of APC the prolongation of the APTT is dependent on the activator, the CaCl2 concentration, the citrate concentration in the sample, and on sample handling. The effect of sample handling can be reduced by calculating the APC-Sensitivity Ratio (APC-SR). The actual prolongation of the APTT is also influenced by low protein S levels (reduction of APC-SR) and by reduced levels of factors V, VIII and IX (increase of APC-SR). The APC-SR is most dramaticly effected by reduced levels of factors II and X, which result often in “unmeasurable” APC-SR’s in plasmas of patients on oral anticoagulant treatment. So at present no reliable APC-SR’s can be measured in these patients. Patients treated with heparin can be tested after treatment of their plasma with Hepzym®. The inter- and intra-assay variation in the APC-SR is 4% and 2%, respectively, when using the same batches of activator and APC. The variation which is introduced in the APC-SR by use of different batches of activator or APC, or by the use of different APC or CaCl2 concentrations, can effectively be avoided by expressing the result of the test in normalized-APC-SR (n-APC-SR).

The diagnosis of APC-resistance is defined by a n-APC-SR < 0.84. Patients who are heterozygous for the Factor V Leiden mutation have a n-APC-SR of 0.45-0.70, while patients who are homozygous for the mutation have a n-APC-SR <0.45.
Effects of Unfractionated and Low Molecular Weight Heparins on Platelet Thromboxane Biosynthesis “In Vivo”
Authors: Landolfi Raffaele , De Candia Erica , Rocca Bianca , Ciabattoni Giovanni , Antinori Armando , Masetti Riccardo , Patrono Carlo
Published: July 2018

Several “in vitro” and “in vivo” studies indicate that heparin administration may affect platelet function. In this study we investigated the effects of prophylactic heparin on thromboxane (Tx)A2 biosynthesis “in vivo”, as assessed by the urinary excretion of major enzymatic metabolites 11-dehydro-TxB2 and 2,3-dinor-TxB2. Twenty-four patients who were candidates for cholecystectomy because of uncomplicated lithiasis were randomly assigned to receive placebo, unfractionated heparin, low molecular weight heparin or unfractionaed heparin plus 100 mg aspirin. Measurements of daily excretion of Tx metabolites were performed before and during the treatment. In the groups assigned to placebo and to low molecular weight heparin there was no statistically significant modification of Tx metabolite excretion while patients receiving unfractionated heparin had a significant increase of both metabolites (11-dehydro-TxB2: 3844 ± 1388 vs 2092 ±777, p <0.05; 2,3-dinor-TxB2: 2737 ± 808 vs 1535 ± 771 pg/mg creatinine, p <0.05). In patients randomized to receive low-dose aspirin plus unfractionated heparin the excretion of the two metabolites was largely suppressed thus suggesting that platelets are the primary source of enhanced thromboxane biosynthesis associated with heparin administration. These data indicate that unfractionated heparin causes platelet activation “in vivo” and suggest that the use of low molecular weight heparin may avoid this complication.
Hereditary Increase of Plasma Histidine-Rich Glycoprotein Associated with Abnormal Heparin Binding (HRG Eindhoven)
Authors: Hoffmann J J M L , Hennis B C , Kluft C , Vijgen M
Published: July 2018

Plasma histidine-rich glycoprotein (HRG) was found to be persistently increased in a patient with a history of recurrent arterial thromboembolic events. The mean concentration was 270% of normal pooled plasma. Increased HRG was found in eight of the 17 relatives studied, but none of them has experienced thrombo-embolism yet. Apparently, increased HRG was hereditary with autosomal dominant inheritance. A significant correlation was found between the increased plasma concentration of the protein and the age of the subjects (P<0.02), whereas no such relation is present in a normal population.

