bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation between Oxidized Low Density Lipoproteins and von Willebrand Factor in Chronic Renal Failure
Authors: Holvoet Paul , Donck Jan , Landeloos Michèle , Brouwers Els , Luijtens Kristel , Arnout Jozef , Lesaffre Emmanuel , Vanrenterghem Yves , Collen Désiré
Published: July 2018

An ELISA specific for a wide spectrum of oxidized apo B-100 in OxLDL was developed and applied to blood samples from 27 control subjects, 20 mild chronic renal failure (MCRF) patients, 21 severe chronic renal failure patients on conservative treatment (SCRF) and 56 severe chronic renal failure patients on maintenance hemodialysis (HEMO). Mean levels of OxLDL were 0.59 mg/dl in controls (95% Cl, 0.52-0.66 mg/dl), and were 2.7-fold (p <0.01), 3.1-fold (p <0.001) and 5.4-fold (p <0.001) higher in MCRF, SCRF and HEMO patients, respectively. Levels of von Willebrand factor, a marker of endothelial injury, were 100 percent in controls (95% Cl, 90-110 percent), and were 1.5-fold (p = NS), 1.6-fold (p <0.01) and 2.1-fold (p <0.001) higher in MCRF, SCRF and HEMO patients, respectively. Multiple regression analysis revealed that the extent of renal failure (F = 14; p = 0.0004) accounted for a significant fraction of the variation in OxLDL levels, also after exclusion of patients with evidence of ischemic atherosclerotic disease (F = 21; p = 0.0001). After adjustment for the extent of renal failure, hemodialysis (F = 5.6; p = 0.021) and LDL cholesterol levels (F = 7.1, p = 0.0095) contributed significantly to the variation in OxLDL levels. Whereas the extent of renal failure contributed only marginally to the individual variations in vWF levels (F = 4.1; p = 0.048), these levels correlated significantly with plasma levels of OxLDL (F=26; p=0.0001). In conclusion, atherogenic OxLDL increase progressively during the development of renal failure suggesting that the oxidation of LDL may be associated with endothelial injury and atherogenesis in these patients.
Bernard-Soulier Syndrome with Severe Bleeding: Absent Platelet Glycoprotein lb alpha Due to a Homozygous One-base Deletion
Authors: Li Chaoyang , Pasquale Dominick N , Roth Gerald J
Published: July 2018

Bernard-Soulier syndrome is a rare congenital platelet disorder that affects a surface membrane adhesion receptor, glycoprotein (GP) Ib-V-IX. Both the genetic defects and the bleeding diatheses associated with the syndrome are heterogeneous due, in part, to the complexity of the involved receptor which consists of four different members, GPs: Ibα-Mr 143 K (contains the von Willebrand factor-binding site), Ibβ-Mr 22 K, V-Mr 83 K and IX-Mr 20 K. We studied a kindred that includes a 40 year-old man with severe Bernard-Soulier syndrome: life-threatening gastrointestinal bleeding, thrombocytopenia, giant platelets and absent ristocetin-dependent platelet aggregation. By Southern blotting, PCR amplification/sequencing, hetero-duplex analysis, and allele-specific oligonucleotide hybridization, the Ib-V-IX genes were analyzed, and the molecular genetic defect was defined as a one-base deletion in the GPIbα gene, involving an adenine of codon 19. The mutation, K19R, homozygous in the propositus and heterozygous in the available unaffected relatives, leads to a frame shift in codons 19-21 and a premature stop codon after codon 21. No functional GPIbα can be produced from the mutant allele, implying that the platelets of the affected patient lack all GPIbα. Within the spectrum of Bernard-Soulier syndrome, this patient’s disorder exemplifies a severe or “classic” extreme; an “experiment of Nature” that illustrates the effect of a complete deficiency of the ligand-binding chain (GPIbα) of the GPIb-V-IX receptor.
Antagonism of SR 90107A/Org 31540-lnduced Bleeding by Protamine Sulfate in Rats and Mice
Authors: Bernat A , Hoffmann P , Herbert J M
Published: July 2018

The neutralization by protamine sulfate of bleeding enhancement induced by the potent anti-factor Xa pentasaccharide SR 90107A/Org 31540 and by heparin has been studied in rats and mice. Bleeding, as measured by transection of the tail of anaesthetised rats or mice, was increased following the administration of heparin and very high doses of SR 90107A/Org 31540. In rats, i.v. doses of 0.6 mg/kg heparin or 15 mg/kg SR 90107A/Org 31540 were required to enhance bleeding time to approximately the same extent (5- or 7-fold increase), whereas in mice a 13-fold increase in blood loss was observed with i.v. doses of 3 mg/kg heparin or 10 mg/kg SR 90107A/Org 31540. Protamine sulfate (10 mg/kg i.v.) reduced bleeding in rats and mice induced by both compounds. It also neutralized the anti-factor Xa activity as well as the antithrombotic activity of heparin as observed in venous thrombosis models in both species. However, protamine sulfate neither affected the anti-factor Xa activity nor the antithrombotic activity of SR 90107A/Org 31540 in rats and mice. The present results suggest that protamine sulfate may be regarded as a potential antidote to neutralize bleeding side-effects in cases of SR 90107A/Org 31540 overdosing.
Increased Levels of Activated Factor VII and Decreased Plasma Protein S Activity and Circulating Thrombomodulin during Use of Oral Contraceptives
Authors: Quehenberger Peter , Loner Uta , Kapiotis Stylianos , Handler Sylvia , Schneider Barbara , Huber Johannes , Speiser Wolfgang
Published: July 2018

