bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hemophilia B with Mutations at Glycine-48 of Factor IX Exhibited Delayed Activation by the Factor VIIa-tissue Factor Complex
Authors: Wu Pai-Chih , Hamaguchi N. , Yu Yi-Shing , Shen Ming-Ching , Lin Shu-Wha 
Published: March 2018

SummaryGly-48 is in the conserved DGDQC sequence (residues 47-51 of human factor IX) of the first EGF (EGF-1)-like domain of factor IX. The importance of the Gly-48 is manifested by two hemophilia B patients; factor IXTainan and factor IX>Malmö27, with Gly-48 replaced by arginine (designated IXG48R) and valine (IXG48V), respectively. Both patients were CRM+ exhibiting mild hemophilic episodes with 25% (former) and 19% (latter) normal clotting activities. We characterize both factor IX variants to show the roles of Gly-48 and the conservation of the DGDQC sequence in factor IX. Purified plasma and recombinant factor IX variants exhibited approximately 26%–27% normal factor IX’s clotting activities with G48R or G48V mutation. Both variants depicted normal quenching of the intrinsic fluorescence by increasing concentrations of calcium ions and Tb3+, indicating that arginine and valine substitution for Gly-48 did not perturb the calcium site in the EGF-1 domain. Activation of both mutants by factor XIa appeared normal. The reduced clotting activity of factors IXG48R and IXG48V was attributed to the failure of both mutants to cleavage factor X; in the presence of only phospholipids and calcium ions, both mutants showed a 4∼7-fold elevation in K
m, and by adding factor VIIIa to the system, although factor VIIIa potentiated the activation of factor X by the mutants factor IXaG48R and factor IXaG48V, a 2∼3-fold decrease in the catalytic function was observed with the mutant factor IXa’s, despite that they bound factor VIIIa on the phospholipid vesicles with only slightly reduced affinity when compared to wild-type factor IXa. The apparent K
d for factor VIIIa binding was 0.83 nM for normal factor IXa, 1.74 nM for IXaG48R and 1.4 nM for IXaG48V. Strikingly, when interaction with the factor VIIa-TF complex was examined, both mutations were barely activated by the VIIa-TF complex and they also showed abnormal interaction with VIIa-TF in bovine thromboplastinbased PT assays. Taken together, our results suggest that mutations at Gly-48 altered the interaction of factor IX with its extrinsic pathway activator (VIIa-TF complex), its macromolecular substrate (factor X), and its cofactor (factor VIIIa).
Transcriptional Activation of the Factor VIII Gene in Liver Cell Lines by Interleukin-6
Authors: Hannant Wendy , Ludlam Christopher , Stirling David 
Published: March 2018

SummaryCirculating factor VIII (fVIII) levels increase during inflammation suggesting that fVIII synthesis or secretion is stimulated during acute inflammation. To examine the mechanisms underlying this increase in circulating factor VIII, we have developed a sensitive and reliable semi-quantitative assay for fVIII mRNA utilising competitive reverse transcriptase-polymerase chain reaction (RT-PCR, and used this to study two human liver cell lines, Hep-G2 and Chang Liver cells. These cells were cultured under basal conditions or following treatment with interleukin-1, -2 and 6 (IL-1, -2, and -6). Following 18h culture with IL-6 (maximum concentration 40U/ml), these levels had risen 6 and 9 fold respectively, with no concomitant rise in control RNA levels. The dose responses for both cell types were similar, with an ED50 of 11 U/ml. The time course of this response was also similar in both cell lines with the increase in fVIII mRNA first reaching significance by 3 h, and reaching maximum levels by 12 h. IL-1 and IL-2 had no effect at any of the doses studied. This study provides the first evidence for regulated expression of fVIII in human cell lines, and suggests that increased plasma fVIII levels during the acute phase response may be due to increased expression of fVIII mRNA.
Relationship between Thyroid Hormones and Plasma D-dimer Levels
Authors: Bruckert Eric , Ankri Annick , Beucler Isabel , Giral Philippe , Turpin Gérard , Chadarevian Rita 
Published: March 2018

