bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Second Laminin G-type Domain of Protein S Is Indispensable for Expression of Full Cofactor Activity in Activated Protein C-catalysed Inactivation of Factor Va and Factor VIIIa
Authors: Evenäs Petra , García de Frutos Pablo , Nicolaes Gerry , Dahlbäck Björn 
Published: March 2018

SummaryVitamin K-dependent protein S is a cofactor to the anticoagulant serine protease activated protein C (APC) in the proteolytic inactivation of the procoagulant, activated factor V (FVa) and factor VIII (FVIIIa). In the FVa degradation, protein S selectively accelerates the cleavage at Arg306, having no effect on the Arg506 cleavage. In the FVIIIa inactivation, the APC-cofactor activity of protein S is synergistically potentiated by FV, which thus has the capacity to function both as a pro- and an anticoagulant protein. The SHBG-like region of protein S, containing two laminin G-type domains, is required for the combined action of protein S and FV. To elucidate whether both G domains in protein S are needed for expression of APC-cofactor activities, chimeras of human protein S were created in which the individual G domains were replaced by the corresponding domain of the homologous Gas6, which in itself has no anticoagulant activity. In a plasmabased assay, chimera I (G1 from Gas6) was as efficient as wild-type recombinant protein S, whereas chimera II (G2 from Gas6) was less effective. The synergistic cofactor activity with FV in the inactivation of FVIIIa was lost by the replacement of the G2 domain in protein S (chimera II). However, chimera I did not exert full APC-cofactor activity in the FVIIIa degradation, indicating involvement of both G domains or the entire SHBG-like region in this reaction. Chimera I was fully active in the degradation of FVa in contrast to chimera II, which exhibited reduced cofactor activity compared to protein S. In conclusion, by using protein S-Gas6 chimeric proteins, we have identified the G2 domain of protein S to be indispensable for an efficient inactivation of both FVIIIa and FVa, whereas the G1 domain was found not to be of direct importance in the FVa-inactivation experiments.
Anticoagulant Activity and Pharmacokinetic Properties of a Sub-cutaneously Administered Mixed Micellar Formulation of Argatroban in Experimental Animals
Authors: Lunven Catherine , Lecoffre Catherine , Lainée Pierre , O’Connor Stephen , André Frédéric , Roger Bénédicte , Garrigou-Gadenne Danielle , Rouchouse Alain , Roome Nigel , Vivan Nathalie , Berry Christopher 
Published: March 2018

SummaryWe have studied the anticoagulant properties of a novel mixed micellar formulation containing 14 mg/ml argatroban administered by the sub-cutaneous (s. c.) route to rats, rabbits, dogs and primates. Blood samples were taken at various times post-treatment for the determination of the thrombin time (TT), Ecarin clotting time (ECT) and the activated partial thromboplastin time (aPTT). Plasma levels of argatroban were determined in the dog and primate.Mixed micelles alone (0.15 M sodium glycocholate and 0.15 M egg lecithin) were without effect on the clotting parameters. The mixed micellar formulation of argatroban dose-dependently increased all three clotting parameters in the rat (1–4 mg/kg), the rabbit (1 and 2 mg/kg), the dog (1 and 2 mg/kg) and the primate (0.25 and 0.5 mg/kg). In each case the TT was the most sensitive parameter, followed by the ECT and the aPTT. The duration of action of argatroban in each species was dose dependent and varied from 3 h in the rat to 6 h in the dog. In the latter, the mixed micelle formulation had a significantly increased plasma half-life and mean residence time without affecting the overall area under the curve. The increases in the clotting time were strongly correlated with the plasma levels of argatroban and were linear across the range of concentrations obtained in the dog and the primate, although the aPTT plasma concentration response curve was very flat. Species differences were noted between the increase in clotting time for a given plasma concentration, with the primate being more sensitive than the dog (e. g. 4.7 times more so in terms of the ECT).Thus, a mixed micellar formulation of argatroban, which markedly enhances its solubility, could be useful as a potential anticoagulant for sub-cutaneous administration.
Tissue Factor Encryption/de-encryption Is not Altered in the Absence of the Cytoplasmic Domain
Authors: Bromberg Michael , Carson Steven 
Published: March 2018

