bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Mouse Thrombin-activatable Fibrinolysis Inhibitor
Authors: Wagenaar Gerry , Reijerkerk Arie , Tiekstra Margriet , van Rossum Agnes , Gebbink Martijn , Meijers Joost , Marx Pauline
Published: March 2018

Based on in vitro studies, thrombin-activatable fibrinolysis inhibitor (TAFI) has been hypothesized as a link between coagulation and fibrinolysis, but the physiological role of TAFI in vivo has not yet been established. To anticipate on the availability of genetically modified mouse models, we studied the endogenous expression of TAFI in mice. Functional TAFI was found in mouse plasma. TAFI mRNA was only detectable in the liver, showing a hepatocyte-specific expression with a pericentral lobular distribution pattern. The murine TAFI cDNA was cloned and sequenced. The deduced amino acid sequence revealed that murine TAFI is highly identical to human TAFI. The murine cDNA was stably expressed and the activated recombinant protein was functionally active; it converted the substrate hippuryl-arginine, and prolonged the clot lysis time of TAFI depleted plasma. We conclude that mice have functional TAFI in plasma, which is highly similar to human TAFI. Therefore, genetically modified mice may provide useful models to study the role of TAFI in vivo.

Abbreviations: TAFI, thrombin-activatable fibrinolysis inhibitor; CPI, carboxypeptidase inhibitor from potato tubers; CPN, carboxypeptidase N; t-PA, tissue-type plasminogen activator; PPACK, H-D-Phe-Pro-Arg-chloromethylketone.
Randomized, Placebo-controlled, Blinded and Cross-matched Study on the Antiplatelet Effect of Inhaled Nitric Oxide in Healthy Volunteers
Authors: Herr Axel , Motsch Johann , Holzmann Alexandra , Weimann Jörg , Taut Friedemann , Erbe Natalie , Bode Christoph , Martin Eike , Gries André
Published: March 2018

The platelet inhibitory effect of 0-40 ppm inhaled nitric oxide (NO) was investigated in healthy men and women. In both groups, ADPand collagen-induced platelet aggregation was significantly inhibited 20 (T20) and 40 min (T40) after the beginning of inhalation of 5, 10, and 40 ppm. Moreover, in both men and women, the in vitro bleeding time was significantly prolonged at T20 and T40 during inhalation of 40 ppm. Inhalation of NO also inhibited P-selectin expression at 5, 10, and 40 ppm and fibrinogen binding to the GPIIb/IIIa-receptor at 40 ppm. In conclusion, in healthy volunteers, the platelet inhibitory effect of inhaled NO was not dose-related, since it was significant at 5 and 10 ppm but did not increase during the administration of higher NO concentrations. In addition, gender-related differences were only observed in ADP-induced platelet aggregation at 10 ppm and in bleeding time prolongation at 40 ppm.
An In-vitro Model to Study Device-induced Thrombosis and Embolism:
Authors: Sukavaneshvar Sivaprasad , Mohammad Syed , Solen Kenneth
Published: March 2018

A bovine in-vitro model was developed to investigate device-induced thromboembolism (TE) and its pharmacological intervention, using a stent as a prototype device. Emboli were assessed continuously using a light-scattering microemboli detector (LSMD). Thrombus on the stent was assessed gravimetrically at the end of the experiment. The contribution of the stent as the predominant source of detectable thromboemboli in this model was verified by placing LSMD probes upstream and downstream of the stent. The effectiveness of ethylenedinitrilo-tetraacetic-acid (EDTA) and three anti-thrombogenic agents (aspirin, dipyridamole, and tirofiban) for mitigating device-induced TE was also assessed. The results show that 1) the model has potential to study device-induced TE and the efficacy of possible interventional strategies, 2) the LSMD is capable of continuous, non-invasive, real-time assessment of embolism, 3) the assessment of embolization may constitute an important part of evaluating hemocompatibility, 4) tirofiban is effective in reducing both stent-induced thrombosis and embolism above certain concentrations.
Use of the PFA-100™ Apparatus to Assess Platelet Function in Patients Undergoing PTCA during and after Infusion of cE3 Fab in the Presence of other Antiplatelet Agents
Authors: Hézard Nathalie , Metz Damien , Nazeyrollas Pierre , Droulle Chantal , Elaerts Jacques , Potron Gérard , Nguyen Philippe
Published: March 2018

The PFA-100™ (Dade) is a new functional whole blood analyzer, the accuracy and reliability of which have been evaluated in von Willebrand disease and during acetyl salicylate acid therapy. This new test has the advantages of rapidity and simplicity. It may be useful to monitor new antiplatelet agents, such as GPIIb/IIIa receptor antagonists. The objective of this study was to assess the PFA-100™ in comparison with aggregometry and with the percentage of blockaded receptors GPIIb/IIIa during and after c7E3 Fab infusion in fifteen patients undergoing PTCA. Our results showed a change of closure time values from normal to abnormal within a small margin of flow cytometric values (60–75% of blockaded receptors), and moreover a variable platelet response to long-term low dose aspirin treatment in agreement with aggregometry. No influence with heparin was observed. In conclusion, this study shows that PFA-100™ may be helpful in the decision making for additional antiaggregant therapy before PTCA or in monitoring long-term GPIIb/IIIa receptor antagonist treatment.
HPA-1 and HPA-3 Polymorphisms of the Platelet Fibrinogen Receptor and Coronary Artery Disease and Myocardial Infarction
Authors: Böttiger Corinna , Koch Werner , Mehilli Julinda , Seidl Holger , Schömig Kathrin , von Beckerath Nicolas , Schömig Albert , Kastrati Adnan
Published: March 2018

Platelet fibrinogen receptor (glycoprotein [GP] IIb/IIIa) plays a fundamental role in atherothrombosis. The human platelet antigen (HPA) -1 and the HPA-3 are the most extensively studied polymorphisms of GPIIIa and GPIIb, respectively. This study was designed to test, in a large population, the hypothesis that these polymorphisms represent a risk factor for the occurrence of coronary artery disease (CAD) and myocardial infarction (MI).

