bluuebunny/crossref_metadata_2025 huggingface dataset:
Insights in the molecular mechanisms of the anti-angiogenic effect of an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase
Authors: Vincent Loïc , Albanese Patricia , Bompais Heidi , Uzan Georges , Pierre Vannier Jean , Gabriel Steg Philippe , Soria Jeannette , Soria Claudine
Published: March 2018

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) reduce the risk of coronary event by cholesterol-lowering dependent and independent mechanisms. We have already described that the inhibitory effect of cerivastatin on angiogenesis contribute to the cholesterol-independent beneficial effect and was due to the inhibition of the cell signaling cascade RhoA/FAK/Akt. In this study, new insights in the molecular mechanism of action were provided. It indicates an inhibition of exposure of alpha V beta 3 integrin on cell membrane and a modification of gene expression. The inhibition of angiogenesis could be related to 1) an increase in genes involved in the inhibition of cell proliferation (p19INK4, p21Waf/Cip1, Wnt-5a), the inhi bition of cell migration (Rho-GDI 1, α E-catenin) and 2) a downregulation of genes involved in angiogenesis (PAI-1, Vitronectin, HoxD3, Notch4) or in cell invasion (Semaphorin E). In addition, DNA repair protein genes (MLH1, XRCC1) were increased.

This study may indicate new biological interest of genes involved in angiogenesis control.
A FV multiallelic marker detects genetic components of APC resistance contributing to venous thromboembolism in FV Leiden carriers
Authors: Legnani Cristina , Lunghi Barbara , Scanavini Daniela , Palareti Gualtiero , Marchetti Giovanna , Mingozzi Federico , Castoldi Elisabetta , Bernardi Francesco
Published: March 2018

Activated protein C resistance (APCR) is a major risk factor for venous thromboembolism (VTE). Although the factor V (FV) Leiden mutation accounts for the vast majority of APCR cases, other polymorphisms may contribute to the APCR phenotype. Genetic components of APCR and thrombophilia were investigated by two dinucleotide repeats, characterized in introns 2 and 11 of the FV gene. Only the intron 11 marker was genetically stable and thus suitable for further analysis. Its allelic frequencies were found to differ significantly (P=0.003) between subjects selected for increased APCR in the absence of the FV R506Q mutation (n=70, normalized ratios ≤0.80), and for increased APC sensitivity (n=98, normalized ratios ≥1.31). Genotype differences were also found (P=0.017) between FV R506Q heterozygotes (n=100) who had experienced previous VTE and those (n=100), who were still asymptomatic for VTE. Significance was mostly driven by the relative over-representation of the 12R allele and to a minor extent by the under-representation of the 15R allele among the symptomatic versus the asymptomatic FV Leiden carriers.

Two SNPs (4070A/G and 2391A/G) were found to underlie the 12R and 15R alleles respectively, and marked extended haplo-types, previously (HR2) or newly (HT2) identified. Only the FV HR2 differed (P=0.002) in frequency between the two groups of FV R506Q heterozygotes, suggesting that it represents the most relevant FV genetic component of APCR or VTE detectable by this experimental and clinical approach. Our analysis indicates that frequent FV genetic components might contribute to shape the risk for VTE in FV Leiden carriers.
Expression of BCL2L12, a new member of apoptosis-related genes, in breast tumors
Authors: Talieri Maroulio , Diamandis Eleftherios , Katsaros Nikos , Gourgiotis Dimitrios , Scorilas Andreas
Published: March 2018

Apoptosis, a normal physiological form of cell death, is critically involved in the regulation of cellular homeostasis. If the delicate balance between cell death and cell proliferation is altered by a defect in the normal regulation of apoptosis signaling, a cell population is able to survive and accumulate, thereby favoring the acquisition of further genetic alterations and promoting tumorigenesis. Dysregulation of programmed cell death mechanisms plays an important role in the pathogenesis and progression of breast cancer, as well as in the responses of tumors to therapeutic intervention. Overexpression of anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-XL has been implicated in cancer chemoresistance, whereas high levels of pro-apoptotic proteins such as Bax promote apoptosis and sensitize tumor cells to various anticancer therapies. Recently, a new member of the Bcl-2 family, BCL2L12, was cloned. The BCL2L12 gene is constitutively expressed in many tissues, suggesting that the encoded protein serves an important function in different cell types. In the present study, the expression of BCL2L12 gene was analyzed by reverse transcription-PCR (PT-PCR) in 70 breast cancer tissues. Our results indicate that BCL2L12 positive breast tumors are mainly of lower stage (I/II) or grade (I/II) (p=0.02 or p=0.04 respectively). Cox regression analysis revealed that BCL2L12 expression is positively related to disease-free (DFS) and overall survival (OS) at both univariate and multivariate analysis (p=0.021, p=0.029, p=0.032, p=0.044 respectively). Kaplan-Meier survival curves also demonstrated that patients with BCL2L12-positive tumors have significantly longer DFS and OS (p=0.002 and p<0.001 respectively). BCL2L12 expression may be regarded as a new independent favorable prognostic marker for breast cancer.