SummarySince the cytoplasmic domain of tissue factor (TF) appears to have a role in TF function beyond coagulation, experiments were conducted to determine whether the cytoplasmic domain also has a role in regulating procoagulant activity of TF present in the cell membrane. TF encryption was quantitated in human YU-SIT1, U87-MG, and mouse 3T3 cells which were transfected for expression of human tissue factor or a construct lacking the cytoplasmic domain (TFCD). Comparison of intact cells (encrypted) with fully disrupted cells (de-encrypted) showed that TF and TFCD were equally encrypted with respect to function in fX activation. Moreover, cells expressing TF and TFCD were indistinguishable in their procoagulant responses to A23187-calcium and varied concentrations of nonionic detergents. TF in membrane vesicles spontaneously shed by U87-MG cells was largely, but incompletely, de-encrypted, and the degree of de-encryption was independent of the cytoplasmic domain. We conclude that the predominant mechanism(s) for encrypting TF procoagulant activity is independent of the cytoplasmic domain.
Role of α5β1 and αvβ3 Integrins on Smooth Muscle Cell Spreading and Migration in Fibrin Gels
Authors: Yee Karen , Schwartz Stephen , Ikari Yuji 
Published: March 2018

SummaryFibrin is found at sites of vascular injury and is one of the major matrix ligands for β3 integrins. Blocking the β3 integrin on smooth muscle cell is hypothesized as a potential target to prevent restenosis because it could inhibit cell attachment and migration into fibrin provisional matrix. Human aortic smooth muscle cells (HNB18E6E7) spread stably in plasma gels within 24 h. Cell spreading was dramatically blocked by simultaneous use of α5β1 and αvβ3 integrin antibodies (P <0.0001), however, blocking of either integrin alone failed to inhibit spreading. GPenGRGDSPCA, which has been considered a specific αvβ3 antagonist, inhibited spreading at 500 µM, suggesting that the peptide blocked both α5β1 and αvβ3. Similarly, invasive migration into fibrin gels was blocked by simultaneous use of both α5β1 and αvβ3 antibodies, however, blocking of either integrin alone failed to effect cell migration. Another migration assay using transwell indicated similar results. In conclusion, both α5β1 and αvβ3 integrins are responsible for smooth muscle cell spreading and migration into fibrin gels. These data suggest that blocking β3 integrin alone would not affect smooth muscle cell interaction with fibrin.
Analysis of the 677 C→T Mutation of the Methylenetetrahydrofolate Reductase Gene in Different Ethnic Groups
Authors: Franco R. F. , Araújo A. G. , Guerreiro J. F. , Elion J. , Zago M. A. 
Published: March 2018

SummaryA recently described mutation in the methylenetetrahydrofolate reductase (MTHFR) gene (a C to T transition at nucleotide 677) is associated with a thermolabile phenotype and decreased enzyme activity. In homozygotes, the mutation is also related to hyperhomocysteinemia and increased risk for atherosclerotic disease and (apparently) venous thrombosis. The prevalence of this mutation in different human populations is unknown. We have investigated the frequency of the 677 C→T mutation in the MTHFR gene in 337 individuals (674 chromosomes) belonging to four ethnic groups: Whites, African and Brazilian Blacks, Asians and Amerindians. The frequencies of the positive allele among Whites and Asians were similar to those previously reported for Caucasian populations. The positive allele seems to be slightly rarer among the Amerindians (frequency 24.0%) in comparison to Whites and Asians, with a heterogeneous distribution among the five Indian tribes analysed. In contrast, the mutation has a very low prevalence in Blacks, especially among the African Blacks, for whom the mutation was absent in homozygosity. Our data indicate that the 677 C→T MTHFR mutation has a significantly heterogeneous distribution among different ethnic groups, a fact that may contribute to explain geographical or racial differences in the risk for vascular disease.
Vitamin K Intake and Sensitivity to Warfarin in Patients Consuming Regular Diets
Authors: Lubetsky Aharon , Dekel-Stern Edit , Chetrit Angela , Lubin Flora , Halkin Hillel 
Published: March 2018