Consecutive, angiographically examined patients with significant coronary stenoses but without symptoms or signs of old or acute MI constituted the group with CAD (CAD, n = 998) and those with old or acute MI constituted the group with MI (MI, n = 793). As controls served subjects, matched with patients for age and sex, with neither angiographic CAD nor symptoms or signs of MI (matched controls [MC], n = 340) as well as a group of blood donors without cardiac symptoms or signs of CAD (BD, n = 104). Genotype distribution was similar across the groups; HPA-1a/a: HPA-1a/b: HPA-1b/b was 75.0%: 22.1%: 2.9% in BD, 72.6%: 24.7%: 2.6% in MC, 70.5%: 26.8%: 2.7% in CAD, and 70.7%: 26.4%: 2.9% in MI; HPA-3a/a: HPA-3a/b: HPA-3b/b was 39.4%: 40.4%: 20.2% in BD, 33.5%: 50.0%: 16.5% in MC, 35.0%: 46.4%: 17.0% in CAD, and 37.1%: 48.0%: 16.5% in MI. There was no interaction between these polymorphisms, nor between each of these polymorphisms and other risk factors.

Thus, the HPA-1 and HPA-3 polymorphisms are neither separately nor in concert associated with any measurable increase of the risk for CAD or MI in angiographically evaluated subjects.
Incomplete Inhibition of Platelet Aggregation and Glycoprotein IIb-IIIa Receptor Blockade by Abciximab: Importance of Internal Pool of Glycoprotein IIb-IIIa Receptors
Authors: Pogatsa-Murray Gisela , Dickfeld Timm , Rüdiger Silja , Taubitz Winfried , Fischer Jörg , Müller Iris , Meier Dieter , Schömig Albert , Gawaz Meinrad , Ruf Andreas , Patscheke Heinrich
Published: March 2018

Resting platelets contain a substantial internal pool of GPIIb-IIIa complexes that is exposed on the surface of activated platelets. Whether the exposure of internal GPIIb-IIIa complexes on the activated platelet surface affects therapy with GPIIb-IIIa antagonists is poorly understood. We addressed this issue in thirteen patients who underwent elective coronary stenting and received abciximab. Platelet aggregation, surface expression of GPIIb-IIIa and P-selectin, receptor blockade of GPIIb-IIIa, and platelet release in response to ADP and thrombin-receptor activating peptide (TRAP) were determined ex vivo by Lumi-aggregometry and flow cytometry before, during and after abciximab administration. We found that inhibition of aggregation and GPIIb-IIIa blockade of ADP-stimulated platelets was almost complete during abciximab administration. In contrast, when TRAP was used to stimulate platelets ex vivo aggregation was only partially inhibited, most likely due to release of internal pool of unblocked GPIIb-IIIa complexes. Using electron microscopy we found that 7E3-occupied GPIIb-IIIa complexes are internalized into the surface connected system (SCS) and the α-granules of washed platelets which was associated with a reduced degranulation of the α-granula membrane protein P-selectin. We conclude, that despite internalization of abciximab into the internal pool of GPIIb-IIIa, upon strong platelet activation with thrombin a significant amount of unblocked internal GPIIb-IIIa can be exposed on the platelet surface and mediate platelet aggregation. Incomplete blockade of the internal GPIIb-IIIa pool may limit clinical efficacy of abciximab.
Treatment with Interferon plus Ribavirin in anti-HIV Negative Patients with Congenital Coagulation Disorders and Chronic Hepatitis C
Authors: Sauleda Silvia , Altisent Carmen , Puig Lluís , Esteban Rafael , Guardia Jaume , Esteban Juan
Published: March 2018

Hepatitis C virus (HCV) infected hemophiliacs respond at low rate to interferon (IFN) monotherapy.

To assess efficacy of IFN and RBV in HIV negative hemophiliacs with chronic hepatitis C and identify early predictive factors of response.

Twenty naive patients were treated with interferon and RBV for twelve months. Response was assessed by both serial ALT and HCV RNA levels.

Normalization of ALT with clearance of HCV RNA occurred in seven (35%) patients. Age and age at infection were the only features associated with a higher likelihood of response. In all responders the viral load had decreased by at least one log within two months of starting treatment.

Combination of interferon and ribavirin is well tolerated by hemophiliacs who achieve similar sustained response rates to non-hemophiliacs. Quantitative assessment of viral load at two months of treatment is a useful method to identify non-responders at an early stage.
Sucrose Formulated Recombinant Human Antihemophilic Factor VIII Is Safe and Efficacious for Treatment of Hemophilia A in Home Therapy
Authors: Gorina E. , Kellermann E. , Vosburgh E. , Abshire T. C. , Brackmann H.-H. , Scharrer I. , Hoots K. , Gazengel C. , Powell J. S. ,   and the International Kogenate-FS Study Group
Published: March 2018

