bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between Thyroid Hormones and Plasma D-dimer Levels
Authors: Bruckert Eric , Ankri Annick , Beucler Isabel , Giral Philippe , Turpin Gérard , Chadarevian Rita
Published: March 2018

High serum levels of cholesterol and triglycerides are risk factors for coronary heart disease and are strongly related to several haemostatic parameters. Thyroid disorders are a frequent feature in hyperlipidemic patients and are also associated with a variety of haemostatic abnormalities. Therefore, we analysed the relationships between free T4 (fT4) levels and Factor VII and VIII activities (FVIIc and FVIIIc), D-Dimers (DDI) and Plasminogen Activator Inhibitor type 1 (PAI-1), in a group of 472 healthy patients referred for hyperlipidemia.

Fourty patients were found to have primary hypothyroidism. A negative correlation was found in the whole study population between fT4 and DDI (p = 0.0001, r = –0.21) and the same results were found after exclusion of the patients with fT4 below the normal range (p = 0.0007, r = –0.17). In a multivariate regression analysis, the relationship between DDI and fT4 was independent of age, Body Mass Index (BMI), gender and total cholesterol. Less impressive correlation coefficients were found with FVIIc (r = –0.10), FVIIIc (r = –0.09) and PAI-1 (r = –0.09).

These results suggest that fT4 may play a physiological role in the regulation of the haemostatic equilibrium in hyperlipidemic patients and that low levels of fT4 are associated with a hypercoagulable state.
Fibrin D-dimer, Tissue Plasminogen Activator, Plasminogen Activator Inhibitor, and the Risk of Major Ischaemic Heart Disease in the Caerphilly Study
Authors: Yarnell J. W. G. , Sweetnam P. M. , Rumley A. , Thomas H. F. , Elwood P. C. , Lowe G. D. O.
Published: March 2018

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

No quantitative, simple and non-radioactive method has been described for measuring the platelet content of experimental thrombi. The aim of the present study was to develop a simple method for quantifying platelets in thrombi formed on thrombogenic surfaces in flowing native human blood. To test the relevance of this new method, the effect of unfractionated heparin on arterial thrombus formation was investigated. Tissue factor (TF)- and collagen-coated coverslips were exposed to non-anticoagulated blood at an arterial wall shear rate (2,600 s–1) for 1 to 4 min. Platelet deposition was quantified by measuring the P-selectin (PS) and β-thromboglobulin (βTG) content of dissolved plasmin-digested thrombi using immunoenzymoassays; fibrin deposition was determined by measuring the D-dimer levels. These results were compared to those established by morphometrical analysis.

Morphometric evaluation showed that fibrin deposition was maximum on TF by 1 min perfusion time. Platelets deposited subsequently and reached a maximum at 3 min. On collagen, platelets deposited directly on the collagen fibrils without detectable fibrin deposit. Platelet deposition increased from 1 to 4 min. Platelet deposition quantified by PS was correlated to the values obtained by morphometry (r = 0.72, r = 0.67, p <0.001, on TF and collagen, respectively). As compared to PS, βTG measurements gave an underestimation of the size of the thrombus platelet number. Unfractionated heparin infused through a mixing device proximal to the perfusion chamber to obtain plasma concentrations of 0.5, 1 and 3 IU/ml, reduced fibrin deposition on TF-coated coverslips in a dose-dependent manner (77% reduction at 3 IU/ml, p <0.01), but had no significant effect on platelet deposition (33% at 3 IU/ml, p >0.05). In contrast, heparin had no effect on fibrin or platelet deposition on collagen-coated coverslips.