Part of this paper was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.
Fibrinogen mediates bladder cancer cell migration in an ICAM-1-dependent pathway
Authors: Roche Yann , Pasquier Dominique , Rambeaud Jean-Jacques , Seigneurin Daniel , Duperray Alain
Published: March 2018

Fibrinogen (fg), present in tumor matrices, has been demonstrated to be determinant in metastatic potential. We have recently shown that fg/ICAM-1 interactions are involved in leukocyte migration across endothelial cell monolayers. Using bladder transitional cell carcinoma as a model, we will show in this study that bladder high grade tumor cell lines express ICAM-1, and that this expression induces an fg-mediated migration. This phenomenon was dependent on ICAM-1/fg interaction as well as RhoA activity. ICAM-1 was concentrated in focal adhesion plaques when tumor cells were allowed to adhere on immobilized fg, suggesting a role in cell migration. The addition of fg induced a 3- to 6-fold enhancement of bladder tumor cell migration through HUVEC monolayers. This process was inhibited by an anti-ICAM-1 antibody blocking fg binding, demonstrating that ICAM-1/fg interaction was involved in the extravasation process. Finally, immunohistological studies revealed that the expression of ICAM-1 was closely associated with an infiltrative histological phenotype.

Part of this paper was originally presented as part of the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.
Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer
Authors: Kotzsch Matthias , Meye Axel , Langerholc Thomaz , Füssel Susanne , Olbricht Natalie , Albrecht Sybille , Ockert Detlev , Muehlenweg Bernd , Friedrich Katrin , Grosser Marianne , Schmitt Manfred , Baretton Gustavo , Magdolen Viktor , Luther Thomas
Published: March 2018

The cellular receptor for urokinase-type plasminogen activator, uPAR, plays a central role in both cell surface-associated proteolysis and cellular adhesion. In the present study, we systematically searched for splice variants of uPAR mRNA in human cells and tumor tissues by qualitative RT-PCR using specific primers for uPAR exons 1 and 6. Beside the wild-type (wt) uPAR mRNA and the previously described splice variant lacking exon 5 (uPAR-del5), a novel splice variant lacking both exons 4 and 5 (uPAR-del4/5) was found predominantly in various cancer cell lines. To elucidate whether alternatively spliced uPAR mRNA may be translated and post-translationally processed, we generated stably transfected Chinese hamster ovary cells, which harbor expression plasmids of wt uPAR and various uPAR variants including uPAR-del5 and uPAR-del4/5. By ELISA, flow cytofluorometry, and Western blot analysis, we confirmed synthesis and secretion of wt uPAR and the uPAR variants by the use of domain-specific monoclonal antibodies against uPAR. For quantification of uPAR mRNA variants, we established two highly sensitive real-time RT-PCR assays based on LightCycler technology. Study of their expression in a representative set of breast cancer tissues indicated that the novel mRNA variant uPAR-del4/5 was expressed very frequently and independently of uPAR mRNA variants covering exon 4 (uPAR-wt and uPAR-del5). Higher uPAR-del4/5 expression was significantly associated with shorter disease-free survival (p = 0.0004) of breast cancer patients. These results suggest that uPAR-del4/5 mRNA may serve as a novel prognostic marker in breast cancer.

Theme paper: Part of this paper was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.
The protective effect of angiotensin converting enzyme inhibition in experimental renal fibrosis in mice is not mediated by bradykinin B2 receptor activation
Authors: Schanstra Joost , Duchene Johan , Desmond Laurence , Neau Eric , Calise Denis , Estaque Serge , Girolami Jean-Pierre , Bascands Jean-Loup
Published: March 2018

Unilateral ureteral obstruction (UUO) is an animal model of accelerated renal tubulointerstitial fibrosis. We have recently shown, using this model, that mice lacking the bradykinin B2-receptor (B2-/-
) were more susceptible than control animals to the development of tubulointerstitial fibrosis. Angiotensin converting enzyme (ACE) inhibition slows down UUO-induced renal fibrosis. Since ACE-inhibition increases bradykinin and decreases angiotensin II concentrations we have verified if bradykinin is involved in the antifibrotic effects of ACE-inhibition using the UUO-model and B2-/-
mice. Surprisingly, although ACE-inhibition significantly reduced renal fibrosis, no difference was observed between the degree of tubulointerstitial fibrosis, macrophage infiltration and cell proliferation between ACE-inhibitor treated B2+/+
and B2-/-
mice suggesting the absence of a role of the B2-receptor in the antifibrotic effects of ACE-inhibition. This was confirmed at the level of bradykinin-induced activity of enzymes involved in the degradation of the extracellular matrix. However in both mouse strains, ACE-inhibitors were more efficient than AT1 receptor antagonists.

Theme paper: Part of this paper was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.
Changes in tissue factor and activated factor XII following an acute myocardial infarction were uninfluenced by high doses of n-3 polyunsaturated fatty acids
Authors: Hetland Øyvind , Nilsen Dennis , Grundt Heidi
Published: March 2018

Few data exist on the effects of n-3 polyunsaturated fatty acids (PUFAs) on the initiators and endstage products of coagulation following an acute myocardial infarction (MI).

We assessed the long-term effects of n-3 PUFAs on postinfarct variations of tissue factor (TF), activated factor XII (FXIIa) and fibrin monomer (FM), and expected additional statin treatment to modify thrombogenicity.

Acute MI patients (n = 300) were randomly allocated to a high dose of n-3 PUFAs or corn oil for at least one year.

Plasma concentrations of TF, FXIIa and FM were unaffected by n-3 PUFAs as compared to corn oil, and were uninfluenced by additional statin treatment in subgroup analyses. TF decreased (p = 0.0001), while FXIIa increased during the first 6 weeks (p = 0.001). FM remained essentially unchanged during the entire observation period.