To add an increased level of safety to antihemophilic factor replacement therapy, a full-length, recombinant Factor VIII (rFVIII) product has been developed without human-derived plasma proteins during purification and formulation and using an additional solvent/detergent viral inactivation step. This first clinical trial of a sucrose-formulated full-length rFVIII (rFVIII-FS) was conducted in previously treated patients (≥100 prior exposure days) with severe (<2% FVIII) hemophilia A in North America (NA) and Europe (EU). Pharmacokinetic profiles for rFVIII-FS were compared with those of currently licensed rFVIII product (Kogenate®) in 35 patients. Safety and efficacy during home therapy were evaluated in 71 patients. The new formulation displayed a pharmacokinetic profile similar to that of rFVIII. Patients on home therapy received a cumulative total of 11,867 exposure days, 12,546 infusions, and 22,443,694 IU of rFVIII-FS. Of 2585 bleeds, 93.5% were treated with 1-2 infusions and 80.5% of responses were rated as excellent or good. No evidence of de novo inhibitor formation was observed. Only 0.27% of infusions were associated with any drugrelated adverse event. Except for an episode of intermittent chest pain with palpitations which ceased after treatment with analgesics, associated adverse events were mild or moderate. Overall, rFVIII-FS provided excellent hemostatic control, was well-tolerated, and caused no significant adverse effects, thus demonstrating safety and efficacy for treatment of bleeds in patients with hemophilia A.
Relationship between Factor VIII Mutation Type and Inhibitor Development in a Cohort of Previously Untreated Patients Treated with Recombinant Factor VIII (Recombinate™)
Authors: Williams Ian , Bray Gordon , Peake Ian , Goodeve Anne ,   for the Recombinate™ PUP Study Group
Published: March 2018

A cohort of 79 previously untreated patients (PUPs) with moderatesevere haemophilia A (baseline Factor VIII <2%) were enrolled in a study to evaluate the safety, efficacy and immunogenicity of recombinant factor VIII (r-FVIII, Recombinate™). Blood samples were obtained retrospectively from a total 55 PUPs who were investigated for the spectrum of FVIII gene mutations responsible for their haemophilia. FVIII gene inversion mutations were found in 27 (49%) patients. Two patients had partial gene deletions. The remaining 26 patients were then screened for mutations in the FVIII gene coding region using conformation sensitive gel electrophoresis. Point mutations were identified in 22 (85%) of the patients and 14 of these mutations were novel. Study subjects were monitored for the development of FVIII inhibitors throughout the study. A total of 23 of the 73 evaluable subjects (including one subject with a low inhibitor titer at baseline) demonstrated an inhibitor on one or more occasions; 11 (15%) were persistent. Inhibitors were detected in patients with partial gene deletions and inversions and in three of eight patients with missense mutations. No inhibitors were found in 11 patients with small insertions or deletions resulting in an alteration of the protein translation reading frame (frameshift mutations). The results corroborate the observation that mutation type is an important determinant of the propensity to develop inhibitory anti-FVIII antibody.
A Monoclonal Antibody Raised against Human β-factor XIIa which also Recognizes α-factor XIIa but not Factor XII or Complexes of Factor XIIa with C1 Esterase Inhibitor
Authors: Esnouf M. , Burgess Annette , Dodds Alister , Sarphie Anna , Miller George
Published: March 2018

A monoclonal antibody (mAb 2/215) against human β-factor XIIa (β-FXIIa), was shown by equilibrium binding studies to have a high affinity for α-factor XIIa (α-FXIIa) (Kd 1.8 nM) and β-FXIIa (Kd 0.65 nM) but no detectable reaction with FXII zymogen or α - esterase inhibitor (C1-INH) complex. Surface plasmon resonance studies showed that the mAb 2/215 bound to immobilized α-FXIIa with high affinity (KD 3.93 ± 1.46 × 10−11 M). Western blots employing mAb 2/215 indicated that human plasma contained small amounts of α-FXIIa but no β-FXIIa. mAb 2/215 did not inhibit the amidolytic activity of β-FXIIa and protected β-FXIIa from inhibition by C1-INH. The recovery by ELISA ,employing mAb 2/215 as the capture antibody, of α-FXIIa added to plasma was 11.3%, 42% after inhibition of α-FXIIa with 3:4dichloroisocoumarin, and 82% when 0.5% TritonX100 was added to the assay. Gel filtration showed that the majority of plasma α-FXIIa existed as a complex (Mr ∼170000). This distinctive mAb increases the capacity to study the contact system in health and disease.
Hereditary Thrombocytopenia due to Reduced Platelet Production
Authors: Tonelli Roberto , Strippoli Pierluigi , Grossi Alberto , Savoia Anna , Iolascon Achille , Savino Maria , Teriaca Maria , Servedio Veronica , Morfini Massimo , Zelante Leopoldo , Borgna-Pignatti Caterina , Rosito Pasquale , Pession Andrea , Paolucci Guido , Bagnara Gian
Published: March 2018

Hereditary thrombocytopenias represent heterogeneous clinical and genetic syndromes. They include a consistent group of families which are considered as a separate clinical entity, characterized by autosomal dominant transmission, incomplete penetrance in females, chronic thrombocytopenia with early age of onset and frequently increased platelet volume, without any other hematologic abnormality. The molecular defect in these families is still unknown. We describe 2 families in 3 generations (10 patients), and report the first study of the TPO/ c-mpl system in autosomal dominant thrombocytopenia. We performed mutational screening of c-mpl coding, flanking introns and promoter regions in 2 probands from the two families by DNA sequencing. The results do not provide evidence of c-mpl sequence alterations in either of the 2 families investigated. Moreover, the normal TPO serum levels detected in 5 patients from each family leads us to exclude the possibility of a defect in TPO production in our families. Finally, the involvement of both c-mpl and TPO genes in the pathogenesis of thrombocytopenia in these two families was excluded by negative results of linkage analysis.
Expression of Vascular Endothelial Growth Factor in Human Umbilical Vein Endothelial Cells Stimulated with Interleukin-1α
Authors: Itaya Hiroyuki , Nasu Satoki , Yoshida Hidemi , Matsubara Yuki , Fujimoto Koji , Matsumiya Tomoh , Kimura Hiroto , Satoh Kei , Imaizumi Tadaatsu
Published: March 2018