Thus, a new quantitative and simple method for measuring platelet deposition in flowing blood has been developed and characterized. Utilizing this system, we have demonstrated that unfractionated heparin did not inhibit arterial thrombus formation either on procoagulant or on proaggregant surface.
Effects of Melagatran, a New Low-molecular-weight Thrombin Inhibitor, on Thrombin and Fibrinolytic Enzymes
Authors: Antonsson T. , Bylund R. , Eriksson U. , Gyzander E. , Nilsson I. , Elg M. , Mattsson C. , Deinum J. , Pehrsson S. , Karlsson O. , Nilsson A. , Sörensen H. , Gustafsson D.
Published: March 2018

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

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

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

Human anticoagulant activated protein C (hAPC) is less potent than the bovine APC (bAPC) molecule and our aims were to elucidate the molecular background for this difference and to create an APC with enhanced anticoagulant activity. In the protease domain of human protein C (hPC), the loop 148 (GWGYHSSREKEAKRN) is four residues longer than the corresponding loop in bovine APC (GWGY RDETKRN). To investigate whether this caused the species difference, the loop in hPC was replaced by the shorter bovine loop, whereas the longer human loop was introduced in bovine protein C. The mutation in hAPC yielded enhanced catalytic activity against chromogenic (4-fold) as well as natural (factors Va and VIIIa) substrates and 2-3-fold increased anticoagulant activity. The opposite effects were obtained with the bovine mutant. As compared to wild-type hAPC, the mutant hAPC was inhibited slightly faster by the protein C inhibitor, whereas the inhibition by α 1-antitrypsin was unaffected by the mutation. A computer model of bAPC was developed in order to analyse further our data. Collectively, our results demonstrate enhanced catalytic efficiency to result from mutagenesis in the loop 148 and show that APC mutant with increased anticoagulant activity can be created.

Abbreviations: PC, protein C; APC, Activated protein C. SDS-PAGE, polyacrylamide gel electrophoresis run in the presence of SDS, The chymotrypsinogen nomenclature for APC and thrombin is used throughout the text while the PC numbering is indicated between brackets whenever appropriate. P2, P1 and P1’, P2’.. designate inhibitor residues amino- and carboxy-terminal to the scissile peptide bond, respectively, and S2, S1 and S1’, S2’.. the corresponding subsites of the protease. APTT, activated partial thromboplastin time; rmsd, root mean square deviation. The coordinates of the bAPC model are freely available.
Elevated Plasma Fibrinogen and Increased Fibrin Turnover among Healthy Women who both Smoke and Use Low-dose Oral Contraceptives
Authors: Vissac Anne-Marie , Kirzin Jean-Michel , Bourgeat Pierre , Amiral Jean , Agher Rachid , Guize Louis , Scarabin Pierre-Yves
Published: March 2018

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

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

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

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

Patients on oral anticoagulant therapy (OAT) may bleed due to a very low factor IX level caused by a mutation at Ala-10 in the propeptide region of the factor IX gene. We evaluated screening of patients on OAT with an APTT to detect patients with this abnormality.

In 734 patients an APTT was assessed. Twenty-three patients had a disproportionately prolonged APTT. In these patients the factor IX level, the mutation at Ala-10 and the frequency of bleeding complications were assessed. No severely lowered factor IX levels were found (1 patient with 5% factor IX). No mutations at Ala-10 were found and bleeding complications were not more frequent in these patients. Conclusion: Routine APTT screening of patients on OAT is not useful to detect patients with increased bleeding or with the Ala-10 mutation in the factor IX gene.
Is a Prolonged Bleeding Time Associated with an Increased Risk of Hemorrhage after Liver Biopsy?
Authors: Brosstad Frank , Egeland Thore , Egge Tor , Schrumpf Erik , Boberg Kirsten
Published: March 2018

Bleeding time determination is not advised as a general preoperative hemostasis screening test, but it might be useful in some patient groups. Patients referred for liver biopsy frequently have coagulation disturbances and are at risk of hemorrhage. In this prospective study 219 liver biopsies were carried out regardless of a prolonged bleeding time, but with minimum requirements for hemoglobin concentration, platelet count, and tests of the internal and external coagulation pathways. The bleeding time was prolonged in the case of 48 (22%) of the biopsies. Significant bleeding as defined by a hemoglobin decrease of ≥2.0 g/dl occurred in nine patients. Three of these patients were bone marrow transplanted. Patients with a prolonged bleeding time carried a five times higher risk of bleeding (odds ratio = 5.0; confidence interval = 1.1-21.8; p = 0.019). We conclude that the bleeding time may give additional information on the risk of bleeding in some patient groups undergoing liver biopsy.
Activation of Plasminogen Activator Inhibitor-1 Synthesis by Phorbol Esters in Human Promyelocyte HL-60
Authors: Lopez Sophie , Peiretti Franck , Morange Pierre , Laouar Amale , Fossat Chantal , Bonardo Bernadette , Huberman Eliezer , Juhan-Vague Irène , Nalbone Gilles
Published: March 2018