In conclusion, TF, FXIIa and FM were unaffected by long-term treatment with high- dosed n-3 PUFAs and by additional statin treatment.
c7E3 Fab Inhibits Low Shear Flow Modulated Platelet Adhesion to Endothelium and Surface-adsorbed Fibrinogen by Blocking Platelet GP IIb/IIIa as well as Endothelial Vitronectin Receptor
Authors: Agneskirchner Jens , Bode Patrick , Spannagl Michael , Wurzinger Laurenz , Reininger Armin
Published: March 2018

The c7E3 Fab reduces ischemic complications in patients undergoing high-risk coronary angioplasty or atherectomy. The present study investigated how c7E3 Fab inhibition of the platelet receptor glycoprotein IIb/IIIa and the endothelial vitronectin receptor affected platelet adhesion to endothelium and surface adsorbed fibrinogen under flow conditions.

Platelet adhesion was examined using a stagnation point flow device with shear stress and shear rates up to 2.2 dynes/cm2 and 170 s−1, respectively. Ex vivo adhesion was compared between two groups of patients with acute myocardial infarction (AMI) treated with angioplasty and stent implantation and a group of healthy controls. Only one AMI group received c7E3 Fab therapy. Patients in both groups were administered acetyl salicylic acid (ASA) and heparin. In AMI patients c7E3 Fab reduced platelet adhesion to adsorbed fibrinogen by 79% compared to AMI patients without c7E3 Fab treatment and by 74% compared to healthy controls. Thirty hours after termination of c7E3 Fab infusion adhesion had slightly recovered with an inhibition of 61% and 52% still present, respectively. Additionally, in vitro platelet adhesion to intact endothelium and to adsorbed fibrinogen was measured during superfusion with ADP stimulated platelet rich plasma of healthy controls to which c7E3 Fab was added at a final concentration fc of 20 µg/ml. In spite of ADP stimulation c7E3 Fab completely blocked platelet adhesion to adsorbed fibrinogen and, moreover, to intact endothelium. Preincubation of endothelial cells with c7E3 (fc = 20 µg/ml) blocked adhesion of ADP-stimulated platelets by approximately 50%.

Apart from the inhibition of platelet aggregation, c7E3 Fab added in vitro and given therapeutically in patients effectively blocks platelet adhesion to components of the injured as well as intact vessel wall under stagnation point flow conditions.
Oxidation of Human α-Thrombin by the Myeloperoxidase-H2O2-chloride System: Structural and Functional Effects
Authors: Landolfi Raffaele , De Cristofaro Raimondo
Published: March 2018

The myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This study investigated the biochemical and functional effects of α-thrombin oxidation by MPOS. This system, in the presence of 100 µM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCl, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human α-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed several changes, arising from a perturbation of the S2-S3 specificity of the enzyme. Ox-thrombin was also characterized by a 5-fold decrease of the kcat/Km value for both fibrinopeptide A and B release from fibrinogen, a 5.8-fold increase of the EC50 value for platelet activation and a 2-fold decrease of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Perturbed interactions with Protein C and the heparin-ATIII complex were the most relevant functional abnormalities of ox-thrombin.

Abbreviations: DNPH: 2,4-dinitrophenylhydrazine; FpA: fibrinopeptide A; FpB: fibrinopeptide B; FRS: Fibrinogen recognition Site; GpIb: Glycoprotein Ib; HBS: Heparin Binding Site; MPO: myeloperoxidase; MPOS: Myeloperoxidase System; Pip: Pipecolynic acid; PMN: polymorphonuclear cells; pNA: para-nitroaniline; PPACK: Phenylalanine-Proline-Arginine-Chloromethyl-Ketone; TM: Thrombomodulin; TNB: 5-thio-2-nitrobenzoic acid; TPCK: N-tosyl-L-Phenylalanine-Chloromethyl-Ketone. Thrombin residues are numbered according to the chymotrypsin numbering system (31).
Venous Thrombotic Risk in Family Members of Unselected Individuals with Factor V Leiden
Authors: Lensen R. P. M. , Bertina R. M. , de Ronde H. , Vandenbroucke J. P. , Rosendaal F. R.
Published: March 2018

The factor V Leiden mutation (FVL) leads to a seven-fold increased risk of venous thromboembolism (VTE). In thrombophilic families, 25% of carriers have experienced thrombosis before the age of 40 years. Aim of our study was to assess the association of FVL with VTE in first-degree family members of unselected symptomatic and asymptomatic carriers of FVL.

We tested 197 relatives of consecutive thrombosis patients with FVL and 36 relatives of asymptomatic carriers on the presence of FVL and the occurrence of VTE.

The incidence of VTE in relatives with FVL of symptomatic carriers was 0.34%/year. This was similar to the incidence in relatives with FVL of asymptomatic carriers. Kaplan Meier analysis in relatives of symptomatic propositi showed that at the age of 58 years, thrombosisfree survival was reduced to 75% in carriers and 93% in non-carriers (P < 0.05). Carriers of FVL had a three times higher thrombotic risk than non-carriers. In combination with environmental risk factors, FVL clearly adds to the risk of VTE. The thrombotic incidence rate in these unselected relatives with FVL, however, is considerably lower than was seen in carriers of thrombophilic families (1.7%/year). Therefore, special care should be paid to individuals with a positive family history of venous thrombosis while exposed to these risk factors.
Tissue Factor and Homocysteine Levels in Ischemic Heart Disease Are Associated with Angiographically Documented Clinical Recurrences after Coronary Angioplasty
Authors: Prisco Domenico , Brunelli Tamara , Pepe Guglielmina , Gori Anna , Fedi Sandra , Capanni Monia , Simonetti Ignazio , Federici Giorgio , Pastore Anna , Abbate Rosanna , Gensini Gian , Marcucci Rossella
Published: March 2018