Vascular endothelial growth factor (VEGF) is a specific mitogen for endothelial cells. We studied the production of VEGF by human umbilical vein endothelial cells (HUVEC) and smooth muscle cells (SMC) in response to the stimulation with interleukin-1α (IL-1α). HUVEC expressed VEGF mRNA in response to IL-1α in doseand time-dependent manners. In HUVEC VEGF protein was detected only in cell lysates whereas in SMC most of the VEGF protein was detected in the conditioned medium. Immunofluorescent staining also confirmed the cell-associated VEGF in HUVEC. IL-1α also induced the expression of mRNA for IL-1α itself in HUVEC. Cycloheximide treatment of HUVEC slightly inhibited the IL-1α-induced expression of VEGF mRNA, and IL-1α may mediate, at least in part, VEGF expression in response to IL-1α. The growth of HUVEC stimulated with IL-1α was inhibited by a neutralizing antibody against VEGF. We conclude that IL-1α and VEGF may play an important role in autocrine growth regulation of HUVEC.
CD9 Gene Deficiency Does not Affect Smooth Muscle Cell Migration and Neointima Formation after Vascular Injury in Mice
Authors: Lupu F. , Collen D. , Le Naour F. , Boucheix C. , Lijnen H. R.
Published: March 2018

The hypothesis that CD9, a member of the tetraspanin family, plays a role in smooth muscle cell (SMC) migration was tested with the use of a vascular injury model in wild-type (CD9+/+) and CD9-deficient (CD9−/−) mice. Neointima formation 3 weeks after electric injury of the femoral artery was not significantly different in CD9+/+ and CD9−/− mice (area of 0.019 ± 0.0034 mm2 versus 0.013 ± 0.0036 mm2; mean ± SEM, n = 6). The medial areas were also comparable, resulting in intima/media ratio’s of 1.3 ± 0.15 and 0.90 ± 0.22, respectively. Nuclear cell counts in cross-sectional areas of the injured region were comparable in media (33 ± 5 versus 27 ± 2) and neointima (135 ± 16 versus 97 ± 17) of CD9+/+ and CD9−/− arteries. Immunocytochemical analysis revealed expression of CD9 in the endothelium, by SMC in the media and by some fibroblasts in the adventitia of non-injured femoral arteries. Three weeks after injury, there appeared to be a gradient of increased CD9 expression from the adventitia to the neointima, in which SMC are abundantly present. Immunogold labeling and electron microscopy with non-injured femoral arteries of CD9+/+ mice confirmed the presence of CD9 at the surface of adventitial fibroblasts and in SMC or pericytes, as well as in the endothelium.

Thus, in this model CD9 is highly expressed by migrating SMC, but deficiency of CD9 does not affect SMC migration or neointima formation after perivascular injury.
Characterization of a Monoclonal Antibody, D73H, that Maps to a Highly Conserved Region on Fibrinogen Bβ Chain
Authors: Rybarczyk B. J. , Pereira M. , Simpson-Haidaris P. J.
Published: March 2018

The primary structure of fibrinogen is highly conserved across species, yet often times monoclonal antibodies produced against the fibrinogen of one species will not crossreact with the fibrinogen of another. Herein, we describe the production and characterization of murine MAb, D73H, raised against human fibrinogen. D73H crossreacts with a highly conserved epitope on the Bβ chain of fibrinogen from human, rat, bovine, guinea pig, and mouse. Western blotting revealed that D73H reacted with the Bβ chain of plasmin fragment D, localizing its epitope to Bβ134-461. A 7 kDa band was identified by D73H in Western blots of reduced fibrinogen CNBr-fragments. N-terminal sequencing mapped this fragment to Bβ243-253, further localizing the epitope to Bβ243-305. In silico analysis indicated that Bβ243-305 is predominantly hydrophilic, and surface probability prediction indicated three potential antigenic determinants corresponding to Bβ252-258, Bβ262-269, and Bβ279-286. Further in silico analysis of the crystal structure of fibrinogen fragment D-D indicated that Bβ262-269 (FGRKWDPY) is predominantly α-helical and located on the surface of the molecule adjacent to a bend imposed in the β chain at residue 260, which is near the junction between the rigid coiled-coil domain and the globular C-terminus. A synthetic peptide corresponding to Bβ261-272 competitively inhibited the binding of D73H to the Bβ chain of denatured intact fibrinogen and reduced and denatured Bβ chain in Western blots, experimentally proving the validity of these predictive algorithms. Together these data indicate that, although plasmin resistant, Bβ chain residues Bβ261-272 comprising the D73H epitope are highly conserved across species, surface exposed, and immunogenic.
Effect of Strenuous Arm Exercise on Oxidized-LDL-Potentiated Platelet Activation in Individuals with Spinal Cord Injury
Authors: Yang Chih , Wong May-Kuen , Chow Shu-Er , Chen Jan-Kan , Wang Jong-Shyan
Published: March 2018