SummaryThe effect of dietary vitamin K intake on warfarin sensitivity is known only from case reports and few small clinical studies. We followed 50 patients commencing warfarin and consuming their regular diets (for 8 weeks) to study this relationship. A one-week recall dietary questionnaire was completed at weeks 2 and 8. Daily intake of nutrients and vitamin K was calculated from standard tables. Warfarin sensitivity index (WSI) was defined as final INR/final warfarin dose (mg/day/m2 of body surface area) (week 8). Vitamin K intake was 17-974 (median: 179) μg/day. Median WSI was 0.82 (0.31-4.47). A WSI value of 1.1 significantly separated excess (≥250 μg/day) from normal (<250 μg/day) vitamin K consumers (16/18 vs. 15/32, respectively, p <0.01). The former had lower day 5 INR (median: 1.9 vs. 3.0, p <0.001), needed more warfarin to achieve INR ≥2.0 (32.0 ± 9.2 mg vs. 25.4 ± 6.4 mg, p = 0.009) and required a higher maintenance steady state warfarin dose (5.7 ± 1.7 mg/day vs. 3.5 ± 1.0 mg/day, p <0.001).We conclude that in 32% (16/50) of anticoagulated patients under usual dietary conditions sensitivity to warfarin is decreased by vitamin K intake ≥250 μg/day.
Solvent Effects on Activity and Conformation of Plasminogen Activator Inhibitor-1
Authors: Egelund R. , Jensen S. , Rodenburg K. W. , Andreasen P. A. 
Published: March 2018

SummaryWe have studied effects of the solvent composition on the activity and the conformation of human plasminogen activator inhibitor-1 (PAI-1) from HT-1080 fibrosarcoma cells. Non-ionic detergents, including Triton X-100, reduced the inhibitory activity of PAI-1 more than 20-fold at 0° C, but less than 2-fold at 37° C, while glycerol partly prevented the detergent-induced activity-loss at 0° C. The activity-loss was associated with an increase in PAI-1 substrate behaviour. Evaluating the PAI-1 conformation by proteolytic susceptibility of specific peptide bonds, we found that the V8-proteinase susceptibility of the Glu332-Ser333 (P17-P16) bond, part of the hinge between the reactive centre loop (RCL) and β-strand 5A, and the endoproteinase Asp-N susceptibility of several bonds in the β-strand 2A-α-helix E region were increased by detergents at both 0 and 37° C. The susceptibility of the Gln321-Ala322 and the Lys325-Val326 bonds in β-strand 5A to papain and trypsin, respectively, was increased by detergents at 0° C, but not at 37° C, showing a strict correlation between proteinase susceptibility of β-strand 5A and activity-loss at 0° C. Since the β-strand 2A-α-helix E region also showed differential susceptibility to endoproteinase Asp-N in latent, active, and reactive centre-cleaved PAI-1, we propose that a detergent-induced conformational change of the β-strand 2A-α-helix E region influences the movements of β-sheet A, resulting in a cold-induced conformational change of β-strand 5A and thereby an increased substrate behaviour at low temperatures. These results provide new information about the structural basis for serpin substrate behaviour.
The Activation of Plasminogen Activator Inhibitor-1 Expression by IL-1β Is Attenuated by Estrogen in Hepatoblastoma HepG2 Cells Expressing Estrogen Receptor α
Authors: Scicchitano Marshall , Kilbourne Edward 
Published: March 2018

