bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Mechanism of Type I Congenital Heparin Cofactor (HC) II Deficiency Caused by a Missense Mutation at Reactive P2 Site: HC II Tokushima
Authors: Kanagawa Yasuhiko , Shigekiyo Toshio , Aihara Ken-ichi , Akaike Masashi , Matsumoto Toshio , Azuma Hiroyuki
Published: March 2018

We found a 66-year-old Japanese patient with type I congenital heparin cofactor (HC) II deficiency manifesting multiple atherosclerotic lesions. To investigate the molecular pathogenesis of our patient, we performed sequencing analysis and expressed recombinant human wild-type and mutant HC II molecules in COS-1 and CHO-K1 cells. Sequencing analysis following amplification of each of all 5 exons and its flanking region showed a single C to T transition at nucleotide position 12,854 in exon 5, which changed a Pro443 codon (CCG) to Leu codon (CTG). Because this mutation generates a new Bbv I site, the Bbv I digestion pattern of the PCR-amplified exon 5 fragments from each family member was analyzed. In all cases, the patterns were consistent with the activities and antigen levels of plasma HC II in those members. Transient transfection, metabolic labeling and pulse-chase experiments followed by immunoprecipitation analysis showed that the recombinant mutant HC II molecules were secreted from COS-1 cells in reduced amounts compared with the wild-type, and that an enhanced intracellular association of the mutant molecules with a chaperone, GRP78/BiP, was observed in CHO-K1 cells. Northern blot analysis indicated that the mutant HC II mRNA was transcribed at a similar level as that of wild-type.

Immunohistochemical staining of the transfected cells revealed that COS-1 cells expressing the mutant HC II molecules were stained mainly in the perinuclear area. We conclude that the impaired secretion of the mutant HC II molecules, due to intracellular degradation, is the molecular pathogenesis of type I congenital HC II deficiency caused by a Pro443 to Leu mutation at reactive P2 site.
Antibodies to Factor XII Are Distinct from Antibodies to Prothrombin in Patients with the Anti-phospholipid Syndrome
Authors: Nicholls P. J. , Donohoe S. , Gallimore M. J. , Winter M. , Jones D. W.
Published: March 2018

Patients with the anti-phospholipid syndrome (APS) have antiphospholipid antibodies (aPA) which are often targeted towards phospholipid binding proteins such as β2-glycoprotein I and prothrombin. Antibodies to factor XII (FXIIabs) have also been identified in some patients with APS. Factor XII (FXII) is a member of the kringle family of proteins which include plasminogen and prothrombin. Antibodies to prothrombin have been associated with myocardial infarction and have been shown to cross react with plasminogen.

Sixteen patients with APS and FXIIabs were investigated for the presence of antibodies to prothrombin, by enzyme linked immunosorbent assay in a calcium (Ca++) independent assay. All sixteen showed different antibody binding patterns than those observed for antibodies to FXII.

Eight patients were further investigated using surface plasmon resonance (SPR) for antibody binding to covalently bound FXII and to covalently bound prothrombin in both Ca++ dependent and independent systems. Of three patients demonstrating antibody binding to FXII by SPR, none demonstrated antibody binding to prothrombin in a Ca++ independent system with one demonstrating antibody binding to prothrombin that was Ca++ dependent. Of five patients who did not bind FXII by SPR, one demonstrated antibody binding to prothrombin in a Ca++ independent system while two demonstrated antibody binding to prothrombin in a Ca++ dependent system. Antibodies to FXII in patients with APS appear to be distinct from antibodies to prothrombin.
Factor VII Activation, Apolipoprotein A-I and Reverse Cholesterol Transport: Possible Relevance for Postprandial Lipaemia
Authors: Cooke C. J. , Nanjee M. N. , Howarth D. J. , Cooper J. A. , Stepanova I. P. , Morrissey J. H. , Miller N. E. , Miller G. J.
Published: March 2018

Postprandial lipaemia is associated with activation of factor VII (FVII) and efflux of cholesterol from tissues to nascent plasma high density lipoproteins (HDL) containing apolipoprotein A-I (apo A-I). To determine whether FVII activation and cholesterol efflux occur together in other situations, the responses to intravenous infusion of HDL-like apo A-I/phosphatidylcholine discs were measured in 10 healthy men. Disc infusion (40 mg apo A-I/kg body weight) over 4 h was followed by increases in HDL cholesteryl ester and plasma apo A-I (p <0.0001). Significant activation of FVII was apparent during infusion in fasting subjects (p = 0.03), activated FVII averaging 123% of baseline value by 12 h (p <0.0001). Plasma thrombin-antithrombin (TAT) complex increased to 156% of baseline level by 12 h (p >0.05) but individual responses differed considerably. Peak TAT post-infusion was associated inversely with peak HDL triglyceride concentration (p = 0.004). The coagulation responses to disc-infusion may be due to transfer of phosphatidylserine to cell surfaces during cholesterol efflux.
Immunoglobulin G from Patients with Antiphospholipid Syndrome Impairs the Fibrin Dissolution with Plasmin
Authors: Kolev Krasimir , Gombás Judit , Váradi Balázs , Skopá Judit , Mede Katalin , Pitlik Ervin , Nagy Zoltán , Machovich Raymund
Published: March 2018