HL-60 cells treated by PMA develop the monocyte adherent pheno-type and synthesize plasminogen activator inhibitor type-1 (PAI-1). We focused our study on the identification of the PMA-activated protein kinase C (PKC) isoform and its downstream transduction pathway activating PAI-1 synthesis. Acquisition of the monocytic phenotype was evidenced by cell adherence (90-95%) and a sharp increase of CD 36 and receptor for urokinase plasminogen activator (uPAR) surface expression. Ro 31-8220, a specific inhibitor of PKC, prevented PMA-induced PAI-1 synthesis (mRNA and protein levels) and cell adhesion. To identify the PKC isoform, we took advantage of the HL-525 cell line, an HL-60 cell variant deficient in PKCβ gene expression. This defect prevents PMA to induce the differentiation process. HL-525 stimulated by PMA did not synthesize PAI-1 nor become adherent. However, in HL-525 cells either pretreated by retinoic acid that reinduces PKCβ gene expression or transfected with PKCβ cDNA, PMA significantly activated PAI-1 synthesis and adhesion of cells. Immunoblotting of active Mitogen Activated Protein Kinase (MAPK) p42/p44 in HL-60 cells showed a preferential and sustained activation of the p42 isoform by PMA over the p44 isoform. Ro 31-8220 significantly attenuated this activation. PD 098059 and U0126, both highly specific MEK inhibitors, efficiently prevented PMA-induced PAI-1 synthesis (mRNA and protein levels) and cell adhesion whereas SB203580, a specific inhibitor of stress-activated MAPK p38, did not. Results obtained from HL-60 and HL-525 cells indicate that the PMA-activated transduction pathway of uPAR expression involves a PKC isoform other than PKCβ.

In conclusion, we propose that the pathway PKCβ-MEK-MAPK p42 is a potential linear route for PAI-1 synthesis leading to morphological changes and adherence linked to PMA-induced differentiation in HL-60 cells.
Platelet Activation Induced by Combined Effects of Anticardiolipin and Lupus Anticoagulant IgG Antibodies in Patients with Systemic Lupus Erythematosus
Authors: Suehisa Etsuji , Kuratsune Hirohiko , Machii Takashi , Koike Takao , Kitani Teruo , Kanakura Yuzuru , Amino Nobuyuki , Nojima Junzo
Published: March 2018

Antiphospholipid antibodies (aPL) are well known to be associated with arterial and venous thrombosis. In a series of 180 patients with systemic lupus erythematosus (SLE), the prevalence of arterial thrombosis was obviously higher in the patients who had both anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) (17/35, 48.6%, p <0.05) (Table 1) than in the other patients bearing aCL or LA alone or neither of them (2/145, 1.4%). Since a substantial fraction of the former group of patients with arterial thrombosis also had thrombocytopenia (12/17, 70.6%), there was a possibility that aCL and LA might have enhanced platelet activation and aggregation. To test this possibility, we studied the in vitro effects of aCL and LA on the enhancement of platelet activation by flow cytometric analysis using anti-CD62P and anti-CD41 monoclonal antibodies directed against platelet activation-dependent granule-external membrane (PADGEM) protein and platelet glycoprotein IIb (GPIIb), respectively. Platelet activation defined by the surface expression of CD62P was not induced by aCL+·LA+ plasma only, but was significantly augmented by aCL+·LA+ plasma in combination with adenosine diphosphate (ADP) at a low concentration that had only a modest effect on platelet activation. In contrast, aCL+·LA–, aCL–·LA+ and aCL–·LA– plasma samples were incapable of enhancing platelet activation in the presence or absence of ADP stimulation. In addition to plasma samples, the purified IgG from aCL+·LA+ plasma (aCL+·LA+-IgG) also yielded apparent enhancement of platelet activation induced by ADP. Furthermore, platelet activation was generated by the mixture of aCL+·LA–-IgG and aCL–·LA+-IgG fractions prepared from individual patients, but not by each fraction alone. These results suggest that aCL and LA may cooperate to promote platelet activation, and may be involved, at least partially, in the pathogenesis of arterial thrombosis and thrombocytopenia in patients with SLE.
Antithrombotic Drugs in the Primary Medical Management of Intermittent Claudication: A Meta-analysis
Authors: Bernardi Enrico , Prins Martin , Wouter ten Cate Jan , Prandoni Paolo , Hettiarachchi Rohan , Marras Elena , Maria Stefani Piero , Girolami Antonio , Büller Harry , Girolami Bruno
Published: March 2018

