bluuebunny/crossref_metadata_2025 huggingface dataset:
Poor Relationship between Phenotypes of Protein S Deficiency and Mutations in the Protein S Alpha Gene
Authors: Hermida José , Mannucci Pier Mannuccio , Faioni Elena
Published: March 2018

By single strand conformational polymorphism, nucleotide sequencing and enzyme restriction, we analyzed the protein S α gene in 17 protein S-deficient probands and in their available family members. The relationship between genotype and phenotype was also evaluated. Twelve different sequence variations were identified in 17 probands. Ten were putative causal mutations distributed in 16 probands: 4 were nonsense, 5 missense and one a splice site mutation. In most families in which a mutation was identified, more than one phenotype of PS deficiency was present. The same splice site mutation (intron j G-A, exon 10+5) was associated with type I deficiency in one family and with type I/III in another unrelated family. A phenotypic discrepancy was also observed for the Arg474Pro, Gly597Asp and Arg410stop mutations. Glu26Ala, previously reported in kindreds with type I deficiencies, was found in association with I, II and III phenotypes in four unrelated kindreds. Phenotypic analysis of protein S deficiency is poorly related to the underlying genetic defect.
Pharmacokinetics of Recombinant Factor VIII (Recombinate) Using One-stage Clotting and Chromogenic Factor VIII Assay
Authors: Owens D. , Bray G. , Giangrande P. , Collins P. , Hay C. , Gomperts E. , Schroth P. , Barrowcliffe T. , Lee C. A.
Published: March 2018

In a study designed to demonstrate the safety and pharmacokinetics of a recombinant factor VIII (Recombinate) manufactured in Andover, MA and Thousand Oaks, CA, two different methods of factor VIII assay (one-stage clotting and Chromogenic substrate) were compared in vivo. The study was performed in four centres in the UK: London, Oxford, Cardiff and Manchester. Two pharmacokinetic studies, at least one week apart, were performed in 30 patients with severe haemophilia A (VIII:C < 2 IU/dl). A dose of 50 IU/kg was administered with sampling pre-infusion, and +0.25, 0.5, 1, 3, 6, 9, 12 and 24 h post-infusion. The aggregate 60 pharmacokinetic study showed a half-life of 12.7 and 13.0 h (p = 0.28) and recovery of 127 and 161 IU/dl (p = 0.0001) using one-stage clotting or chromogenic substrate respectively. In a supplementary experiment, 20 post-infusion samples were re-assayed by 1-stage and chromogenic assay using two plasma (20th British plasma standard and an “in-house” pooled normal plasma) and two concentrate standards, derived from the same type, but different batch of infused concentrate (Recombinate) and pre-diluted in either individual pre-infusion sample or in pooled commercial haemophilic plasma. The use of the Recombinate concentrate standard overcame the significant difference in FVIII levels between 1-stage and chromogenic assay methods when a plasma standard was used (p <0.0001). It is concluded that where potency dosing designation is carried out by an assay system different to that used in the clinical situation, the use of the recombinant concentrate as a standard in post-infusion plasma samples is likely to give more reliable and reproducible results.
Mutations in the αIIb and β3 Genes that Cause Glanzmann Thrombasthenia Can Be Distinguished by a Simple Procedure Using Transformed B-Lymphocytes
Authors: Rosenberg Nurit , Dardik Rima , Rosenthal Ester , Zivelin Ariella , Seligsohn Uri
Published: March 2018

Glanzmann thrombasthenia (GT) is caused by a defect in either glycoprotein (GP) IIb (αIIb) or GPIIIa (β3) genes and therefore screening of both genes is required for mutation identification. The β subunit of the GPIIb/IIIa complex (β3) forms a complex with another α subunit (αv) yielding the αvβ3 vitronectin receptor (VnR). GT patients with mutations in the GPIIIa gene that cause diminished synthesis of GPIIIa are deficient in both GPIIb\IIIa and VnR, whereas patients with mutations in the GPIIb gene are deficient in GPIIb\IIIa, yet express normal or increased VnR in their platelets. The presence or absence of VnR in platelet membranes of GT patients has therefore been used for distinguishing between mutations in the GPIIb gene and mutations in the GPIIIa gene. However, the method of assessing VnR in platelets is cumbersome and use of fresh platelets is indispensible. In the present work we devised a procedure for detection of the VnR in B-lymphocytes transformed by Epstein-Bar virus (EBV). The transformed lymphocytes transcribed GPIIIa mRNA but not GPIIb mRNA and expressed VnR on their surface. Using flow cytometry analysis or immuno-precipitation and western blotting VnR was found in B-lymphocytes of GT patients bearing a well characterized mutation in the GPIIb gene. In contrast, in B-lymphocytes of GT patients bearing 2 different mutations in the GPIIIa gene no VnR was detectable. Thus, for determining which gene is mutated in a GT patient, EBV-transformed B-lymphocytes are useful and can as well be used for analyses of GPIIIa mRNA and genomic DNA. Ten ml of blood are sufficient for the procedure.
IgA Anticardiolipin Antibodies – Relation with other Antiphospholipid Antibodies and Clinical Significance
Authors: Ordi-Ros Josep , Monegal-Ferran Francesc , Martinez Nuria , Cortes-Hernandez Fina , Vilardell-Tarres Miquel , Selva-O’Callaghan Albert
Published: March 2018