The plasma HRG of the proposita and 9 of her family members displayed abnormal binding to heparin, as assessed in a crossed affinity immuno-electrophoresis system: the usual increase in mobility after binding to heparin was absent. The binding of this variant HRG to plasminogen was normal. This case represents the first abnormal HRG variant reported and it is proposed to designate it: HRG Eindhoven.
The Role of β-Fibrinogen Genotype in Determining Plasma Fibrinogen Levels in Young Survivors of Myocardial Infarction and Healthy Controls from Sweden
Authors: Green Fiona , Hamsten Anders , Blombäck Margareta , Humphries Steve
Published: July 2018

β-fibrinogen G/A-455 polymorphism genotype was determined in 123 young survivors of myocardial infarction and 86 healthy controls from Sweden. Frequency of the A-455 allele was 0.25 (95% Confidence Interval 0.21–0.29) in both patients and controls, significantly higher than that reported previously (0.19, 95% Cl 0.16–0.22) in 292 men from the UK (1). Mean plasma fibrinogen level was significantly higher in patients (3.6 g/1) than in controls (3.1 g/1), with p <0.001. Genotype was significantly associated with (adjusted) plasma fibrinogen level in controls (p = 0.003). This effect was confined to the smokers (n = 50) where mean fibrinogen level was significantly (p = 0.001) higher in G/A-455 and A/A-455 individuals (3.5 g/1) compared with G/G-455 homozygotes (2.9 g/1). There was a significant interaction between smoking status and genotype in determining plasma fibrinogen levels in the controls. These data provide independent confirmation of the association between β-fibrinogen G/A-455 genotype and plasma fibrinogen levels previously observed in healthy men from the UK (1).
Interaction of Immobilised Unfractionated and LMW Heparins with Proteins in Whole Human Plasma
Authors: Zammit Adrian , Pepper Duncan S , Dawes Joan
Published: July 2018

The profile of proteins bound to immobilised heparins in hirudin-anticoagulated human plasma was analysed. In molar terms, antithrombin III was the most abundant protein bound to therapeutic doses of unfractionated heparin (Mr = 12,000), whereas heparin cofactor II constituted <1% of the protein bound. Histidine-rich glycoprotein was the only plasma protein likely to influence anticoagulant activity by direct competition with antithrombin III, though significant quantities of complement Factor H, fibrinogen, fibronectin, vitronectin and apolipo-protein B were also detected. Only traces of von Willebrand factor, complement factor I, inter-α-trypsin inhibitor, a2-macro-globulin, serum amyloid P and transferrin were identified, and neither thrombospondin nor platelet factor 4 were measurable. Binding of both antithrombin III and histidine-rich glycoprotein varied with the ratio of heparin to plasma. Clexane (Mr = 4,500) also bound anti thrombin III, but both histidine-rich glycoprotein and vitronectin were quantitatively significant neutralising proteins. Neutralising proteins dominated the binding profile for Oligo H (Mr = 2,200).
The Influence of Oral Contraceptives on the Time-Integral of Thrombin Generation (Thrombin Potential)
Authors: Rotteveel R C , Roozendaal K J , Eijsman L , Hemker H C
Published: July 2018

The thrombin potential (TP) has been defined as the time integral of (i.e. the area under) the thrombin generation curve. It has been shown that this parameter decreases with all types of anticoagulant treatment and increases with ATIII deficiency.

We evaluated the use of this parameter for detection of the hypercoagulative state known to accompany oral contraception.

In fresh frozen control plasma the TP could be determined with high reproducibility (n = 82, c.v. 2.9%). The TP was linearly diminished by mixing fresh frozen plasma with prothrombin deficient plasma while a high coefficient of correlation was observed (r = 0.997).

Women using oral contraceptives showed a significantly (p <0.0001) higher TP (TP = 569 nM·min, SD = 55, n = 17), compared to men or, to women not using oral contraceptives (TP = 484 nM·min, SD = 52, n = 41).