Immunoglobulin G (IgG) isolated from normal human blood plasma stabilizes the structure of perfused crosslinked fibrin and prolongs the time for its dissolution with plasmin, when the fibrin surface is exposed to 500 s-1 shear rate flow. The IgG from patients suffering in antiphospholipid syndrome with thrombotic complications exerts even stronger antifibrinolytic effect. A patient, whose IgG does not affect the fibrin dissolution with plasmin, displays a bleeding tendency. The shear stress-induced disassembly of the fibrin clots containing IgGs with antifibrinolytic potency occurs at a much more advanced stage of fibrin digestion, as evidenced by the electrophoretic pattern of the ureatreated samples. The antifibrinolytic effects are also produced under static conditions and these are caused by the variable portion of the IgG molecules (fragment Fab), whereas the constant part (fragment Fc) has no inhibitory effect. The IgGs with antifibrinolytic properties do not affect directly the plasmin activity in amidolytic assay, but the IgGs from APS patients obliterate the competition of the fibrin and the peptidyl-p-nitroanilide for the protease in the same assay system suggesting interference of the IgGs with the plasmin action on the fibrin substrate. Thus, the correlation of the clinical symptoms with the effect of the isolated IgG on the dissolution of perfused fibrin clots supports a physiological and a pathological role of IgG in the fibrinolytic process related to the variability of the cross-reactions of immunoglobulins with fibrin, fibrin degradation products or fibrin-plasmin complexes.
Distinguishing Features of Anti-β2 Glycoprotein I Antibodies between Patients with Leprosy and the Antiphospholipid Syndrome
Authors: Renaudineau Y. , Mane I. , Perraut R. , Krilis S. A. , Youinou P. , Arvieux J.
Published: March 2018

Anticardiolipin (ACA), anti-β2 glycoprotein I (β2GPI), and antiprothrombin antibodies of IgG and IgM classes were quantitated by enzyme-linked immunosorbent assays in 176 untreated leprosy patients across the histopathological spectrum. Positivity rates ranged from 21% (IgG ACA) to 30% (IgM anti-prothrombin) versus 4% in healthy controls (p <10-2 to 10-3). Levels of IgM anti-β2GPI and IgG ACA were significantly higher in lepromatous leprosy and multibacillary patient subgroups. IgG3 was the most common subclass reactive to both β2GPI and prothrombin in selected high-titer leprosy sera, unlike antibodies from patients with the antiphospholipid syndrome (APS) largely restricted to IgG2. In leprosy patients, but not in the APS control group, there was no statistical correlation between ACA and anti-β2GPI antibody levels. Likewise, a large fraction of anti-β2GPI positive sera (36/45 and 28/44 for IgG and IgM, respectively) were unreactive in the standard ACA assay. Most assayed anti-β2GPI antibodies from leprosy patients showed (i) ability to recognize both human and bovine β2GPI immobilized on non-irradiated polystyrene plates, (ii) concentrationdependent inhibition of binding by cardiolipin, and (iii) relatively high avidity binding to fluid-phase β2GPI, thereby differing from those found in APS. Finally, the location of the major epitopic region on the β2GPI molecule targeted by autoantibodies was different in leprosy and APS, as assessed by direct binding to domain Iand V-deleted mutants and competition with the mouse monoclonal antibody 8C3, directed at domain I. Thus, leprosy-related antiphospholipid antibodies comprise persistent IgG and IgM anti-β2GPI that differ from APS-related ones with respect to IgG subclass, avidity and epitope specificity, possibly reflecting distinct pathophysiological significance.
Differential Effects of Fibroblast Growth Factors on Expression of Genes of the Plasminogen Activator and Insulin-like Growth Factor Systems by Human Breast Fibroblasts
Authors: Martens John , Dorssers Lambert , Klijn Jan , Foekens John , Sieuwerts Anieta
Published: March 2018

In breast stroma urokinase plasminogen activator (uPA) is predominantly expressed by fibroblasts located in the near vicinity of tumor cells, and fibroblast-derived insulin-like growth factor-1 (IGF-1) may be involved in inhibiting the expression of uPA in these fibroblasts. To investigate a possible role for fibroblast growth factors (FGFs), we evaluated the expression of components of the PA system and the IGF system in normal and tumor-tissue-derived human breast fibroblasts exposed to various FGFs in vitro. mRNA analysis revealed that FGF-1, FGF-2 and FGF-4 induced the mRNA expression levels of uPA, tPA, uPAR, PAI-1 and PAI-2, and reduced those of IGF-1, IGF-1R, IGF-2R and IGFBP-4, without significantly affecting the levels of IGFBP-3, IGFBP-5 and IGFBP-6 mRNA. Concerning the expression of IGF-2 mRNA, the effects mediated by FGF-1, FGF-2 and FGF-4 were divergent. In general, the effects elicited by FGF-1 on the various mRNA levels studied were rapid and short-term. Those mediated by FGF-2 overall lagged behind but were longer-lasting. For FGF-4 an in between pattern was observed. Blocking transcription and translation demonstrated that a) both the FGF-1 and FGF-2 induced effects were the result of altered gene transcription or mRNA stability, b) the short-term effects mediated by FGF-1 and FGF-2 required de novo protein synthesis, and c) the long-term effects elicited by FGF-2 did not depend on de novo protein synthesis during the first 24 h, but were triggered by proteins produced or made available thereafter. The data presented propose that of the FGFs studied (FGF-1, -2, -4, -5, and -7), FGF-2 is the most attractive target for therapeutical strategies aimed at diminishing the contribution of stromal fibroblasts in the PA-directed breast tumor proteolysis.
Fibrinogen Adsorption, Platelet Adhesion and Thrombin Generation at Heparinized Surfaces Exposed to Flowing Blood
Authors: Willems George , Morra Marco , Keuren Jeffrey , Wielders Simone , Lindhout Theo
Published: March 2018