Objective: To evaluate the usefulness of IgA antiphospholipid antibodies as markers of thrombosis and/or antiphospholipid antibody syndrome. Patients and Methods: A cross-sectional study design in a tertiary, university-based, autoimmune reference hospital. Seven-hundred ninety-five patients classified into five different groups – autoimmune diseases (255), deep vein thrombosis (153), transitory ischemic attacks (108), obstetric complications (196), infectious diseases (83) and controls (81) – were tested for IgA, IgG and IgM aPL, and lupus anticoagulant. Plasma and serum samples were drawn for detection of aPL using an internationally standardized ELISA method and LA was carried out using coagulometric assays. Results: True IgA aPL were found only in two patients with systemic lupus erythematosus; these patients were also positive to IgG aPL. Conclusion: The incidence of true positivity to IgA anticardiolipin antibodies is extremely low. Their determination was not helpful in diagnosing the antiphospholipid syndrome or in explaining thrombotic events or aPL related manifestations – fetal loss – in the groups studied.
Patterns of Hepatitis G Viraemia and Liver Disease in Haemophiliacs Previously Exposed to Non-virus Inactivated Coagulation Factor Concentrates
Authors: Jarvis Lisa , Hayes Peter , Lee Amanda , Simmonds Peter , Ludlam Christopher , Hanley John
Published: March 2018

Hepatitis G virus (HGV), a novel flavivirus, has been implicated as a cause of posttransfusion hepatitis. We have performed a longitudinal study in a cohort of haemophiliacs (n = 68) who previously received non-virus inactivated coagulation factor concentrates to assess both patterns of HGV viraemia and any associated liver disease. Hepatitis C virus (HCV) RNA was present in 58/68 and co-infection with human immunodeficiency virus (HIV) was present in 15/68.

HGV RNA was detected in 17/68 (25%) samples from the mid-1980s. There was no association between either HIV infection (p = 0.74) or co-infection with a particular HCV genotype (p = 0.62). However, there was a relationship between HGV viraemia and the severity of haemophilia (p = 0.0004) with HGV RNA detected in 5/19, 9/16 and 3/32 patients with mild, moderate and severe haemophilia respectively.

A longitudinal study was performed in 15/17 haemophiliacs with HGV viraemia using stored serum samples from the 1980s and 1990s. HGV viraemia persisted in 8/15 and cleared in 7/15 over a variable period of time. A Weibull model was constructed to estimate the duration of HGV viraemia in the study group. The 75th and 90th percentiles for the duration of HGV were estimated to be 8.7 years (95%, confidence interval 4.8-15.7) and 23.6 years (95% confidence interval 11.8-47.1) respectively. Laparoscopic liver inspection/biopsy was performed in 25/68. There was no association between severity of liver disease and HGV viraemia (p = 0.43). This study demonstrates considerable variation in patterns of HGV viraemia in haemophiliacs. We found little evidence to implicate HGV as a major cause of chronic liver disease in haemophiliacs.
Cloning, Expression, and Characterization of Mouse Tissue Factor Pathway Inhibitor (TFPI)
Authors: Monroe Dougald , Oliver Julie , Liles Darla , Roberts Harold , Chang Jen-Yea
Published: March 2018

Tissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor Xa/ TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA sequences of TFPI from several different species have been previously reported. A high level of similarity is present among TFPIs at the molecular level (DNA and protein sequences) as well as in biochemical function (inhibition of factor Xa, VIIa/tissue factor). In this report, we used a PCR-based screening method to clone cDNA for full length TFPI from a mouse macrophage cDNA library. Both cDNA and predicted protein sequences show significant homology to the other reported TFPI sequences, especially to that of rat. Mouse TFPI has a signal peptide of 28 amino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, like that of other species, consists of three tandem Kunitz type domains. Recombinant mouse TFPI was expressed in the human kidney cell line 293 and purified for functional assays. When using human clotting factors to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factors VIIa, IXa, as well as factor XIa. Cloning and expression of the mouse TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.
Modulation of Pro- and Antifibrinolytic Properties of Human Peritoneal Mesothelial Cells by Transforming Growth Factor β1 (TGF- β1), Tumor Necrosis Factor α (TNF-α) and Interleukin β1 (IL-1β)
Authors: Elbrecht Anne , Schauerte Carsten , Klosterhalfen Bernd , Amo-Takyi Baffour , Gehlen Johanna , Winkeltau Gerd , Mittermayer Christian , Handt Stefan , Tietze Lothar
Published: March 2018

A decreased fibrinolytic activity of serosal surfaces appears to be a major factor in the development of peritoneal fibrous adhesions. Serosal fibrinolysis is regulated by mesothelial release of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor types 1 and 2 (PAI-1 and PAI-2). We investigated the influence of tumor necrosis factor alpha (TNF-α), transforming growth factor β (TGF- β1) and interleukin 1β (IL-1β) on pro- and antifibrinolytic properties of mesothelial cells (HOMC) using a cell/fibrin clot assay. TGF-β1, TNF-α and IL-1β induced a dose dependent 2.9, 2.3 and 1.9-fold increase of PAI-1 antigen, respectively, whereas t-PA concentrations decreased to one third of the control values. This modified PAI-1/t-PA secretion pattern leads to a significant delay of fibrinolysis. Analysis of m-RNA levels revealed increased PAI-1 m-RNA concentrations after 12 h and decreased m-RNA concentrations for t-PA after 6 h. Serosal hypofibrinolysis during peritonitis may be explained at least in part by cytokine effects which thus may favor adhesion formation.
Effect of Antihypertensive Treatment with Doxazosin on Insulin Sensitivity and Fibrinolytic Parameters
Authors: Christ Günter , Gabriel Harald , Mundigler Gerald , Beckmann Renate , Binder Bernd , Huber Kurt , Zehetgruber Manfred
Published: March 2018