This suggests that the thrombin potential indicates the prethrombotic state known to exist in women using oral contraceptives.
Degranulation of Human Platelets by the Thrombin Receptor Peptide SFLLRN: Comparison with Degranulation by Thrombin
Authors: Kinlough-Rathbone R L , Perry D W , Guccione M A , Rand M L , Packham M A
Published: July 2018

A new, simplified method of degranulating human platelets using the thrombin receptor peptide SFLLRN (20 |iM) is described; released fibrinogen cannot be converted to fibrin, and the platelets are not exposed to a proteolytic enzyme, as they are when thrombin is used for degranulation. The peptide-degranulated platelets regain their disc shape and are recovered as single platelets which have released approximately 90% of the contents of their dense granules. Their procoagulant activity is greater than that of control platelets, but somewhat less than that of thrombin-degranulated platelets. Without added fibrinogen, the peptide-degranulated platelets aggregate slightly in response to 50 μM SFLLRN, and to collagen, arachidonic acid, the thromboxane A2 mimetic U46619, platelet activating factor, ADP, and the divalent cation ionophore A23187; added fibrinogen enhances aggregation caused by these agonists. Extensive aggregation of peptide-degranulated platelets is caused by thrombin in the absence of added fibrinogen; it may be that the alternative thrombin receptor that is not activated by SFLLRN is responsible for the strong response to thrombin. Aggregation responses to most of the agonists are greater than those observed previously (10) with thrombin-degranulated platelets. By this method, platelets are obtained that have been degranulated in a way that is similar to in vivo degranulation. They are useful for studies of platelet responses without the complicating effects of released granule contents, and for investigation of the characteristics and functions of platelets that have come in contract with release-inducing agents in vivo.
Thapsigargin Amplifies the Platelet Procoagulant Response Caused by Thrombin
Authors: Smeets E F , Heemskerk J W M , Comfurius P , Bevers E M , Zwaal R F A
Published: July 2018

The platelet procoagulant response involves an increase in surface-exposed phosphatidylserine, which allows binding and assembly of enzyme complexes of the coagulation pathway resulting in acceleration of the clotting process. This response essentially requires the presence of extracellular Ca2+, and varies in extent with the type of agonist used. In the present paper we demonstrate that the moderate procoagulant response of human platelets caused by thrombin is strongly amplified by the presence of thapsigargin, an inhibitor of the microsomal Ca2+-ATPase. Thapsigargin, like thrombin, has only a weak effect on procoagulant activity. The large increase in procoagulant activity observed with the combined action of these two agonists is associated with increased shedding of microvesicles from the platelet plasma membrane as well as with inhibition of transport of a fluorescent-labeled analog of phosphatidylserine from the outer to the inner leaflet of the plasma membrane by the aminophospholipid translocase. The latter two observations support current concepts regarding the mechanism of development of procoagulant activity.

Although the synergistic effect of thapsigargin on thrombin-induced procoagulant activity is at least in part due to the high levels of intracellular [Ca2+] evoked by these agonists, the data clearly indicate that a rise of the intracellular [Ca2+] is insufficient to completely explain this response. The present findings suggest that additional factors control expression of procoagulant activity upon stimulation of platelets by thrombin.
The Effect of Interleukin-4 on Tumour Necrosis Factor-Alpha Induced Expression of Tissue Factor and Plasminogen Activator Inhibitor-1 in Human Umbilical Vein Endothelial Cells
Authors: Martin N B , Jamieson A , Tuffin D P
Published: July 2018