Background In ischemic heart disease (IHD) patients high plasma levels of Tissue Factor (TF), the trigger of coagulation cascade, are present. Homocysteine (Hcy) is a risk factor for coronary artery disease, and several different pathophysiological mechanisms by which Hcy may play a role in thrombus formation have been postulated in “in vitro” studies. We investigated the “in vivo” role of Hcy in affecting plasma levels of TF, its inhibitor Tissue Factor Pathway Inhibitor (TFPI) and hypercoagulability. Methods and Results We investigated 119 IHD patients who underwent PTCA and compared them with 103 healthy subjects. TF, TFPI, Thrombin-Antithrombin complexes (TAT) and Hcy levels were significantly higher in the patients than in the controls. A positive correlation was found between Hcy and TF (r = 0.54; p < 0.0001), Hcy and TFPI (r = 0.26; p < 0.05) as well as Hcy and TAT (r = 0.33; p < 0.0001) levels. An inverse correlation existed between folate intake and Hcy levels (r = −0.28; p = 0.001). Hcy levels within the first quartile and in the highest quartile were associated with a lower (p < 0.001) and higher (p <0.0001) rate of clinical recurrences, respectively. Patients with TF values in the first quartile had a lower rate of angiographically documented clinical recurrences as compared to those in the fourth quartile (p < 0.01); those in the highest quartile of TF showed a higher rate of recurrences (p = 0.001). Multivariate analysis confirmed these results (first quartile of Hcy: OR 0.02, Cl 0.002–0.27; fourth quartile of Hcy: OR 36.5, Cl 3.6-365/first quartile of TF: OR 0.006, Cl 0.001–0.44; fourth quartile of TF: OR 16.4, Cl 3.0 – 90.0), also after adjustment for risk factors and Hcy and TF respectively. Conclusions In this study we show that TF, TFPI and TAT levels are correlated with Hcy plasma levels in IHD patients, providing evidence of an “in vivo” pathophysiological mechanism of hyperhomocysteinemia. The observed association between angiographically documented clinical recurrences and TF and Hcy values awaits confirmation in studies designated to evaluate this issue on a larger number of patients.
Clinical Evaluation of a Monoclonal Antibody-based Enzyme Immunoassay for Fibrin Degradation Products in Patients with Clinically Suspected Pulmonary Embolism
Authors: de Monyé Wouter , Lijmer Jeroen , Nieuwenhuizen Willem , Büller Harry , Huisman Menno , Brandjes Dees , Gillavry Melvin R. ,   the ANTELOPE-Study Group
Published: March 2018

We prospectively evaluated the diagnostic accuracy of the Fibrinostika® FbDP assay in 304 consecutive patients with suspected pulmonary embolism and examined potentially useful cut-off points at which the disease can be excluded. The prevalence of pulmonary embolism was 31%. The assay generated an area under the Receiver Operating Characteristic curve of 0.79 (95% CI 0.73–0.84). A cut-off point of 0.05 µ,g/ml yielded a sensitivity, specificity, negative predictive value and an exclusion efficiency of 100% (95% CI 96–100), 5% (95% CI 2–9), 100% (95% CI 69–100) and 3% (95% CI 2–6), respectively. A clinically useful cut-off point seems to be 0.11 µ,g/ml which corresponded with a sensitivity, specificity, negative predictive value and an exclusion efficiency of 96% (95% CI 90–99), 27% (95% CI 24–28), 93% (95% CI 84–98) and 20% (95% CI 16–25), respectively. We conclude that the assay has potential clinical utility for the exclusion of pulmonary embolism, but it cannot be used as a sole test.
Elastase Mediated Fibrinolysis in Acute Promyelocytic Leukemia
Authors: Oudijk Erik-Jan , Nieuwenhuis H. , Bos Rogier , Fijnheer Rob
Published: March 2018

The bleeding syndrome of acute promyelocytic leukemia (APL) is complex and consists of disseminated intravascular coagulation (DIC) and hyperfibrinolysis. Elastase, derived from malignant promyelocytes, is believed to mediate the fibrinogeno- and fibrinolysis by aspecific proteolysis. In this study we measured the role of elastase in fifteen patients with APL by using an assay for elastase degraded fibrin(ogen) and the results were compared with those obtained in patients with sepsis induced DIC.

High levels of elastase were observed in sepsis and APL. The levels of fibrinogen and fibrin degradation products were significantly higher in APL patients compared to patients with sepsis induced DIC. Nevertheless, the level of elastase degraded fibrin(ogen) was higher in the sepsis group (635.3 ng/ml, compared to 144.3 ng/ml in APL; p <0.0001). So, the enormous increase in fibrin and fibrinogen degradation products in APL cannot be explained by elastase activity. This study suggests a minor role for elastase mediated proteolysis in the hemorrhagic diathesis in APL patients.
Fibrinogen, Fibrin and Crosslinking in Aging Arterial Thrombi
Authors: McBane Robert , Ford Monique , Karnicki Krzysztof , Stewart MaryLou , Owen Whyte
Published: March 2018