This study investigates how strenuous arm exercise affects oxidizedlow density lipoprotein (Ox-LDL) mediated-platelet activation in patients with SCI. Ten patients with SCI and ten age- and sex-matched healthy subjects exercised strenuously using an arm crank ergometer. The following measurements were taken both when the subjects were at rest, and immediately after exercise: plasma lipid profile, Ox-LDL mediated platelet aggregability and [Ca2+]i, urinary 11-dehydro-thromboxane B2 (11-dehydro-TXB2) and 8-iso-prostaglandin F2α. (8-iso-PG F2α.) contents, and plasma NO metabolite (nitrite plus nitrate) level. Based on these measurements, the major findings of this study can be summarized as follows: 1) the SCI group had higher urinary 8-iso- PGF2α. and 11-dehydro-TXB2 contents, but a lower plasma nitrite plus nitrate level than the control group; 2) at rest, the SCI group had a higher platelet aggregability and [Ca2+]i, and Ox-LDL-potentiated platelet activation than the control group; 3) Ox-LDL-potentiated platelet aggregation was enhanced by strenuous arm exercise in both groups, but the effect of exercise was more pronounced in the SCI group than in the control group; 4) treating the platelet with L-arginine inhibited Ox-LDL-potentiated platelet activation in both groups. The study concludes that individuals with SCI had more extensive resting and exercise-enhanced Ox-LDL-potentiated platelet activation and greater amounts of preformed lipid peroxides than those without SCI. Therefore, supplementation therapy with antioxidants may be needed for patients with SCI, especially in a strenuous arm exercise period.
Local and Systemic Effects of Intra-arterial Desmopressin in Healthy Volunteers and Patients with Type 3 von Willebrand Disease
Authors: Stewart Andrew , Witherow Fraser , Grieve Susan , Dawson Pamela , Fox Keith , Webb David , Ludlam Christopher , Newby David
Published: March 2018

Intra-arterial desmopressin caused dose and time dependent increases (p <0.001 for all) in forearm blood flow (all doses) and plasma tissue plasminogen activator (t-PA) concentrations (desmopressin ≥70 ng/min). Although plasma t-PA concentrations rose in both forearms, there was a modest local release of t-PA in the infused forearm (14 ng/100 mL of tissue/min, p <0.05). At desmopressin doses ≥300 ng/min, plasma von Willebrand factor (vWf) and Factor VIII:C concentrations rose in both forearms (p <0.001) and correlated with the rise in interleukin-6 concentrations (r = 0.92, p <0.001; r = 0.85, p = 0.002 respectively). Neither desmopressin nor substance P caused t-PA, vWf or Factor VIII:C release in the patients, although desmopressin increased plasma interleukin-6 concentrations as in healthy volunteers. We conclude that desmopressin releases t-PA, vWf and Factor VIII:C predominantly via systemic mechanisms, possibly mediated by cytokine release. Patients with type 3 vWD appear to have a generalised failure to release t-PA acutely despite a normal interleukin-6 response to desmopressin infusion.
The Factor VIII Acute Phase Response Requires the Participation of NFκB and C/EBP
Authors: Notley Colleen , Tinlin Shawn , Sawyer Lisa , Begbie Megan , Lillicrap David
Published: March 2018

Coagulation Factor VIII is an acute phase protein in humans that has recently been shown to be transcriptionally responsive to interleukin-6. In this study, we have demonstrated that the human Factor VIII promoter is activated in cultured hepatocytes exposed to bacterial lipopolysaccharide (LPS). Deletion analysis has narrowed the LPS-responsive element of the Factor VIII promoter to a small region which contains two C/EBP binding sites and an adjacent NFκB binding site. Mutation of the downstream C/EBP site reduces LPS-responsiveness by ∼50%, while mutation of the NFκB binding site completely eliminates LPS-responsiveness. While binding of C/EBPβ and NFκB is still observed in gel retardation studies using acute phase nuclear extracts and a probe containing mutations to the downstream C/EBP site, neither NFκB nor C/EBP appear to bind to a probe in which the NFκB site has been mutated. Conservation of this region of the Factor VIII promoter in species which exhibit an increase in Factor VIII levels in response to inflammatory stimuli suggests that these transcription factor binding sites are important for normal regulation of the Factor VIII gene under conditions of stress.
Fibrinogen Caracas V, an Abnormal Fibrinogen with an Aα 532 Ser→Cys Substitution Associated with Thrombosis
Authors: Marchi Rita , Lundberg Ulf , Grimbergen Jos , Koopman Jaap , Torres Argimiro , de Bosch Norma , Haverkate Frits , Piñango Carmen
Published: March 2018

A new dysfibrinogenemia associated with thrombophilia has been identified in a Venezuelan kindred. Thrombin and Reptilase times were prolonged and the accelerating capacity of the patient’s fibrin on the t-PA-induced plasminogen activation was decreased. In addition the affinity of fibrinogen for plasminogen was diminished. Permeability and electron microscopy studies revealed that the abnormal clot was made up of thin and densely packed fibres giving rise to a reduced fibrin gel porosity. This was confirmed by turbidity studies showing a decreased fibre mass/length ratio. Affected members were heterozygous for an Aα 532 Ser→ Cys mutation as demonstrated by genetic analyses. This abnormal fibrinogen has been designated as Fibrinogen Caracas V. The family study showed a convincing association between the mutation and thrombotic manifestations. The thrombotic tendency may be ascribed to lack of accelerating capacity of fibrin to induce fibrinolysis caused by an abnormal clot structure with thin fibres and reduced porosity.
A Novel Point Mutation of the Splicing Donor Site in the Intron 2 of the Plasmin Inhibitor Gene
Authors: Yoshinaga Haruhiko , Chung Dong , Miyasaka Nobuyuki , Aoki Nobuo , Favier Remi , Hirosawa Shinsaku
Published: March 2018