The pro-inflammatory cytokine tumour necrosis factor-a (TNF-α) is able to alter the haemostatic balance of human umbilical vein endothelial cells (HUVECs) towards that of a procoagulant and anti-fibrinolytic state. Treatment of HUVECs in culture with human recombinant TNF-α (0.5-50 U/ml; 6 h) significantly increased total cell expression of tissue factor (TF) 10-fold from 40 mU/well to 400-500 mU/well. Levels of plasminogen activator inhibitor-1 (PAI-1) antigen secreted from HUVECs also increased up to 2-fold in concentration-dependent fashion following addition of TNF-α (10-100 U/ml; 24 h). TNF-α induced total and cell surface expression of TF on HUVECs was significantly inhibited when the cells were pre-incubated with interleukin-4 (IL-4; p <0.001). This effect was time and concentration dependent. Pretreatment of HUVECs with IL-4 for 4 h had no significant effect, but increasing inhibition of total TF expression occurred after 8 and 16 h pre-incubations. Treatment with IL-4 at 20 and 200 U/ml significantly inhibited cell surface TF responses induced by TNF-α, whereas a low concentration (0.2 U/ml) was without effect. In contrast, the production of PAI-1 from HUVECs stimulated by TNF-α (50 U/ml) was unaffected by the presence and/or prior incubation with 200 U/ml IL-4. Thus, IL-4 may regulate the pro-coagulant but not the antifibrinolytic effects of TNF-α at sites of vascular inflammation.
Effect of Type IIB von Willebrand Disease Mutation Arg(545)Cys on Platelet Glycoprotein Ib Binding – Studies with Recombinant von Willebrand Factor
Authors: Inbal Aida , Kornbrot Nurit , Harrison Paul , Randi Anna M , Sadler J Evan
Published: July 2018

Type IIB von Willebrand disease (vWD) is characterized by a selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma due to their abnormally enhanced reactivity with platelets. Several missense mutations in the platelet glycoprotein lb (GPIb) binding domain of vWF were recently characterized that cause type IIB vWD. The effect of type IIB mutation Arg(545)Cys on vWF binding to platelet GPIb was studied using recombinant wild type (rvWFWT) and mutant rvWFR545C expressed in COS-7 cells. In the absence of ristocetin, 50% of rvWFR545C bound spontaneously to platelet GPIb and the binding increased to 70% in the presence of 0.2 mg/ml ristocetin; rvWFWT did not bind significantly under either condition. Botrocetin-induced binding of rvWFR545C was only slightly increased compared to rvWFWT. These data demonstrate that the Arg(545)Cys mutation increases the affinity of vWF for GPIb, resulting in the characteristic gain-of-function type IIB vWD phenotype.
Chrono-Pharmacological Study of Once Daily Curative Dose of a Low Molecular Weight Heparin (200IU antiXa/kg of Dalteparin) in Ten Healthy Volunteers
Authors: Mismetti P , Reynaud J , Tardy-Poncet B , Laporte-Simitsidis S , Scully M , Goodwyn C , Queneau P , Decousus H
Published: July 2018

Low molecular weight heparin (LMWH) is currently prescribed for the treatment of deep vein thrombosis at the dose of 100 IU antiXa/kg twice daily or at a dose of 175 IU antiXa/kg once daily with a similar efficacy. We decided to study the chrono-pharmacology of curative dose of LMWH once daily administrated according to the one previously described with unfractionated heparin (UFH).

Ten healthy volunteers participated in an open three-period crossover study according to three 24 h cycles, separated by a wash-out interval lasting 7 days: one control cycle without injection, two cycles with subcutaneous injection of 200 IU antiXa/kg of Dalteparin (Fragmin®) at 8 a.m. or at 8 p.m. Parameters of heparin activity were analysed as maximal values and area under the curve.

Activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and tissue factor pathway inhibitor (TFPI) were higher after 8 p.m. injection than after 8 a.m. injection (p <0.05) while no chrono-pharmacological variation of anti factor Xa (AXa) activity was observed. Thus the biological anticoagulant effect of 200 IU antiXa/kg of Dalteparin seems to be higher after an evening injection than after a morning injection.

A chrono-therapeutic approach with LMWH, as prescribed once daily, deserves further investigation since our results suggest that a preferential injection time may optimise the clinical efficacy of these LMWH.
Role of the Liver and Kidney in the Desulphation of Heparin in vivo
Authors: Wells Xanthe E , Dawes Joan
Published: July 2018

