bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ins405AsnPro Mutation in the von Willebrand Factor Propeptide in Recessive Type 2A (IIC) von Willebrand’s Disease
Authors: Karpman D. , Isaksson C. , Kristoffersson A. C. , Lethagen S. , Schneppenheim R. , Holmberg L. 
Published: March 2018

SummaryThe molecular defects of the von Willebrand factor (vWF) have been studied in the patient in whom the von Willebrand disease phenotype IIC was originally described. A six nucleotide insert, AATCCC, was found in exon 11 of the vWF gene, predicting the insertion of the amino acids asparagine and proline between phenylala-nine 404 and threonine 405 of the vWF propeptide. The mutation was present in one allele. Analysis of amplification products derived from platelet vWF mRNA showed the other allele to be silent. The patient is thus a compound heterozygote for a null allele and the IIC allele, in accord with the recessive mode of inheritance of the IIC phenotype. Family studies indicated the IIC mutation to have occurred de novo, possibly as a result of a duplication event. In vitro mutagenesis and expression in COS-7 cells confirmed the detrimental effect of the mutation on vWF multimer assembly. Taken together with those of earlier studies the present findings suggest that the IIC phenotype may well be exclusively caused by mutations which result in changes of the amino acid sequence in certain regions of the vWF propeptide. Although in the recently revised classification of von Willebrand’s disease variants, the IIC type is included in the 2A category, obviously it constitutes a very distinct subtype.
Weight-Loss Induced Changes in Plasma Factor VII Coagulant Activity and Relation to the Factor VII Arg/Gln353 Polymorphism in Moderately Obese Adults
Authors: Folsom Aaron , Shahar Eyal , Tsai Michael , Jeffery Robert , Wing Rena , Pankow James 
Published: March 2018

SummaryElevated plasma factor VII coagulant activity (factor VIIc) may be an independent risk factor for coronary heart disease. Several cross-sectional studies suggest that a polymorphism of the factor VII gene (ArgGln353) interacts with plasma triglyceride level in determining factor VIIc, but prospective data are lacking. Factor VII genotype, factor VIIc, and triglyceride level were measured in moderately obese adults aged 25 to 45 who underwent a six-month clinical trial to evaluate strategies for weight loss. A total of 48 men and 50 women who experienced substantial weight loss (mean: 10 kg) provided samples for genetic analysis. Overall, 78% of participants were homozygous for the Arg353 allele, while the remaining 22% were heterozygous (Arg/Gln353). At the baseline examination, heterozygotes had lower mean factor VIIc than Arg353 homozygotes (92% vs. 112%; p<0.001), and genotype explained 18% of the variance of factor VIIc. Average six-month weight loss was similar in both genotypes; mean reductions in factor VIIc following weight loss were greatest among Arg353 homozygotes with high initial values (>120%). Cross-sectional and longitudinal associations between plasma factor VIIc and triglyceride level were not dependent on genotype. These data confirm that the Gln353 allele is associated with lower factor VII coagulant activity in moderately obese adults, but they do not support the hypothesis that the Arg-Gln353 polymorphism interacts with plasma triglyceride level in determining factor VIIc.
Monoclonal Antibody-based Immunoassays for the Specific Quantitation of Rat PAI-1 Antigen and Activity in Biological Samples
Authors: Ngo Thu-Hoa , Verheyen S. , Knockaert I. , Declerck P. J. 
Published: March 2018