In the present study the effect of oral contraceptive (OC) treatment on selected factors involved in the activation, i.e. circulating activated factor VII (cFVIIa), and in the inhibition of blood coagulation, i.e. plasma protein S activity and circulating thrombomodulin (cTM), were for the first time measured in OC users in a prospective study. Beside other coagulation variables, these parameters were measured during treatment with three low estrogen formulations containing different gestagen components (norgestimate, gestodene). During OC treatment increases in the activation markers prothrombin fragment F1+2 and D-Dimer were found, suggesting an increased activation of blood coagulation and fibrinolysis. Along with elevated plasma levels of FVII antigen, cFVIIa was also found increased in all three treatment groups, while inhibitory components of blood coagulation, plasma protein S activity and cTM, significantly and similarily decreased during treatment in all three treatment groups. We conclude that low dose estrogen pills induce similar changes in the plasma levels of main regulatory components of blood coagulation, despite differences in their gestagen components. Increased levels of activators and decreased activities of inhibitors may contribute to arterial and venous thrombotic complications seen in predisposed OC users.
Detection of Factor V Leiden from a Drop of Blood by PCR-SSCP
Authors: Corral Javier , Iniesta Juan A , González-Conejero Rocio , Vicente Vicente
Published: July 2018

Recently, a point mutation in the coagulation Factor V gene (G to A in position 1691) has been identified which makes the mutant Factor V (called Factor V Leiden) resistant to activated protein C. This defect is a well established genetic risk factor in venous thrombosis. Because of the high prevalence of Factor V Leiden in normal population (2-7%), it would be reasonable to perform a rapid and simple method for screening the genetic abnormality in population at risk.

We have developed a simple, reproducible, rapid and cheap procedure that, using PCR and SSCP, allows the identification of the mutation responsible for Factor V Leiden. Specificity of this method has been tested in 319 samples: 304 normal, 14 heterozygous and 1 homozygous for Factor V Leiden. This assay allows non-isotopic Factor V Leiden identification by using frozen whole blood in 3 h. All these features make this test adequate for routine screening of this mutation in a large number of samples.
The Mechanism of Platelet Aggregation Induced by HLA-Related Antibodies
Authors: Brandt John T , Julius Carmen J , Osborne Jeanne M , Anderson Clark L
Published: July 2018

Immune-mediated platelet activation is emerging as an important pathogenic mechanism of thrombosis. In vitro studies have suggested two distinct pathways for immune-mediated platelet activation; one involving clustering of platelet FcyRIIa, the other involving platelet-associated complement activation. HLA-related antibodies have been shown to cause platelet aggregation, but the mechanism has not been clarified. We evaluated the mechanism of platelet aggregation induced by HLA-related antibodies from nine patients. Antibody to platelet FcyRIIa failed to block platelet aggregation with 8/9 samples, indicating that engagement of platelet FcyRIIa is not necessary for the platelet aggregation induced by HLA-related antibodies. In contrast, platelet aggregation was blocked by antibodies to human C8 (5/7) or C9 (7/7). F(ab’)2 fragments of patient IgG failed to induce platelet activation although they bound to HLA antigen on platelets. Intact patient IgG failed to aggregate washed platelets unless aged serum was added. The activating IgG could be adsorbed by incubation with lymphocytes and eluted from the lymphocytes. These results indicate that complement activation is involved in the aggregation response to HLA-related antibodies. This is the first demonstration of complement-mediated platelet aggregation by clinical samples. Five of the patients developed thrombocytopenia in relationship to blood transfusion and two patients developed acute thromboembolic disease, suggesting that these antibodies and the complement-dependent pathway of platelet aggregation may be of clinical significance.
The Antiplatelet Effects of a New Nitroderivative of Acetylsalicylic Acid - An In Vitro Study of Inhibition on the Early Phase of Platelet Activation and on TXA2 Production
Authors: Lechi Clara , Andrioli Giuseppe , Gaino Stefania , Tommasoli Rosamaria , Zuliani Valeria , Ortolani Riccardo , Degan Maurizio , Benoni Giuseppina , Bellavite Paolo , Lechi Alessandro , Minuz Pietro
Published: July 2018

We studied in vitro the antiplatelet activity of a new nitroderivative chemically related to acetylsalicylic acid: 2 acetoxybenzoate 2-[l-nitroxy-methyl]-phenyl ester (NCX 4016), in order to identify any effects due to the release of nitric oxide and the blockade of cyclooxygenase

The effects of scalar doses of NCX 4016 on the early phase of platelet activation, platelet aggregation and thromboxane A2 production were investigated. We observed inhibitory effects of NCX 4016 on platelet adhesion (IC50 = 7.3 × 10−5 M), platelet cytosolic calcium concentration, assayed by fluorescent probe Fura 2, and the expression of glycoprotein IMIIa (CD41 / αIIbβ3) (IC50 = 3.4 × 10−5 M) and P-selec-tin (CD62 / GMP-140) (IC50 = 4.9 × 10−5 M) measured by flow cytometry. NCX 4016 also prevented thrombin-induced platelet aggregation (IC50 = 3.9 × 10−5 M). None of these parameters were affected by acetylsalicylic acid. These inhibitory activities of NCX 4016 were abolished by oxyhaemoglobin and methylene blue. Intracellular cyclic GMP observed during thrombin-induced aggregation was increased by incubation with NCX 4016. These results appear to be attributable to the release of nitric oxide, which activates soluble platelet guanylyl-cyclase and promotes intracellular cyclic GMP increase. NCX 4016 almost completely inhibited platelet thromboxane A2 production and arachidonic acid-induced platelet aggregation. This also occurred in the presence of oxyhaemoglobin and methylene blue, indicating that its antiplatelet activity can be attributed not only to nitric oxide release but also to cyclo-oxygenase inhibition.
Biological Consequences of Thrombin Receptor Deficiency in Mice
Authors: Darrow Andrew L , Fung-Leung Wai-Ping , Ye Richard D , Santulli Rosemary J , Cheung Wai-Man , Derian Claudia K , Burns Carol L , Damiano Bruce P , Zhou Lubing , Keenan Charlotte M , Peterson Per A , Andrade-Gordon Patricia
Published: July 2018