Thrombus formation at an artificial surface in contact with blood is a complex process that encompasses accretion of platelets from flowing blood and fibrin deposition. Platelet adhesion and fibrin formation are intimately intertwined reactions that are triggered by different sets of surface adsorbed plasma proteins. To dissect the contribution of protein adsorption and platelet adhesion to thrombin formation, a coherent study was performed with non-coated (NC) and heparin-coated (HC) surfaces. Thrombin production in whole blood, platelet adhesion and protein adsorption were studied using an amidolytic thrombin assay, a dynamic platelet adhesion assay and ellipsometry, respectively. Thrombin generation in flowing whole blood exposed to HC surfaces was greatly diminished when compared with NC surfaces. However, separate platelet adhesion and protein adsorption studies with anticoagulated whole blood revealed that platelets do not adhere because fibrinogen is not available in the protein layer that was deposited during the perfusion. These findings indicate that the in vitro thrombogenicity of a material cannot be predicted from platelet adhesion and protein adsorption data when these measurements are performed with anticoagulated blood or platelet rich plasma. Preincubation of NC and HC surfaces with fibrinogen or 2000-fold diluted plasma resulted in similar amounts of surface-bound fibrinogen and mediated massive platelet adhesion from flowing whole blood. These results indicate that a) platelet adhesion correlates with the availability of surface-bound fibrinogen and b) NC and HC surfaces are indistinguishable with respect to protein (fibrinogen) adsorption and platelet adhesion. It is apparent that the heparinized surface used in our studies exerts its anti-thrombogenic properties by neutralizing locally formed thrombin and not by reducing fibrinogen-dependent platelet adhesion.
Inherited Thrombophilia and First Venous Thromboembolism during Pregnancy and Puerperium
Authors: De Stefano Valerio , Taioli Emanuela , Paciaroni Katia , Rossi Elena , Pier Mannucci , Martinelli Ida
Published: March 2018

Venous thromboembolism is a rare but threatening complication of pregnancy. Little conclusive information is available on the actual risk of venous thromboembolism during pregnancy or puerperium in women with inherited thrombophilia, particularly in carriers of factor V Leiden and of the G20210A prothrombin gene mutation. To determine the pregnancy-related and puerperium-related risk of venous thromboembolism in women with inherited thrombophilia, we performed a case-control study on 119 women who had a first episode of deep vein thrombosis and/or pulmonary embolism during pregnancy or puerperium and 232 healthy women who had at least one pregnancy without thrombosis. Inherited thrombophilia was diagnosed in 47 patients (39.5%) and 15 controls (6.5%). The relative risk of venous thromboembolism was 10.6 (95% CI, 5.6-20.4) for heterozygous carriers of factor V Leiden, 2.9 (95% CI, 1.0-8.6) for heterozygous carriers of the prothrombin mutation and 13.1 (95% CI, 5.0-34.2) for those with antithrombin, protein C or protein S deficiency taken together. Sixty-eight of the 119 women (57%) had thrombosis after delivery, confirming the puerperium as a particularly high-risk period. When women were divided into two groups of those with antenatal or postnatal thrombosis, the relative risks associated with each type of inherited thrombophilia were of similar magnitude. In conclusion, women with inherited thrombophilia have an increased risk of venous thromboembolism during pregnancy. Among thrombophilic abnormalities, the prothrombin mutation was the weakest risk factor. Thrombosis occurred more frequently in puerperium than in pregnancy, whether or not thrombophilia was diagnosed.
Absence of Placental Transfer of Pentasaccharide (Fondaparinux, Arixtra®) in the Dually Perfused Human Cotyledon in vitro
Authors: Vergnes C. , Brun J.L. , Paolucci F. , Nadal T. , Leng J.J. , Saux M.C. , Banwarth B. , Lagrange F.
Published: March 2018

The synthetic pentasaccharide, fondaparinux, is the first of a new antithrombotic class: selective factor Xa inhibitors. Comparative clinical trials of fondaparinux versus heparins in prevention and treatment of venous thromboembolism are ongoing. Little is known about fondaparinux during pregnancy, as women of child-bearing potential were excluded from clinical trials. No particular safety issue, for either mother or fetus, has been reported for heparins. The objective of this study was to compare in vitro the steady state placental transfer of fondaparinux and enoxaparin at the plasma concentrations reached during acute treatment of venous thromboembolism (1.75 µg/mL and 1 anti- Xa IU/mL respectively), using antipyrine (20 mg/L) as reference. No biological activity was detectable in the fetal venous effluent during perfusion of enoxaparin-antipyrine, fondaparinux-antipyrine or control media. Furthermore, fetal venous samples did not differ significantly from fetal arterial samples. This apparent absence of placental transfer supports further evaluation of fondaparinux in pregnant women.
European Concerted Action on Anticoagulation
Authors: van den Besselaar A.M.H.P. , Tripodi A. , Shiach C. , Jespersen J. , Poller L. , Keown M. , Chauhan N. , Conard J. , Dias D. , Egberg N. , Iriarte J.A. , Kontopoulou-Griva I. , Otridge B.
Published: March 2018

A procedure for using citrated fresh plasmas for International Sensitivity Index (ISI) calibration of two types of whole blood point-of-care test (POCT) prothrombin time (PT) monitor systems has been assessed in a multicentre study.

The CoaguChek Mini and TAS PT-NC systems gave higher ISI with whole blood samples than with fresh plasma calibrations. However, there was good agreement between whole blood and fresh plasma monitor system International Normalised Ratio (INR) and the reference INR of target samples.