The effects of the selective alpha-1-adrenoceptor antagonist doxazosin on metabolic and fibrinolytic parameters were studied in hypertensive patients with various degrees of fasting plasma insulin levels (Group A: 22.5 ± 3 μU/ml, Group B: 8.1 ± 1.5 μU/ml; p <0.01) to disclose a potential link between a doxazosin-induced alteration of insulin and/or lipid metabolism and possible changes of these parameters on the fibrinolytic system. Doxazosin treatment resulted in a dose-dependent reduction of basal insulin levels in group A to 16 ± 3 μU/ml; p <0.05. This finding was paralleled by a dose-dependent increase in t-PAmass concentration in the same patient group (basal t-PAmass from 9.7 ± 1 to 15.5 ± 2 ng/ml; p <0.05). As PAI-1 “active” as well as total antigen levels were not altered in parallel, the net effect on the endogenous fibrinolytic system is an increase of the fibrinolytic potential.
In Vitro Anticoagulant Properties of a Minimum Functional Fragment of Human Thrombomodulin and In Vivo Demonstration of its Benefit as an Anticoagulant in Extracorporeal Circulation Using a Monkey Model
Authors: Suzuki Makoto , Yamamoto Syuji , Mohri Mitsunobu
Published: March 2018

Recently, we demonstrated that the last three consecutive epidermal growth factor-like structures of human thrombomodulin form the minimum functional fragment for protein C activating activity. In this study, the recombinant minimum functional fragment of human thrombomodulin (E456) had similar activities for protein C activation and clotting time compared to the recombinant extracellular domains of human thrombomodulin (rhsTM) previously described. E456 had a shorter half-life (6-9 min) in animals, compared to rhsTM (5 h). To evaluate whether or not E456 is a suitable anticoagulant for extra-corporeal circulation, the effects of E456 were investigated in the extracorporeal circulation model in monkeys. The effect of E456 at a dose of 0.03 mg/kg plus 0.113 mg/kg/h on circuit pressure was as potent as that of low molecular weight heparin, Fragmin, at a dose of 30 IU/kg plus 15IU/kg/h. E456, however, also more effectively suppressed an increase of thrombin-antithrombin III complex than Fragmin. Also the APTT quickly returned to the pre-treatment level following E456, but did not when using Fragmin. Moreover, E456 had no influence on the plasma free fatty acid levels, while Fragmin significantly increased them. Thus, E456 appears to have anticoagulant properties which make it more suitable anticoagulant in extracorporeal circulation than Fragmin.
BCH-2763, a Novel Potent Parenteral Thrombin Inhibitor, Is an Effective Antithrombotic Agent in Rodent Models of Arterial and Venous Thrombosis – Comparisons with Heparin, r-Hirudin, Hirulog, Inogatran and Argatroban
Authors: Finkle Carolyn , Pierre Annie , Leblond Lorraine , Deschenes Isabelle , DiMaio John , Winocour Peter
Published: March 2018

Current clinical use of heparin as an antithrombotic agent is limited by suboptimal efficacy and safety considerations. Thrombin’s central role in thrombosis makes it an attractive target to develop more effective and safer antithrombotic agents. BCH-2763 is a novel, potent (Ki: 0.11 nM), low molecular weight (1.51 kDa), bivalent direct thrombin inhibitor. The antithrombotic efficacy of BCH-2763 in vivo following i.v. bolus plus infusion in rats was compared in arterial and venous thrombosis models with two other bivalent direct thrombin inhibitors, r-hirudin and hirulog, with two catalytic site-directed thrombin inhibitors, inogatran and argatroban, and with heparin. In vivo efficacy was related to inhibition in vitro of fibrin clot formation, thrombin-induced aggregation of rat or human washed platelets and activity of free and plasma clot-bound thrombin. All the direct thrombin inhibitors were effective on both arterial and venous thrombosis at markedly lower fold aPTT increases than heparin. The antithrombotic doses of all inhibitors against venous thrombosis were less than against arterial thrombosis. The rank order of potency based on doses (mg/kg/h) required for full efficacy against arterial thrombosis was BCH-2763 (1.2) inogatran (1.5) r-hirudin (1.8) hirulog (3.3) argatroban ( 3.0); heparin required a markedly higher dose (5.7). In venous thrombosis the doses required for full efficacy were substantially lower for the bivalent (BCH-2763: 0.12; r-hirudin: 0.12; hirulog: 0.18) than for the catalytic site-directed (inogatran: 0.48; argatroban: 0.90) thrombin inhibitors; the dose required for heparin was 0.19. All the direct thrombin inhibitors caused similar shifts in aPTT at doses required to inhibit arterial thrombosis, but BCH-2763 inhibited venous thrombosis at lower aPTT fold increases. In vivo antithrombotic efficacy of direct thrombin inhibitors correlated with their inhibitory activity in vitro against fibrin clot formation and platelet aggregation. In contrast to heparin, all the direct thrombin inhibitors inhibited plasma clot-bound thrombin, but the relative IC50s did not correlate with their antithrombotic efficacy. In summary, direct thrombin inhibitors are more effective than heparin in inhibiting arterial and venous thrombosis in rats with less aPTT increases. BCH-2763 is effective at lower doses than the other direct thrombin inhibitors and for venous thrombosis at a smaller aPTT increase. BCH-2763 may offer an improved therapeutic index in the treatment of thromboembolic complications over heparin and other direct thrombin inhibitors.
Comparative Studies of an Orally-active Factor Xa Inhibitor, YM-60828, with other Antithrombotic Agents in a Rat Model of Arterial Thrombosis
Authors: Sato Kazuo , Sakai Yumiko , Hirayama Fukushi , Koshio Hiroyuki , Taniuchi Yuta , Matsumoto Yuzo , Kawasaki Tomihisa
Published: March 2018