The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetylcholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2 Our results indicate that ThrR deficiency has a strong impact on fetal development; however, ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type
Post Discharge Clinically Overt Venous Thromboembolism in Orthopaedic Surgery Patients with Negative Venography -an Overview Analysis
Authors: Ricotta Serena , lorio Alfonso , Parise Pasquale , Nenci Giuseppe G , Agnelli Giancarlo
Published: July 2018

A high incidence of post-discharge venous thromboembolism in orthopaedic surgery patients has been recently reported drawing further attention to the unresolved issue of the optimal duration of the pharmacological prophylaxis. We performed an overview analysis in order to evaluate the incidence of late occurring clinically overt venous thromboembolism in major orthopaedic surgery patients discharged from the hospital with a negative venography and without further pharmacological prophylaxis. We selected the studies published from January 1974 to December 1995 on the prophylaxis of venous thromboembolism after major orthopaedic surgery fulfilling the following criteria: 1) adoption of pharmacological prophylaxis, 2) performing of a bilateral venography before discharge, 3) interruption of pharmacological prophylaxis at discharge in patients with negative venography, and 4) post-discharge follow-up of the patients for at least four weeks. Out of 31 identified studies, 13 fulfilled the overview criteria. The total number of evaluated patients was 4120. An adequate venography was obtained in 3469 patients (84.1%). In the 2361 patients with negative venography (68.1%), 30 episodes of symptomatic venous thromboembolism after hospital discharge were reported with a resulting cumulative incidence of 1.27% (95% C.I. 0.82-1.72) and a weighted mean incidence of 1.52% (95% C.I. 1.05-1.95). Six cases of pulmonary embolism were reported. Our overview showed a low incidence of clinically overt venous thromboembolism at follow-up in major orthopaedic surgery patients discharged with negative venography. Extending pharmacological prophylaxis in these patients does not appear to be justified. Venous thrombi leading to hospital re-admission are likely to be present but asymptomatic at the time of discharge. Future research should be directed toward improving the accuracy of non invasive diagnostic methods in order to replace venography in the screening of asymptomatic post-operative deep vein thrombosis.
Lupus Anticoagulant is the Strongest Risk Factor for both Venous and Arterial Thrombosis in Patients with Systemic Lupus Erythematosus
Authors: Horbach Daniëlle A , Oort Erica V , Donders Richard C J M , Derksen Ronald H W M , de Groot Philip G
Published: July 2018

Antiphospholipid antibodies (aPL) characterize patients at risk for both arterial and venous thrombotic complications. Recently it has been recognized that the presence of plasma proteins such as β2-glycoprotein I (β2GPI) and prothrombin are essential for the binding of aPL to phospholipids and that these proteins are probably the real target of aPL. The discovery of these new antigens for aPL introduces the possibility of new assays to detect the presence of aPL. However, it is not known whether these assays improve the identification of patients at risk for thrombosis.

In this retrospective study we compared the value of the classic assays LAC (lupus anticoagulant) and ACA (anticardiolipin antibodies) to detect aPL associated with thrombotic complications, with new assays which are based on the binding of aPL to the plasma proteins prothrombin and P2GPI. To do so, we have used these assays in a group of 175 SLE patients and correlated the positivity of the different assays with the presence of a history of venous and arterial thrombosis. Control groups were patients without SLE but with LAC and/or ACA and thrombosis (n = 23), patients with thrombosis without LAC and ACA (n = 40) and 42 healthy controls.

In the univariate analysis, in which no distinction has been made between high and low antibody levels, we confirmed LAC and ACA to be related to both arterial and venous thrombosis. Anti-β2GPI- and anti-prothrombin-antibodies, both IgG and IgM correlate with venous thrombosis and anti-β2GPI-IgM with arterial thrombosis. Multivariate analysis showed that LAC is the strongest risk factor (OR 9.77; 95% CI 1.74-31.15) for arterial thrombosis. None of the other factors is a significant additional risk factor. For venous thrombosis LAC is the strongest risk factor (OR 6.55; 95% Cl 2.36-18.17), but ACA-IgM above 20 MPL units also appeared to be a significant (p = 0.0159) risk factor (OR 3.90; 95% Cl 1.29-11.80). Furthermore, the presence of anti-β2GPI- and/or anti-prothrombin-antibodies in LAC positive patients (n = 60) does not increase the risk for thrombosis.