Reliable INR can therefore be obtained with both whole blood and plasma samples on these two POCT systems based on their respective ISI. With the CoaguChek Mini system, the plasma calibration ISI can also be used to derive reliable INR with whole blood PT results. This was not possible with the TAS PT-NC system.
Platelet Aging In Vivo Is Associated with Activation of Apoptotic Pathways: Studies in a Model of Suppressed Thrombopoiesis in Dogs
Authors: Soto Mónica , Palomo Iván , Ocqueteau Mauricio , Coetzee Lindi-Marie , Astudillo Smiljan , Aranda Eduardo , Mezzano Diego , Pereira Jaime
Published: March 2018

The mechanism(s) involved in the clearance of senescent platelets are largely unknown. We have recently demonstrated that platelet aging in vivo is associated with loss of membrane phospholipid asymmetry, a universal phenomenon in cells undergoing apoptosis. Thus, we postulated that senescent platelets may exhibit programmed cell death changes, which may trigger their removal from circulation. Since platelets contain the apoptosis machinery as well as mitochondria, a key organelle in the regulation of apoptosis, we studied the appearance of apoptotic-like changes during platelet aging in vivo. To investigate this, we assessed changes in mitochondrial membrane potential (Δψ) in circulating canine platelets during decline in platelet count after suppression of thrombopoiesis by estradiol injection, a validated model to obtain circulating platelets of increasing mean age. Phosphatidylserine (PS) exposure was determined by flow cytometry by binding of FITC-labeled annexin V. Mitochondrial Δψ was studied with the cationic lipophilic dye DIOC6 (3) and the J-aggregate-forming cation JC-1 and analysis by flow cytometry. The proportion of platelets with exposed PS rose significantly with age, from 2.88% before to 6.7%, 8 days after estradiol injection. By flow cytometry it was demonstrated a significant decreased in DIOC6 (3) fluorescence (median fluorescence intensity 791 ± 98 vs 567 ± 102 day 0 vs day 8 post injection of estradiol, respectively; n: 11; p <0.01), consistent with mitochondrial Δψ collapse. JC-1 has the unique property of forming J-aggregates under high mitochondrial Δψ (red fluorescence, FL2) whereas the monomeric form fluoresces in green (FL1). Aged platelets in vivo, loaded with JC-1, exhibited a significant increase in FL1/FL2 ratio (2.5 ± 1.7 vs 4.7 ± 1.6, day 0 vs day 8 post injection of estradiol, respectively; n: 13; p <0.05), confirming the mitochondrial Δψ alteration.

The results show that platelet aging in vivo is associated with a decrease in mitochondrial Δψ and PS exposure. In conclusion, our data provide for the first time, evidence that platelet senescence is associated with changes characteristics of apoptosis, which may promote their removal from circulation.
Factor V Leiden and Prothrombin Gene G20210A Mutation in Children with Venous Thromboembolism
Authors: Hepner Mirta , Sciuccati Gabriela , Pieroni Graciela , Feliú-Torres Aurora , Mardaraz Claudia , Frontroth Juan , Bonduel Mariana
Published: March 2018

To determine whether factor V Leiden (FVL) and/or prothrombin gene G20210A mutation (PT20210A) are risk factors for venous thromboembolism (VTE) in Argentinean children. One hundred and thirty consecutive children with VTE were prospectively assisted at a single centre. Blood samples were available from 110 of them for detailed haematological analysis. The prevalence of both mutations was compared with a control group. The odds ratio for VTE was significantly increased in patients with FVL (OR 3.64; 95% CI: 1.14-11.6, p <0.029) whereas odds ratio for VTE was not significantly increased in patients with PT20210A (OR 1.06; 95% CI: 0.24-4.73, p = 0.938). Combined disorders were found in 5 of the 10 children with the aforementioned mutations. In 21 children (19%) without these mutations other inherited and acquired disorders were detected. Our data show that FVL is a risk factor for VTE whereas PT20210A does not seem to be a risk factor in our paediatric population.
Tissue Factor Activity Is Upregulated in Human Endothelial Cells Exposed to Oscillatory Shear Stress
Authors: Silacci Paolo , Bouzourene Karima , Daniel François , Brunner Hans , Hayoz Daniel , Mazzolai Lucia
Published: March 2018

Hemodynamic forces play a critical role in the pathogenesis of atherosclerosis as evidenced by the focal nature of the disease. Oscillatory shear stress characterizes the hemodynamic environment of plaque-prone areas as opposed to unidirectional shear stress typical of plaque-free areas. These particular flow conditions modulate atherosclerosis-related genes. Tissue factor (TF) initiates blood coagulation, contributes to vascular remodeling, and is therefore a potential contributor in the development/progression of atherosclerosis. We investigated the effect of oscillatory and unidirectional flows on TF using an in vitro perfusion system. Human endothelial cells exposed for 24 h to oscillatory shear stress, significantly increased TF mRNA, and TF protein expression (1.5-and 1.75-fold, respectively, p <0.01), and surface TF activity (twofolds-increase). Expression of TF inhibitor (TFPI), mRNA and protein, remained unchanged as compared to static conditions. Conversely, cells exposed to unidirectional shear, showed a decrease in TF activity with a significant increase in TFPI mRNA and protein expression (1.5-and 1.8-fold, respectively, p <0.01). These results show for the first time that pulsatile oscillatory shear stress induces a procoagulant phenotype of endothelial cells which may favor formation/progression of atherothrombotic lesions.
Intercellular adhesion molecule-1 (ICAM-1) expression is upregulated by thrombin in human monocytes and THP-1 cells in vitro and in pregnant subjects in vivo
Authors: Jordan Fiona , Pearson Charles , D.Walker Isobel , Sattar Naveed , Ellison Joanne , A.Greer Ian , Clark Peter
Published: March 2018

Monocytes play a pivotal role in both the inflammatory and coagulation responses, which may be mediated through a variety of adhesion molecules on the cell surface, including intercellular adhesion molecule-1 (ICAM-1). Monocytes also possess thrombin receptors. In the current study, we have demonstrated that thrombin can upregulate ICAM-1 mRNA and induce ICAM-1 expression on the monocyte in vitro and that, in vivo, higher monocyte ICAM-1 expression is observed in pregnancy (which is characterised by a physiological increase in thrombin generation). In pregnant subjects, a positive correlation between monocyte ICAM-1 expression and a number of markers of vascular/thrombotic disease (including blood group, acquired activated protein C resistance and non-fasting plasma triglyceride levels) was observed. We also observed a significant relationship between monocyte ICAM-1 expression and soluble plasma ICAM-1 levels, which would be consistent with a contribution of monocytic ICAM-1 to the levels of free ICAM-1 observed in plasma during pregnancy.