Heparin radiolabelled with 125I was given intravenously to intact, bilaterally nephrectomised or completely hepatectomised rats in the presence and absence of 1 mg unlabelled heparin/kg. Plasma samples were collected and analysed by gel filtration chromatography and affinity chromatography on Polybrene-Sepharose, which binds sulphated glycosaminoglycans. Radiolabel in the plasma was associated with both intact heparin and fully desulphated macromolecular carbohydrate chains. Levels of intact heparin in plasma from control rats decreased with time at both doses with a concomitant increase in desulphated material. Livers accumulated the greatest amounts of radiolabel on a per organ basis. Hepatectomy both increased levels of radiolabel in plasma and decreased amounts of desulphated heparin produced, indicating that the liver was the major site of desulphation. Even after removal of the liver, the desulphated metabolite comprised approximately 15% of radiolabelled material at all times and doses, suggesting an additional site of GAG desulphation which may be the vascular endothelium. The kidneys contributed significantly to in vivo desulphation of heparin only at the high dose.
Generation and Mechanism of Action of a Potent Inhibitor of Factor XIII Function
Authors: Reed G L , Lukacova D
Published: July 2018

Although known to play a critical role in thrombosis, the precise role of factor XIII in other processes like wound healing and gestation remains to be elucidated. Because a specific, potent inhibitor could help define the function of factor XIII in these processes, or determine the potential benefits of factor XIII suppression in thrombotic disease, we have derived and characterized a monoclonal antibody inhibitor of factor XIII activation. This immunoinhibitor, 9C11, reacts specifically with both plasma and platelet forms of factor XIII. When added to human plasma, either as whole immunoglobulin or as Fab fragments, 9C11 completely prevented thrombin-activated factor XIII activity with small molecular weight substrates like 14C-putrescine. In addition, when clotted with plasma, 9C11 ablated the gamma chain crosslinking of fibrin catalyzed by factor XIII and markedly accelerated the fibrinolysis of plasma clots by urokinase. Studies of the mechanism of action showed that 9C11 inhibited the cleavage and activation of factor XIII by thrombin, but did not affect the catalytic function of previously activated factor XIII. Although it inhibited thrombin activation, experiments indicated that it bound comparably to both factor XIII zymogen and the thrombin-cleaved zymogen, and did not bind to the thrombin cleavage site of the molecule. Taken together these experiments indicated that 9C11 acted as an inhibitor of the thrombin cleavage and activation of the factor XIII A-subunit. Its potency and specificity make it a useful agent for studying thrombin-activatable factor XIII function in biological systems.
Assembly and Activation of the Intrinsic Fibrinolytic Pathway on the Surface of Human Endothelial Cells in Culture
Authors: Lenich Catherine , Pannell Ralph , Gurewich Victor
Published: July 2018

Factor XII has long been implicated in the intrinsic pathway of fibrinolysis, but the mechanism by which it triggers plasminogen activation and targets fibrinolysis has not been established. In the present study, the assembly and function of activated Factor XII (F.XIIa), prourokinase (pro-u-PA), high molecular weight kininogen (H-kininogen), and prekallikrein on human umbilical vein endothelial cells (HUVEC) was investigated. 125I-prekallikrein was shown to bind to HUVEC via receptor-bound H-kininogen in the presence of 50 μM ZnCl2. After the addition of F.XIIa, 78% of the 125I-prekallikrein initially bound to HUVEC was converted to 125I-kallikrein. However, only 6% of the HUVEC-bound 125I-pro-u-PA was thereby activated. This discrepancy was shown to be related to rapid dissociation (>50% within 15 min) of prekallikrein/kallikrein, but not pro-u-PA, from HUVEC. Increasing the level of cell-bound kallikrein increased the portion of cell-bound pro-u-PA activated, indicating that their co-localization was important for this pathway. Finally, F.XIIa was shown to trigger plasminogen activation on HUVEC via this pathway. This assembly of reactants on the endothelium suggests a mechanism whereby local fibrinolysis may be triggered by blood coagulation.
Does Low Protein Concentration of Tissue-type Plasminogen Activator Predict a Low Risk of Spontaneous Deep Vein Thrombosis?
Authors: Gram Jørgen , Sidelmann Johannes , Jespersen Jørgen
Published: July 2018

Many reports have demonstrated an abnormal fibrinolysis in a subset of patients with deep vein thrombosis. We have studied systemic global fibrinolytic activity and protein concentrations of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) in plasma of 25 young patients with a previous instance of spontaneous deep vein thrombosis documented by phlebography and in 50 healthy controls. The two populations were comparable with respect to a number of base-line variables (age, height, weight, etc.), while the patients had significantly lower fibrinolytic activity (p <0.02), and significantly higher protein concentrations of t-PA (p <0.0001) and PAI-1 (p <0.0006).