The assumption that fibrin and crosslinked fibrin impart irreversibility to arterial thrombi is explored with procedure developed for measuring changes in platelet function, morphology and fibrinogen metabolism in aging occlusive thrombi, in which the condition of stasis is imposed uniformly. Arterial thrombi containing autologous 111In labeled platelets were generated in vivo by bilateral mechanical injury of porcine carotid arteries. Vessels containing the platelet-rich thrombi were harvested and incubated intact (37° C) for intervals ranging from 30 min to 12 h. The isolated vessels were then bisected and agitated in culture medium containing tick anticoagulant and hirudin for 60 min. Disaggregated platelets were evaluated for yield (from 111In radioactivity) viability (dense body ATP secretion) and morphology (electron microscopy). Western analysis of fibrin(ogen) in thrombus extracts was performed using anti-fibrinogen Bβ- and γ-chain monoclonal antibodies for thrombi at each time point. A stable recovery of nearly 50% of platelets was observed during 12 h of thrombus aging. As thrombi aged, viability of disaggregated platelets gradually decreased with platelet necrosis the predominant feature beyond 6 h. By western analysis of thrombus extracts, nearly 50% of fibrinogen was cleaved to fibrin and extensively crosslinked within 30 min of injury with no evidence of fibrinolysis. With the exception of a declining proportion of γ-monomer, these features remain relatively constant during 12 h of thrombus maturation. It is concluded that neither fibrin nor crosslinked fibrin are dominant factors imparting cohesion within platelet thrombi. Furthermore, under conditions of complete arterial occlusion imposed by this experimental design, there is no evidence of endogenous fibrinolysis.
Comparison of the O’Brien Filter Test and the PFA-100 Platelet Analyzer in the Laboratory Diagnosis of von Willebrand’s Disease
Authors: Schlammadinger Agota , Kerenyi Adrienne , Muszbek Laszlo , Boda Zoltan
Published: March 2018

Von Willebrand’s disease (vWD) is the most common congenital haemorrhagic diathesis, characterized by the quantitative or qualitative disorder of von Willebrand factor (vWF). A number of methods have been used for the diagnosis of the disease, and the bleeding time determination is widely accepted as a screening test in spite of its low sensitivity. Our aim was to evaluate and compare the performance of two high shear systems (the O’Brien filter test and the PFA-100 device) in the screening and diagnosis of vWD. Thirty patients (n=13 type 1 with mild symptoms, n = 9 type 1 with severe symptoms, n = 2 type 2A, n = 3 type 2B and n = 3 type 3 vWD) and twenty controls were investigated. In mild vWD the platelet retention in the second phase of the filter test with citrated blood showed the highest sensitivity (91.6%). The sensitivity of the PFA-100 method with collagen-epinephrine cartridges in this group was 76.9%, while the bleeding time was prolonged only in 15.4% of the cases. In severe type 1, in type 2A and type 3 all functional tests reflected the bleeding tendency of the patients. In type 2B disease the bleeding time was prolonged only when the patient was thrombocytopenic, but both high shear systems revealed the disease independently of the presence of thrombocytopenia. The overall sensitivity of the bleeding time determination was 50% compared to the 80-90% sensitivity of the O’Brien filter test and the PFA-100 system. The sensitivity values of the filter test and the PFA-100 device with collagen-epinephrine cartridges were in the same range, but the collagen-ADP cartridges showed a lower (65.5%) sensitivity, though the results were specific and had high positive predictive value. We conclude that both high shear systems are suitable for the screening of vWD, and that they are superior to the traditional bleeding time determination in case of mild disease or type 2B vWD.
Role of the Leucine-Rich Domain of Platelet GPIbα in Correct Post-translational Processing – The Nancy I Bernard-Soulier Mutation Expressed on CHO Cells
Authors: Ulsemer Philippe , Baas Marie-Jeanne , Schwartz Agnès , Ravanat Catherine , Briquel Marie-Elisabeth , Cranmer Sue , Jackson Shaun , Cazenave Jean-Pierre , de la Salle Corinne , Lanza François
Published: March 2018

The mechanisms governing the biosynthesis and surface expression of platelet adhesive receptors on parent megakaryocytes are as yet poorly understood. In particular, the assembly and processing of the multisubunit glycoprotein (GP) Ib-IX-V complex, a receptor for von Willebrand factor (vWf) is not fully understood. In the present work, these questions were addressed by reproducing a natural mutation of GPIbα found in a variant case of Bernard-Soulier syndrome (Nancy I), due to the deletion of leucine 179 in the seventh leucine-rich repeat of the polypeptide. Wild type and mutated GPIbα were transfected into CHO cells expressing GPIbβ and GPIX. Flow cytometry showed surface expression of the three subunits of both GPIb-IX complexes, but GPIbαΔLeu was present at lower levels (20-40%) and was recognized only by a sub class of monoclonal antibodies which epitopes were not modified by the mutation. These properties reproduce the defect found in the patient’s platelets, demonstrating the causative nature of the mutation and validate the use of the CHO cells model. Biochemical studies were performed in an attempt to elucidate the mechanism of the conformational change of GPIbαΔLeu. They unexpectedly revealed a major glycosylation deficiency of the mutated GPIbα leading to a 40% decrease in molecular weight. The other two subunits of the complex were however normal and present at the plasma membrane. The deletion led to complete functional deficiency with lack of vWf binding of CHOαΔLeu transfected cells in the presence of botrocetin and defective adhesion to a vWf coated surface under static conditions. Finally, in contrast to normal CHOαβIX cells, which displayed rolling and deceleration when perfused over a vWf surface, CHOαΔLeuβIX cells were unable to roll over or attach to a vWf substratum. These results show that the integrity of the leucine-rich region of GPIbα is essential for normal processing and function of the GPIb-IX complex. In addition, these results obtained in a cellular system supported the suspected role of the macroglycopeptide region of GPIbα in maintaining a suitable conformation of this multisubunit receptor to perform its adhesive function.
Homozygous Pro74 → Arg Mutation in the Platelet Glycoprotein Ibβ Gene Associated with Bernard-Soulier Syndrome
Authors: Tomiyama Yoshiaki , Honda Shigenori , Fukunishi Mariko , Hara Junichi , Inoue Chikako , Kamiya Tadashi , Saito Hidehiko , Kunishima Shinji
Published: March 2018