SummaryA low estrogen status in postmenopausal women is associated with elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1). In this study, the ability of estrogen compounds to regulate PAI-1 expression was determined in a hepatocyte HepG2 cell line made to stably express estrogen receptor α (ERα). In both the wild type and ER expressing HepG2 cells, estrogen had no effect on basal PAI-1 expression. However, in the ER expressing cells the ability of IL-1β to increase PAI-1 mRNA and protein levels was attenuated by 17β-estradiol, tamoxifen and twelve estrogen components of Premarin. In contrast, the mixed agonist/antagonist raloxifene had weak agonist activity and like the pure antagonist ICI 182780, it dose dependently blocked the effect of 17β-estradiol on IL-1β stimulated PAI-1 levels. These results suggest that estrogen agonists may lower PAI-1 levels in vivo by inhibiting cytokine activated PAI-1 expression by an ER dependent mechanism.
Function of Glycoprotein VI and Integrin α2β1 in the Procoagulant Response of Single, Collagen-Adherent Platelets
Authors: Siljander Pia , Vuist Wim , Breikers Githa , Reutelingsperger Chris , Barnes Michael , Graham Knight C. , Lassila Riitta , Farndale Richard , Heemskerk Johan 
Published: March 2018

SummaryVarious collagen-based materials were used to assess the structural requirements of collagen for inducing the procoagulant response of adhering platelets, as well as the collagen receptors involved. Cross-linked or monomeric collagen-related peptide (CRP), Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly was highly adhesive for platelets in a glycoprotein VI- (GpVI-)dependent manner. Adhesion was followed by a prolonged increase in cytosolic [Ca2+]i, formation of membrane blebs, exposure of phosphatidylserine (PS) and generation of prothrombinase-stimulating activity. Fibrils of type-I collagen were less adhesive but, once adhered, many of the platelets presented a full procoagulant response. Monomeric type-I collagen was unable to support adhesion, unless Mg2+-dependent integrin α2β1 interactions were facilitated by omission of Ca2+ ions. With all surfaces, however, post-addition of CaCl2 to adhering platelets resulted in a potent Ca2+-influx signal, followed by PS exposure and bleb formation. The procoagulant response elicited by binding to CRP was inhibited by anti-GpVI Fab fragments, but not by impeding integrin α2β1-mediated events. With fibrillar collagen, it was inhibited by blocking either the GpVI- or integrin α2β1- mediated interactions. This suggests that the triple-helical Gly-Pro-Hyp repeat in CRP and analogous sequences in fibrillar collagen stimulate the procoagulant response of adherent platelets by acting as ligands for GpVI. Influx of Ca2+ is required for this response, and adhesion via integrin α2β1 serves to potentiate the signaling effects of GpVI.
Eicosanoids and Iso-Eicosanoids: Constitutive, Inducible and Transcellular Biosynthesis in Vascular Disease
Authors: Folco Giancarlo , Patrono Carlo , Maclouf Jacques 
Published: March 2018