We used probit scale plots to identify the consequence of different cut-off points to separate patients from controls. Reasonable separation could be obtained for t-PA with a cut-off point of 5.2 ng/ml and for PAI-1 18 ng/ml. The sensitivity and specificity for these cut-off points were for t-PA 73% (95% confidence interval 63%-84%) and for PAI-1 67% (confidence interval 55%-77%). The negative predictive value with a cut-off point t-PA concentration of 5.2 ng/ml was 85% (95% confidence interval 70%-94%). We observed a significantly negative association between concentration of t-PA and fibrinolytic activity (rs = -0.47; p <0.005) and also between PAI-1 and fibrinolytic activity (rs = -0.78; p <0.005).

We conclude that a young healthy population is characterized by low protein concentration of t-PA (and PAI-1) compared with young patients with a previous instance of spontaneous vein thrombosis, and we tentatively state that a low protein concentration of t-PA predicts a low risk of spontaneous deep vein thrombosis.
Isolation and Characterization of jararaca GPIb-BP, a Snake Venom Antagonist Specific to Platelet Glycoprotein lb
Authors: Fujimura Y , Ikeda Y , Miura S , Yoshida E , Shima H , Nishida S , Suzuki M , Titani K , Taniuchi Y , Kawasaki T
Published: July 2018

A platelet glycoprotein lb-binding protein (GPIb-BP) was isolated from the snake venom of Bothrops jararaca. Jararaca GPIb-BP showed a single band with Mr of 30,000, and two distinct bands with Mr. of 17,000/13,000 under non-reducing and reducing conditions, respectively, on SDS-polyacrylamide gel electrophoresis. Jararaca GPIb-BP itself induced neither platelet aggregation nor serotonin release from platelets, but specifically bound to GPIb (40,629 ± 2,521 molecules per normal platelet, with Kd 39.1 ± 2.4 nM at saturation). The purified venom protein completely inhibited ristocetin- or botrocetin-induccd von Willebrand factor (vWF) binding, and blocked the bovine vWF binding to GPIb, with IC50 values ranging from 28 to 42 nM, without affecting the platelet aggregation induced by ADP or α-thrombin. 1251-jararaca GPIb-BP binding to GPIb was not altered by the presence of human α-thrombin. Jararaca GPIb-BP at a final concentration of 104 nM totally abolished vWF-dependent shear- induced platelet aggregation (SIPA) at a high shear stress, but had no effect on SIPA at a low shear stress. Reduced and S-carboxyamidomethylated jararaca GPIb-BP lost its inhibitory activity on SIPA. The NH2-terminal amino acid sequences of the subunits revealed a high degree of homology with those of several Ca2+-dependent lectins, especially to those of two functionally opposite venom proteins, botrocetin (a vWF-modulator) and alboaggregin-B (a GPIb- modulator).
Prolonged Inhibition of Acute Arterial Thrombosis by High Dosing of a Monoclonal Anti-platelet Glycoprotein IIb/IIIa Antibody in a Baboon Model
Authors: Kotzé H F , Badenhorst P N , Lamprecht S , Meiring M , Wyk V Van , Nuyts K , Stassen J M , Vermylen J , Deckmyn H
Published: July 2018