Consistent with a role in fibrinogen binding, our preliminary in vivo results suggest that monocyte ICAM-1 expression may be a useful marker of the thrombotic/inflammatory response, although further work is required to assess the relationship of monocyte ICAM-1 expression in thrombotic disorders.
Distinct accumulation patterns of soluble forms of E-selectin, VCAM-1 and ICAM-1 upon infusion of TNFα in tumor patients
Authors: Lieuw-a-Fa Mariska , Schalkwijk Casper , van Hinsbergh Victor
Published: March 2018

The transmigration of leukocytes across the endothelium is a prerequisite for the inflammatory process. Leukocyte-endothelium interaction is regulated by several endothelial adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Their expression is enhanced by inflammatory mediators, such as TNFα. In vivo a small part of these adhesion molecules is shed by proteases, and can be detected in the circulation. In this study, the time course of the TNFα-induced accumulation in the blood of three circulating soluble adhesion molecules, sE-selectin, sICAM-1 and sVCAM-1, was studied in plasma samples of tumor patients enrolled in a phase I trial receiving TNFα. Two different cohorts were studied. Eleven patients received a continuous 24-hour TNFα infusion while 10 other patients received a 5-day continuous TNFα infusion. After 24 hours of TNFα infusion sE-selectin levels increased by 1985 ± 312 %, sVCAM-1 by 301± 19 % and sICAM-1 by 445 ± 82 %. Differences in accumulation patterns were observed after 5 days of continuous TNFα infusion. sVCAM-1 and sICAM-1 levels showed an increase during the infusion with a maximum at 3 to 5 days and stayed elevated after discontinuation of the TNFα infusion. In contrast, sE-selectin reached its peak at day 1 and declined thereafter. In conclusion, sVCAM-1 and sICAM-1 show a different accumulation pattern upon TNFα infusion as compared to sE-selectin in man.
Recombinant human tissue plasminogen activator protects the basal lamina in experimental focal cerebral ischemia
Authors: Burggraf Dorothe , Martens Helge , Jäger Gabriele , Hamann Gerhard
Published: March 2018

While recombinant tissue plasminogen activator (rt-PA) is successfully used in human ischemic stroke, it may also cause hemorrhagic complications. Animal experiments have shown that hemorrhages are related to microvascular basal lamina damage. We investigated the effects of different doses of rt-PA on the brain microvasculature. Experimental cerebral ischemia in rats was induced for 3 h and followed by 24 h reperfusion (suture model). Each group of rats (n = 6) received either treatment (0.9, 9, or 18 mg rt-PA/kg body weight) or saline (control group) at the end of ischemia. The loss of microvascular basal lamina antigen collagen type IV was measured by Western blot of the ischemic and non-ischemic basal ganglia and cortex. Compared with the contralateral non-ischemic area, collagen type IV was significantly reduced in the ischemic area: (basal ganglia/cortex) 43% +/- 9% / 64% +/- 4 %. Low/moderate doses of rt-PA had a protective effect: 0.9 mg 79% +/- 3% / 89% +/-6%, 9 mg 72% +/- 9%/ 81% +/- 12% (p < 0.05). Higher doses of rt-PA (18 mg) had a similar effect as seen in untreated controls: 57% +/- 11% / 59% +/- 9% (p < 0.05, Anova). MMP-9 and MMP-2, measured by gelatine zymography, steadily increased over higher doses of rt-PA: MMP-9 (basal ganglia/cortex): control 115% +/- 4% / 123% +/- 3% compared with 18 mg rt-PA 146% +/- 5%/ 162% +/- 6% (p < 0.05) and MMP-2: control 109% +/-4%/ 116% +/- 5% and 18 mg rt-PA 222% +/- 15%/ 252% +/- 2% (p < 0.05). Low to moderate doses of rt-PA protect the microvascular basal lamina, whereas high doses of rt-PA have the opposite effect, probably due to increased coactivation of MMP-2 and MMP-9.
Assessment of the clinical significance of serum matrix metalloproteinases MMP-2 and MMP-9 in patients with various chronic liver diseases and hepatocellular carcinoma
Authors: Kuyvenhoven Johan , van Hoek Bart , Blom Eric , van Duijn Wim , Hanemaaijer Roeland , Verheijen Jan , Lamers Cornelis , Verspaget Hein
Published: March 2018

Matrix metalloproteinases (MMPs) have the ability to degrade basement membranes and may thus play an important role in extracellular matrix turnover in liver fibrosis and carcinogene-sis. Serum levels of MMPs have been suggested as diagnostic markers in these processes.

We measured serum MMP-2 and MMP-9 by ELISA in 91 patients with chronic liver disease, including 25 patients with hepatocellular carcinoma (HCC), and in 60 controls.

MMP-2 was significantly higher in patients with chronic liver disease compared to controls, and increased with Child-Pugh class. There was a significant correlation between MMP-2 and liver function (bilirubin, albumin, and prothrombin time), and a strong opposite correlation between MMP-9 and these parameters.

MMP-2 levels in patients with HCC were significantly higher than in controls, but comparable to patients with chronic liver disease without this malignancy. MMP-9 yielded no significant differences between patients with or without HCC and controls.

Serum MMP-2 and to a lesser extent MMP-9 correlate with the severity of liver disease and may reflect changes in extracellular matrix remodeling. Due to a considerable overlap in patients with chronic liver disease with or without HCC, MMP-2 and MMP-9 can not be used as a diagnostic marker for HCC.