SummaryHigh serum levels of cholesterol and triglycerides are risk factors for coronary heart disease and are strongly related to several haemostatic parameters. Thyroid disorders are a frequent feature in hyperlipidemic patients and are also associated with a variety of haemostatic abnormalities. Therefore, we analysed the relationships between free T4 (fT4) levels and Factor VII and VIII activities (FVIIc and FVIIIc), D-Dimers (DDI) and Plasminogen Activator Inhibitor type 1 (PAI-1), in a group of 472 healthy patients referred for hyperlipidemia.Fourty patients were found to have primary hypothyroidism. A negative correlation was found in the whole study population between fT4 and DDI (p = 0.0001, r = –0.21) and the same results were found after exclusion of the patients with fT4 below the normal range (p = 0.0007, r = –0.17). In a multivariate regression analysis, the relationship between DDI and fT4 was independent of age, Body Mass Index (BMI), gender and total cholesterol. Less impressive correlation coefficients were found with FVIIc (r = –0.10), FVIIIc (r = –0.09) and PAI-1 (r = –0.09).These results suggest that fT4 may play a physiological role in the regulation of the haemostatic equilibrium in hyperlipidemic patients and that low levels of fT4 are associated with a hypercoagulable state.
Fibrin D-dimer, Tissue Plasminogen Activator, Plasminogen Activator Inhibitor, and the Risk of Major Ischaemic Heart Disease in the Caerphilly Study
Authors: Yarnell J. W. G. , Sweetnam P. M. , Rumley A. , Thomas H. F. , Elwood P. C. , Lowe G. D. O. 
Published: March 2018

SummaryPlasma levels of fibrin D-dimer, tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI) have been associated with ischaemic heart disease (IHD). However their associations with incident IHD in samples of the general population are not established. D-dimer antigen, tPA antigen and PAI activity were measured in stored, fasting plasma samples from 1,998 men aged 45-65 examined between 1984 and 1988, during the first re-examination of the Caerphilly Study cohort. These variables were related to major IHD events (myocardial infarction or IHD death) which occurred in 129 men during a follow-up period which averaged 61 months. Mean fibrin D-dimer was higher in men who developed IHD events (90 vs. 71 ng/ml; age-adjusted logarithmic mean difference 0.21; 95% CI 0.11, 0.30; p <0.0001). This association remained after adjusting for baseline IHD and for other risk factors including fibrinogen: the adjusted relative odds of IHD in the highest fifth of D-dimer were 3.5 (95% CI 1.8, 6.9; p = 0.0003). Mean tPA antigen was also higher in men who developed IHD (12.6 vs. 11.6 ng/ml; mean difference 0.9; 95% CI 0.2, 1.7; p = 0.02); however this difference largely disappeared after adjusting for other risk factors. PAI activity was not associated with risk of IHD.
A New Method for Quantifying Platelet Deposition in Flowing Native Blood in an Ex Vivo Model of Human Thrombogenesis
Authors: Bossavy J. P. , Sakariassen K. S. , Barret A. , Boneu B. , Cadroy Y. 
Published: March 2018

SummaryNo quantitative, simple and non-radioactive method has been described for measuring the platelet content of experimental thrombi. The aim of the present study was to develop a simple method for quantifying platelets in thrombi formed on thrombogenic surfaces in flowing native human blood. To test the relevance of this new method, the effect of unfractionated heparin on arterial thrombus formation was investigated. Tissue factor (TF)- and collagen-coated coverslips were exposed to non-anticoagulated blood at an arterial wall shear rate (2,600 s–1) for 1 to 4 min. Platelet deposition was quantified by measuring the P-selectin (PS) and β-thromboglobulin (βTG) content of dissolved plasmin-digested thrombi using immunoenzymoassays; fibrin deposition was determined by measuring the D-dimer levels. These results were compared to those established by morphometrical analysis.Morphometric evaluation showed that fibrin deposition was maximum on TF by 1 min perfusion time. Platelets deposited subsequently and reached a maximum at 3 min. On collagen, platelets deposited directly on the collagen fibrils without detectable fibrin deposit. Platelet deposition increased from 1 to 4 min. Platelet deposition quantified by PS was correlated to the values obtained by morphometry (r = 0.72, r = 0.67, p <0.001, on TF and collagen, respectively). As compared to PS, βTG measurements gave an underestimation of the size of the thrombus platelet number. Unfractionated heparin infused through a mixing device proximal to the perfusion chamber to obtain plasma concentrations of 0.5, 1 and 3 IU/ml, reduced fibrin deposition on TF-coated coverslips in a dose-dependent manner (77% reduction at 3 IU/ml, p <0.01), but had no significant effect on platelet deposition (33% at 3 IU/ml, p >0.05). In contrast, heparin had no effect on fibrin or platelet deposition on collagen-coated coverslips.Thus, a new quantitative and simple method for measuring platelet deposition in flowing blood has been developed and characterized. Utilizing this system, we have demonstrated that unfractionated heparin did not inhibit arterial thrombus formation either on procoagulant or on proaggregant surface.
Effects of Melagatran, a New Low-molecular-weight Thrombin Inhibitor, on Thrombin and Fibrinolytic Enzymes
Authors: Antonsson T. , Bylund R. , Eriksson U. , Gyzander E. , Nilsson I. , Elg M. , Mattsson C. , Deinum J. , Pehrsson S. , Karlsson O. , Nilsson A. , Sörensen H. , Gustafsson D. 
Published: March 2018

