bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Heparin on TAFI-Dependent Inhibition of Fibrinolysis: Relative Importance of TAFIa Generated by Clot-Bound and Fluid Phase Thrombin
Authors: Pentimone Anna , Binetti Bianca , Cramarossa Marialisa , Piro Donatella , Semeraro Nicola , Colucci Mario 
Published: March 2018

SummaryHeparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 µg/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through “localized” TAFIa generation.
Transient Platelet Interaction Induces MCP-1 Production by Endothelial Cells via IκB Kinase Complex Activation
Authors: Nothdurfter Caroline , Weber Marion , Fischer Claudia , Ungerer Martin , Gawaz Meinrad , Page Sharon , Massberg Steffen , Brand Korbinian 
Published: March 2018

SummaryActivated platelets alter endothelial chemotactic and adhesive properties. Transient interaction of α-thrombin-activated platelets with endothelial cells is sufficient to induce secretion of the NF-κBregulated chemokine MCP-1. To evaluate upstream signaling events in platelet-induced NF-κB activation, we compared the effect of platelets, IL-1β or TNF-α on IκB kinase (IKK) complex activation and IκB phosphorylation/proteolysis. Kinase assays demonstrated that platelets induced a slow increase in IKK activity over 30 min, which was similar, but not identical, to IL-1β, whereas TNF-α elicited a rapid induction of IKK. Differential effects were also found on IκB-α/ε degradation, while IKK levels were unaffected. Furthermore, overexpression of kinase-inactive IKK-βKA, as well as NIKKA, inhibited plateletor IL-1β-induced κB-, MCP-1or VCAM-1-dependent transcription. Using adeno-associated virus particles for cell transduction, we found that IKK-βKA substantially reduced stimulus-induced MCP-1 secretion. Platelet-induced signaling and resulting endothelial gene expression may play a role in early atherogenesis as well as plaque progression/destabilization.Meinrad Gawaz, Sharon Page, and Steffen Massberg contributed equally to this work.
Overexpression of Fibrinogen in ApoE*3-Leiden Transgenic Mice Does not Influence the Progression of Diet-Induced Atherosclerosis
Authors: Rezaee F. , Gijbels M. J. , Offerman E. H. , van der Linden M. , De Maat M. P. M. , Verheijen J. H. 
Published: March 2018

SummaryAlthough many epidemiological studies have shown an association between hyperfibrinogenemia and atherosclerosis, it is not established whether elevated fibrinogen has an etiological role in the pathogenesis or is only a reflection of the ongoing disease.We have studied the contribution of fibrinogen to the development of atherosclerosis in atherosclerosis-prone ApoE*3-Leiden mice that have been cross-bred with transgenic mice overexpressing fibrinogen. Genetic compound offspring were used to evaluate the progression of atherosclerotic lesions after being fed an atherogenic diet for 7 weeks. It was observed that the lesion area of the plaques as well as the severity of the lesions in the aortic valve was comparable in control single transgenic ApoE*3-Leiden mice and in double transgenic apoE*3-Leiden mice overexpressing fibrinogen. No thrombus or fibrin deposition was observed in atherosclerotic lesions in either group of mice.These results indicate that elevated plasma fibrinogen concentrations in ApoE*3-Leiden transgenic mice do not affect the progression of diet-induced atherosclerotic lesions.
Venous Thrombosis Following the Use of Intermediate Purity FVIII Concentrate to Treat Patients with Von Willebrand’s Disease
Authors: Colvin B. , Gupta V. , Shields M. L. , Smith M. P. , Makris M. 
Published: March 2018

SummaryWe describe four patients with von Willebrand’s disease (VWD) who experienced venous thrombosis after treatment with an intermediate purity factor VIII (FVIII) concentrate (Haemate P®) was used to cover invasive or surgical procedures. Most patients had additional risk factors for venous thromboembolism (VTE) and it is difficult to be certain of the contribution of the concentrate to the VTE. In view of the recognised association between high factor VIII activity (FVIII:C) levels and VTE there is a physiological basis for this complication and it is important to consider this when administering FVIII containing concentrates to VWD patients.
Novel Fibrinogen Truncation with Deletion of Bβ Chain Residues 440-461 causes Hypofibrinogenaemia
Authors: Brennan Stephen , Ockelford Paul , George Peter , Homer Vivienne 
Published: March 2018