Theme paper: Part of this paper was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.
Systemic coagulation reactivation in recurrence of colorectal cancer
Authors: Thorlacius-Ussing Ole , Iversen Lene
Published: March 2018

At time of diagnosis, most cancer patients present with laboratory evidence of systemic coagulation activation. After treatment with curative intent, these hemostatic alterations seemingly disappear as seen in colorectal cancer with regard to prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and soluble fibrin (SF).

The aim of this study was to investigate whether coagulation activation recurs with cancer recurrence and to study whether preoperative coagulation tests have any prognostic value in colorectal cancer.

Plasma F1+2, TAT, and SF levels were prospectively recorded from 113 patients followed after curative resection of colo-rectal cancer. The patients were seen in clinic after 3, 6, 12, and 18 months, and after 2, 3, 4, and 5 years.

Coagulation reactivation was observed at the time of recurrence, as demonstrated by significantly increased plasma TAT and SF, along with a non-significant increase (P = 0.09) in F1+2.

Preoperative values of F1+2,TAT, and SF did not show association with prognosis.
Tissue factor pathway inhibitor deficiency enhances neointimal proliferation and formation in a murine model of vascular remodelling
Authors: Singh Ripudamanjit , Pan Shuchong , Mueske Cheryl , Witt Tyra , Kleppe Laurel , Peterson Timothy , Caplice Noel , Simari Robert
Published: March 2018

Tissue factor (TF) is a small-molecular-weight glycoprotein that initiates the extrinsic coagulation pathway but may have important noncoagulation vascular functions as well. Tissue factor pathway inhibitor (TFPI) is a major physiological inhibitor of TF-initiated coagulation. Enhancement of vascular TFPI either by overexpression using gene transfer or delivery of protein to the vessel has been shown to reduce neointimal formation. However, the inherent role of TFPI in this process has not been defined. To do so, we utilized a murine model of vascular remodeling using flow cessation in mice, which are heterozygous for a genetic deletion of the first Kunitz domain of TFPI or wild type littermates. The heterozygotic mice had 50% of wild type TFPI activity in plasma as well as vascular homogenates. To study the effect of TFPI deficiency on neointimal formation, age matched TFPIK1+/- and wildtype littermates underwent unilateral common carotid artery ligation. Mice were sacrificed at 4 weeks and the ligated carotid arteries were analyzed. There was a significantly greater neointima to media ratio and less luminal area in the TFPIK1+/- mice compared to their TFPIK1+/+ littermates. The proliferative index of intimal cells in TFPIK1+/-mice at 1 week was significantly higher compared to TFPIK1+/+mice. We conclude that TFPI deficiency enhances neointimal formation and proliferation associated with flow cessation. This suggests that TFPI may regulate vascular remodeling primarily through modulation of neointimal formation.

Theme paper: Part of this paper was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.
Prevention of thromboembolism in patients with mitral stenosis and associated atrial fibrillation: effectiveness of low intensity (INR target 2) oral anticoagulant treatment
Authors: Barbero Fabio , Biasiolo Alessandra , Pegoraro Cinzia , Noventa Franco , Iliceto S. , Pengo Vittorio
Published: March 2018

Mitral stenosis (MS) in association with atrial fibrillation (AF) is a clinical condition at high risk for systemic thromboembolism. Although oral anticoagulants greatly reduce the incidence of thromboembolism in these patients, the optimal intensity of treatment has never been tested in specific clinical trials, and current recommendations are derived from studies of nonrheuma-tic AF. In this study we tested the effectiveness of two different intensities. The study design was carried out as an open randomized prospective study in an anticoagulation clinic.

We randomized 103 patients with MS and AF to a low (target INR = 2) or moderate (target INR = 3) anticoagulation regimen. The primary end points were systemic thromboembolism, major bleeding and vascular death.

During a mean follow-up of 4.5 years, 1 systemic embolism occurred in the low intensity group (0.41 per 100 pt/yrs, CI 0.01-2.3), and 1 minor stroke occurred in the moderate intensity group (0.40 per 100 pt/yrs, CI 0.01-2.3; p = ns). Major bleeding occurred in 8 patients, with 3 in the low intensity (1.25 per 100 pt/yrs) and 5 in the moderate intensity group (2.0 per 100 pt/yrs, Incidence Rate Ratio 0.6, CI 0.1-3.1; p = ns). Total events (systemic embolism, major bleeding and vascular death) occurred in 7 low intensity patients and 8 moderate intensity patients. As expected, minor bleeding was more frequent in the moderate intensity group of patients, who actually had more intense treatment and required closer monitoring of oral anticoagulant treatment.

These data suggest that low intensity anticoagulation, as performed in an anticoagulation clinic, is effective and safe in high risk patients with MS and AF.
D-dimer Assays for the Exclusion of Venous Thromboembolism: Which Test for which Diagnostic Strategy?
Authors: de Moerloose P.
Published: March 2018

D-dimer measurement has proven to be very useful to rule out deep vein thrombosis (DVT) and pulmonary embolism (PE) in symptomatic outpatients (1). The problem faced by many physicians is the choice and the position of the D-Dimer tests in the diagnostic work-up of patients suspected of venous thromboembolism (VTE). In the last and present issues of Thrombosis and Haemostasis, two very interesting studies addressing these questions were published.