Plasmin inhibitor (PI) is a major physiological inhibitor of plasminmediated fibrinolysis; hence, its deficiency results in a severe haemorrhagic diathesis. We analyzed the PI gene of a French boy apparently homozygous for PI deficiency and his heterozygous parents. Both alleles of the homozygous patient had a novel G to A transition at the consensus splicing donor site in the intron 2 of the PI gene. In an expression assay using the heterologous cells transfected with the mutant PI expression vector, 3 types of aberrant transcripts using a cryptic splicing donor site within the intron 2 were detected. All of these mRNAs had a stop codon upstream of the cryptic splicing site and encode only 25 amino acids, comprising the first 21 amino acids of the signal peptide (27 amino acids) plus 4 new amino acids. This mutant was designated as PI-Paris-Trousseau.
Molecular Characterization of a Multiethnic Group of 21 Patients with Type 3 von Willebrand Disease
Authors: Cozzi Giovanna , Canciani Maria , Peyvandi Flora , Srivastava Alok , Federici Augusto , Mannucci Pier , Baronciani Luciano
Published: March 2018

Type 3 von Willebrand disease is a rare autosomal disorder characterized by unmeasurable levels of von Willebrand factor and severe hemorrhagic symptoms. We studied a multiethnic group of 37 patients, from Italy (n = 14), Iran (n = 10) and India (n = 13) to identify the molecular defects and to evaluate genetic heterogeneity among these populations. Twenty-one patients (6 Italians, 9 Iranians and 6 Indians) were fully characterized at the molecular level. Twenty-four different gene alterations were identified, 20 of which have not been described previously. The majority of the mutations caused null alleles, 11 being nonsense mutations (Q218*, W222*, R365*, R373*, E644*, Q706*, S1338*, Q1346*, Y1542*, R1659*, E2129*), 4 small deletions (437delG, 2680delC, 6431delT, del 8491-8499), 3 possible splice site mutations [IVS9(-1)g→a, IVS29(+10)c→t, IVS40(-1)g → c], 3 candidate missense mutations (C275S, C2174G, C2804Y), 2 small insertions (7375insC, 7921insC) and 1 large gene deletion. The latter mutation was associated with the development of alloantibodies to VWF, but this complication was also found in a patient homozygous for a nonsense mutation (Q1346*). Due to the ethnic origin of the patients most of them were the offspring of consanguineous marriages and so were homozygous for the mutations found (18/21). Our results indicate that molecular defects responsible for type 3 VWD are scattered throughout the entire VWF gene (from exon 3 to 52), and that there is no prevalent and common gene defect in the three populations studied by us.
Ethnic Heterogeneity of the Factor XIII Val34Leu Polymorphism
Authors: Attié-Castro Flávia , Zago Marco , Lavinha João , Elion J. , Rodriguez-Delfin L. , F.Guerreiro João , Franco Rendrik
Published: March 2018

A polymorphism in the coagulation factor XIII gene (FXIII Val34Leu) has been recently described to confer protection for arterial and venous thrombosis and to predispose to intracerebral hemorrhage. At present it is known that FXIII Val34Leu is prevalent in Caucasians, but information upon its distribution in different ethnic groups is scarce. We investigated the prevalence of FXIIIVal34Leu in 450 unrelated subjects of four ethnic groups: 97 Caucasians (Brazilians of European descent and Portuguese), 149 Blacks (Brazilians, and Africans from Cameroon, Zaire and Angola), 40 Asians (Japanese descendents) and 164 Amerindians from South America. PCR amplification of exon 2 of FXIII gene followed by MseI restriction-digestion was employed to define the genotypes. FXIIIVal34Leu was detected in 44.3% of the Caucasians, in 28.9% of the Blacks, in 2.5% of the Asians and in 51.2% of the Amerindians. These data confirm that FXIII Val34Leu is highly prevalent in Caucasians and indicate that it is rarer in populations of African origin. The very high frequency among Amerindians indicates that FXIII Val34Leu is not absent among Asians, and since it has a very low prevalence in Japanese, a heterogeneity in its distribution in Asia may be inferred. Taken together, our data showed that FXIII Val34Leu exhibits a significant ethnic heterogeneity, a finding that is relevant for studies relating this polymorphism with thrombotic and bleeding disorders.
The Effect of DDAVP Infusion on Thrombin Generation in Platelet-rich Plasma of von Willebrand Type 1 and in Mild Haemophilia A Patients
Authors: Keularts Irene , Hamulyak K. , Hemker H.C. , Béguin Suzette
Published: March 2018

In von Willebrand disease (vWD) type 1 and mild haemophilia A patients we studied the effect of an infusion of DDAVP (0.3 µg/kg body weight) on thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Baseline thrombin generation in PRP was diminished both in the haemophilia A and vWD patients. It was normal in vWD plasma when sufficient procoagulant phospholipids were present, either via adding phospholipid vesicles to PPP or via scrambling of the platelet membrane with ionomycin in PRP. In haemophilia A plasma, thrombin generation did not normalize by providing procoagulant phospholipids. Treatment with DDAVP temporarily restored thrombin generation in PRP to normal in both diseases.

To investigate the individual roles of von Willebrand factor (vWF) and factor VIII, we also studied the effect of factor VIII infusion on thrombin generation in a severe haemophilia patient. It appears that at a fixed normal vWF concentration, <25% factor VIII is sufficient for normal thrombin generation in PRP. At a sufficient factor VIII concentration, however, thrombin generation is still lower than normal in vWD patients; ∼40% of vWF is required for half-normal thrombin generation in PRP.