We examined the antithrombotic activity of a novel synthetic inhibitor of factor Xa, YM-60828, in an electrically-induced carotid artery thrombosis model in rats. In the first experiment, the antithrombotic activity of YM-60828 after i.v. infusion was compared with those of heparin, darteparin and argatroban. Test drug was administered by i.v. infusion from 30 min before electrical stimulation to the end of the experiment. YM-60828 at 1 mg/kg/h significantly improved patency status, prolonged the time to occlusive thrombus formation and duration of patency. Heparin at 300 U/kg/h also improved these parameters, but were accompanied by a marked increase in systemic coagulation time. In the second experiment, the antithrombotic activity of YM-60828 after oral administration was compared with those of ticlopidine, cilostazol, aspirin, beraprost, ethyl icosapentate and warfarin. Test drug was orally administered to fasted rats 60 min before electrical stimulation. YM-60828 at 30 mg/kg p.o., but not ticlopidine, cilostazol, aspirin, beraprost, ethyl icosapentate or warfarin, significantly reduced the incidence of occlusion and improved carotid arterial patency. These results suggest that YM-60828 may be a promising antithrombotic agent for the treatment and prevention of arterial thrombosis which can be given by oral as well as intravenous administration.
Predictive Value of Plasma Thrombomodulin in Preeclampsia and Gestational Hypertension
Authors: Valsecchi Luca , Fausto Alfonso , Gozin Danielle , Vigano’ D’Angelo Silvana , Safa Omid , Castiglioni Maria Teresa , Amiral Jean , D’Angelo Armando , Boffa Marie-Claire
Published: March 2018

In a prospective longitudinal study, 130 primigravidae at risk for preeclampsia were examined and plasma sampling performed in 45 of them. Plasma thrombomodulin (pTM) was sequentially measured at weeks 12, 24 and 32 of gestation and after delivery in 20 primigravidae who developed either mild preeclampsia (n = 8) or gestational hyper-tension (n = 12) between weeks 32 and 39 of gestation and in 25 (age-matched) primigravidae who had uneventful pregnancies. pTM elevations were not observed until week 32 in uneventful pregnancies, but were present by week 24 (p = 0.002) in patients who later developed hypertensive complications. A net individual pTM increase ≥4.2 ng/ml between weeks 12 and 24 (more than 8 times that of normotensive primigravidae) and/or pTM level ≥47.5 ng/ml at week 32 predicted the development of hypertensive complications with 80% accuracy. Serial pTM determinations can be useful to select pregnancies who may benefit from early pharmacological intervention.
Elevation of Serum Thrombopoietin Precedes Thrombocytosis in Kawasaki Disease
Authors: Ishikita Takashi , Shimbo Toshikazu , Matsubara Kousaku , Baba Kunizou , Hayashi Yasuhide , Naritaka Shin-ichi , Nakahata Tatsutoshi , Ishiguro Akira
Published: March 2018

Kawasaki disease (KD) is an acute systemic vasculitis causing coronary arterial aneurysms and myocardial infarction in young children. Prominent thrombocytosis with increased megakaryocytes develops during the convalescent period. To clarify the mechanisms of thrombocytosis, we studied serum levels of thrombopoietin (TPO) and other thrombopoietic cytokines in 40 patients with KD (149 samples) and 106 age-matched controls using ELISA. TPO values in the controls were 1.94 ± 0.69 fmol/ml (mean ± SD) with a 95% reference interval of 0.85 to 3.27 fmol/ml. In the first week of KD, platelet counts were normal but TPO values increased (~ 15.5 fmol/ml). TPO levels peaked on day 6 ± 2 at 5.94 ± 2.64 fmol/ml and then fell gradually. When platelet counts peaked in the second to third weeks, TPO levels were still high or comparable with the controls. TPO levels in KD patients with normal platelet counts were significantly higher than control levels. Interleukin (IL)-6 levels in the first week rose, but neither IL-11 nor leukemia inhibitory factor was detectable. These results suggest that TPO contributes to thrombocytosis in KD in conjunction with IL-6 and TPO production may be enhanced during the acute phase.
Significant Correlations between Tissue Factor and Thrombin Markers in Trauma and Septic Patients with Disseminated Intravascular Coagulation
Authors: Nanzaki Satoshi , Sasaki Shigeyuki , Kemmotsu Osamu , Gando Satoshi
Published: March 2018

To determine the role of plasma tissue factor on disseminated intravascular coagulation (DIC) in trauma and septic patients, and also to investigate the relationships between tissue factor and various thrombin markers, we made a prospective cohort study. Forty trauma patients and 20 patients with sepsis were classified into subgroups according to the complication of DIC. Plasma tissue factor antigen concentration (tissue factor), prothrombin fragment F1+2 (PF1+2), thrombin antithrombin complex (TAT), fibrinopeptide A (FPA), and D-dimer were measured on the day of admission (day 0), and on days 1, 2, 3, and 4 after admission. The levels of plasma tissue factor in the DIC group were more elevated than those of the non-DIC group in both the trauma and the septic patients. In patients with sepsis, tissue factor levels on days 0 through 4 in the non-DIC group showed markedly higher values than those in the control patients (135 ± 8 pg/ml). Significant correlations between tissue factor and PF1+2, TAT, FPA, and D-dimer were observed in the DIC patients, however, no such correlations were found in the non-DIC patients. These results suggest that elevated plasma tissue factor in patients with trauma and sepsis gives rise to thrombin generation, followed by intravascular coagulation.
Characterization of a Factor VII Molecule Carrying a Mutation in the Second Epidermal Growth Factor-like Domain
Authors: Kavlie Anita , Örning Lars , Grindflek Anne , Stormorken Helge , Prydz Hans
Published: March 2018