SummaryTwo enzyme-linked immunosorbent assays (ELISAs) for the quantitation of rat plasminogen activator inhibitor-1 (PAI-1) antigen and activity, respectively, in biological fluids were developed using monoclonal antibodies raised against recombinant rat PAI-1. These assays had a lower limit of sensitivity in plasma of 0.3 and 0.15 ng/ml, respectively. The intra-assay, inter-assay and inter-dilution coefficients of variation were 9, 14 and 9%, respectively, for the antigen assay and 8, 17 and 13%, respectively for the activity assay. Assay recoveries of recombinant rat PAI-1 (5 to 20 ng/ml) added to plasma were 73 to 88% and 89 to 93% for the antigen and the activity assay, respectively. The level of PAI-1 antigen in rat plasma was 1.8 ± 0.9 ng/ml (mean ± SD, n = 18), with a corresponding value of 1.0 ± 0.5 ng/ml for PAI-1 activity. In lysed platelet-rich rat plasma PAI-1 antigen was 6.0 ± 1.0 ng/ml (n = 8) and PAI-1 activity was 2.3 ± 0.4 ng/ml (n = 8). Endotoxin injection (0.5 mg/kg) induced a time-dependent increase of both PAI-1 antigen and PAI-1 activity levels in rat plasma, eventually resulting in a 100- to 200-fold increase (p <0.0001 vs. baseline). A linear correlation was found between PAI-1 antigen and activity levels in normal plasma (r = 0.63, n = 18, p <0.01) and in plasma from endotoxin-treated rats (r = 0.90, n = 35, p <0.001). Application of these assays for the analysis of gel filtration experiments of plasma from endotoxin-treated rats demonstrated that PAI-1 antigen eluted as two peaks (with corresponding Mr of ~430 kDa and 61 kDa) whereas PAI-1 activity eluted as a single peak corresponding with the high molecular weight antigen form.Thus, these unique assays allowing the specific determination of rat PAI-1 antigen and rat PAI-1 activity may constitute important tools for further investigations on the pathophysiological role of PAI-1 in a variety of experimental rat models.
Multiple Inhibition of Platelet Activation by Aurintricarboxylic Acid Prevents Vascular Stenosis after Endothelial Injury in Hamster Carotid Artery
Authors: Kozawa Osamu , Niwa Masayuki , Tanabe Kumiko , Ichimaru Katsuhiro , Takiguchi Yoshiharu , Yokota Masayuki , Hayashi Hideharu , Uematsu Toshihiko , Matsuno Hiroyuki 
Published: March 2018

SummaryActivated platelets are instrumental in restenosis due to their role in thrombus formation. Aurintricarboxylic acid (ATA) has been reported to prevent platelet activation by inhibiting von Willebrand factor binding to platelet glycoprotein (GP) Ib. We investigated the effects of ATA in vitro and in vivo in hamsters. ATA inhibited the in vitro platelet aggregation induced by ADP, botrocetin and thrombin, but not by collagen. The IC50 values during the ex vivo platelet aggregation by ADP, botrocetin and thrombin were 8.2 ± 1.8 μM, 0.9 ± 0.4 μg/ml and 2.4 ± 0.8 unit/ml, respectively. The platelet retention time to collagen-coated beads of hamster blood samples was inhibited by ATA (0.1, 0.3 and 1.0 mg/kg per hour) in a dose-dependent manner. Continuous administration of ATA (0, 0.1, 0.3, 1.0, 3.0 and 10.0 mg/kg per h) via an infusion pump produced dose-dependent antithrombotic effects: the time to occlude the carotid artery after vascular injury with a modified catheter was prolonged. Only when infused at doses of 3.0 and 10.0 mg/kg per hour, bleeding times were significantly prolonged. The continuous treatment with ATA (1.0 mg/kg per h) using a 2ML1 Alzet infusion pump for 2 weeks, resulted in a decrease in neointimal area by 22.2 ± 6.8% when measured 2 weeks after injury induction. DNA synthesis using DDT1MF2 hamster SMCs was decreased by ATA in a dose-dependent manner. ATA reduced the number of platelets adhering on the injured area, as detected by electron microscopy. These results indicated that treatment with ATA inhibited platelet adhesion but also SMC proliferation. These observations may explain the effect of ATA on neointima formation.
Very Low Activated Factor VII and Reduced Factor VII Antigen in Familial Abetalipoproteinaemia
Authors: Mitropoulos K. A. , Nanjee M. N. , Howarth D. J. , Martin J. C. , Esnouf M. P. , Reeves B. E. A. , Miller N. E. , Cooper J. A. , Miller G. J. 
Published: March 2018