The results showed that (i) the LAC assay correlates best with a history of both arterial and venous thrombosis and (ii) neither the anti-P2GPI ELISA nor the anti-prothrombin ELISA gives additional information for a thrombotic risk in SLE patients.
Nucleotide Structure and Characterization of the Murine Blood Coagulation Factor VII Gene
Authors: Idusogie Esohe , Rosen Elliot D , Carmeliet Peter , Collen Desire , Castellino Francis J
Published: July 2018

The gene encoding murine coagulation factor VII (fVII) has been cloned. Seven introns and eight exons are present, with the introns positioned as splice junctions between the major domain units of the protein. A total of 11,748 bp of the gene was sequenced, and included 1,077 bp of a 5’-flanking region, in which several high probability binding sites for liver transcription factors were present, as well as a CCAAT sequence and possible GC boxes. Primer extension analysis revealed that the major transcription start site was positioned only 9 residues upstream of the ATG initiation codon, thus providing a very short 5’-untranslated region of the gene. The sequence of the CAP site in the murine fVII gene matched exactly the consensus eukaryotic sequence. A total of 1,484 bp of 3’-flanking nucleotides included a probable polyadenylation site (ATTAAA) and an appropriately positioned downstream consensus sequence (AGTGTTTC) for the efficient formation of a 3’ terminus of mRNA. These results indicate that all elements are present for liver-based transcription of the gene for murine factor VII. The sequence and restriction endonuclease map of this gene will facilitate construction of fVII deficient mice and mice containing mutant fVII genes.
Age and Sex Dependent Regulation of the Factor IX Gene in Mice
Authors: Kurachi Sumiko , Hitomi Eri , Kurachi Kotoku
Published: July 2018

Plasma factor IX and liver factor IX mRNA levels in two normal mouse strains (B6D2F1 and BALB/CJNIA) were determined in relation to aging and sex of the animals. With male B6D2F1 mice, mean plasma factor IX activity levels for the 14 and 21-22 month-old animals were found to be 124% and 226%, respectively, of the 5 month-old group. Similarly, liver factor IX mRNA levels for the same age animal groups were 145% and 227%, respectively, of the reference group. Mean plasma factor IX levels for the same age female animals were 132% and 175%, respectively, and were accompanied by similarly elevated liver factor IX mRNA levels, 119 and 175%, respectively, of the 5 month-old female group. Factor IX activity and mRNA levels for the 5,14 and 21-22 month-old female animal groups were lower than those of the corresponding male age groups by 25, 20 and 37%, and 20,36 and 38%, respectively. With BALB/CJNIA mice, similar correlation was observed between the advancing age and substantial elevations in the factor IX mRNA level as well as on the unequal factor IX mRNA levels in females and males.

These results indicate that the plasma factor IX level in both male and female mice is greatly elevated with aging, in general agreement with a similar phenomenon observed for human populations, and that this increase is due to a similar elevation in the factor IX mRNA level in the liver. In mice, both factor IX activity and mRNA levels are significantly higher in males than in females, which has not been described for humans.
Distinct Patterns of Urokinase Receptor (uPAR) Expression by Leukemic Cells and Peripheral Blood Cells
Authors: Jardi Mercè , Inglés-Esteve Julia , Burgal Maria , Azqueta Carmen , Velasco Francisco , López-Pedrera Chary , Miles Lindsey , Félez Jordi
Published: July 2018

The urinary type plasminogen activator, urokinase (uPA) is localized on the cell surface through the binding of a specific receptor, the uPA receptor (uPAR). The uPA localization enhances plasmin formation on the cell surface and facilitates cell migration. The cellular and tissue distribution of uPAR is not fully established. We have analyzed uPAR expression in nine leukemic cell lines of distinct lineages and maturational states and correlated this with expression of plasminogen receptors, tissue-type plasminogen activator (tPA) receptors and LDL receptor-related protein (LRP). The most immature and least differentiated cell line (an erythro-myeloid cell line) and cells of lymphoid lineage, did not express uPAR, whereas cells differentiated along the myelo-monocytic pathway displayed this receptor. Plasminogen and tPA receptors were expressed by all leukemic cell lines and by all nucleated peripheral blood cells but B and T lymphocytes were negative for cell surface expression of both uPAR and LRP while monocytes and neutrophils were positive for expression of both uPAR and LRP. PMA stimulation induced surface expression of uPAR in lymphocytes but did not induce expression of LRP by these cells. In contrast, lymphoid cell lines were negative for uPAR expression even after PMA stimulation, indicating differences in regulation of uPAR expression between lymphocytes and lymphoid cell lines. The pattern of uPAR expression on leukemic cell lines was also studied on bone marrow blast cells from leukemic patients. Only the most mature myeloid cells expressed uPAR on their surfaces. In contrast, M3 leukemic cells and other blast cells displaying lymphoid markers such as TdT (+) and/or CD2 (+) did not express intracellular or cell-surface associated uPAR, indicating an heterogeneity among these promyelo-cytic cells and suggesting that uPAR may be a useful marker for leukemia typing. Myeloid blast cells from some patients contained intracellular pools of uPAR but displayed no receptor on the cell surface, suggesting that translocation may be a mechanism regulating uPAR expression in these cells. The comparison of uPAR expression between these cell lines and peripheral blood cells and it correlation with plasminogen receptors, tPA receptors and LRP expression offers new insights regarding potential mechanisms for regulation of uPA-uP-AR-mediated pericellular proteolysis.
PAICA: A Method for Characterizing Platelet-Associated Antibodies - Its Application to the Study of Idiopathic Thrombocytopenic Purpura and to the Detection of Platelet-bound c7E3
Authors: Macchi Laurent , Clofent-Sanchez Gisèle , Marit Gérald , Bihour Claude , Durrieu-Jais Catherine , Besse Pierre , Nurden Paquita , Nurden Alan T
Published: July 2018