SummaryMelagatran, a new, competitive and rapid inhibitor of thrombin with a molecular mass of 429 Da is described. Melagatran is well tolerated when administered in very high doses, and the oral bioavailability in the dog is relatively high. The aim of the study was to determine, in the preclinical setting, the degree of selectivity against the fibrinolytic system required for entering the clinical development phase. Melagatran was compared with two structurally similar thrombin inhibitors, inogatran and H 317/86. The potent inhibition of thrombin by melagatran was demonstrated by a low inhibition constant (Ki) for thrombin (0.002 μmol/l) and prolongation of clotting time to twice the control value in coagulation assays at low concentrations (0.010, 0.59 and 2.2 μmol/l for thrombin time, activated partial thromboplastin time and prothrombin time, respectively). Furthermore, thrombin-induced platelet aggregation was inhibited at the same concentration (IC50-value 0.002 μmol/l) as the Ki-value for thrombin. In two assays of global fibrinolysis, inhibition was observed at a concentration of 1.1 μmol/l in a euglobulin plasma fraction model, while no inhibition was observed at a concentration of ≤10 μmol/l in a plasma model. In an in vivo model of endogenous fibrinolysis in the rat, inhibition of fibrinolysis was observed at ≥1.0 μmol/l. In all assays, except the Ki-ratio determinations, the compounds could be graded with regard to selectivity against the fibrinolytic system: inogatran > melagatran > H 317/86. For melagatran, inhibition of fibrinolysis was not observed at concentrations below the upper limit of the proposed therapeutic plasma concentration interval (<0.5 μmol/l). Thus, melagatran seems to have a sufficient selectivity against the fibrinolytic system, while H 317/86 was considered to be insufficient for clinical development.
High Levels of Tissue Factor Pathway Inhibitor in Patients with Nephrotic Proteinuria
Authors: Moia M. , Rivolta E. , Ponticelli C. , Mannucci P. M. , Ariëns R. A. S. 
Published: March 2018

SummaryAcquired deficiency of naturally occurring anticoagulant proteins, due to loss in the urine, has been proposed as one of the major thrombogenic alterations in nephrotic proteinuria. The aim of this study was to investigate if proteinuria may induce deficiency of tissue factor pathway inhibitor (TFPI). TFPI, protein C (PC) and antithrombin (AT) were measured in 31 patients with nephrotic proteinuria, compared with 62 age- and sex-matched controls. Plasma levels of TFPI activity, total TFPI antigen and free TFPI antigen were significantly higher in patients with nephrotic proteinuria than in controls, and none of the patients had TFPI deficiency. Intravenous injection of 7500 IU unfractionated heparin induced a significant further increase of TFPI in two patients with high pre-heparin levels. Also plasma levels of PC were significantly higher in patients than in controls. Mean AT antigen levels were not significantly different between patients and controls, and AT activity was only marginally increased with borderline significance. Three out of 31 patients had substantial acquired AT deficiency. In conclusion, proteinuria is not associated with TFPI deficiency, but with a marked increase of this anticoagulant protein. The acquired thrombophilic diathesis of patients with nephrotic proteinuria can therefore not be attributed to TFPI deficiency.
A Common Splice Site Mutation Is Shared by Two Families with Different Type 2N von Willebrand Disease Mutations
Authors: Hampton Kingsley , Preston F. Eric , Peake Ian , Goodeve Anne , Nesbitt I. Mandy 
Published: March 2018

SummaryUsing an ELISA-based method to detect type 2N von Willebrand disease (VWD), we found two individuals with absent FVIII binding. Direct sequencing of the FVIII binding region of the von Willebrand factor (VWF) gene showed that one individual had an R854Q substitution whilst the other had a T791M substitution. The very low FVIII binding and the VWF:Ag levels in both individuals suggested a second defect on the other VWF allele. Conformation sensitive gel electrophoresis of polymerase chain reaction amplified DNA was used to detect an additional change in the VWF gene of each patient. Direct sequencing confirmed a previously unreported G to A transition in the donor splice site in intron 25 of both individuals which should result in a null allele. This was confirmed by mRNA analysis. These two individuals therefore have compound heterozygous VWD in which the only expressed allele has a type 2N mutation. In our population, such compound heterozygosity appears to be a significant cause of type 2N VWD.
Interspecies Loop Grafting in the Protease Domain of Human Protein C Yielding Enhanced Catalytic and Anticoagulant Activity
Authors: Shen Lei , Villoutreix Bruno , Dahlbäck Bjorn 
Published: March 2018