Background. There is no consensus on the efficacy of the antithrombotic drugs available for patients with intermittent claudication. Methods. A Medline and manual search was used to identify relevant publications. Uncontrolled or retrospective studies, double reports or trials without clinical outcomes were excluded. Included studies were graded as level 1 (randomised and double- or assessor-blind), level 2 (open randomised), or level 3 (non-randomised comparative). Mortality, cerebro- or cardiovascular events, amputations, arterial occlusions or number of revascularization procedures performed in the lower limbs, pain-free and total walking distance, ankle brachial index and calf blood flow, were the main outcomes considered. When feasible, end of treatment results, either continuous or binary, were combined with appropriate statistical methods. Results. Mortality was significantly decreased by ticlopidine compared to placebo (common odds ratio 0.68, 95% C.I., 0.49 - 0.95); clopidogrel decreased vascular events in comparison to aspirin (odds ratio 0.76, 95% C.I., 0.63 - 0.92) in level 1 studies. Arterial occlusions and the number of revascularization procedures performed were statistically significantly decreased by aspirin and ticlopidine, respectively. A small but statistically significant improvement in pain-free walking distance was determined by picotamide, indobufen, low molecular weight heparins, sulodexide and defibrotide, in small studies. Conclusions. Clopidogrel and ticlopidine do reduce clinically important events in patients with intermittent claudication and could be added to the primary medical treatment of these patients. The use of aspirin in these patients cannot be based on direct evidence, but only on analogy with coronary and cerebral atherosclerosis, where it has documented efficacy. Other antithrombotic drugs were not properly evaluated in patients with intermittent claudication.
Prevalence of Prothrombin G20210A, Factor V G1691A (Leiden), and Methylenetetrahydrofolate Reductase (MTHFR) C677T in Seven Different Populations Determined by Multiplex Allele-specific PCR
Authors: Luhm Robert , Pearson Steven , Endean Debra , Friedman Kenneth , Montgomery Robert , Hessner Martin
Published: March 2018

Individuals belonging to six racial groups (African American, Asian Indian, Caucasian, Hispanic, Korean, Native American), and a seventh group comprised of referred patients with thrombosis were genotyped for the prothrombin G20210A mutation, the factor V G1691A (Leiden) mutation, and the methylenetetrahydrofolate reductase (MTHFR) C677T mutation by multiplexed allele-specific PCR. The prothrombin 20210A and factor V 1691A allele frequencies in the thrombosis patients, 3.2% and 9.5%, were significantly higher than those in the random Caucasians, 1.3% and 1.8%, (p = 0.043 and p <0.001, respectively). The relative risk of venous thrombosis was determined to be 2.4-fold for carriers of the prothrombin 20210A allele (odds ratio = 2.54; 95% confidence interval = 0.94, 6.82) and 4.5-fold for carriers of the factor V 1691A allele (odds ratio = 5.06; 95% confidence interval = 2.25, 11.36). Among the seven populations, significant differences were observed in the MTHFR 677T allele distribution, however this mutation was not determined to be a risk factor for venous thrombosis in the patient group studied, either alone or in combination with the pro-thrombin 20210A and/or the factor V 1691A allele(s).
FXII (46C→T) Polymorphism and In Vivo Generation of FXII Activity
Authors: Kohler H. P. , Futers T. S. , Grant P. J.
Published: March 2018