In idiopathic thrombocytopenic purpura (ITP), autoantibodies reacting with antigens on the platelet membrane bring about accelerated platelet destruction. We now report PAICA (“Platelet-Associated IgG Characterization Assay”), a method for detecting autoantibodies bound to specific membrane glycoproteins in total platelet lysates. This monoclonal antibody (MAb) capture assay takes into account the fact that antibodies on circulating platelets may be translocated to internal pools as well as being on the surface. A total of twenty ITP patients were examined by PAICA, and the results compared with those obtained by measuring (i) serum antibodies bound to paraformaldehyde-fixed control platelets by ELISA, (ii) IgG bound to the surface of the patient’s own platelets by flow cytometry (PSIgG), (iii) total platelet-associated IgG (PAIgG) by ELISA and (iv) serum antibodies reacting with control platelets by MAIPA (“Monoclonal Antibody-specific Immobilization of Platelet Antigens”). Of twelve patients with elevated PAIgG, nine had increased PSIgG yet eleven reacted positively in PAICA. Of these, eight possessed antibodies directed against GP Ilb-IIIa, two against GP Ib-IX and one patient possessed antibodies directed against GP Ilb-IIIa and GP Ia-IIa respectively. Only seven of the patients possessed serum antibodies detectable by MAIPA. PAICA was also able to detect platelet-associated c7E3 (the chimeric form of Fab fragments of the MAb 7E3) following its infusion during antithrombotic therapy, when it proved more sensitive over a seven-day period than a MAIPA assay adapted for assessing surface-bound antibody. We propose that PAICA provides added sensitivity to the detection of platelet-associated antibodies in immune thrombocytopenias or following therapy with humanized MAbs.
A Novel Regulatory Epitope Defined by a Murine Monoclonal Antibody to the Platelet GPIIb-IIIa Complex (αIIbβ3 Integrin)
Authors: Tokuhira Michihide , Handa Makoto , Kamata Tetsuji , Oda Atsushi , Katayama Masahiko , Tomiyama Yoshiaki , Murata Mitsuru , Kawai Yohko , Watanabe Kiyoaki , Ikeda Yasuo
Published: July 2018

We characterized a murine monoclonal antibody, PT25-2 (IgG1), raised against washed human platelets. The antibody and its Fab fragments were both capable of inducing platelet aggregation in a fibrinogen-dependent manner and induced 125I-fibrinogen binding to unstimulated platelets (120,000 molecules/platelet at a 100 nM IgG concentration). The antibody immunoprecipitated the αIIbβ3 complex from lysates of iodinated platelets but did not react with the respective subunits when complex formation was disrupted by treatment with 5 mM EDTA at 37°C for 30 min. However, simply removing the extracellular divalent cation with EDTA had no effect on antibody binding indicating that the antibody’s epitope depends upon a conformational structure maintained by αβ subunit association. Antibody binding to unstimulated, washed platelets yielded binding parameters (Kd = 40 nM, Bmax = 100,000 molecules/platelet), which were found to be virtually unchanged when binding was performed using thrombin or RGDS-peptide-stimulated platelets. Thus, the PT25-2 antibody defines a novel regulatory epitope expressed by the αIIbβ3 integrin on unstimulated, quiescent platelets.
Platelets from Bleeding Simmental Cattle Have a Long Delay in both ADP-activated Expression of GpllB-IIIA Receptors and Fibrinogen-dependent Platelet Aggregation
Authors: Frojmovic Mony M , Wong Truman , Searcy Gene P
Published: July 2018

We have previously reported that platelets from bleeding Simmental cattle do not aggregate in vitro in response to ADP, collagen and calcium ionophore A23187, though calcium mobilization and myosin light chain phosphorylation do occur. The aggregation abnormality, measured by aggregometry, was ascribed to abnormal cytoskeletal expression, with the maximal numbers of activated GpIIb-IIIa receptors per platelet no different from that seen in normal bovine platelets activated with ADP. We have therefore sought to compare the kinetics of microaggregation with the rate of expression of GpIIb-IIIa receptors required for mediating fibrinogen (Fg)-dependent platelet aggregation, to provide a more direct molecular explanation for the aggregation abnormality. We compared aggregation kinetics of ADP-activated platelets using both aggregometry and particle counting to monitor microaggregation. Fibrinogen receptor expression was monitored with FITC-labelled human Fg and with the reporting antibody for activated GpIIb-IIIa, FITC-PAC1, using flow cytometry. The affected platelets show a marked delay in onset of microaggregation for ADP-activated platelets stirred with human Fg, paralleded by an unusual delay in activated GpIIb-IIIa receptor expression (DARE) for otherwise competent Fg binding. The on-rates for Fg binding to platelets maximally pre-activat-ed with PMA are identical for normal and affected platelets, whether comparing the binding of human or bovine Fg. The unique DARE syndrome explains the observed delay in aggregation of platelets from affected Simmental cattle and predicts the bleeding problems due to delayed binding of adhesive proteins.
Two Distinct Novel Splice Site Mutations in a Compound Heterozygous Patient with Protein S Deficiency
Authors: Yamazaki Tomio , Katsumi Akira , Okamoto Yoshihiro , Takafuta Toshio , Tsuzuki Shinobu , Kagami Kazuo , Sugiura Isamu , Kojima Tetsuhito , Fujimura Kingo , Saito Hidehiko
Published: July 2018