SummaryHuman anticoagulant activated protein C (hAPC) is less potent than the bovine APC (bAPC) molecule and our aims were to elucidate the molecular background for this difference and to create an APC with enhanced anticoagulant activity. In the protease domain of human protein C (hPC), the loop 148 (GWGYHSSREKEAKRN) is four residues longer than the corresponding loop in bovine APC (GWGY RDETKRN). To investigate whether this caused the species difference, the loop in hPC was replaced by the shorter bovine loop, whereas the longer human loop was introduced in bovine protein C. The mutation in hAPC yielded enhanced catalytic activity against chromogenic (4-fold) as well as natural (factors Va and VIIIa) substrates and 2-3-fold increased anticoagulant activity. The opposite effects were obtained with the bovine mutant. As compared to wild-type hAPC, the mutant hAPC was inhibited slightly faster by the protein C inhibitor, whereas the inhibition by α 1-antitrypsin was unaffected by the mutation. A computer model of bAPC was developed in order to analyse further our data. Collectively, our results demonstrate enhanced catalytic efficiency to result from mutagenesis in the loop 148 and show that APC mutant with increased anticoagulant activity can be created.
Abbreviations: PC, protein C; APC, Activated protein C. SDS-PAGE, polyacrylamide gel electrophoresis run in the presence of SDS, The chymotrypsinogen nomenclature for APC and thrombin is used throughout the text while the PC numbering is indicated between brackets whenever appropriate. P2, P1 and P1’, P2’.. designate inhibitor residues amino- and carboxy-terminal to the scissile peptide bond, respectively, and S2, S1 and S1’, S2’.. the corresponding subsites of the protease. APTT, activated partial thromboplastin time; rmsd, root mean square deviation. The coordinates of the bAPC model are freely available.
Elevated Plasma Fibrinogen and Increased Fibrin Turnover among Healthy Women who both Smoke and Use Low-dose Oral Contraceptives
Authors: Vissac Anne-Marie , Kirzin Jean-Michel , Bourgeat Pierre , Amiral Jean , Agher Rachid , Guize Louis , Scarabin Pierre-Yves 
Published: March 2018

SummaryAmong users of low-dose oral contraceptives (OC), cardiovascular diseases occur mainly in smokers. The mechanisms by which OC and smoking increase the risk for arterial thrombotic risk have not been adequately explained. Epidemiological evidence suggests that changes in blood coagulation and fibrinolysis may play an important role as determinants of thrombotic events. Therefore, we have investigated the associations of OC and smoking with haemostatic variables among 194 premenopausal healthy women. Fourty women were current users of low-dose OC and 62 women were smokers. After adjustment for age and body mass index, mean values of factor XIIa, factor VII activity and antigen, fibrinogen, D-dimer, global fibrinolytic capacity were significantly higher in OC users than in non-users. Mean levels of PAI activity and t-PA antigen were significantly lower in OC users than in non-users. Smokers had significantly higher mean values of fibrinogen than non-smokers. Two-way analysis of variance showed that the differences in mean levels of fibrinogen and D-dimer between OC users and non users were restricted to smokers. The positive and significant interactions between OC use and smoking in their effects on haemostatic variables were consistent with respect to age and type of OC. These preliminary data suggest that elevated plasma levels of fibrinogen and intravascular fibrin deposition may play a role in the pathogenesis of arterial thrombotic disease among women who are both low-dose OC users and smokers.
Compound Heterozygosity for One Novel and One Recurrent Mutation in a Thai Patient with Severe Protein S Deficiency
Authors: Poort Swibertus , Vos Hans , Bertina Rogier , Mahasandana Chularatana , Tanphaichitr Voravarn , Veerakul Gavivann , Kankirawatana Suthida , Suvatte Vinai , Pung-amritt Parichat 
Published: March 2018