Increased Factor XIIa concentrations have been found in association with coronary artery disease. Recently, a common 46 C to T point mutation in exon 1 of the factor XII gene has been described which is associated with lower FXII clotting activity and lower zymogen levels in relation to possession of the T allele. It is not known whether this polymorphism relates to the phenotypes of FXIIa in vivo or to coronary artery disease. The aim of the study was to investigate the interaction of this polymorphism with FXIIa plasma levels and to study the prevalence of the polymorphism in 266 patients with suspected coronary artery disease characterised by angiography and in 185 healthy controls. FXIIa levels were strongly associated with FXII genotype with lower levels with increasing numbers of T alleles (p <0.0001). There was no difference between the prevalence of this polymorphism in patients with MI compared to those without MI and controls and between all patients and controls (p ≥0.2, chi-square test). There was no association between extent of coronary artery disease (0, 1, 2, and 3 vessel disease) and FXII genotype. In conclusion, the common 46 C to T point mutation is strongly associated with FXIIa but the present study did not show an association with coronary artery disease. The role of this polymorphism in other thrombotic disorders such as ischemic stroke and venous thrombosis and its clinical significance in FXII deficient states remains to be investigated.
Fibrinogen Matsumoto III: a Variant with γ275 Arg→Cys (CGC→TGC) – Comparison of Fibrin Polymerization Properties with those of Matsumoto I (γ364 Asp→His) and Matsumoto II (γ308 Asn→Lys)
Authors: Terasawa Fumiko , Higuchi Yumiko , Ishikawa Shinsuke , Tozuka Minoru , Ishida Fumihiro , Kitano Kiyoshi , Katsuyama Tsutomu , Okumura Nobuo
Published: March 2018

Fibrinogen Matsumoto III (M-III) is a dysfibrinogen identified in a 66-year-old woman with rectal cancer. The fibrinogen level determined by the thrombin-time method was markedly decreased in preoperative coagulation tests of her plasma. Three fibrinogen polypeptide-chain gene fragments from the proposita were amplified by the polymerase chain reaction method, then sequenced. The triplet CGC encoding the amino acid residue γ275 was replaced by TGC, resulting in the substitution of Arg→Cys. There have been previous reports of nine families with the same alteration, nine families with an Arg→His variant and one family with an Arg→Ser variant in this residue, which has been shown to be one of the most important amino acids in the ’D:D’ interaction site. In addition, there are three silent mutations in the Aα-chain gene and two mutations in the intron of the Bβ-chain and the γ-chain gene. However, none of these mutations is thought to be the cause of the dysfunctional fibrinogen. The thrombin-catalyzed fibrin polymerization in the presence of 1 mM Ca ions was markedly delayed in purified M-III. Its lag period was longer than those of Matsumoto II (M-II; γ308Asn→Lys) and Matsumoto I (M-I; γ364Asp→His). γ364Asp is one of the most important residues in the polymerization pocket of the ’D:E’ interaction site and γ308Asn is located in the vicinity of a high affinity Ca2+ binding site in the D-domain, γ311-336. The maximum slope of the polymerization curve for M-III was about 4-fold steeper than that for M-I but less steep than that for M-II. These results may suggest that the tertiary structure of the polymerization pocket plays a more important role in the lateral aggregation of protofibrils than that of the ’D:D’ interaction site.
The P2Y1 Receptor Is Normal in a Patient Presenting a Severe Deficiency of ADP-induced Platelet Aggregation
Authors: Léon Catherine , Vial Catherine , Ohlmann Philippe , Hechler Béatrice , Cazenave Jean-Pierre , Lecchi Anna , Cattaneo Marco , Gachet Christian
Published: March 2018