Genetic analysis revealed two distinct novel splice site mutations in a compound heterozygous patient with protein S deficiency. The paternal mutation was a G-to-T transition at position -1 of the acceptor splice site of intron N (Mutation I), and the maternal mutation was a G-to-C transversion at position -1 of the donor splice site of intron C (Mutation II). Both splice site mutations decreased the mutated mRNA accumulation to the same extent, approximately 40% of the normal mRNA. However, the mutations were associated with different phenotypical expressions: the paternal mutant protein S was not detected in vivo, while the maternal mutant protein S was present in the plasma in reduced quantity. Because Mutation I caused a cryptic splicing in the mutated mRNA, resulting in a reading frameshift and premature termination, the predicted mutant protein S might be highly unstable. In contrast, Mutation II led to the substitution of Val46 by Leu, which might be much less deleterious for the synthesis, secretion and stability of the predicted mutant protein S. It was supposed that the different post-translational metabolisms produced the distinct phenotypical expressions of the mutations.
Thromboprophylaxis with Low Molecular Weight Heparin (Fragmin) in High Risk Pregnancies
Authors: Hunt Beverley J , Doughty Heidi-Ann , Majumdar G , Copplestone Adrian , Kerslake Sian , Buchanan Neil , Hughes Graham , Khamashta Munther
Published: July 2018

Venous thromboembolic disease remains the commonest cause of maternal death. The management of thromboprophylaxis in high risk women during pregnancy is contentious. Low molecular weight heparins (LMW) have theoretical advantages compared with unfractionated heparin and warfarin but have been poorly studied in pregnancy. We report on the use of LMW heparin (Fragmin) as thromboprophylaxis in thirty four high risk pregnancies.

All the women had a previous thrombosis or a thrombosis in their current pregnancy +/- a recognised thrombophilic state (eleven had the antiphospholipid syndrome).

Fragmin was given subcutaneously to maintain trough anti-Xa activity of 0.15-0.2 U/ml and 2 h post injection levels of 0.4-0.6 U/ml. The levels were checked monthly during pregnancy. Most women required 5,000U Fragmin once daily during the first trimester unless they were greater than 100 kg at the start of pregnancy. The mean time for dosage increase was 20.5 week (S.D. 8.2). 26/34 pregnancies (76%) required twice daily at the end of pregnancy. Epidural anaesthesia was managed by omitting Fragmin dose or inserting the needle 6 hours after the previous Fragmin injection. There were no thromboembolic events, thrombocytopenias or excessive haemorrhage. One woman had osteoporotic vertebral collapse post partum, she had no other risk factors for osteoporosis.

LWM heparin (Fragmin) appears to be efficacious in preventing recurrent thromboembolic disease in pregnant women at high risk, but it is notable that osteoporotic fractures occurred post partum in one woman. Further trials are required to determine optimal dosage and safety.
Tissue Factor (TF) and Urokinase Plasminogen Activator Receptor (uPAR) and Bleeding Complications in Leukemic Patients
Authors: López-Pedrera Chary , Jardí Merce , del Mar Malagón Maria , Inglés-Esteve Julia , Dorado Gabriel , Torres Antonio , Félez Jordi , Velasco Francisco
Published: July 2018

Tissue factor (TF) and urokinase receptor (uPAR) are key cellular receptors triggering, respectively, coagulation and fibrinolysis. Bleeding complications among leukemic patients have been related to an abnormal expression of TF by blast cells and/or to an abnormal fibrinolytic response. In this study the expression of TF and uPAR has been assessed in 18 acute non-lymphoblastic and 8 lymphoblastic leukemic blast cells using several methodological approaches. TF mRNA was evaluated by in situ hybridization and TF and uPAR antigen were evaluated immunologically in cell lysates and on the cell surface by flow cytometry. In addition, TF-procoagulant activity was measured in coagulation-based assays. The reliability of these methods was corroborated in six leukemic cell lines of different lineages and states of maturation. Disseminated intravascular coagulation was detected in two M3 leukemia patients whose blast cells expressed high amounts of TF. Hyperfibrinolysis was detected in one M1 and two M2 patients, whose blast cells displayed a high content of uPAR antigen, but no TF. Furthermore, M5 leukemia blast cells expressed both TF and uPAR, although no hemostatic defects or bleeding complications were detected in these patients. Taken together, although a limited number of patients was included in this study, these data suggest that in leukemia patients exhibiting bleeding, either TF or uPAR are expressed by their blast cells. However, the presence of these receptors does not necessarily imply the existence of a hemostatic disorder.
Genetic Diagnosis of Factor V Leiden Using Heteroduplex Technology
Authors: Bowen D J , Standen G R , Granville S , Bowley S , Wood N A P , Bidwell J
Published: July 2018

A new genetic test has been developed for detection of the mutation known as factor V Leiden. The test employs heteroduplex technology and comprises a single PCR reaction followed immediately by PCR product analysis. It therefore represents the minimum practical route from blood/tissue sample to genetic result. A cohort of 100 patients with a history of thrombosis have been screened using both the new heteroduplex test and a previously described PCR-restriction endonuclease test. Results gave 100% correlation: normals 75 (75%), heterozygotes 24 (24%) and homozygotes 1 (1%). The heteroduplex test has been shown to give straightforward diagnosis in three different analytical systems: standard polyacrylamide gel electrophoresis (PAGE), mini-gel PAGE and capillary electrophoresis. The latter system is semiautomated, therefore rapid through-put of large sample numbers is now · possible.
Utilization of Dilute Russell’s Viper Venom Time to Detect Autoantibodies against β2-Glycoprotein I which Express Anticoagulant Activity in the Presence but not in the Absence of Exogenous Phospholipids
Authors: Pengo V , Balestrieri G , Tincani A , Spatola L , Biasiolo A , Brocco T
Published: July 2018