SummaryA 24-year-old male with hepatitis C was initially diagnosed with hypofibrinogenaemia during investigations prior to a liver biopsy. He had a low functional and gravimetric fibrinogen concentration of 1.0 mg/mL and DNA sequencing of all exons, exon-intron boundaries and promoter regions of the fibrinogen Aα, Bβ, and γ genes revealed a single heterozygous g→a mutation at nucleotide 8035 of the Bβ gene. This creates a premature stop at the Trp 440 codon and results in a 22-residue truncation of the Bβ chain. Analysis of purified plasma fibrinogen by SDS PAGE, reverse phase HPLC and ESI MS, however, failed to detect any of the truncated chains in the plasma fibrinogen. The non-expression of aberrant molecules was further confirmed by functional analysis, which revealed normal fibrin polymerisation. The principal structural feature of the independently folding βD domain is its five-stranded anti-parallel β sheet. The deletion here of residues 440 to 461 removes the second strand from this sheet structure and appears to impact on the viability of the nascent chain and its ability to be incorporated into mature fibrinogen molecules. The mutation does not however provoke the formation of hepatic inclusion bodies.
A Reassessment of the Relationship between International Reference Preparations for Human and Rabbit Thromboplastins
Authors: Tripodi A. , van den Besselaar M. H. P. A. 
Published: March 2018

SummaryTwo established international reference preparations (IRP) for thromboplastins, i.e. RBT/90 (rabbit, plain) and rTF/95 (recombinant human, plain) have been calibrated against each other in a 7-centre exercise performed in 2000. The purpose of the study was to compare the calibration results with those of the original calibration study performed in 1995. The international sensitivity index (ISI) for rTF/95 was calculated relative to the ISI for RBT/90. The mean ISI for rTF/95 was 0.961 (between-lab SD 0.028) which was 2% greater than the historical value determined in 1995. There is no indication that the two IRP have deteriorated, but further monitoring of the calibration relationship is recommended.
Mice with a Severe Deficiency of the Endothelial Protein C Receptor Gene Develop, Survive, and Reproduce Normally, and Do not Present with Enhanced Arterial Thrombosis after Challenge
Authors: Liang Zhong , Volkir S. , Haalboom Erijka , Martin J. , Sandoval-Cooper Mayra , Rosen Elliot , Castellino Francis 
Published: March 2018

SummaryThe endothelial cell Protein C receptor (EPCR) functions to enhance activation of anticoagulant Protein C (PC) by the thrombin/ thrombomodulin (Tm) complex on the surface of the endothelium. This overall system functions in anticoagulation, profibrinolytic, and antiinflammatory responses. Mice with a severe targeted deficiency of this receptor have been generated by integration of exogenous DNA elements into the 5’-untranslated region of the EPCR gene. Despite the retention of the entire endogenous EPCR coding sequence in the altered EPCR gene locus, only very low EPCR message contents were detected in mice by quantitative RT-PCR during embryogenesis and up to at least early adulthood. Immunohistochemical analysis of various regions of the arterial tree of mice up to 4 months of age, employing an anti-murine EPCR antibody, confirmed that undetectable levels of this protein were present in arterial regions during these periods. Despite this, these mice are not more prone to arterial thrombosis after challenge in a FeCl3 carotid artery thrombosis model. Small amounts (<10% of wild-type) of this protein were found in other tissues. Matings of mice homozygous for this deficiency led to normal births and survival of the offspring, in contrast to results by others demonstrating early embryonic lethality of a total EPCR deficiency. These data further show that minimal levels of EPCR are able to support male and female virility, as well as embryonic development, birth, and survival to adulthood.
Role of Microthrombus Formation in the Development of Ischemia/Reperfusion-induced Liver Injury in Rats
Authors: Harada Naoaki , Kushimoto Shigeki , Uchiba Mitsuhiro , Okajima Kenji 
Published: March 2018