A missense mutation at codon 100 in the second epidermal growth factor-like domain, resulting in Gln100→Arg, was detected in 19 out of 21 available severely factor VII (FVII) deficient patients in Norway. Seventeen patients were homozygous, and the two remaining were compound heterozygotes. In the homozygous patients, FVII antigen was measured to 10-28%, and activity to 0.6-6.5% of that in normal pooled plasma. Recombinant FVII containing the mutation was expressed transiently in CHO cells to a mean antigen level of 57% of the wild type FVII protein, and with a specific activity of 6% of wild type. The mutant protein had a 14-fold reduction in affinity for tissue factor (TF), whereas binding of FX seemed unaffected. In line with the experimental data, molecular modelling of the mutation based on the coordinates of the tissue factor/FVIIa complex showed that substituting arginine for glutamine disrupts the interface between the catalytic and second epidermal growth factor-like domains.
New Splicing Mutations in the Human Factor XIIIA Gene, Each Producing Multiple Mutant Transcripts of Varying Abundance
Authors: Miloszewski Krzysztof , Markham Alexander , Anwar Rashida
Published: March 2018

Coagulation factor XIII, a transglutaminase which stabilises blood clots by covalently cross-linking fibrin, is essential for normal haemostasis. FXIII deficiency results in a life-long bleeding disorder with added complications in wound healing and tissue repair. Sequence changes in the human FXIIIA gene, largely missense mutations, are primarily responsible for inherited FXIII deficiency. We have carried out molecular analysis of the FXIIIA gene in two unrelated FXIII deficient individuals and identified three splice site mutations; a g→a at the exon 6 acceptor splice site, a g→a at the exon 7 donor splice site and a coding sequence T→G at the exon 8 donor splice site. We have also examined the FXIIIA mRNA in these patients and find that each mutation gives rise to multiple transcripts which vary in their relative abundance. The precise molecular mechanisms which result in these variant transcripts, and their relative abundance in our FXIII deficient patients, are discussed.
Nitric Oxide and Prostacyclin Are Involved in Antithrombotic Action of Captopril in Venous Thrombosis in Rats
Authors: Chabielska Ewa , Golatowski Janusz , Azzadin Arsalan , Buczko Włodzimierz , Pawlak Robert
Published: March 2018

The long-term administration of captopril to patients with a left ventricular dysfunction after myocardial infarction reduces the rate of recurrent coronary thrombosis. Thus, in the present study we investigated the influence of angiotensin-converting enzyme inhibitors (ACE-Is) on experimental venous thrombosis in normotensive rats and the involvement of NO and PGI2 in this effect. Animals were treated with captopril (1.5, 5 or 25 mg/kg twice daily, CAP), enalapril (15 mg/kg once daily, ENA) or distilled water for 10 days, per os. After ligation of the vena cava the thrombus weight decreased in both CAP and ENA treated rats. The effect was most pronounced in animals given the highest dose of CAP (p<0.0001 vs. control) and was significantly stronger than observed in ENA treated animals (CAP vs. ENA p<0.01). The mean blood pressure measured by the “tail cuff” method and platelet aggregation were not altered by either of the ACE-Is. The antithrombotic activity of CAP was reduced by indomethacin (2.5 mg/kg, s.c.) and independently by the NO-synthase inhibitor NG-nitro L-arginine methyl ester (3 mg/kg i.v. bolus + 3 mg/kg/h i.v. infusion, L-NAME). In the latter case CAP regained its antithrombotic properties in rats pretreated with L-Arginine (300 mg/kg i.v. + 300 mg/kg/h i.v.) before administration of L-NAME (p<0.05 vs. control). Moreover, the concomitant administration of indomethacin and L-NAME failed to completely abolish the antithrombotic action of captopril. Similar effects were observed in respect to the incidence of venous thrombosis. Our study documents a novel and important effect of ACE-Is on the vein thrombotic process and demonstrates the involvement of NO and PGI2 in this phenomenon.
Factor XI and Protection of the Fibrin Clot against Lysis – a Role for the Intrinsic Pathway of Coagulation in Fibrinolysis
Authors: von dem Borne Peter , Meijers Joost , Bouma Bonno
Published: March 2018

Introducation

Blood coagulation is an important mechanism that maintains the integrity of the vascular system to prevent blood loss after injury. The conceptions on the working mechanism of coagulation are based on the waterfall or cascade model, which was already proposed more than 30 years ago, independently by Davie and Ratnoff (1) and MacFarlane (2). Blood coagulation was viewed as a series of linked proteolytic reactions in which zymogens are converted into serine proteases, ultimately leading to the formation of thrombin, which converts soluble fibrinogen into insoluble fibrin. Coagulation was thought to proceed via two pathways, an extrinsic and an intrinsic pathway. Activation of the extrinsic pathway of coagulation occurs by the exposition of tissue factor at the site of injury (3) whereas the intrinsic system is activated after exposure of plasma to an activating surface. Although the in vivo activating surface is unknown, the contact system was believed to play a role in the initiation of the intrinsic pathway. This system consists of factor XII, prekallikrein, high molecular weight kininogen and factor XI. The physiological relevance of the contact system is unclear, since a deficiency of factor XII, prekallikrein or 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 (urinary tract, nose, oral cavity, tonsils) (4, 5). This suggested there was an alternative route for the activation of factor XI, and recently such a route was described (6, 7). Thrombin was found to activate factor XI, even in the absence of a negatively charged surface (6-11), and factor XI was shown to play a role in the protection of the fibrin clot against lysis (9). In plasma the possibility cannot be excluded that the activation of factor XI by thrombin takes place via an intermediary component. Recently, it was shown that meizothrombin was capable of activating factor XI (12).
Comparative Studies on the Antiplatelet Effects of a Humanized Anti-Platelet Glycoprotein IIb/IIIa Antibody (YM337) and ReoPro under Flow Conditions
Authors: Kuwahara Mitsuhiro , Tsuji Shizuko , Miyata Shigeki , Nishio Kenji , Kaku Seiji , Suzuki Ken-ichi , Kawasaki Tomihisa , Yoshioka Akira , Sugimoto Mitsuhiko
Published: March 2018