In the first paper (2), de Groot and colleagues evaluated, in a management study, the clinical utility of incorporating the SimpliRED assay in the diagnostic work-up of patients with suspected PE. Of the 245 study subjects, 59 did not receive anticoagulant therapy on the basis of a nondiagnostic lung scan, a normal D-dimer and a non-high clinical probability of PE. In the follow-up, only one patient experienced a thromboembolic event (which can be compared with the 6% of subsequent rate of VTE in the follow-up of the 54 patients with a normal perfusion lung scan). However, if SimpliRED D-dimer would have been used alone as a first exclusion step, 6 of 61 patients with proven PE had been missed (9.8%, 95% CI 3.7-20.2).
The Factor V R2 Allele: Risk of Venous Thromboembolism, Factor V Levels and Resistance to Activated Protein C
Authors: Luddington Roger , Jackson Andrew , Pannerselvam Sabapathy , Brown Karen , Baglin Trevor
Published: March 2018

Case-control studies have yielded conflicting results regarding the relative risk of venous thromboembolism associated with the factor V R2 allele. We calculated odds ratios in 581 patients and 469 agematched controls. The odds ratio for the R2 allele in patients relative to controls was 1.21 (95% CI 0.84 to 1.74). These results do not support the hypothesis that the R2 allele is a risk factor for venous thromboembolism. There was no relationship between factor V levels and R2 carrier status. Normalised APC sensitivity ratios were not lower in carriers of the R2 allele. In an in vitro model progressive APC resistance was observed with factor V levels of 60% and less but ratios less than 2.4 (equivalent to a normalised ratio of 0.73) did not occur until factor V levels were less than 20%. The relationship between APC resistance and factor V level was not observed in a factor VIII-independent model.
Relationship of Cardiac Troponin T and Procoagulant Activity in Unstable Angina
Authors: Bardorff Margit , Schweikart Sybille , Wiegand Uwe , Kampmann Markus , Katus Hugo , Giannitsis Evangelos
Published: March 2018

The present study sought to determine the incidence of increased procoagulant activity in patients with unstable angina (UAP), and to evaluate the relationship between cardiac troponin T (cTnT) and molecular markers of hemostatic activation.

We studied 44 patients with UAP further classified by plasma cTnT levels. All patients received an antithrombotic therapy consisting of therapeutic doses of unfractionated heparin and acetylsalicylic acid. Quantitative levels of cTnT and plasma concentrations of fibrin monomers (FM), prothrombin fragments F1+2, thrombin antithrombin III complexes (TAT), plasminogen and α2-antiplasmin were sampled serially within the first 48 h.

Increased plasma concentrations of FM were detected in 45.5% of patients and were more frequently present among those with cTnT concentrations >0.1 ng/ml (13 of 18 vs 7 of 26 patients, p = 0.003). In these patients, mean plasma concentrations of FM were significantly higher than in patients with cTnT <0.1 ng/ml (7.93 ± 2.3 vs 3.12 ± 0.6 µg/ml, p = 0.02). There was a close relationship between plasma levels of cTnT and FM (r = 0.74, p <0.004), prothrombin fragments F1+2 (r = 0.71, p = 0.046) and a trend to significance was noted for TAT (r = 0.42, p = 0.055). No significant correlation was observed with markers of the fibrinolytic system (plasminogen and α2-antiplasmin). Plasma levels of cTnT >0.1 ng/ml identified a concomitant increase of hemostatic markers with a sensitivity, specificity and positive predictive value of 65, 79, and 72% for FM, 63, 76, and 67% for prothrombin fragments F1+2, and 58, 66, and 39% for TAT, respectively.

In patients with UAP, cTnT identifies patients with increased procoagulant activity and is closely related to plasma levels of molecular markers of hemostatic activation. Therefore, cTnT alone or in combination with one of these markers may be helpful to identify patients requiring more potent antithrombin or antiplatelet therapy.
The Impact of Glu102Lys on the Factor X Function in a Patient with a Doubly Homozygous Factor X Deficiency (Gla14Lys and Glu102Lys)
Authors: Forberg Eva , Huhmann Iris , Jimenez-Boj Ester , Watzke Herbert
Published: March 2018

Two homozygous point mutations were found in a patient with factor X (FX) deficiency; One results in substitution of Lys for Gla+14 and the second causes a Lys substitution for Glu102. The proposita has a severely reduced FX coagulant activity in the extrinsic (<1% of normal) and in the intrinsic (30% of normal) system of coagulation and after activation with Russel’s viper venom (18% of normal). The FX antigen is reduced in this patient to 20% of normal. The substitution of Lys for Glu102 in FX deficiency has been reported previously in a heterozygous state in conjunction with a Lys for Gla+14 substitution and with a Pro for Ser334 substitution. The contribution of the Lys for Glu102 substitution in the observed combined FX defect in these patients was unclear. The mutation causing the Glu102Lys substitution was introduced by site directed mutagenesis into a wild-type FX cDNA, and recombinant protein was expressed in HEK 293 cells. Compared to the wild-type FX cDNA, the mutant construct had a 67% activity upon activation in the extrinsic system, 93% activity upon activation in the intrinsic system and 72% after activation with RVV. The data presented show that the substitution of Lys for Glu102 results in a minor functional defect of the FX molecule.
An Automated Heteroduplex Assay for the PlA Polymorphism of Glycoprotein IIb/IIIa, Multiplexed with Two Prothrombotic Genetic Markers
Authors: O’Connor Fiona , Fitzgerald Desmond , Murphy Ronan
Published: March 2018

Screening for the PlA polymorphism has been made faster and simpler with the advent of heteroduplex technology. Simultaneous screening for three common prothrombotic polymorphisms PlA, factor V Leiden, and MTHFR(C677T) has been achieved with multiplex heteroduplex analysis. We describe a quick and simple method for PlA heteroduplex probe production. The probe was multiplexed with heteroduplex probes for MTHFR(C677T) and factor V Leiden polymorphisms in a one tube assay, allowing rapid automated genotyping of all three. This automated multiplex assay was applied to a cohort of 165 patients and showed excellent correlation with gel-based assays, both PAGE and RFLP. This approach will facilitate the analysis of multiple polymorphisms in complex disease in large populations.
Bovine Protein C Inhibitor Has a Unique Reactive Site and Can Transiently Inhibit Plasmin
Authors: Yuasa Hiroyuki , Tanaka Hitoshi , Hayashi Tatsuya , Wakita Toshiaki , Nakamura Hideaki , Nishioka Junji , Kawarada Yoshifumi , Suzuki Koji
Published: March 2018