It thus appears that vWF is also a clotting factor, in the sense that it is required for normal thrombin generation. This underlines the importance of the interaction between coagulation and the platelets in normal haemostasis. Thrombin generation in PRP appears to be a suitable test to reflect the combined function.
Cerivastatin, an Inhibitor of HMG-CoA Reductase, Inhibits Urokinase/Urokinase-receptor Expression and MMP-9 Secretion by Peripheral Blood Monocytes
Authors: Ganné Florence , Vasse Marc , Beaudeux Jean-Louis , Peynet Jacqueline , François Arnaud , Mishal Zohar , Chartier Antoine , Tobelem Gérard , Vannier Jean-Pierre , Soria Jeannette , Soria Claudine
Published: March 2018

It is now recognised that acute myocardial infarction results from the rupture of atherosclerotic plaques. Lymphocytes and macrophages, which infiltrate rupture sites, contribute to plaque degradation by expressing urokinase (u-PA) bound to cell membrane by urokinase receptor (u-PAR) and by secreting metalloproteinase MMP-9.

We have previously demonstrated that the uptake of oxidised LDL (ox-LDL) by monocytes induces an increase of u-PA and u-PAR expression. The present study shows that the expression of u-PA and u-PAR induced by ox-LDL on monocyte surface is suppressed by cerivastatin (a synthetic inhibitor of HMG-CoA reductase, Bayer) from 2 nM. This leads to reduced plasmin generation and monocyte adhesion to vitronectin. Furthermore, higher concentrations of cerivastatin (50-100 nM) reduce the expression of u-PA and u-PAR on unstimulated monocytes. It also inhibits MMP-9 secretion but has no effect on TIMP-1 secretion, suggesting that the decrease in MMP-9 has a real protective effect on plaque stabilisation. The inhibitory effect of cerivastatin on u-PA expression and MMP-9 secretion can be explained by the inhibition of NF-kappa B translocation into the nucleus, as shown by immunofluorescence. As farnesyl-pyrophosphate reverses the effect of cerivastatin, it is postulated that these effects could also be due to the inhibition of Ras prenylation. This was confirmed by confocal microscopy, which shows the Ras delocalisation from the monocyte membrane. The cerivastatin-induced effects on monocyte functions could explain, at least in part, the protective effect of this drug against atherothrombotic events.
The Effect of Very-low-dose Warfarin on Markers of Hypercoagulation in Metastatic Breast Cancer: Results from a Randomized Trial
Authors: Levine M. N. , Consonni R. , Gritti G. , Delaini F. , Oldani E. , Julian J. A. , Barbui T. , Falanga A.
Published: March 2018

Malignancy is a risk factor for thromboembolism and anti-cancer chemotherapy can increase this risk. Prophylaxis of thrombosis with very-low-dose warfarin given concurrently with chemotherapy has a significantly reduced rate of thromboembolism in a randomized trial in women with stage IV breast cancer. In a group of 32 patients randomized in one center (16 subjects on warfarin and 16 on placebo), we have prospectively studied the plasma levels of: 1. Markers of ‘in vivo’ clotting activation (thrombin-antithrombin complex [TAT], prothrombin fragment 1+2 [F1+2] and D-dimer), 2. Factor VII (FVII), and 3. Natural anticoagulants (protein C [PC] and antithrombin [AT]). The aims of this study were: 1. to examine whether laboratory tests predicted those patients who developed thrombosis, and 2. to evaluate the effect of very-low-dose warfarin on hemostatic variables. The patients’ hemostatic parameters were evaluated before entry into the study and after starting chemotherapy ± prophylaxis, before each course for nine courses. Before-treatment results were compared to those of a sex and age-matched non-cancer control group. There was a significant elevation of plasma levels of TAT (p <0.001), F1+2 (p <0.001), D-dimer (p <0.0001) and FVIIa (p <0.05), as well as an increase of FVII proteolysis (p <0.05), whereas plasma PC and AT concentrations were not different from controls. After starting chemotherapy, markers of clotting activation were progressively lower in the group receiving warfarin prophylaxis compared to the group on placebo. Differences between the groups became statistically significant (p <0.01) after the 4th course of chemotherapy. Deep vein thrombosis occurred in two patients in the placebo arm. The results of this study indicate that before therapy, an hypercoagulable state is present in stage IV breast cancer, and after starting chemotherapy, abnormalities of hypercoagulation markers persist, however they are reduced by very-low-dose-warfarin. None of the laboratory variables could predict thrombosis in the single patient.
Factor V Leiden and Thrombotic Complications in Heparin-induced Thrombocytopenia
Authors: Lee David , Denomme Gregory , Lagrotteria Danny , Kelton John , Warkentin Theodore
Published: March 2018

To determine whether factor V Leiden is associated with thrombotic events in patients with heparin-induced thrombocytopenia (HIT), we evaluated 165 patients with serologically confirmed HIT for the presence of factor V Leiden and determined the incidence of venous or arterial thrombosis during the period of HIT. Factor V Leiden was detected in 16 of 165 HIT patients (9.7%). HIT-associated venous thrombosis occurred in 11 of 16 factor V Leiden positive subjects and 94 of 149 factor V Leiden negative subjects (69% vs. 63%; p = 0.79). Arterial thrombosis occurred in 1 of 16 factor V Leiden positive subjects and 21 of 149 factor V Leiden negative subjects (6% vs. 14%; p = 0.70). There was no difference in the incidence of proximal limb DVT, pulmonary embolism, venous limb gangrene, local skin reactions, hemorrhagic adrenal infarction, stroke, or myocardial infarction between the groups. No difference in the severity of venous thrombosis between Leiden positive and negative subjects was detected. Our data suggest that in the acute prothrombotic milieu of HIT, heterozygous factor V Leiden is not an important additional risk factor for thrombosis.
Differences in Immune Response between HCV Positive, HIV Negative Haemophilia A and B Patients
Authors: Smid W. M. , Verspiek S. P. J. , Smit J. W. , van der  Meer J. , Meijer K.
Published: March 2018

We measured numbers of lymphocytes and subsets in seven HIV negative, HCV positive severe haemophilia B patients, before and after substitution was changed from prothrombin complex concentrate to monoclonally purified concentrate. Data were compared with controls and our previous findings in haemophilia A.