Bernard-Soulier syndrome (BSS) is an autosomal recessive bleeding disorder due to quantitative or qualitative abnormalities in the glycoprotein (GP) Ib/IX/V complex, the platelet receptor for von Willebrand factor. This complex is composed of four subunits, GPIbα, GPIbβ, GPIX and GPV. We describe here the genetic basis of the disorder in a patient with BSS. Flow cytometric analysis of the patient’s platelets showed greatly reduced GPIbα and GPIX surface expression. Immunoblot analysis disclosed absence of GPIbα, GPIbβ and GPIX in the platelets. DNA sequencing analysis revealed a novel missense mutation in the GPIbβ gene that converts Pro (CCG) to Arg (CGG) at residue 74. Homozygosity of the mutation was confirmed by allele-specific restriction analysis, chromosome 22 microsatellite analysis and quantitative Southern blotting. The mutant GPIbβ was normally transcribed. Transient transfection studies confirmed that mutant GPIbβ impairs surface expression of GPIb/IX, showing that the mutation is responsible for a BSS phenotype observed in the patient.
Persistently Elevated Levels of von Willebrand Factor Antigen in HIV Infection
Authors: Bjørnsen S. , Lunden B. , Otterdal K. , Ng E. C. , Ameln W. , Ueland T. , Müller F. , Solum N. O. , Brosstad F. , Frøland S. S. , Aukrust P.
Published: March 2018

Levels of circulating von Willebrand factor (vWf) antigen are thought to reflect endothelial involvement in various disorders. In the present study we found markedly elevated plasma levels of vWf in HIV-infected patients demonstrated on both cross-sectional and longitudinal testing. Notably, we found that a persistent rise in vWf antigen was associated with progression of HIV-related disease. This elevation of vWf antigen represented functionally normal vWf as evaluated by plasma FVIII, ristocetin cofactor assay and vWf multimer analyses. While HIV-infected patients showed enhanced platelet activation, platelets did not contribute substantially to the increased vWf levels. The high vWf levels were significantly correlated with high viral load, and during HAART, the pronounced decline in HIV RNA levels was accompanied by a corresponding decrease in vWf. The persistent elevation of functionally normal vWf during HIV infection, most probably reflecting a persistent endothelial cell activation, may have an important role in the pathogenesis of HIV infection.
Normalisation of Tissue Factor Pathway Inhibitor Activity after Glycaemic Control Optimisation in Type 1 Diabetic Patients
Authors: Rigla Mercedes , Mateo Jose , Fontcuberta Jordi , Souto Juan , de Leiva Alberto , Pérez Antonio
Published: March 2018

Increased plasma concentrations of various markers of endothelial damage have been observed in type 1 diabetic patients, particularly in those with microangiopathy. Objective. To evaluate the effect of nearnormalisation of glycaemic control on different markers of endothelial injury involved in haemostasis in poorly-controlled type 1 diabetic patients. Material and Methods. TFPI, thrombomodulin (TM), plasminogen activator inhibitor, tissue-type plasminogen activator and von Willebrand factor were measured in 14 poorly-controlled type 1 diabetic patients free of diabetes-related complications (8 men, 6 women; mean age 29.8 ± 9.9 years) before (baseline) and after 3 months of intensive therapy and in 14 sex-, age-and BMI-matched control subjects. Results. After a mean follow-up of 107 ± 49 days (56-210), Hb A1c decreased from 11.2 ± 2.3 to 6.7 ± 0.7% (p <0.0001). TFPI activity at baseline was higher than in the control group (126.9 ± 34 vs 92.0 ± 13%, p <0.005) and decreased after good glycaemic control was achieved (p <0.005), becoming similar to that in the control group (91.0 ± 16.5%). The TFPI descent correlated with the variations observed in HbA1c (p <0.05; r = 0.54). TM levels at baseline were significantly higher than in the control group (42.3 ± 9.1 vs 29.00 ± 10.9; p <0.005) and did not change. The remaining parameters studied were similar between patients and controls and did not change after glycaemic optimisation. Conclusions. Optimisation of glycaemic control normalises the increased activity of TFPI but not the higher TM levels observed in poorly-controlled type 1 diabetic patients without chronic complications.
Activity of a Sub-cutaneously Administered Novel Mixed Micellar Formulation of Argatroban in Rat and Rabbit Models of Venous Thrombosis
Authors: Visconte Catherine , Lecoffre Catherine , Lochot Sylvette , Girard Denise , Berry Christopher
Published: March 2018

We studied the antithrombotic activity of a mixed micellar formulation containing 14 mg/ml argatroban administered by the subcutaneous (s. c.) route in rat and rabbit models of venous thrombosis. The effects on bleeding time in the rat tail transection bleeding time test were also studied. In a tissue factor-dependent arterio-venous shunt model, argatroban treatment led to dose-dependent reduction in thrombus weight with an estimated ID50 of 1.8 mg/kg s. c. In the same model, heparin had an estimated ID50 of 179 IU/kg. The antithrombotic activity of argatroban was accompanied by increases in the thrombin and ecarin clotting times but not the aPTT, whereas heparin increased the thrombin time and aPTT but not the ecarin clotting times. Argatroban also inhibited thrombus formation in a rabbit model of thromboplastin + stasis induced thrombosis in the rabbit jugular vein with an estimated ID50 of 1 mg/kg s. c. When tested in the rat tail transection bleeding time test, the mixed micellar formulation of argatroban caused significant increases in the bleeding time as from 8 mg/kg s. c., while heparin significantly increased the bleeding time at 800 U/kg. Mixed micellar argatroban appears to have a superior safety margin to heparin in terms of antithrombotic efficacy and bleeding risk.