Protein C inhibitor (PCI) regulates the anticoagulant protein C pathway by neutralizing activated protein C and thrombin-thrombomodulin complex in the human hemostatic system. In this study, we cloned a full-length bovine PCI cDNA encoding a putative 19-residue signal peptide and a 385-residue mature protein; this showed 70.6%, 70.6%, 57.5% and 59.6% amino acid sequence homology with the human, rhesus monkey, rat and mouse PCIs, respectively. Bovine PCI mRNA (2.1 kb in size) was expressed strongly in the liver, and moderately in the kidney and testis, but not in other tissues tested. Bovine PCI has a putative reactive site peptide bond, Lys-Ser, that is different from the reactive site sequence (Arg-Ser) of other species’ PCI. We found that bovine PCI transiently inhibits bovine plasmin, but not human plasmin. Western blot analysis showed that the reactive site of bovine PCI is cleaved during the course of complex formation with bovine plasmin; degraded PCI is released from the complex gradually concomitant with the recovery of plasmin activity. These findings suggest that bovine PCI plays a role not only in the protein C pathway but also in the fibrinolytic activity of bovine hemostatic system.

Abbreviations: PCI, protein C inhibitor, Serpin, serine protease inhibitor, APC, activated protein C, TM, thrombomodulin.
Cellular Degradation of Free and Inhibitor-bound Tissue-type Plasminogen Activator
Authors: Camani Chantal , Gavin Olivier , Kruithof Egbert
Published: March 2018

The low density lipoprotein receptor-related protein (LRP) is a multiligand clearance receptor that removes free tissue-type plasminogen activator (t-PA) or complexes of t-PA with plasminogen activator inhibitor type 1 (PAI-1) from the blood circulation or the pericellular space. Co-receptors are essential for LRP-mediated clearance of several ligands (e.g. glycosaminoglycans for thrombin/protease nexin and lipoprotein lipase, and the urokinase receptor for urokinase/PAI-1 complexes). The present study was undertaken to investigate whether LRP-mediated t-PA clearance requires a co-receptor as well.

In five cell lines from different organs and species degradation of t-PA and t-PA/PAI-1 was mediated by LRP (or LRP-like receptors). No degradation of t-PA and t-PA/PAI-1 occurred in THP-1 or U-937 human monocyte-like cells, despite the presence of functional LRP. As glycosaminoglycans can bind t-PA and PAI-1 we investigated whether they are involved in t-PA/PAI-1 degradation. Pre-treatment of COS cells or HT1080 cells with chlorate, an inhibitor of glycosaminoglycan sulfation, did not decrease t-PA/PAI-1 degradation. Furthermore, CHO cells genetically deficient in glycosaminoglycans efficiently degraded t-PA/PAI-1. Thus it is unlikely that glycosaminoglycans are co-receptors for degradation of t-PA or t-PA/PAI-1.

This study indicates that THP-1 and U-937 cells lack a critical component (co-receptor?) for the LRP-mediated degradation of t-PA.

Abbreviations: LRP, low density lipoprotein receptor-related protein; PAI-1, plasminogen activator inhibitor type 1; RAP, receptor-associated protein; t-PA, tissue-type plasminogen activator; u-PA, urokinase; u-PAR, urokinase receptor.
Homocysteine and Oxidized Low Density Lipoprotein Enhance Platelet Adhesion to Endothelial Cells under Flow Conditions: Distinct Mechanisms of Thrombogenic Modulation
Authors: Varon D. , Tamarin I. , Zivelin A. , Salomon O. , Shenkman B. , Savion N. , Dardik R.
Published: March 2018

We investigated the effects of two well established risk factors for cardiovascular disease, homocysteine and oxidized low density lipoprotein (ox-LDL), on endothelial cell thrombogenicity. For this purpose we studied platelet adhesion to human endothelial cells (EC) under flow conditions at a shear rate of 350 s−1 following EC treatment with either homocysteine or ox-LDL. Treatment of EC with either homocysteine (1 or 10 mmol/L for 16 h) or ox-LDL (100 µg/ml for 16 h) resulted in a 2-3 fold enhancement in platelet adhesion. The enhancement in platelet adhesion induced by 1 mmol/L homocysteine, but not that induced by 10 mmol/L homocysteine, was absolutely dependent on fibrin formation. Homocysteine treatment has significantly increased the cell surface tissue factor (TF) activity and slightly reduced the expression of the intercellular adhesion molecule I (ICAM-1). In contrast, ox-LDL treatment upregulated ICAM-1 expression and had no significant effect on endothelial TF activity. Neither homocysteine nor Ox-LDL affected surface expression of the αvβ3 integrin. The homocysteine-induced enhancement in platelet adhesion was almost completely abolished by blockade of the EC TF activity by a polyclonal antibody. The enhancing effect of homocysteine was also greatly reduced by inhibition of the EC αvβ3 integrin, but was not affected by blockade of EC ICAM-1. On the other hand, ox-LDL-induced enhancement in platelet – EC adhesion was greatly inhibited by blocking ICAM-1 or αvβ3, but remained unaffected by inhibition of TF activity. Preincubation of platelets with the glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist Reo-Pro has virtually abolished the enhancing effect of both homocysteine and ox-LDL. Our results suggest that homocysteine and ox-LDL might increase endothelial thrombogenicity by distinct mechanisms: homocysteine – by inducing TF activity, and ox-LDL – by upregulating ICAM-1, both of which enhance GPIIb-IIIa/fibrinogen dependent platelet adhesion to EC. The αvβ3 integrin, although not affected by EC stimulation, seems to play a crucial role in platelet-EC interaction regardless of the mechanism of EC perturbation.