The in vivo activity of MA-16N7C2, the first monoclonal antibody that contains an echistatin-like RGD-sequence and inhibits platelet glycoprotein (GP)IIb/IIIa function, was determined in baboons. A dosefinding study assessing haemostatic variables such as bleeding time and ex vivo platelet aggregation showed that doses of as low as 0.2-0.3 mg/kg resulted in a pronounced effect. The effects were dose-dependent and lasted for several days, implying that MA-16N7C2 is a potent and long-acting GPIIb/IIIa inhibitor. Following the initial studies, the antithrombotic effect of 0.1 and 0.3 mg/kg of the antibody, given as a bolus, was determined in a baboon model of platelet-dependent, arterial-type thrombus formation. In these studies, a thrombogenic device consisting of Dacron vascular graft material was inserted as extension segments into a permanent arteriovenous shunt. The results confirmed the potent and long-lasting antithrombotic effect of MA-16N7C2. Surprisingly, the antithrombotic effect was stronger 48 h after a dose of 0.3 mg/kg administration than on the day of treatment with 0.1 mg/kg, despite the fact that comparable numbers of GPIIb/IIIa receptors were occupied on resting platelets. We postulate that with the high dose of MA-16N7C2 and after an extended period, occupied GPIIb/IIIa may be internalised by the platelets. Upon platelet activation, these receptors become reexposed but arc unable to participate in thrombus formation. This is in contrast to unoccupied internal GPIIb/IIIa receptors early after a low dose of MA-16N7C2.
Characterization of Endothelial Cell Differential Attachment to Fibrin and Fibrinogen and Its Inhibition by Arg-Gly-Asp-Containing Peptides
Authors: Chang Mei-Chi , Wang Bih-Ru , Huang Tur-Fu
Published: July 2018

We investigated the adhesion of human umbilical vein endothelial cells (HUVECs) to fibrin(ogen) molecule of varying structure for identifying sites that mediate cell attachment. Fibrin was prepared either with ancrod which liberates only FPA from fibrinogen, or with thrombin, which liberates both FPA and FPB. Both fibrin preparations equally supported HUVEC attachment. GRGDS, RGD-containing peptides of snake venoms, and monoclonal antibodies against αvβ3 (23C6 and 7E3) inhibited the attachment of HUVECs to fibrin by 65–75%. In contrast, the attachment of HUVECs to fibrinogen was less effective and was almost completely inhibited by both RGD-containing peptides and by antibodies against integrin αvβ3 (85-95% inhibition). The C-terminal dodecapeptide of fibrinogen γ chain (residues 400–411) inhibited minimally the attachment of HUVECs to fibrin. Additionally, the binding of RGD-containing snake venom peptides to HUVECs was both RGD- and divalent-cation-dependent. The IC50s for inhibition of HUVEC attachment to fibrin were 0.09 μM (rhodostomin), 1.54 μM (trigramin) and 1.64 μM (halysin).

These results indicate that fibrin mediated support of cell attachment is independent of the cleavage of FPB from fibrinogen. HUVEC attachment to fibrinogen was almost completely inhibited by RGD-containing peptides and by antibodies against αvβ3. In contrast, the attachment to fibrin was partially resistant to RGD-containing peptides and to the monoclonal antibodies against integrin αvβ3. However, αvβ3 is the major receptor mediating HUVEC attachment to fibrin.
Increased Expression of Inducible Cyclooxygenase-2 in Human Endothelial Cells by Antiphospholipid Antibodies
Authors: Habib Aïda , Martinuzzo Marta E , Carreras Luis O , Lévy-Toledano Sylviane , Maclouf Jacques
Published: July 2018