SummaryAbetalipoproteinaemia is a rare disorder of apolipoprotein B metabolism associated with extremely low plasma concentrations of triglyce-ride. To discover whether the general positive association between factor VII and triglyceride levels extends to this condition, 5 patients were compared with 18 controls. All patients had a triglyceride below 100 μmol/l. Plasma unesterified fatty acid concentration was normal. Although factor IX activity was only slightly reduced (mean 88% standard) and factor IX antigen was normal, mean activated factor VII in patients was strikingly reduced to 34% of that in controls, a level similar to that found in haemophilia B. The patients’ mean factor VII activity and factor VII antigen were also significantly reduced to 54% and 63% of those in controls, respectively. Mean factor XI activity and tissue factor pathway inhibitor activity were reduced in patients to 70% and 75% of control values respectively, while factor XII, factor XI antigen, factor X, prothrombin and protein C were normal.
Treatment with a GPIIb/IIIa Antagonist Inhibits Thrombin Generation in Platelet Rich Plasma from Patients
Authors: Keularts I. M. L. W. , Béguin S. , Zwaan C. de , Hemker H. C. 
Published: March 2018

SummaryInfusion of the GPIIb/IIIa-inhibitor MK383 inhibits thrombin generation in platelet rich plasma by interfering with the production of platelet procoagulant phospholipid exposure. The effect is similar to that of 0.2 U/ml of heparin. Heparin infusion, well known to inhibit thrombin generation by fostering antithrombin activity, inhibits the formation of platelet-derived procoagulant microparticles, probably by decreasing the formation of free thrombin, which, under our circumstances, is the main platelet activator.
Proteolysis of Tissue Factor Pathway Inhibitor (TFPI) by Plasmin: Effect on TFPI Activity
Authors: Li Anguo , Wun Tze-Chein 
Published: March 2018

SummaryAn important regulator of the initiation of blood coagulation is the plasma glycoprotein, tissue factor pathway inhibitor (TFPI). TFPI inhibits factor Xa and factor VIIa/tissue factor complex, thereby dampens the proteolytic cascade of the tissue factor pathway. Plasma clot lysis is primarily mediated by the fibrinolytic enzyme, plasmin, which is generated through limited proteolysis of plasminogen by endogenous or exogenously administered plaminogen activators. In this study, the interaction of plasmin with recombinant E. coli-derived TFPI (rTFPI) was examined. Plasmin was found to cause a time and concentration dependent proteolysis of rTFPI, resulting in the decrease of anti-factor Xa (measured by chromogenic substrate assay) and anticoagulant (measured by tissue factor-induced clotting assay) activities. Amino-terminal sequencing of the proteolytic fragments revealed that plasmin cleaved rTFPI at K86-T87, R107-G108, R199-A200, K249-G250, and K256-R257. Western blot analysis showed that proteolysis of exogenously added rTFPI also occurred in plasma supplemented with urokinase, and this is accompanied by decrease of anticoagulant activity. These changes were abolished by addition of aprotinin, an inhibitor of plasmin. These data indicate that TFPI is susceptible to proteolysis when plasma fibrinolytic system is activated. The results taken together suggest that plasmin degradation of TFPI may contribute to rethrombosis after thrombolysis, and may contribute to the variability of the efficacy of TFPI in various thrombolysis/reocclusion studies reported previously.
Quantitative Analysis of von Willebrand Factor and Its Propeptide in Plasma in Acquired von Willebrand Syndrome
Authors: Boertjes Ria , van Mourik Jan , van Genderen Perry 
Published: March 2018

SummaryMeasurement of the von Willebrand factor (vWF) propeptide, also known as von Willebrand antigen II, has been suggested to be helpful in the discrimination of congenital von Willebrand disease type I from type 2 and in assessing the extent of activation of the endothelium. We performed a quantitative analysis of mature vWF and its propeptide in plasma in 8 patients with acquired von Willebrand syndrome (AvWS) and in 20 normal individuals. Mature vWF levels were significantly lower in AvWS as compared with normal individuals (13.4 ± 3.5 vs 35.6 ± 3.3 nM, p <0.001). In contrast, propeptide levels were significantly higher in AvWS (11.4 ± 1.1 vs 4.7 ± 0.2 nM, p < 0.001), probably reflecting a compensatory increase in vWF synthesis or increased perturbation of the endothelium in AvWS. After treatment with DDAVP, propeptide and mature vWF levels rose 5-fold in AvWS, whereas propeptide levels were not altered by the infusion of a vWF concentrate or treatment with high dose intravenous immunoglobulins, indicating that plasma propeptide levels are a reliable reflection of vWF synthesis. Measurement of propeptide levels may provide additional information in AvWS as to whether decreased levels of mature vWF in the circulation are due to a decrease in synthesis or due to an accelerated removal of vWF from the circulation.
Intratracheal Administration of Recombinant Human Factor IX (BeneFix™) Achieves Therapeutic Levels in Hemophilia B Dogs
Authors: Russell K. E. , Read M. S. , Bellinger D. A. , Leitermann K. , Rup B. J. , McCarthy K. P. , Keith J. C. , Khor S. P. , Schaub R. G. , Nichols T. C. 
Published: March 2018