At baseline, haemophilia B patients did not differ from controls. After two years, T helper cells showed an increase (p = 0.028), while a rise in B cells approached statistical significance (p = 0.063). Haemophilia A patients showed increased numbers of activated non-B lymphocytes (p = 0.003) and lowered numbers of B cells (p = 0.001) at baseline. After two years activated non-B lymphocytes decreased (p = 0.004), as did the CD4/CD8 ratio (p = 0.002), due to increasing numbers of CD8 positive cells (p = 0.087).

Our data suggest minor inhibition of the immune system in haemophilia B patients, which recovers after changing therapy to a monoclonally purified product. These findings contrast with the excessive immune stimulation in haemophilia A. The observed differences might be due to the administered concentrates.
Observer and Biological Variation of a Rapid Whole Blood D-Dimer Test
Authors: van Beek Edwin , Büller Harry , Turkstra Franktien
Published: March 2018

In consecutive patients with suspected venous thromboembolism the interobserver variability of the SimpliRED D-dimer test was evaluated by two observers who independently scored one plate, the between assay variation was performed simultaneously by a third independent observer, who assessed a second plate. The biological variation was studied, 1-4 hours later by an independent evaluation.

A total of 155 patients entered the study, venous thromboembolism was present in 42 patients (28%). The interobserver variability was 2/83 samples, with a kappa of 0.95 (95% confidence interval 0.88-1.0). The between assay variation was 2/98, with a kappa value of 0.96 (95% confidence interval 0.90-1.0). When testing the biological variation the observers disagreed in 2 of 69 patients (3%).

The SimpliRED D-dimer assay has a good to excellent interobserver variability, between assay variation and reproducibility.
Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain
Authors: Takamiya Osamu
Published: March 2018

Murine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.
Pharmacokinetics, Pharmacodynamics and Tolerability of a Potent, Non-peptidic, GP IIb/IIIa Receptor Antagonist following Multiple Oral Administrations of a Prodrug Form
Authors: Modi Nishit , Bullens Sherron , Pater Cheryl , Lipari Michael , Robarge Kirk , Blackburn Brent , Beresini Maureen , Weller Thomas , Steiner Beat , Bunting Stuart , Refino Canio
Published: March 2018

Ro 44-3888 is a potent and selective antagonist of GP IIb/IIIa. Following IV administration to rhesus monkeys, the (mean ± SD.) clear ance, volume of distribution and terminal half-life of Ro 44-3888 were 4.4 ± 1.8 ml/min/kg, 0.8 ± 0.4 l/kg and 2.5 ± 0.8 h respectively. Oral administration of Ro 48-3657 (1 mg/kg), a doubly protected prodrug form, produced peak concentrations of Ro 44-3888 (152 ± 51 ng/ml), 4.2 ± 2.2 h after dosing. Terminal half-life and estimated bioavailabil ity were 5.1 ± 1.6 h and 33 ± 6% respectively. No effect on blood pressure, heart rate or platelet counts were seen. Adenosine diphosohate (ADP) induced platelet aggregation (PA) and cutaneous bleeding times (CBT) were determined prior to and after the last of 8 daily oral administrations of Ro 48-3657 (0.25 or 0.5 mg/kg) to eight rhesus monkeys. Peak and trough plasma concentrations were proportional to dose and steady state was achieved after the second administration. Inhibition of PA and prolongation of CBT were concentration dependent. The ex vivo IC50 (82 nM) for ADP-mediated PA correlated with a value (58 nM) determined in vitro. The CBT response curve was displaced to the right of the PA curve. CBT was prolonged to ≥25 min when levels of Ro 44-3888 exceeded 190 nM and PA was >90% inhibited. Therefore, in rhesus monkeys, Ro 48-3657 is reproducibly absorbed and converted to its active form, is well tolerated, and has a concentration-dependent effect on PA and CBT. These properties make Ro 48-3657 an attractive candidate for evaluation in patients at high risk for arterial thrombosis.
Association of a Common Polymorphism in the Factor XIII Gene with Myocardial Infarction
Authors: Kohler Hans , Stickland Max , Ossei-Gerning Nicholas , Carter Angela , Mikkola Hanna , Grant Peter
Published: March 2018

Factor XIII when activated by thrombin, crosslinks fibrin, however its role in thrombotic disorders is unknown. A common point mutation (G T) in exon 2 of the A-subunit gene which codes for an amino acid change three amino acids from the thrombin activation site (Factor XIIIVal34Leu) is a candidate for a role in the pathogenesis of acute myocardial infarction. Factor XIII genotype frequencies were determined in a case-control study of 398 caucasian patients and 196 healthy controls. Patients had undergone angiography for investigation of coronary artery disease and were evaluated for a history of myocardial infarction. The prevalence of the mutation was lower in patients with myocardial infarction than without (32% vs. 50%), p = 0.0009 and than in controls (32% vs. 48%), p = 0.005. Patients possessing the mutation with a history of myocardial infarction had higher PAI-1 concentrations (mean, 27.9 vs. 16.7 ng/ml, p = 0.004) and the PAI-1 4G/4G genotype was commoner (43% vs. 26%, p = 0.03). There was no difference in PAI-1 4G/4G genotype (33% vs. 32%) and PAI-1 levels (mean, 21.0 vs. 20.9 ng/ml) in patients possessing wild type with MI compared to those without MI. These results indicate that the G T mutation coding for factor XIIIVal34Leu is protective against myocardial infarction and suggest a mechanism whereby elevated levels of PAI-1 may contribute to vascular risk.