Recent clinical studies have shown that the interruption of platelet function appears to be effective for treatment of coronary occlusive diseases. For this purpose, a Fab fragment of humanized anti-platelet glycoprotein (GP) IIb/IIIa monoclonal antibody (YM 337) that exhibited an in vivo antithrombotic effect without prolongation of bleeding time in monkeys was previously characterized. In this study, the effect of YM 337 under physiological flow conditions with high or low shear rate was evaluated. The antiplatelet effects of YM 337 under varying wall shear rates were examined with the whole blood flow system. This technique allows real time visualization of the formation of fluorescence-labeled platelet thrombi on a collagen surface in a parallel plate flow chamber mounted on an epifluorescence microscope. The process of thrombus growth was also recorded in video tape and subjected to computer-assisted image analysis. We found that YM 337 displayed a high shear-preferential antiplatelet effect, while ReoPro, a control anti-GP IIb/IIIa antibody, did not show such shear dependency. The present findings therefore suggest that YM 337 might be a useful antiplatelet agent which can block pathological thrombotic events occurring under high shear, such as in coronary occlusive diseases.
Prevalence of 20210 A Allele of the Prothrombin Gene in Venous Thromboembolism Patients
Authors: Mercier Bernard , Oger Emmanuel , Chenu Eric , Abgrall Jean-François , Férec Claude , Mottier Dominique , Leroyer Christophe
Published: March 2018

Background. The 20210 A allele variation in the 3’ -untranslated region of the prothrombin gene was recently identified as a risk factor as regards deep venous thrombosis. Aim. To assess the frequency of the variation in unselected patients with a proven venous thromboembolism (VTE). Methods. The presence of the prothrombin variation was determined in all consecutive patients referred from July 1994 to August 1997 for a clinical suspicion of VTE, and in whom the diagnosis was confirmed. A control group consisted of bone marrow volunteer donors. Results. Of the 366 patients included, 17 (4.6%) were carriers of the 20210 A allele (95% CI, 2.4% to 6.7%). The mutation was present in 1.0% of the 400 controls. Odds ratio for having VTE in the presence of the 20210 A allele was 4.8 (95% CI, 1.5 to 19.8). Forty-six (12.5%) patients had the mutation of the factor V gene and five (1.4%) patients shared both mutations. After excluding the carriers of the factor V mutation, odds ratio for having VTE in the presence of the 20210 A allele was 3.7 (95% CI, 1.1 to 13.6). Mean age at admission as well as mean age of the first VTE episode were both significantly higher in patients free from the two mutations studied, as compared to carriers of the 20210 A allele (p = 0.04 and 0.01, respectively). Conclusion. Our findings in a large series of patients (1) confirm the 20210 A allele prothrombin gene as a risk factor for VTE. (2) suggest that its association with the factor V Leiden is not uncommon.
Factor XII Does not Initiate Prekallikrein Activation on Endothelial Cells
Authors: Røjkjær Rasmus , Hasan Ahmed , Motta Guacyara , Schousboe Inger , Schmaier Alvin
Published: March 2018

It is well known that on artificial surfaces, binding and autoactivation of factor XII (FXII) is the initiating event of plasma prekallikrein (PK) activation. We performed investigations to examine whether this mechanism was true for FXII activation on endothelial cells (HUVEC). Activation of PK on HUVEC required an optimal substrate and Zn2+ concentration, the latter of which varied with the buffer’s carrier protein. Maximal PK activation required the addition of 250 μM or 10 μM Zn2+ to buffers containing bovine serum albumin (BSA) or gelatin, respectively. However, the actual free Zn2+ concentration in these buffers was the same at 8 μM. In both BSA- and gelatin-containing buffers and using two different chromogenic substrates for FXII, no autoactivation of FXII on HUVEC was seen when incubated for up to 60 min. Rather, initiation of FXII enzymatic activity required the presence of PK. FXII activation after PK activation contributed to the extent of measured enzymatic activity, but its role was secondary because treatment with corn trypsin inhibitor or a neutralizing antibody to FXIIa did not abolish the measured enzymatic activity. They also reduced the activity to the level seen with PK activation alone. Alternatively, soybean trypsin inhibitor abolished the proteolytic activity associated with PK and FXII activation on HUVEC. Further, only normal human and FXII-deficient plasmas, not PK-deficient plasma, had the ability to generate proteolytic activity when incubated over endothelial cells. In a purified system, maximal PK activation was measured after a 10-15 min incubation depending upon the concentration of reactants. When FXII was added with the PK, maximal activation occurred within 7.5-10 min. In normal human or FXII-deficient plasmas, but not in PK-deficient plasma, maximal activation was seen in 4 min. These data indicate that on HUVEC, unlike artificial surfaces, PK activation when bound to HK is the initiating activation event in this system. FXII activation is secondary to PK activation and contributes to the extent of measured enzymatic activity. These data challenge the accepted dogmas of “contact activation” and suggest that on biologic membranes a new notion as to how this system is activated needs to be considered.
Cleavage Requirements of Factor V in Tissue-factor Induced Thrombin Generation
Authors: Thorelli Elisabeth , Kaufman Randal , Dahlbäck Björn
Published: March 2018