SummaryHomozygous or compound heterozygous protein S (PS) deficiency is a very rare disorder in the anticoagulant system, that can lead to life-threatening thrombotic complications shortly after birth. This report describes the results of the genetic analysis of the PROS1 genes in a Thai girl patient. She was reported in 1990 as the first case with homozygous PS deficiency and neonatal purpura fulminans. In the present report, we identified the mutations in this patient by direct sequencing of PCR products representing all 15 exons of the PROS1 gene and their flanking intronic regions. The patient turned out to be compound heterozygous for two null mutations. One allele contained a novel sequence variation, an A-insertion in an A5-tract covering codon 146 and 147, that results in a frameshift and a stop codon (TAA) at position 155. The other allele contained a nonsense mutation in exon 12 by a transition at codon 410 CGA (Arg) to TGA (stop). Cosegregation of PS deficiency with these two genetic defects was observed in her family.
Pharmacological Effects of a Novel Recombinant Hirudin, CX-397, In Vivo and In Vitro: Comparison with Recombinant Hirudin Variant-1, Heparin, and Argatroban
Authors: Inoue Yoshifumi , Goto Yuso , Fukazawa Tominaga , Hayashi Hideya , Komatsu Yasuhiko 
Published: March 2018

SummaryThe novel recombinant hirudin analog CX-397 was investigated with respect to its pharmacological activity and antithrombin profiles in vivo and in vitro. In three different types of thrombosis models in rats, including stasis and thrombin-induced venous, glass surface-activated arterio-venous shunt, and ferric chloride-induced arterial thrombosis models, CX-397 and rHV-1 elicited potent antithrombotic effects, where the minimum effective doses of rHV-1 tended to be higher than those of CX-397 in the arterio-venous shunt and arterial thrombosis models. The hemorrhagic risk of CX-397 in template bleeding in rats was not higher than that of rHV-1, indicating that CX-397 is superior to rHV-1 for treating the platelet-dominant type of thrombosis. However, no differences were detected between CX-397 and rHV-1 in their effects on in vitro coagulation times and thrombin-induced platelet aggregation, suggesting the possibility that some unknown mechanisms other than simple thrombin inhibition are also involved in their anti-thrombotic actions.
Secreted Dense Granule Adenine Nucleotides Promote Calcium Influx and the Maintenance of Elevated Cytosolic Calcium Levels in Stimulated Human Platelets
Authors: Weiss Harvey , Lages Bruce 
Published: March 2018

SummaryEvidence that secreted dense granule adenine nucleotides mediate part of the agonist-induced cytosolic calcium ([Ca2+]i) responses in human platelets was obtained from comparisons of fura-2-loaded platelets from normal subjects and from patients with a form of platelet storage pool deficiency (SPD) in which the secretory dense granules and their contents are virtually absent. SPD platelets had normal initial [Ca2+]i in creases induced by thrombin and the endoperoxide analog U46619, but a significantly enhanced decay of elevated [Ca2+]i levels following the initial increases. With thrombin, this enhanced [Ca2+]i decay was associated with decreased Ca2+ influx, as measured by Mn2+ quench of fura-2 fluorescence. Addition of micromolar concentrations of ADP, alone or together with ATP, after stimulation reversed the enhanced [Ca2+]I decay and increased Mn2+ quench in SPD platelets, but had no effect on these responses in normal platelets, while addition of 100-fold higher concentrations of ATP or apyrase before stimulation increased [Ca2+]I decay and decreased Mn2+ quench in normal platelets, but had little effect in SPD platelets. ATP and α,β-methylene ATP, a specific agonist for P2X1 receptors, at micromolar concentrations also increased Mn2+ quench, but to lesser extents than did ADP, in SPD platelets isolated and loaded with fura-2 in the presence of apyrase. Similar effects of ADP and excess ATP were seen in U46619-stimulated platelets, but decreased Ca2+ influx could not be measured directly in SPD platelets, presumably due to the very transient influx response seen with U46619. These results suggest that secreted dense granule ADP and ATP contribute to the maintenance of elevated [Ca2+]i levels, but not to the initial [Ca2+]i increases, in stimulated human platelets, most likely via a nucleotide-specific component of Ca2+ influx which may be mediated by interactions with both P2X1 and P2Y1 purinoceptors.
No Indication for APTT Screening in Patients on Oral Anticoagulant Therapy
Authors: Vos Hans , Rosendaal Frits , van der Meer Felix 
Published: March 2018

SummaryPatients on oral anticoagulant therapy (OAT) may bleed due to a very low factor IX level caused by a mutation at Ala-10 in the propeptide region of the factor IX gene. We evaluated screening of patients on OAT with an APTT to detect patients with this abnormality. In 734 patients an APTT was assessed. Twenty-three patients had a disproportionately prolonged APTT. In these patients the factor IX level, the mutation at Ala-10 and the frequency of bleeding complications were assessed. No severely lowered factor IX levels were found (1 patient with 5% factor IX). No mutations at Ala-10 were found and bleeding complications were not more frequent in these patients. Conclusion: Routine APTT screening of patients on OAT is not useful to detect patients with increased bleeding or with the Ala-10 mutation in the factor IX gene.