Thus, a mixed micellar formulation of argatroban, which markedly enhances its solubility, could be useful as a potential antithrombotic agent for subcutaneous administration.
Homozygous 2bp Deletion in the Human Factor VII Gene: A Non-Lethal Mutation that Is Associated with a Complete Absence of Circulating Factor VII
Authors: Mannucci Pier , Jenkins P. , Lee Anselm , Coppola Raffaella , Perry David , Peyvandi Flora
Published: March 2018

We report the case of a 5-year-old boy with severe factor VII deficiency. The affected child presented at the age of 8 months and again at 18 months with bleeding from the gastrointestinal tract but the diagnosis of factor VII deficiency was not made until the age of 3 years. He was treated with fresh frozen plasma and subsequently factor VII concentrates and to date remains well. To identify the causative mutation, the factor VII gene was screened by SSCP and direct sequence analysis. A single homozygous 2bp deletion (-CT) mutation was identified in exon 1a removing nucleotides 27/28 (codons 52/53). Both parents, who were first cousins, were heterozygous for the mutation. The mutation located in the prepropeptide of factor VII, results in a complete absence of factor VII in plasma. This case indicates that a complete absence of plasma factor VII is not necessarily a lethal condition.
Sensitization of CD4+ T Cells to Coagulation Factor VIII: Response in Congenital and Acquired Hemophilia Patients and in Healthy Subjects
Authors: Reding Mark , Wu Huiyun , Krampf Mark , Okita David , Diethelm-Okita Brenda , Christie Beverly , Key Nigel , Conti-Fine Bianca
Published: March 2018

Antibodies (Ab) that inhibit factor VIII (fVIII) may develop in patients with hemophilia A and rarely in individuals without congenital fVIII deficiency (acquired hemophilia). Synthesis of fVIII inhibitors requires CD4+ T cells. We investigated the proliferative response of blood CD4+ cells from 11 patients with congenital or acquired hemophilia and 12 healthy subjects, to recombinant human fVIII, and to pools of overlapping synthetic peptides spanning the sequences of individual fVIII domains. All patients had CD4+ cells that responded to fVIII. The intensity of the responses fluctuated over time: several patients had brief periods when they did not respond to fVIII. All healthy subjects had transient CD4+ responses to fVIII, that were significantly lower than those of hemophilia patients. Also, healthy subjects responded to fVIII less frequently and for shorter periods than hemophilia patients. All patients and healthy subjects recognized several fVIII domains: the A3 domain was recognized most strongly and frequently. The transient sensitization of CD4+ cells to fVIII in healthy subjects suggests that inadequate tolerization of CD4+ cells to fVIII, due to lack of endogenous fVIII, is an important factor in the development of clinically significant anti-fVIII antibodies in hemophilia A.
The Occurrence of Subsequent Malignancy in Patients Presenting with Deep Vein Thrombosis: Results from a Historical Cohort Study
Authors: Rajan Raghu , Gent Michael , Hirsh Jack , Geerts William , Skingley Peter , Julian Jim , Levine Mark
Published: March 2018

Background: Several studies have reported that patients who present with idiopathic deep vein thrombosis (DVT) have an increased risk of subsequently developing cancer. A clinical trial had previously been conducted examining the optimal duration of oral anticoagulant therapy following initial heparin treatment in patients with proximal DVT.

Methods: A historical cohort study was performed on patients enrolled in the duration of anticoagulant trial. Patients known to have cancer at the time of entry into the trial were excluded. The qualifying DVTs were classified as idiopathic (no known associated risk factors) or secondary without knowledge of subsequent recurrent venous thrombosis or cancer. The patients were then followed for the development of cancer.

Results: Thirteen (8.6%) of the 152 patients in the idiopathic cohort subsequently developed cancer compared to eight (7.1%) of 112 patients in the secondary cohort, P = 0.86. Two (5.4%) of 37 patients with recurrent venous thromboembolism and 19 (8.4%) of 227 patients without recurrent thromboembolism developed cancer, P = 0.7.

Conclusion: Our study did not detect an increased risk of subsequent cancer in patients presenting with idiopathic DVT compared to secondary DVT; nor did we detect an increased incidence of cancer in patients with recurrent venous thromboembolism. Further studies are required prior to pursuing a policy of aggressive screening for cancer in patients with idiopathic venous thromboembolism.
Dominant C2 Domain Epitope Specificity of Inhibitor Antibodies Elicited by a Heat Pasteurized Product, Factor VIII CPS-P, in Previously Treated Hemophilia A Patients without Inhibitors
Authors: Sawamoto Yoshikatsu , Prescott Richard , Zhong Degang , Saenko Evgueni , Mauser-Bunschoten Eveline , Peerlinck Kathelijne , van den Berg Marijke , Scandella Dorothea
Published: March 2018

From June, 1990, to November, 1991, in The Netherlands and Belgium, 16 previously treated severe hemophilia A patients (PTP) developed inhibitors after exposure to factor VIII CPS-P, a new heat pasteurized product. A previously untreated patient (PUP) also developed an inhibitor to CPS-P. In inhibitor neutralization assays with recombinant fVIII C2 and A2 domain polypeptides, plasmas from 14 PTPs were ≥79% neutralized by C2 and <10% by A2, but the PUP plasma was partially neutralized by C2 (48%) and A2 (28%). Immunoprecipitation assays of the PTP and PUP plasmas with the fVIII heavy chain and with recombinant C2 and A3-C1 polypeptides confirmed that the C2 dominant immune response to CPS-P was found only in the PTPs. Competition of the binding of 2 inhibitors to 125I-CPS-P by unlabeled CPS-P and another plasma fVIII was similar, demonstrating that the antibody response was not directed to epitopes only present in CPS-P. We propose that the immunogenicity of the CPS-P C2 domain was altered by heat pasteurization.
Fibrillin Containing Elastic Microfibrils Support Platelet Adhesion under Dynamic Shear Conditions
Authors: McIntire Larry , Moake Joel , Kuo Huey-Ju , Qian Rui-Qing , Glanville Robert , Schwartz Elaine , Rand Jacob , Ross Julia
Published: March 2018