Factor V (FV) activation is the result of cleavages at Arg709, Arg1018 and Arg1545 by thrombin or FXa. The relative importance of these cleavages in tissue factor (TF) induced thrombin generation in plasma and in a purified system was elucidated with recombinant FV in which the three sites had been eliminated one by one or in combinations. The mutants were analyzed with a clotting assay using FV-deficient plasma and in a TF induced thrombin generation system using plasma or purified components. Surprisingly, in the standard FV clotting assay, all mutants gave similar clotting activities and the thrombin generation curves obtained with wild-type and thrombin-resistant FV were similar. Differences in clotting activities and thrombin generation patterns between wild-type and thrombin-resistant FV were only observed when lower TF concentrations were used. The thrombin generation curve obtained in plasma containing wt FV was characterized by a short lag phase and a subsequent phase of rapid thrombin generation (propagation phase). The Arg709 to Gln mutation yielded a slightly prolonged lag phase and the rate of thrombin generation during the propagation phase was approximately 5-fold lower than that observed with wt FV. The Arg1018 to Ile mutation only slightly affected the thrombin generation curve, whereas the Arg1545 to Gln mutation yielded a prolonged lag phase and decreased maximum thrombin activity. Thrombin-resistant FV (mutated at all three sites) yielded a prolonged lag phase and poor thrombin generation during the propagation phase. The purified system further demonstrated the importance of the three cleavage sites for rapid and sustained thrombin generation. The results demonstrate that cleavages at positions 709, 1018 and 1545 are not required for assembly of a FXa-FV complex expressing low but significant prothrombinase activity but that all three sites in different ways are important for the creation of a FVa which maximally supports the FXa-mediated activation of prothrombin.
Correlates of Antithrombin, Protein C, Protein S, and TFPI in a Healthy Elderly Cohort
Authors: Sakkinen Pamela , Cushman Mary , Psaty Bruce , Kuller Lewis , Bajaj S. Paul , Sabharwal Arun , Boineau Robin , Macy Elizabeth , Tracy Russell
Published: March 2018

The majority of fatal acute myocardial infarctions occur in the elderly. Since these events are predominantly thrombotic, we studied the cross-sectional associations of the anticoagulant proteins Anti-thrombin, Protein C, Protein S, and Tissue Factor Pathway Inhibitor (TFPI) in a subgroup (n = 400) of the Cardiovascular Health Study (a study of healthy men and women ≥65 years) free of clinical cardiovascular disease (CVD).

We did not observe any strong age-associated trends, although Protein C was lower in older women (p ≤0.001), and TFPI was higher in older men (p ≤0.01). The inhibitors were highly intercorrelated, and were associated with increased levels of inflammation-sensitive proteins (e.g., fibrinogen, plasminogen), lipids (especially total and LDL-cholesterol), and coagulation factors, such as Factors VIIc, IXc, and Xc. None was associated with the procoagulant markers Prothrombin Fragment F1-2 or Fibrinopeptide A. Only TFPI was associated with subclinical atherosclerosis: ankle-arm index and internal carotid artery stenosis, p trend ≤0.01; and carotid wall thickness, p trend ≤0.05. In multivariate analysis the independent predictors of TFPI were levels of fibrinogen; the fibrinolytic marker plasminantiplasmin complex; LDL-cholesterol; and carotid wall thickness (R2 for the model = 0.35).

In summary, the inhibitors did not appear to increase with age, and were predominantly associated with inflammation markers and lipids. Since markers of thrombin production do increase with age, we hypothesize that an age-related hemostatic imbalance may ensue, with associated increased thrombotic risk. Only TFPI was associated with subclinical CVD, suggesting that it may more closely reflect endothelial damage. (250)
Native Atherosclerosis and Vein Graft Arterialization: Association with Increased Urokinase Receptor Expression In Vitro and In Vivo
Authors: Golden Michael , Raghunath P. N. , Tomaszewski John , David Mary , Kuo Alice , Kariko Katalin , Barnathan Elliot , Okada S. Steve
Published: March 2018

Interaction of proteases with cell surface receptors may modulate cell adhesion, migration, invasion, and matrix degradation. Since the plasminogen activator system has been hypothesized to play a role in intimal thickening after various types of vascular injury, we first studied the expression of urokinase receptor (u-PAR) protein and mRNA by smooth muscle cells (SMC) grown in explant cultures from normal and diseased vessels. Using equilibrium binding studies with radiolabeled 125I-labeled single chain urokinase-type plasminogen activator (scu-PA), we determined that SMC cultured from atherosclerotic arteries expressed a higher maximal number of binding sites/cell (3.6 ± 0.4 × 105 sites/cell vs. 2.1 ± 0.3 × 105, ± SEM, p <0.05) with a similar affinity (Kd = 1.5 ± 0.1 vs. 1.2 ± 0.2 nM, p = ns). However, SMC subcultured from diseased saphenous vein grafts expressed the highest levels of u-PAR compared to SMC from normal saphenous vein (4.8 ± 0.6 × 105 sites/cell vs. 1.6 ± 0.9 × 105, ± SEM, p <0.05). Using binding studies and Northern analysis, we demonstrated a dose and time dependent upregulation of u-PAR protein and mRNA expression respectively in human SMC in response to serum stimulation. Using a rabbit specific u-PAR cDNA probe, we demonstrated a similar upregulation of u-PAR mRNA both in rabbit aortic SMC in culture in response to serum stimulation and up to a 20 fold increase in u-PAR mRNA in rabbit jugular veins in response to implantation as arterial grafts in vivo. Finally, to confirm that u-PAR mRNA is upregulated in human vessels after injury, we performed immunohistochemistry and in situ hybridization studies on coronary arteries, normal saphenous veins and saphenous veins from 10 weeks to 13 years after implantation as grafts. u-PAR mRNA was found mainly in the periadventitial microcirculation in normal veins, but was found to be upregulated in the neointima and media of thickened veins in both macrophages and smooth muscle cells. SMC near the internal elastic laminae in diseased coronary arteries appeared to express increased u-PAR mRNA. These data suggest that this increased expression of u-PAR may contribute to early lesion development.
Fibrino(geno)lytic Properties of Purified Hementerin, a Metalloproteinase from the Leech Haementeria depressa
Authors: Chudzinski-Tavassi Ana Marisa , Kelen Eva Maria , Rosa Ana Paula de Paula , Loyau Stephane , Sampaio Claudio , Bon Cassian , Anglés-Cano Eduardo
Published: March 2018