Lupus anticoagulant (LA) is a general term to define immunoglobulins interfering with phospholipid-dependent coagulation tests. It is now clear that the phospholipid-dependence of some LA is related to the presence of the phospholipid-binding plasma protein β2-glycoprotein I β2-GPI) and that autoantibodies to β2-GPI might represent a specific category of LA. To verify this hypothesis we have purified IgG autoantibodies to β2-GPI from plasma of 6 patients with antiphospholipid antibody syndrome, by means of agarose-immobilized human β2-GPI. All 6 preparations tested positive in anti-β2-GPI IgG antibody ELISA and showed a marked LA activity by prolonging dilute Russell Viper Venom Time (dRVVT) from a minimum of 5.3 s in patient # 1 to a maximum of 41.1 s in patient # 3. These IgG preparations behaved as typical LA, with this activity tending to disappear in the presence of increasing phospholipid (PL) concentrations. Moreover, the LA activity of the IgG preparations was not detectable in the absence of PL, in which case the ratio between dRVVT obtained in the presence and absence of IgG autoantibodies to β2-GPI was close to 1. This pattern was confirmed by using plasma from patients with antiphospholipid antibody syndrome testing positive for anti-β2-GPI IgG antibodies. These findings suggest that dRVVT performed both in the presence and absence of PL might constitute a sensitive screening test to detect specific antibodies with LA activity.
Effect of a 15-Minute Infusion of DDAVP on the Pharmacokinetics and Pharmacodynamics of ™REVASC during a Four-Hour Intravenous Infusion in Healthy Male Volunteers
Authors: Amin Dipti M , Mant Timothy G K , Walker Stephen M , Kerry Roger , Lloyd Peter , Lefèvre Gilbert , Close Philippe
Published: July 2018

Plasma pharmacokinetics, effect on coagulation parameters, and safety and tolerability of an intravenous infusion of ™REVASC before, during and after a DDAVP infusion were investigated.

Twelve healthy volunteers were given an intravenous bolus dose followed by a constant rate four-hour infusion of ™REVASC. Fifteen-minute infusions of 0.9% saline and DDAVP were started two and three hours respectively after the start of the ™REVASC infusion.

Plasma ™REVASC concentrations were not affected by either the saline or DDAVP infusion. ™REVASC infusion produced an increase in APTT which plateaued between 0.5 and 3 hours. After the DDAVP infusion there was a tendency towards a new lower plateau whilst the ™REVASC infusion continued. There were no serious adverse events or bleeding episodes throughout the study.

In conclusion, the co-administration of intravenous DDAVP has no effect on the plasma pharmacokinetics of ™REVASC and partially reverses the ™REVASC-induced increase in APTT. This may represent a role for DDAVP in the partial reversal of anticoagulation induced by ™REVASC.
Protein Tyrosine Phosphatase SHP-1 Fails to Associate with Cytoskeleton but is Normally Phosphorylated upon Thrombin Stimulation of Thrombasthenic Platelets
Authors: Li Ruo Ya , Gaits Frédérique , Ragab-Thomas Jeannie M F , Maclouf Jacques , Caen Jacques P , Lévy-Toledano Sylviane , Chap Hugues
Published: July 2018

SHP-1 is a cytoplasmic protein tyrosine phosphatase predominantly expressed in hematopoietic cells. Upon thrombin stimulation of human platelets, SHP-1 is rapidly phosphorylated on both serine and tyrosine residues, and becomes associated with the cytoskeleton, where it could participate in the formation of multiprotein signalling complexes. In order to discriminate between signalling events occurring downstream of G-protein-coupled thrombin receptor and those subsequent to integrin αIIbβ3engagement, SHP-1 behaviour was examined in platelets from two patients lacking integrin αIIbβ3
(Glanzmann’s thrombasthenia). Upon thrombin stimulation, phosphorylation of SHP-1 occurred normally in thrombasthenic platelets, whereas association with the cytoskeleton was abolished. Moreover, inhibition of normal platelet aggregation with the tetrapeptide arg-gly-asp-ser (RGDS), which impairs fibrinogen binding to integrin aIIb(33, did not alter significantly SHP-1 phosphorylation. It is concluded that SHP-1 phosphorylation is not a consequence of integrin signalling but might rather occur downstream of thrombin receptor and heterotrimeric G-proteins.
Abnormal Proteolytic Processing of von Willebrand Factor Arg611 Cys and Arg 611 His
Authors: Nishikubo Toshiya , Christophe Olivier , Lavergne Jean-Maurice , Obert Bernadette , Nonami Kyoko , Takahashi Yukihiro , Yoshioka Akira , Meyer Dominique , Girma Jean-Pierre
Published: July 2018