SummaryThe purpose of this paper was to establish proof of concept for administration of human recombinant F.IX (rF.IX) by inhalation for therapy of hemophilia B. The pharmacokinetics of intratracheal (IT) administration of rF.IX was studied in nine hemophilia B dogs randomized into 3 groups that received 200 IU/kg IT, 1000 IU/kg IT, or 200 IU/kg intravenously (IV). IT rF.IX produced therapeutic levels of F.IX antigen and activity and the pharmacokinetic parameters were consistent with a slow release from a depot site within the lungs. Bio-availability compared to IV administration was 11% for 200 IU/kg IT and 4.9% for 1000 IU/kg. The whole blood clotting time began to shorten at 2 h but F.IX bioactivity was not detected until 8 h post infusion in both IT groups. In all groups, F.IX activity was detected through 72 h post administration. These data demonstrate that biologically active rF.IX can reach the systemic circulation when given IT. Aerosolization of rF.IX may provide a needle-free therapeutic option for delivery of rF.IX to hemophilia B patients.
Neutrophil Proteases Can Inactivate Human PAR3 and Abolish the Co-receptor Function of PAR3 on Murine Platelets
Authors: Cumashi Albana , Ansuini Helenia , Celli Nicola , Blasi Antonio De , O’Brien Peter , Brass Lawrence , Molino Marina 
Published: March 2018

SummaryThree members of the protease-activated receptor family, PAR1, PAR3 and PAR4, are activated when thrombin cleaves the receptor N-terminus, exposing a tethered ligand. Proteases other than thrombin can also cleave PAR family members and, depending upon whether this exposes or removes the tethered ligand, either activate or disable the receptor. For example, on human platelets PAR1 is disabled by cathepsin G, although aggregation still occurs because cathepsin G can activate PAR4. The present studies examine the interaction of cathepsin G and a second neutrophil protease, elastase, with PAR3 using two model systems: COS-7 cells transfected with human PAR3 and mouse platelets, which express PAR3 and PAR4, but not PAR1. In contrast to human platelets, cathepsin G did not aggregate murine platelets, and prevented their activation only at low thrombin concentrations. Elastase had no effect on thrombin responses in mouse platelets, but when added to COS cells expressing human PAR3, both cathepsin G and elastase prevented activation of phospholipase C by thrombin. Notably, this inhibition occurred without loss of the binding sites for two monoclonal antibodies that flank the tethered ligand on human PAR3. We therefore conclude that 1) exposure to cathepsin G disables signaling through human PAR3, and prevents murine PAR3 from serving its normal role, which is to facilitate PAR4 cleavage at low thrombin concentrations, 2) elastase disables human, but not murine, PAR3, 3) in contrast to human PAR4, mouse PAR4 will not support platelet aggregation in response to cathepsin G, and 4) the inactivation of human PAR3 by cathepsin G and elastase involves a mechanism other than amputation of the tethered ligand domain. These results extend the range of possible interactions between PAR family members and proteases, and provide further support for species-specific differences in the interaction of these receptors with proteases other than thrombin.
Factor VIII-Factor IX Interactions: Molecular Sites Involved in Enzyme-Cofactor Complex Assembly
Authors: Celie Patrick , Kolkman Joost , Lenting Peter , Mertens Koen 
Published: March 2018