The fibrino(geno)lytic protein designated hementerin contained in crude extracts of the salivary complex of Haementeria depressa leeches was purified to apparent homogeneity by gel filtration, ion exchange chromatography and preparative SDS-PAGE. It is a single-chain 80 kDa, PhMeSO2F-resistant, calcium-dependent, metalloproteinase, which specifically degrades fibrin(ogen) through a plasminogen-independent pathway. The amino terminal sequence of 8 residues shows 80% similarity with hementin, another fibrino(geno)lytic protein purified from Haementeria ghilianii leeches. However, their activities differ somewhat in terms of kinetics and with regard to the structure of the fibrin(ogen) fragments they may produce. Cleavage by hementerin of fibrinogen Aα, γ and Bβ chains, in that order, produces 270 kDa to 67 kDa fragments which differ from those produced by plasmin. Hementerin was also able to degrade cross-linked fibrin although at a lower rate as compared to fibrinogen. In conclusion, hementerin is a plasminogen-independent fibrino(geno)lytic metalloproteinase that degrades fibrinogen faster than fibrin, prevents blood coagulation and destroys fibrin clots in vitro.
Deletion Polymorphism in the Angiotensin-converting Enzyme Gene as a Thrombophilic Risk Factor after Hip Arthroplasty
Authors: Dilley Anne , Saidi Parvin , Evatt Bruce , Austin Harland , Zawadsky Joseph , Harwood David , Ellingsen Dorothy , Barnhart Elizabeth , Phillips Donald , Hooper William Craig , Philipp Claire
Published: March 2018

Despite thromboprophylaxis, deep vein thrombosis is a common complication of major orthopedic surgery. Predisposing genetic risk factors are unknown. In this case-control study, we investigated the association of the insertion (I)/deletion (D) angiotensin converting enzyme (ACE) gene polymorphism, Factor V Leiden (R506Q) mutation, and 5,10 methylenetetrahydrofolate reductase (MTHFR) gene polymorphism with post-operative venous thrombosis in 85 patients who underwent elective total hip arthroplasty. The odds of a thrombotic event following hip surgery among subjects with the DD genotype of the ACE gene was increased more than 10-fold compared to subjects with the II genotype (odds ratio 11.7 [95% confidence interval 2.3-84.5]); it was increased 5-fold in subjects with the ID genotype compared to the II genotype (odds ratio 5.0 [95% confidence interval 1.1-34.9]). Mean plasma ACE level in control subjects not on ACE inhibitors at the time of study (n = 43) was lowest in persons homozygous for the I allele (18.9 ± 7.95 U/l), intermediate in patients with the ID genotype (31.6 ± 10.8 U/l) and highest in subjects homozygous for the D allele (44.0 ± 7.14 U/l). Mean plasma ACE level among cases was higher (33.0 U/l, n = 25) than among controls (29.4 U/l, n = 43) but this difference was not statistically significant. Neither the Factor V Leiden mutation nor MTHFR gene polymorphism increased the risk of thrombosis following hip replacement. These results demonstrate that the I/D ACE gene polymorphism is a potent risk factor for thrombosis in subjects undergoing total hip arthroplasty.
Hyperhomocysteinemia and Venous Thrombosis: A Meta-analysis
Authors: Heijer Martin den , Rosendaal Frits , Blom Henk , Gerrits Wim , Bos Gerard
Published: March 2018

Hyperhomocysteinemia is an established risk factor for atherosclerosis and vascular disease. Until the early nineties the relationship with venous thrombosis was controversial. At this moment ten case-control studies on venous thrombosis are published. We performed a meta-analysis of these reports.

We performed a MEDLINE-search from 1984 through June 1997 on the keywords “homocysteine” or “hyperhomocysteinemia” and “venous thrombosis”, which yielded ten eligible case-control studies.

We found a pooled estimate of the odds ratio of 2.5 (95% CI 1.8-3.5) for a fasting plasma homocysteine concentration above the 95th percentile or mean plus two standard deviations calculated from the distribution of the respective control groups. For the post-methionine increase in homocysteine concentration we found a pooled estimate of 2.6 (95% CI 1.6-4.4).

These data from case-control studies support hyperhomocysteinemia as a risk factor for venous thrombosis. Further research should focus on the pathophysiology of this relationship and on the clinical effects of reducing homocysteine levels by vitamin supplementation.
No Association between the 20210 G/A Prothrombin Gene Mutation and Premature Coronary Artery Disease
Authors: Eikelboom J. W. , Parsons R. , Taylor R. R. , van Bockxmeer F. M. , Baker R. I.
Published: March 2018

The 20210 G/A prothrombin gene mutation is associated with an increased risk of venous thrombosis but whether there is an association of the mutation with premature coronary artery disease and acute myocardial infarction remains unclear.

To further assess the role of the G/A genotype as a risk factor for arterial vascular disease, we performed a case-control study of 644 patients aged less than 50 years with angiographically proven coronary artery disease, 402 of whom had myocardial infarction, and 679 unrelated healthy control subjects aged less than 50 years, randomly selected from the electoral roll.

The prevalence of the G/A genotype was 2.5% in patients with coronary artery disease, and 3.2% in control subjects (odds ratio 0.8; 95% confidence interval 0.35 to 1.83). The mutation was not more frequent among patients with a history of myocardial infarction (2.2%, odds ratio 0.7; 95% confidence interval 0.27 to 2.05), and there was no evidence of an interaction between the prothrombin mutation and conventional cardiovascular disease risk factors. There was no association between genotype and extent of angiographic coronary artery disease (p = 0.73).

We conclude that the 20210 G/A prothrombin gene mutation is not a major risk factor for premature coronary artery disease in our predominantly Caucasian Australian population.