The vascular subendothelium contains macromolecular structures called microfibrils. Type VI collagen is one protein found in microfibrils that supports platelet adhesion and aggregation and we have previously evaluated the roles of platelet receptors and vWf involved in these processes under physiological shear conditions. Here we investigate the ability of fibrillin containing elastic microfibrils to support mural thrombus formation. Our results show that elastic microfibril surfaces support platelet adhesion under low shear conditions at a level similar to collagen VI tetramers. However, the degree of aggregation on the elastic microfibril surface is much higher. Both adhesion and aggregation were shown to be mediated by the GPIIb-IIIa platelet receptor. Elastic microfibrils do not support the formation of mural thrombi under high shear conditions. These results suggest roles for both collagen VI and fibrillin containing elastic microfibrils in modulating the platelet response to blood vessel injury.
Antagonism of vWF Inhibits both Injury Induced Arterial and Venous Thrombosis in the Hamster
Authors: Yamamoto Hiroshi , Vreys Ingrid , Stassen Jean Marie , Yoshimoto Ryota , Vermylen Jos , Hoylaerts Marc
Published: March 2018

von Willebrand factor (vWF) is instrumental in arterial but has also been implicated in venous thrombogenesis. To address its role in venous thrombosis, experimental thrombosis was induced in the carotid artery and the femoral vein of hamsters, following which thrombus prevention by two different antagonists of vWF was studied. The first antagonist was the anti-human vWF monoclonal antibody AJvW-2, which inhibits the botrocetin and ristocetin induced aggregation of human blood platelets. AJvW-2 reacts with an epitope present in the A1 domain of vWF in very different species (human, pig, rabbit, dog, Guinea pig and rat). This epitope was found to be conformational and overlapping with vWF binding sites for aurin tricarboxylic acid (ATA), but not for botrocetin and heparin. AJvW-2 has affinities for vWF in the absence (Kd = 0.5 ± 0.03 nmol/l in solution) and in the presence of shear stress (Kd = 3.3 ± 0.6 nmol/l during perfusion at 1,300 s–1 over subendothelial matrix associated vWF) sufficiently elevated to neutralize vWF. During perfusion of subendothelial matrix with anticoagulated human blood, the surface covered by adhering platelets was reduced by AJvW-2, with IC50s equal to 6.6 ± 0.34 μg/ml at 1,300 s–1 and to 1 ± 0.01 μg/ml at 2,700 s-1. As a second antagonist, molecular size gel filtered ATA was selected. Fractionated ATA inhibited platelet adhesion to matrix with IC50s equal to 0.27 ± 0.09 mmol/l at 1,300 s –1 and 0.16 ± 0.008 mmol/l at 2,700 s –1. When administered to hamsters, AJvW-2 prevented thrombosis in the injured carotid artery dose-dependently (ED50 = 0.15 ± 0.01 mg/kg). Thrombosis in the similarly injured femoral vein was however also inhibited (ED50 = 0.37 ± 0.06 mg/kg). Likewise, fractionated ATA completely inhibited carotid artery thrombosis (ED50 = 0.42 ± 0.13 mg/kg), but also interfered with femoral vein thrombosis (apparent ED50 between 2 and 3 mg/kg). We conclude that antagonizing the vWF A1 domain by AJvW-2 and to a lesser extent also by fractionated ATA, inhibits thrombosis not only in the arterial but also in the venous circulation. Since venous thrombi were prevented at only 3-5-fold higher doses of antagonist, vWF participates in injury induced venous thrombosis.
Inhibitory Effect of Piracetam on Platelet-rich Thrombus Formation in an Animal Model
Authors: Stockmans F. , Deberdt W. , Nyström Å. , Nyström E. , Stassen J. M. , Vermylen J. , Deckmyn H.
Published: March 2018

Intravenous administration of piracetam to hamsters reduced the formation of a platelet-rich venous thrombus induced by a standardised crush injury, in a dose-dependent fashion with an IC50 of 68 ± 8 mg/kg. 200 mg/kg piracetam also significantly reduced in vivo thrombus formation in rats. However, in vitro aggregation of rat platelets was only inhibited with piracetam-concentrations at least 10-fold higher than plasma concentrations (6.2 ± 1.1 mM) obtained in the treated animals. No effects were seen on clotting tests.

In vitro human platelet aggregation, induced by a variety of agonists, was inhibited by piracetam, with IC50’s of 25-60 mM. The broad inhibition spectrum could be explained by the capacity of piracetam to prevent fibrinogen binding to activated human platelets. Ex vivo aggregations and bleeding times were only minimally affected after administration of 400 mg/kg piracetam i.v. to healthy male volunteers, resulting in peak plasma levels of 5.8 ± 0.3 mM.

A possible antiplatelet effect of piracetam could be due to the documented beneficial effect on red blood cell deformability leading to a putative reduction of ADP release by damaged erythrocytes. However similarly high concentrations were needed to prevent stirring-induced “spontaneous” platelet aggregation in human whole blood.

It is concluded that the observed antithrombotic action of piracetam cannot satisfactorily be explained by an isolated direct effect on platelets. An additional influence of piracetam on the rheology of the circulating blood and/or on the vessel wall itself must therefore be taken into consideration.