IntroductionThe activation of factor X is one of the steps in the coagulation cascade that is driven by the assembly of an activated serine protease with a membrane-bound cofactor. In the initial phase of coagulation, factor X is activated by the complex of activated factor VII (factor VIIa) and tissue factor. Subsequently, during the so-called propagation phase, factor X activation is catalyzed by the complex of activated factor IX (factor IXa) and activated factor VIII (factor VIIIa). In these complexes, factor VIIa and factor IXa are the factor X-activating enzymes, whereas tissue factor and factor VIIIa serve as non-enzymatic cofactors.1 Factors VIIa and IXa are highly homologous to other cofactor-dependent enzymes, such as activated factor X (factor Xa) and activated protein C, both in amino acid sequence, domain organization, and three-dimensional structure.2 Factor VIIa and IXa further share low or negligible activity towards their natural substrate factor X, unless in complex with their physiological cofactors.Although tissue factor and factor VIIIa serve similar roles as biological amplifiers, they are structurally different. Tissue factor is a small, transmembrane protein with an extracellular part comprising 219 amino acids. Factor VIII is much larger (2,332 amino acids), circulates in plasma, and requires proteolytic processing to exert its biological activity.3 When cofactors are assembled with their respective enzymes, a dramatic increase in enzymatic activity occurs. The underlying molecular mechanism, however, remains poorly understood.During the past few years, remarkable progress has been made in understanding the molecular details of enzyme-cofactor assembly within the coagulation cascade. Crystallography has provided high-resolution structures of tissue factor4 and the various cofactor-dependent coagulation enzymes.2 Moreover, the crystal structure of the factor VIIa—tissue factor complex has been resolved and has allowed the identification of the molecular sites involved in enzyme-cofactor interaction.5,6 Such details are still lacking, however, for the factor IXa—factor VIIIa complex. Current views are derived from three-dimensional models generated by homology modeling based on structurally-related proteins, such as nitrite reductase,7 ceruloplasmin,8 and galactose oxidase.9 Despite their inherent limitations, these models greatly facilitate the interpretation of previous functional studies on factor X activation. As such, the availability of molecular models may be considered an important step toward resolving the structure of the factor IXa—factor VIIIa complex and understanding the role of complex assembly and defects thereof. This chapter provides an overview of the current developments in this field.
Fibrinolysis and the Contact System: A Role for Factor XI in the Down-Regulation of Fibrinolysis
Authors: Meijers Joost , Bouma Bonno 
Published: March 2018

IntroductionExposure of blood to negatively-charged surfaces, such as collagen, kaolin, or glass, results in the activation of the contact system of the intrinsic pathway of coagulation. Prekallikrein, factor XII, high molecular weight kininogen, and factor XI are the proteins involved in this contact reaction. The assembly of these components on a negatively-charged surface leads to the activation of factor XI, thereby propagating the intrinsic coagulation pathway. Simultaneously, several other reactions occur, such as the activation of factor VII and the initiation of the fibrinolytic system, kinin-forming pathway, and renin-angiotensin pathway.The first step in the contact phase is to bind factor XII to the negatively-charged surface, making it highly susceptible for proteolysis by kallikrein.1-3 Activated factor XII (α-factor XIIa) is formed in a process that may involve autoactivation.4-7 Prekallikrein is bound to high molecular weight kininogen in plasma. High molecular weight kininogen associates with a negatively-charged surface, thereby localizing prekallikrein to the surface. Limited proteolysis by α-factor XIIa converts prekallikrein to kallikrein. Kallikrein can dissociate from the surface and act on surface-bound factor XII at distant sites, thereby propagating the reciprocal cycle.7 Factor XI circulates plasma in a complex with high molcular weight kininogen. High molecular weight kininogen links factor XI to a negatively charged surface where it is activated by surface bound:α-factor XIIa. Although the in vivo, activating, negatively-charged surface is unknown, assembly and activation of the contact system on biological membranes of endothelial cells, platelets, neutrophils, and monocytes can take place, suggesting that these surfaces are the actual activating surfaces in vivo.8
The physiological significance of the contact system in blood coagulation remains unclear, however, because a deficiency of factor XII, prekallikrein, and high molecular weight kininogen does not result in a bleeding disorder. In contrast, patients deficient in factor XI, most common among Ashkenazi Jews, do suffer from variable bleeding abnormalities, especially from tissues with high local fibrinolytic activity (e.g., urinary tract, nose, oral cavity, tonsils).9,10 This suggested that there was an alternative route for the activation of factor XI, and recently, such a route was described.11,12 Thrombin was found to activate factor XI even in the absence of a negatively-charged surface,11-15 and factor XI was shown to play a role in the downregulation of fibrinolysis.16 In this article, the role of the contact system, with an emphasis on factor XI in the regulation of the fibrinolytic system, will be described.