The structural and functional properties of plasma and platelet vWF were studied in 8 patients (5 unrelated families) with vWD demonstrating a mutation at position 611 (R611C or R611H). Following reduction, electrophoresis and immunoblotting with a polyclonal anti-reduced vWF antibody, abnormal proteolysis of vWF was demonstrated in plasma and to a lesser extent in platelets from all patients, leading to the formation of a unique 209 kDa fragment undetectable in control as well as in type 2A, 2B or 2N vWF. Immunoblotting with MoAbs to reduced vWF showed that the C-terminal end of the 209 kDa fragment was located beyond residue 1744 of the subunit and that its N-terminus was between residues 523 and 1114. Multimeric analysis of patients vWF showed an abnormal pattern in both plasma and platelets, with a moderate decrease of the HMW multimers together with a significant increase of the lowest MW forms. The specific sensitivity of vWF R611C and vWF R611H to proteolysis was further evidenced using V-8 protease. In all patient’s samples the enzyme produced a unique monomeric 80 kDa fragment, absent in V-8 digested normal vWF, which overlapped the N-terminal part of the subunit. The functional analysis of vWF showed a markedly decreased affinity of mutated plasma vWF for platelet GPIb in the presence of ristocetin. Infusion of DDAVP in two of these patients did not lead to significant platelet count change. It induced a limited increase of the HMW multimers in plasma together with a poor correction of the vWF binding to platelet GPIb. In conclusion, our data demonstrate that in addition to a normal proteolysis, vWF mutated at position 611 undergoes a specific cleavage in plasma and platelets. In contrast to the increased proteolysis observed in type 2A and 2B patients’ plasma, this additional cleavage produced a unique 209 kDa species but maintained a HMW multimer-like structure of vWF R611C and R611H.
Two New Closely Related Rat Models with Relevance to Arterial Thrombosis – Efficacies of Different Antithrombotic Drugs
Authors: Vogel Gerard M T , van Amsterdam Ronald G M , Zandberg Pieter , van Houwelingen Piet , Kop Wim J , van Mensvoort Frans W J , Meuleman Dirk G
Published: July 2018

Two thrombosis models in rats are described in which mixed type thrombi are formed at arterial and venous flow rates. The models, containing a silk thread in the aorta and vena cava, respectively, were characterised for the activity of three platelet inhibitors, three thrombin active site inhibitors and five glycosaminoglycans (GAGs).

In the two models a similar highly platelet-dependent thrombus developed both in size and composition during the first 10 min after insertion of the silk thread. The thrombotic processes were self-limiting, thus maintaining blood flow, but persisted twice as long in the vena cava model. In both models the thrombus consisted for more than 65% of platelets. Thrombus development under arterial as well as under venous flow conditions was inhibited dose dependently by all tested compounds including aspirin and the synthetic α-methyl glycoside copy of the ATIII binding pentasaccharide within heparin, Org31540/SR90107A. Simultaneous fibrin deposition and platelet activation, which represents an essential element of arterial thrombosis, initially dominated in both models. The gradual thrombus outgrowth, in the cava model, was more sensitive to factor Xa selective anticoagulants, as is venous thrombosis.
Haemostatic Properties of Human Pulmonary and Cerebral Microvascular Endothelial Cells
Authors: Grau Georges E , de Moerloose Philippe , Bulla Oana , Lou Jinning , Lei Zheng , Reber Guido , Mili Nabil , Ricou Bara , Morel Denis R , Suter Peter M
Published: July 2018

Little is known on the haemostatic profiles of human microvascular endothelial cells (MVEC) from different tissues. In addition it is not known whether MVEC from patients display the same haemostatic pattern as MVEC coming from healthy controls. To address these questions MVEC from human lung and brain were isolated and stimulated with tumour necrosis factor α (TNF) and E. coli lipopolysaccharide (LPS) for 24 h. The level and the kinetics of procoagulant activity (PCA) and thrombomodulin (TM) expression were found to be different depending on the tissue of origin and on the agonist used. In particular, the inducible PCA was higher in lung than in brain MVEC, an observation that may be related to the frequency of lung involvement in septic shock. Differences were also observed for tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) with MVEC supernatants or cell lysates. These variables were then measured in lung MVEC purified from patients with acute respiratory distress syndrome (ARDS) and compared to controls. Cells from ARDS patients constitutively expressed more PCA and PAI-1 than controls. The fibrinolytic potential, expressed as t-PA/PAI-1 ratio, was lower in ARDS than in lung MVEC. It is concluded that MVEC display different haemostatic features depending on the tissue they come from and that lung MVEC from ARDS patients present a procoagulant profile when compared with those from controls.
A Multicenter Study of Recombinant Factor VIII (RecombinateTM) in Previously Treated Patients with Hemophilia A
Authors: White G C , Courter S , Bray G L , Lee M , Gomperts E D ,   The Recombinate Previously Treated Patient Study Group
Published: July 2018

A prospective, open-label multicenter investigation has been conducted to compare pharmacokinetic parameters of recombinant DNA-derived FVIII (rFVIII) and plasma-derived FVIII concentrate (pdFVIII) and to assess safety and efficacy of long-term home-treat- ment with rFVIII for subjects with hemophilia A. Following comparative in vivo pharmacokinetic studies, 69 patients with severe (n = 67) or moderate (n = 2) hemophilia A commenced a program of home treatment using rFVIII exclusively for prophylaxis and treatment of all bleeding episodes for a period of 1.0 to 5.7 years (median 3.7 years). The mean in vivo half-lives of rFVIII and pdFVIII were both 14.7 h. In vivo incremental recoveries at baseline were 2.40%/IU/kg and 2.47%/IU/kg, respectively (p = 0.59). The response to home treatment with rFVIII was categorized as good or excellent in 3,195 (91.2%) of 3,481 evaluated bleeding episodes. Thirteen patients received rFVIII for prophylaxis for twenty-four surgical procedures. In all cases, hemostasis was excellent. Adverse reactions were observed in only 13 of 13,591 (0.096%) infusions of rFVIII; none was serious. No patient developed an inhibitor to r FVIII.