ADP is a key stimulus inducing platelet shape change and aggregation, a rise in internal calcium and inhibition of adenylyl cyclase. These signaling pathways are thought to be activated by three independent receptors, but to date only the P2Y1 receptor responsible for calcium mobilization and the ionotropic P21 receptor have been identified. We report here the characteristics of the P2Y1 receptor in a patient presenting a selective deficiency of ADP-induced aggregation. Cloning of the P2Y1 gene revealed that the patient’s DNA and mRNA were normal. Pharmacological studies showed that the P2Y1 receptor was expressed and functional in patient’s platelets. Hence, the P2Y1 receptor is not the cause of the impaired ADP-induced platelet aggregation in this patient. The P21 mRNA was also found to be present and normal. These findings add evidence to previous observations suggesting that a third P2 receptor coupled to adenylyl cyclase may be involved in ADP-induced platelet aggregation.
Temporal and Topographic Matrix Metalloproteinase Expression after Vascular Injury in Mice
Authors: Lupu F. , Moons L. , Carmeliet P. , Goulding D. , Collen D. , Lijnen H. R.
Published: March 2018

Temporal and topographic expression of matrix metalloproteinases (MMPs) after perivascular electric injury was studied in wild-type (WT) and urokinase-deficient (u-PA-/-) mice. Neointima formation after injury of the femoral artery was significantly reduced in u-PA-/- mice as compared to WT mice (area of 0.002 ± 0.0007 mm2 versus 0.008 ± 0.002 mm2 at 3 weeks after injury; p <0.001), associated with impaired cellular migration (nuclear cell counts of 44 ± 5 versus 82 ± 9 in cross-sectional areas; p <0.001).

Zymographic and/or microscopic analysis indicated that MMP expression gradually increased to reach a maximum at 1 to 2 weeks after vascular injury. In general, MMP levels were lower in u-PA-/- than in WT mice. In non-injured arteries, MMP-2 (gelatinase A) and MMP-3 (stromelysin-1) were produced mainly by adventitial fibroblasts and/or non-contractile smooth muscle cells (SMC). One week after injury, MMP-2 and MMP-3 levels were enhanced due to an increased number and size of producing cells; 2 to 3 weeks after injury, MMP-2 and MMP-3 were produced also by some contractile SMC, which stained with α-actin antiserum. MMP-9 (gelatinase B), MMP-12 (metalloelastase) and MMP-13 (collagenase-3) were found in macrophages located mainly in the adventitia. Immunogold electron microscopic examination revealed that MMP-2 was located predominantly in association with the cell surface of fibroblasts or SMC, while MMP-9 and MMP-12 were located in well defined storage granules within macrophages. MMP-2, MMP-3 and MMP-13, but not MMP-9 or MMP-12, were also found extracellularly, associated with elastin-containing structures (MMP-2), with the basement membrane and occasionally with collagen fibres (MMP-3), or with proteoglycans, collagen and elastin (MMP-13).

The temporal and topographic expression pattern of MMPs after vascular injury, coinciding with smooth muscle cell migration and neointima formation, thus is compatible with a role in vascular remodeling.
Increased Expression of Protease Activated Receptor-2 (PAR-2) in Balloon-Injured Rat Carotid Artery
Authors: D’Andrea Michael , de Garavilla Lawrence , Cheung Wai-man , Andrade-Gordon Patricia , Damiano Bruce
Published: March 2018