The effect of IgGs from 4 patients with antiphospholipid antibodies and elevated excretion of urinary 11-dehydro-thromboxane B2 was evaluated on the production of prostacyclin by human endothelial cells in culture. After 6 h incubation, there was no change in 6-keto-prosta-glandin F1α in the supernatant. However patients’ IgGs induced a marked increase in cyclooxygenase (Cox) activity compared to IgGs from 2 normal individuals or a commercial pool of IgGs from normal donors, tested by adding exogenous arachidonic acid. Western blot analysis of the cellular Cox content using antibodies specific for the different forms of the enzymes revealed that patients’ IgGs stimulated the synthesis of the newly described inducible Cox-2 without affecting the constitutive Cox-1. This effect was partially neutralized by preincubating the IgGs with phospholipids. The induction was dependent on the amount of IgGs; it was visible at 2 h and persisted up to 24 h. Analysis of mRNA levels showed a pattern of variation in good agreement with the results obtained for protein. The protein kinase inhibitor H-7 or long-term incubation of cells with PMA strongly reduced the induction. These results suggest that antiphospholipid antibodies may not prevent the potential of the vascular cells from generating higher amounts of prostacyclin in response to acute episodes of thrombosis.
Evaluation of Some Platelet Parameters in a Group of Elderly People
Authors: Sie P , Montagut J , Blanc M , Boneu B , Caranobe C , Cazard J C , Biermé R
Published: July 2018

Platelet production time (PPT), plasmatic β-thromboglobulin (β-TG), platelet density distribution, and mepacrine labelled granular content of the platelets were simultaneously determined in a group of elderly subjects. Vascular deterioration, assessed by the history and clinical examination, was variable in extension and severity. PPT was slightly but not significantly shortened, whereas the raised β-TG level and diminished platelet density and granular content suggested an in vivo release reaction process. The results are not correlated with the apparent severity of vascular deterioration, and the release reaction may not be solely related to enhanced platelet-vessel wall interactions.
Platelet Activation in Myeloproliferative Disorders
Authors: Cortelazzo S , Viero P , Barbui T
Published: July 2018

Beta-thromboglobulin (beta-TG) and platelet factor four (PF4) are specific platelet proteins released when a process of platelet activation occurs.

The present study was undertaken in order to measure beta-TG and PF4 both as absolute plasma value and ratio to platelet number in 69 patients with myeloproliferative disorders (MD). The aim was to establish whether the increase of the two proteins could depend on platelet number or indicated an “in vivo” platelet activation.

In 74% of patients beta-TG was found elevated and PF4 was high in 68% of cases. However in 34.7% and in 31.9% of cases respectively, the elevation of the two platelet markers was correlated to platelet number and the ratio was normal. Only in about one third of cases an “in vivo” platelet activation could be admitted and this finding provides a more rational use of antiaggregating agents.
Studies on the Degradation of Fibrinogen by Proteolytic Enzymes from the Larvae of Lanomia achelous (Cramer)
Authors: Arocha-Piñango C L , Perales J , Carvajal Z
Published: July 2018

Fibrinogen degradation products formed by the action of purified haemolymph and saliva of a Satumidae caterpillar of the Lonomia genus were studies by immunoelectrophoresis and polyacrylamide/SDS gel electrophoresis.

The pattern of degradation differ form the one described for plasmin, trypsin, brinase and ochrase. The most striking difference being the rapid loss of the a chain in spite of the presence of the protease inhibitor aprotinin, and/or denaturalizing agents such as 8 M Urea and 2% SDS.
The Inhibitor of Prothrombin Conversion in Plasma of Patients on Oral Anticoagulant Treatment
Authors: Bertina R M , Westhoek-Kuipers M E J , Alderkamp G H J
Published: July 2018

Pooled plasma of patients under stable oral anticoagulation has been analysed with respect to the presence of the vitamin-K dependent factors (factors II, VII, IX and X). Of all factors 1.5-2 times more antigen than procoagulant activity was present. The concentration of factors II, X (measured spectrophotometrically) and VII is about 0.25 U/ml while factor IX is slightly higher. Coagulation assays of factor X always gave lower values than the spectrophotometric assay. This discrepancy was not influenced by the removal of either factor II-factor VII- or factor IX antigen. However, when the factor X antigen was replaced by normal factor X, all factor X assays gave identical results, indicating that PIVKA X is responsible for these discrepancies. Using the technic of the Thrombotest-dilution curve it was shown that PIVKA X is the factor that causes the abnormal prolongation of ox-brain prothrombin time in these plasmas.