SummaryAlthough tumor necrosis factor-α (TNF-α) has been shown to play a critical role in the pathologic process leading to ischemia/reperfusion (I/R)-induced liver injury in rats by activating neutrophils, it is not clear whether or not microthrombus formation induced by TNF-α contributes to the liver injury. In the present study, we investigated the role of microthrombus formation in I/R-induced liver injury in rats. Hepatic tissue levels of TNF-α were significantly increased after reperfusion, and these were higher in animals subjected to 120 min-hepatic I/R than in those subjected to 60 min-hepatic I/R. Fibrin deposition was observed histologically in the hepatic sinusoidal space only in animals subjected to 120 min-hepatic I/R. Both the decrease in hepatic tissue blood flow and the extent of liver injury in animals subjected to 60 minand 120 min-hepatic I/R were significantly inhibited by pretreatment with anti-rat TNF-α antibody. Although neutrophil elastase inhibitors inhibited the decrease in hepatic tissue blood flow and reduced liver injury in animals subjected to 60 min-hepatic I/R, anticoagulants did not show any effects. Both anticoagulants and neutrophil elastase inhibitors inhibited the decrease in hepatic tissue blood flow and reduced liver injury in animals subjected to 120 min-hepatic I/R. Therapeutic effects of anti-rat TNF-α antibody on the120 min-I/R-induced liver injury were more marked than those of each anticoagulant or each neutrophil elastase inhibitor, and were comparable to those of combined use of anticoagulants and neutrophil elastase inhibitors. These observations strongly suggest that TNF-α induces I/R-induced liver injury primarily by activating neutrophils, and it exacerbates liver injury by inducing microthrombus formation when the production of TNF-α is further increased.
Relationships between Fibrinolytic and Inflammatory Parameters in Human Adipose Tissue: Strong Contribution of TNFα Receptors to PAI-1 Levels
Authors: Bastelica Delphine , Mavri Alenka , Verdier Monique , Berthet Bruno , Juhan-Vague Irène , Alessi Marie-Christine 
Published: March 2018

SummaryPlasminogen activator inhibitor type 1 (PAI-1), a risk marker of atherosclerosis, is highly expressed in adipose tissue from obese subjects. PAI-1 is also considered as an acute phase protein. Recently, adipose tissue has been described as a source of inflammatory cytokines. Therefore, our aim was to study the relationships between PAI-1, and IL-6, TNF, TNF receptors (TNFRSF1s) and TGFβ1, in plasma and adipose tissue from obese (n = 60) and lean (n = 28) subjects. Study has been extended to plasminogen activators (t-PA and u-PA).Compared to lean subjects, obese subjects exhibited higher plasma levels of all the studied parameters (except for TGFβ1) whereas in adipose tissue only PAI-1, t-PA and TGFβ1 antigen levels differed. In the obese population, plasma PAI-1 levels were weakly associated with circulating TNF, and this relationship disappeared after adjustment for plasma t-PA. Adipose tissue PAI-1 levels were positively associated with TNFRSF1s and TGFβ1, the strongest relationship being observed with TNFRSF1A, which explained 82% of PAI-1 variability. TNF and IL-6 were the main contributors to t-PA variability in plasma and in adipose tissue, respectively.Our results argue on the relevance of TNFRSF1s in the regulation of PAI-1 expression by adipose tissue. Association between t-PA, which is mainly produced by endothelial cells, and IL-6 or TNF suggest that inflammation might be involved in angiogenesis in adipose tissue.Abbreviations: act: activity; ag: antigen; BMI: Body mass index; IL-6: Interleukin-6; PAI-1: plasminogen activator inhibitor type 1; TGFβ1: Transforming Growth Factor beta 1; TNF: Tumor Necrosis Factor alpha; TNFRSF1A and 1B: Tumor Necrosis Factor receptor superfamilly 1A and 1B (TNFRI and II respectively); t-PA: tissue type plasminogen activator; u-PA: urokinase type plasminogen activator
Collagen Response and Glycoprotein VI Function Decline Progressively as Canine Platelets Age in vivo
Authors: Alberio L. , Friese P. , Clemetson K. J. , Dale G. L. 
Published: March 2018