IntrouductionCurrent understanding of the mechanisms underlying plaque development (1-3) assigns a key role to oxidative modifications of specific phospholipids that are carried into the subendothelial space with low-density lipoproteins (LDL). These variably oxidized lipids in turn trigger a chronic inflammatory response, largely orchestrated by monocytes/macrophages (1-3). Thrombotic occlusion of a major coronary or cerebral vessel can complicate the sudden fissuring or rupture of a plaque and lead to myocardial infarction or ischemic stroke, respectively. The determinants of lipid oxidation, plaque fragility and of the hemostatic response to plaque rupture, as well as the hemodynamic factors influencing the multifactorial process of atherothrombosis represent targets of pharmacological interventions aimed at reducing the risk of cardiovascular and cerebrovascular complications.The oxidative modifications of the arachidonic acid backbone, that lead to the formation of enzymatic (eicosanoids) and non-enzymatic (iso-eicosanoids) derivatives (Fig. 1), can provide autacoid mechanisms modulating activation of the major cellular players of atherothrombosis, including platelets, endothelial cells, neutrophils and monocytes/macrophages (4-7). The remarkable clinical effects of low-dose aspirin in reducing the risk of stroke and myocardial infarction by 20 to 50% in various clinical settings (8) provide perhaps the most convincing evidence for the pathophysiologic importance of eicosanoid mechanisms in modulating the thrombotic outcome of plaque fissuring. That interference with a single mechanism of amplification of platelet activation, such as that provided by thromboxane (TX) A2-thromboxane receptor (TP) interactions on the platelet membrane, can lead to clinically detectable consequences is perhaps surprising, but certainly encouraging in the search of novel targets for pharmacological intervention.Considerable progress has been made in the eicosanoid field during the last ten years, well beyond the thromboxane/prostacyclin balance hypothesis (9), with the elucidation of at least three distinct modalities of eicosanoid biosynthesis, i.e., constitutive, inducible and transcellular. In this review, we shall discuss the molecular, cellular and pharmacological aspects of eicosanoid and iso-eicosanoid biosynthesis as they relate to the multifactorial process of atherothrombosis, with the aim of suggesting novel pathophysiologic mechanisms as well as potential therapeutic targets.
Sex Differences in Coagulation and Fibrinolysis in Subjects with Coronary Artery Disease
Authors: Ossei-Gerning Nicholas , Wilson Ian , Grant Peter 
Published: March 2018

SummaryWomen with coronary artery disease (CAD) have a prognosis at least as bad and possibly worse than men. Differences in classical risk factors do not fully account for these findings and there is evidence that circulating levels of haemostatic factors may predict CAD risk. In this study sex differences in haemostatic risk factors were examined in relation to coronary stenosis.609 (420 men, 69%) subjects admitted for coronary angiography for suspected CAD were recruited. Levels of Factor VII:C (FVII:C), fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF) were estimated in 296 subjects from one centre. Of these, women (n = 107) had higher levels of FVII:C (134% vs 117%, p <0.0005), and fibrinogen (3.4 g/l vs 3.2 g/l p = 0.01) than men (n = 189) and these differences remained after adjusting for other covariates. In subjects with angiographically significant atheroma these differences in haemostatic factors (n = 50 for women vs n = 147 for men) were exaggerated, (FVII:C 139% vs 117, p <0.0001, fibrinogen 3.7 g/l vs 3.3 g/l p = 0.003), PAI-1 (26.2 ng/ml vs 19.7 ng/ml, p = 0.02) with a trend towards higher levels of vWF in the women. Women with significant atheroma at angiography (n = 50) had higher levels of PAI-1 (25.0 ng/ml vs 13.4 ng/ml p <0.0001) and vWF (1.25 IU/ml vs 1.06 IU/ml, p = 0.02) and a trend towards higher levels of both fibrinogen and FVII:C than women with normal or in significant coronary vessel disease (n = 57).Elevated circulating levels of PAI-1, vWF, fibrinogen and FVII:C in women with angiographically proven CAD may contribute to an adverse cardiovascular risk factor profile and the poorer prognosis in females than male patients with proven coronary artery disease.
High Sensitivity Detection of Activated Factor IX: Application to the Analysis of Different Therapeutical Factor IX Concentrates and Prothrombin Complexes
Authors: Kusch Manuela , Seitz Rainer , König Herbert 
Published: March 2018