Protease-induced cell signaling is mediated by specific receptors such as the emerging family of protease activated receptors (PARs). Since proteases are involved in various aspects of vascular injury, we assessed expression of PAR-2, a protease-activated receptor closely related to the thrombin receptor (PAR-1) but activated by an unknown protease, in vascular injury. Rat carotids were subjected to balloon-catheter injury and perfusion fixed at 1, 3, 7 or 14 days after injury. Sections of injured and normal carotid arteries were immunohisto-chemically labeled with a polyclonal antibody raised against the N-terminal residues 37-53 of human PAR-2. Sections were also labeled with antibodies to factor VIII-related antigen, smooth muscle actin and a proliferating cell nuclear antigen (PCNA). In normal vessels, PAR-2 labeling was diffuse and patchy in medial smooth muscle and endothelium. At one and three days after injury, before appearance of neointima, PAR-2 labeling increased in cells adjacent to damaged or necrotic smooth muscle cells. In addition, proliferating adventitial myofibroblasts labeled strongly for PAR-2. At 7 and 14 days after injury, the media and neointima of injured vessels had increased PAR-2 labeling which was most intense at the luminal edge of the neointima. Double immunohistochemical labeling confirmed the greatest expression of PAR-2 in areas with the greatest density of PCNA-positive cells. In addition, PAR-2 mRNA localization using in situ hybridization paralleled PAR-2 expression. The data suggest an upregulation of PAR-2 in response to vascular injury which is associated with medial smooth muscle damage, proliferating adventitial myofibroblasts and smooth muscle cells of the neointima, particularly those at the proliferating luminal edge of the neointima. Possible functional consequences of this receptor upregulation and its role in the response to vascular injury remain to be determined.
The Utility of Animal Models in the Preclinical Study of Interventions to Prevent Human Coronary Artery Restenosis: Analysis and Recommendations
Authors: Griggs T. R. , Badimon L. , Johnson G. J.
Published: March 2018

Small animal models have several advantageous characteristics, but those used in preclinical restenosis research have lacked efficacy in predicting the success of interventions to inhibit restenosis in humans.

Large animal models have been more successful than small animal models in predicting efficacy of interventions to inhibit restenosis in humans, but the results of studies carried out with these models have not been uniformly predictive.

Confirmation of the results of small animal studies in large animals has not always yielded information predictive of success in humans; however, the absence of such confirmation has had strong negative predictive value.

Small animal models used for evaluation of interventions to inhibit luminal narrowing following arterial instrumentation have failed to closely simulate human atherosclerosis and the stenotic lesions subjected to instrumentation in humans.

Transgenic, atherosclerotic animals hold promise for the development of more useful small animal models to study mechanisms of the response of diseased arteries to angioplasty and stents.

The pig has been the most useful large animal to study stenosis/ restenosis, but more information is needed to overcome the limitations of this model.
Increased C-reactive Protein Levels during Short-term Hormone Replacement Therapy in Healthy Postmenopausal Women
Authors: van Baal Marchien , Kenemans Peter , van der Mooren Marius , Kessel Hilda , Emeis Jef , Stehouwer Coen
Published: March 2018

Objective: To study the short-term effect of unopposed oestradiol (E2) and sequentially combined hormone replacement therapy (E2 + P) on C-reactive protein (CRP) in healthy postmenopausal women. Design: Prospective, randomised, placebo-controlled 12-week study. Sixty healthy, normotensive, non-hysterectomised postmenopausal women received either placebo (N = 16) or daily 2 mg micronised oestradiol, either unopposed (N = 16, E2 group) or sequentially combined with a progestagen on 14 days of each cycle (N = 28, E2+P group). Data were collected at baseline and at 4 and 12 weeks. Results: CRP levels increased significantly during the 12 weeks in the E2 and the E2+P groups compared to placebo. No differences were found between the E2 group and the E2+P group [E2 and E2+P group together (N = 44) versus placebo: P = 0.01; E2 versus E2+P: P = 0.75]. To give a quantitative estimate of the increase, the median change calculated from baseline in both treatment groups together was +87% (P = 0.02) at 4 weeks, and +114% (P = 0.08) at 12 weeks, as compared to the placebo group. Conclusion: In healthy postmenopausal women, short-term treatment with E2 or E2+P was associated with a rapid rise in CRP concentrations. These observations raise the possibility that the increased risk of cardiovascular events is related to an initial increase in CRP levels after starting hormone replacement therapy.
Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function
Authors: Corral J. , González-Conejero R. , Rivera J. , Ortuño F. , Aparicio P. , Vicente V.
Published: March 2018

The variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.
Titration of Antiplatelet Treatment in Pregnant Women at Risk of Preeclampsia
Authors: Clark N. A. C. , Swiet M. de , Nelson-Piercy C. , Elder M. G. , Sullivan M. H. F.
Published: March 2018