SummaryClinical and experimental observations suggest that platelet function deteriorates quickly with cell age. However, efforts to define agedependent alterations have detected only modest biochemical changes occurring late in the cell life span. In this report, we demonstrate two significant alterations of the collagen response occurring during in vivo aging of canine platelets: a progressive increase in the EC50 for collagen types I, III and V and the emergence of a population of aged platelets which are refractory to collagen. Experiments with convulxin, a specific agonist for the collagen receptor glycoprotein VI (GPVI), also demonstrate an age-dependent decline in activation and the appearance of a non-reactive, aged population as observed with native collagens. Our studies indicate that canine platelets have two distinct binding levels for FITC-labeled convulxin and that the higher binding level disappears upon cell aging. During these studies one dog (#428) was identified whose platelets not only failed to demonstrate an age-dependent decrease in convulxin reactivity but also maintained a high convulxinbinding ability throughout their otherwise normal life span. Transfusion of biotinylated platelets from control dogs into dog #428 showed that the expected changes in collagen response and GPVI function did not occur in the transfused platelets. These observations demonstrate that the canine platelet response towards collagen is strongly dependent upon cell-age and suggest that this functional decline is at least partly due to an extrinsic-mediated alteration, possibly proteolytic, of GPVI.This work was supported in part by grants HL53585 and HL68129 from the National Institutes of Health (GLD) and the Swiss National Science Foundation (SSMBS grant, LA; grant 31-52396.97, KJC). This work has been presented at the XVIII Congress of the International Society on Thrombosis and Haemostasis (Paris, 2001).
Platelet Associated u-PA Up-regulates u-PA Synthesis by Endothelial Cells
Authors: Camoin-Jau L. , Pannell R. , Anfosso F , Bardin N. , Sabatier F. , Sampol J. , Gurewich V. , Dignat-George F. 
Published: March 2018

SummaryAdhesion of platelets to endothelium has been shown to induce important changes in endothelial properties. In this study, we examined the effect of platelet-endothelial cell interactions on the expression of urokinase-type plasminogen activator (u-PA) by human microvascular endothelial cells. After incubation of endothelial cells with platelets, a dose-dependent increase in the expression of u-PA Ag was observed and reached a plateau for a ratio of 300 platelets per endothelial cells. The u-PA Ag upregulation resulted from an increase in u-PA mRNA that originated from a synthesis by endothelial cells since no u-PA mRNA was detected in platelets. The platelet-induced u-PA synthesis was inhibited when the endothelial cells were pre-treated with phospholipase C to remove the u-PA receptor, or when the platelets were incubated with an antibody that blocks the binding of u-PA to u-PAR. Taken together, these data indicate that u-PA present on the platelet surface interacts with u-PAR on the endothelial cells and induces the u-PA synthesis. This mechanism may represent a physiological control of platelet-mediated intravascular fibrin deposition.
The circulatory half-lives of α -profibrin and α-fibrin monomer, and comparisons with other fibrin(ogen) derivatives
Authors: DiBello Patricia , Shainoff John 
Published: March 2018

SummaryPrevious studies showed that α-fibrin monomer (lacking both A-fibrinopeptides, FPA) is normally cleared from the circulation before it assembles into a clot. Recent studies indicate that substantial quantities of an intermediate, α-profibrin lacking only one of the two FPA are produced in the course of conversion of human fibrinogen to fibrin. Since clearance of the α-fibrin monomer is saturable and receptor mediated, the extent to which α-profibrin or other fibrin(ogen) derivatives might compete for monomer uptake was deemed important. We compared plasma decay of injected human α-fibrin, fibrinogen, and α-profibrin in rabbits using rabbit anti-human fibrinogen for assays. The circulatory half-life of human α-fibrin monomer was short (t1/2 = 2.3 h) and followed a simple exponential decay curve, as anticipated from clearance of rabbit α-fibrin. It was absorbed as fast as it permeated the extravascular space with no redistribution. Human fibrinogen had a long half-life (t1/2 = 39.5 h), calculated from the double exponential plasma decay curves (redistribution + catabolism) observed over 28 h. The α-profibrin had an intermediary half-life (t1/2 = 11 h) determined from double exponential decay curves. Since redistribution accompanied the slow clearance of α-profibrin, its binding by the fibrin receptor(s) must be weak, probably too weak to compete with the clearance of α-fibrin monomer. The initial production of α-fibrin monomer is only partially dependent on prior formation of α-profibrin, as recently shown. Thus, it is the slow clearance and the weak competition from α-profibrin that underlie the occurrence of substantial levels of α-profibrin unaccompanied by detectable levels of α-fibrin monomer in many subjects with vascular disease.
Elucidation of the paratope of scFv-8H9D4, a PAI-1 neutralizing antibody derivative
Authors: Verbeke Koen , Gils Ann , Stassen Jean-Marie , Declerck Paul 
Published: March 2018