SummaryA very sensitive and highly reliable test system for the detection of activated coagulation factor IX (FIXa) has been established. This assay system is based on the cleavage of a fluorogenic substrate by activated factor X (FXa) which is generated by FIXa. This assay can be used to process a large number of samples at a time and, being based on the convenient microtiter plate format, can easily be adapted to automated processing for routine screening of large sample numbers.With this assay at hand we determined the FIXa content of different commercially available therapeutic FIX sources, such as high purity FIX (HPFIX) and prothrombin complex concentrates (PCC). Here we demonstrate that PCC from several suppliers do not contain significantly higher levels of FIXa as compared to HPFIX from the same supplier. In fact, there is a tendency for HPFIX to contain more FIXa than PCC. Moreover, HPFIX from certain manufacturers who do not produce PCC are characterized by an exceptionally high content of FIXa. Therefore, the higher thrombogenic potential of PCC which is well documented clinically cannot be explained solely – if at all – by an increased content of FIXa. Rather, it will be necessary to identify other components responsible for this phenomenon.
Influence of Lipoprotein (a) Levels and Isoforms on Fibrinolytic Activity – Study in Families with High Lipoprotein (a) Levels
Authors: Falcó Cristina , Dalmau Jaime , España Francisco , Aznar Justo , Estellés Amparo 
Published: March 2018

SummaryIncreased levels of lipoprotein (a) [Lp(a)] have been considered an independent risk factor for cardiovascular disease, but the mechanism behind this relationship is not completely understood. A high concentration of Lp(a) may interfere with fibrinolysis because of the structural similarity between apo(a) and plasminogen. The aim of the present study was to examine the influence of apo(a) levels and isoforms on fibrinolytic activity in 82 subjects from 24 families in which the Lp(a) levels were ≥30 mg/dl in at least one child and one parent. Several fibrinolytic parameters, including plasmin generation by fibrin-bound tissue plasminogen activator, the lipid profile and apo(a) isoforms were studied. Subjects with high circulating Lp(a) levels (n = 44) had significantly reduced plasmin generation compared with their relatives with normal Lp(a) levels (n = 38). A significant inverse correlation between Lp(a) levels and plasmin generation was observed. The individuals with a combination of high levels of plasma Lp(a) and a major apo(a) isoform ≤580 kD molecular weight show the lowest fibrinolytic activity. A high correlation was found between the levels of apo(a) isoforms in children and the levels of the corresponding parental apo(a) iso-forms. We conclude that the antifibrinolytic effect of Lp(a) in subjects with two apo(a) isoforms may depend not only on the total plasma level of Lp(a) but also on the relative concentration of the small apo(a) isoform.
Measurement of Platelet Microparticles during Cardiopulmonary Bypass by Means of Captured ELISA for GPIIb/IIIa
Authors: Miyamoto Shinji , Marcinkiewicz Cezary , Edmunds L. Henry , Niewiarowski Stefan 
Published: March 2018

SummaryA captured enzyme-linked immunosorbent assay (ELISA) using the disintegrin, kistrin, is described and used to measure platelet microparticles (PMP) generated during open heart surgery. This ELISA detects 75 ng/ml of glycoprotein IIb/IIIa (GPIIa/IIIa) in solution and is more sensitive and less variable than flow cytometry and radioimmunoassay. By ELISA, mean values of GPIIb/IIIa in PMP are 14.2 ± 7.9 μg/ml for outdated platelets and 0.28 ± 0.1 μg/ml in fresh blood from healthy donors. Normal washed platelets (108) contain 8.8 μg of GPIIb/IIIa.In 12 cardiac surgical patients, PMP measured by ELISA significantly increased (p = 0.039) to 0.58 ± 0.3 μg/ml at the end of cardio-pulmonary bypass, but the increase measured by flow cytometry (1207 to 1447 events in PMP gate) was not significant. Neither heparin nor protamine alter PMP. After cardiopulmonary bypass, PMP concentrations return to baseline values before protamine is given. Concentrations of PMP in pericardial blood are greater than in simultaneous perfusate. This ELISA is more sensitive and accurate than alternate methods for measuring PMP and shows the PMP production and rapid clearance during open cardiac surgery.Present address: Dr. Shinji Miyamoto, Department of Cardiovascular Surgery, Oita Medical University, 1-1 Idaigaoka, Oita, Japan