We recruited 111 patients who were considered to be at significantly increased risk of preeclampsia on the basis of previous obstetric history or preexisting medical disorders. All patients were treated with low dose aspirin (75 mg/day) from the first occasion the patient attended the antenatal clinic, regardless of gestational age. If the maternal mean platelet volume (MPV) increased significantly (by >0.8 fl) from the baseline, antiplatelet treatment was increased. Five pregnancies were lost during the second trimester and 106 of the treated patients had live infants. The incidence of neonatal death (3/106 infants) was much lower than in the previous pregnancies in these patients (32/134 infants). Patients who were treated from the first trimester of pregnancy (group A, 89 patients) did substantially better than those treated from the second trimester (group B, 17 patients) as assessed by the incidence of pre-eclampsia or intrauterine growth restriction (IUGR), gestational age and birthweight at delivery. These data suggest that longitudinal monitoring of the MPV may identify the women who could benefit from increased antiplatelet treatment, and that antiplatelet treatment may be more effective when initiated in the first trimester rather than later in pregnancy.
Factor VIII Inhibitors in Mild and Moderate-severity Haemophilia A
Authors: Ludlam C.A. , Colvin B.T. , Hill F.G.H. , Preston F.E. , Wasseem N. , Bagnall R. , Peake I.R. , Berntorp E. , Bunschoten E.P. Mauser , Fijnvandraat K. , Kasper C.K. , White G. , Santagostino E. , Hay C.R.M. ,   the UK Haemophilia Centre Directors Organisation
Published: March 2018

Twenty six patients with mild or moderate haemophilia A and inhibitors are described. The inhibitor was detected at a median age of 33 years, after a median of 5.5 bleeding episodes. This usually following intensive replacement therapy. The median presenting inhibitor titre was antihuman 11.6 BU/ml, antiporcine 1.45 BU/ml. Plasma basal factor VIII level declined from a median of 0.08 IU/ml to 0.01 IU/ml following the inhibitor development. This caused spontaneous bleeding in 22 and a bleeding pattern similar to acquired haemophilia in 17. Bleeding was often severe and caused two deaths.

The inhibitor disappeared spontaneously, or following immune tolerance induction, in 16 cases after a median of 9 months (range 0.5-46), with a return to the original baseline VIIIC level and bleeding pattern accompanied inhibitor loss. The inhibitor persisted in the remainder of the cases over a median period of 99 months (range 17-433 months) of follow-up. Inhibitors are an uncommon complication of mild haemophilia which frequently persist and may be associated with severe, life-threatening, haemorrhage.

Forty-one percent of treated haemophilic family members had a history of factor VIII inhibitors, suggesting a familial predisposition to develop inhibitors in these kindreds. Sixteen patients from 11 families were genotyped. Seven different missense mutations affecting the light chain were detected and two in the A2 domain. Five patients from three families had a mutation causing a substitution of Trp2229 by Cys in the C2 domain which appears to predispose to inhibitor formation since 7 of the 18 affected individuals have a history of inhibitor development.
Simple and Reliable Factor V Genotyping by PNA-Mediated PCR Clamping
Authors: Behn Moira , Schuermann Marcus
Published: March 2018

Resistance to activated protein C (APC resistance) is the most common cause of thrombophilia and linked to a single point mutation in the factor V gene (G>A transition at nucleotide 1691). In the past, several PCR based methods have been proposed to determine the allelostatus of individual patients from small amounts of blood DNA including PCR followed by restriction fragment length polymorphism detection (PCR-RFLP), PCR using sequence-specific primers (PCR-SSP) and oligonucleotide ligation assay (OLA). Here, we present a novel approach based on the method of peptide nucleic acid(PNA)-mediated PCR clamping which is extremely sensitive to base pair mismatches. If PNAs specific for the two allelic variants are applied separately in each case a clear discrimination between a heterozygous or homozygous normal or homozygous Factor V Leiden status is possible and no further confirmation step is required. In a prospective study, 60 patients with suspected venous thrombosis events were tested and compared to the conventional PCR-RFLP technique. The concordance between both methods was 100%. PNA-based factor V genotyping, therefore, should be considered for large scale screening of those patients considered to be at risk for deep venous thrombosis.