SummaryInterfering with increased levels of plasminogen activator inhibitor-1 (PAI-1) might offer new therapeutic strategies for a variety of cardiovascular diseases. Inactivation of PAI-1 can be accomplished by a number of monoclonal antibodies (MA), including MA-8H9D4. In a previous study, a single-chain variable fragment (scFv-8H9D4) was cloned and found to have the same properties as the parental MA-8H9D4. In the present study, we identified the residues of scFv-8H9D4 that contribute significantly to the paratope. The complementarity determining region 3 from the heavy (H3) and the light (L3) chain were analysed through site-directed mutagenesis. Out of twelve mutations, only four residues appeared to contribute to the paratope. The affinity of scFv-8H9D4-H3-L97D for PAI-1 was 38-fold decreased (KA = 4.8 ± 0.2 × 107 M–1 vs. 1.8 ± 0.7 × 109 M–1 for scFv-8H9D4) whereas scFv-8H9D4-H3-R98Y did not bind to PAI-1. The affinities of scFv-8H9D4-L3-Y91S and scFv-8H9D4-L3-F94D for PAI-1 were 9- and 5-fold reduced, respectively, whereas the combined mutation resulted in an 86-fold decreased affinity (KA = 2.1 ± 0.2 × 107 M–1).In accordance with the affinity data, these mutants had no, or a reduced, PAI-1 inhibitory capacity, confirming that these four particular residues form the major interaction site of scFv-8H9D4 with PAI-1. In combination with the three-dimensional structure, these data contribute to the rational design of PAI-1 neutralizing compounds.
Stabilization of monocyte chemoattractant protein-1-mRNA by activated protein C
Authors: Marx Antje , Weiler Hans , Liebe Volker , Lang Siegfried , Kaden Jens , Zieger Wolfgang , Borggrefe Martin , Huhle Guenter , Haase Karl , Brueckmann Martina 
Published: March 2018

SummaryThe activated protein C (APC) pathway has been suggested to be a common link between coagulation and inflammation. APC may function to restore hemostasis via modulation of cytokine expression. We investigated the effect of APC on the endothelial expression of monocyte chemoattractant protein-1 (MCP-1), a chemokine that is controlled by the activation of central proinflammatory transcription factors, such as nuclear factor-kappa B (NF-κ B).We found that human APC (2.5-10 μ g/ml) upregulated the amount of MCP-1-mRNA in human umbilical vein endothelial cells (HUVEC) and caused a time- and dose-dependent increase in MCP-1 protein production (p <0.001 for APC 2.5 μg/ ml at 4 up to 24 h). In this cell culture model MCP-1 induced an improvement of cell migration and wound repair after injury to endothelial monolayers. After stimulation of MCP-1-mRNA-transcription with TNF-α (0.1-1 ng/ml), HUVEC’s were washed and an inhibitor of gene transcription, Actinomycin D (1 μg/ml), was added in the presence or absence of APC. HUVEC’s receiving APC contained more MCP-1-mRNA than controls after one hour and up to eight hours suggesting an inhibitory effect of APC on MCP-1-mRNA degradation (with APC: 753 ± 56 atto mol of MCP-1-mRNA per ml of cell lysate vs. 263 ± 60 atto mol/ml without APC at t =4 h; p <0.001). Electrophoretic mobility shift assays revealed that APC attenuated NF- κB DNA-binding capacity implying that NF- B may not be involved in the upregulatory effect of APC on MCP-1 production.The ability of APC to upregulate the production of MCP-1, most likely by increasing the stability of MCP-1-mRNA rather than by transcriptional activation via NF- B, identifies a novel immunomodulatory pathway, by which APC may control the local inflammatory reaction, thereby initiating wound repair and modulating the extent of endothelial injury.