bluuebunny/crossref_metadata_2025 huggingface dataset:
Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis
Authors: Vischer Ulrich M , Wollheinn Claes B
Published: July 2018

von Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.
Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on Tissue Factor Pathway Inhibitor: In Vitro and In Vivo Studies
Authors: Nagase Hideki , Enjyoji Kei-ichi , Kamikubo Yu-ichi , Kitazato Keiko T , Kitazato Kenji , Saito Hidehiko , Kato Hisao
Published: July 2018

Depolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicusSelenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor Vila by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor Vila inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitroactivity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.
Identification of PDGF Receptors on Human Megakaryocytes and Megakaryocytic Cell Lines
Authors: Yang M , Khachigian L M , Hicks C , Chesterman C N , Chong B H
Published: July 2018

Platelet-derived growth factor (PDGF) is a potent chemotactic and mitogenic factor implicated to play important roles in a variety of normal and pathophysiologic settings. We investigated PDGF receptor expression on human megakaryocytes and several megakaryocytic cell lines (CHRF, DAMI, Meg-01, M-07e) using enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunocytochemical staining. Both PDGF receptor subtypes were identified on CHRF, DAMI, and Meg-01 cells by ELISA; PDGF beta-receptor levels exceeded alpha-receptor levels. Flow cytometry revealed that beta-receptor levels on CHRF and DAMI cells exceeded those on Meg-01 cells, and that M-07e expressed neither receptor. Immunocytochemical staining confirmed these findings and determined that bone marrow megakaryocytes also expressed PDGF receptors. Exposure of megakaryocytes to PDGF-BB dramatically induced the expression of the immediate-early gene, c-fos, within 30 min. Moreover, PDGF-BB significantly stimulated CHRF proliferation and colony formation. The present study demonstrates the presence of functional PDGF receptors on human megakaryocytes and their ability to mediate a mitogenic response.
Platelet Counts and Aggregation Measures in the Incidence of Ischaemic Heart Disease (IHD)
Authors: Meade Thomas W , Cooper Jackie A , Miller George J
Published: July 2018

Although studies in those who have already experienced clinical episodes of ischaemic heart disease (IHD) have suggested properties of platelets influencing recurrence, there is limited information on the value of platelet tests in predicting first episodes of IHD. One study has suggested that a raised platelet count and increased aggregability in response to adenosine diphosphate (ADP) may increase IHD incidence but the numbers of IHD events involved were small. The larger Northwick Park Heart Study (NPHS) included platelet count and both ADP and adrenaline-induced aggregation and this paper presents their associations with subsequent IHD. Platelet counts were performed in 1369 white NPHS men aged between 40 and 64 at recruitment, of whom 181 subsequently experienced a major episode of IHD over a follow-up period of 16.1 years. Platelet count was unrelated to the incidence of IHD. ADP-induced aggregation was performed in a random sample of 740 men in whom 66 IHD events occurred during the subsequent 10.1 years, aggregability being measured both as ED50, the ADP dose at which aggregation occurred at half its maximum velocity, and also as EMR, the maximum rate of aggregation achieved. Neither measurement showed any association with IHD incidence, nor did similar measurements in 460 men in whom adrenaline-induced aggregation was also carried out. There are at least three possible explanations for the lack of any association between the measures of aggregability used and IHD. First, the large within-person variability of platelet aggregation tests may make the demonstration of any associations difficult, though the study had reasonable power to show effects with ADP. Secondly, the tests used may not be a valid index of the contribution of platelet function to thrombosis and IHD. However, the clear effect of several personal and demographic influences associated with IHD on the tests used brings this explanation into question. Thirdly, the role of platelets in thrombogenesis may be determined mainly by plasma influences such as fibrinogen, rather than by intrinsic properties of platelets themselves. Platelet counts within the physiological range and the aggregation tests used in this and in some other studies are of no value as indices of the risk of first episodes of IHD.
Relationship of Anti β2-giycoprotein I and Anti Prothrombin Antibodies to Thrombosis and Pregnancy Loss in Patients with Antiphospholipid Antibodies
Authors: Forastiero Ricardo R , Martinuzzo Marta E , Cerrato Graciela S , Kordich Lucía C , Carreras Luis O
Published: July 2018

The lupus anticoagulant (LA) and anticardiolipin antibodies (aCL) are clinically relevant because of their association with thrombosis and pregnancy loss. The group of antiphospholipid antibodies (aPL) includes antibodies primarily directed against various phospholipid-binding proteins, mainly β2-glycoprotein I (β2GPI) and prothrombin. Some studies suggest that there is an association between the presence of anti β2GPI antibodies (a(β2GPI) of IgG isotype and thrombosis. Therefore, aPL defined according to the plasma protein to which they are directed appear to be more appropriate for the evaluation of their clinical importance. Using home-made ELISAs we evaluated the presence of a(β2GPI and antiprothrombin antibodies (anti-II) of both isotypes (IgG and IgM) in a group of 233 patients with LA and/or aCL. Forty-four women had a history of pregnancy loss, 45 patients had a history of venous thrombosis (VT) and 32 of arterial thrombosis (AT). Patients from the autoimmune group (systemic lupus erythematosus and antiphospholipid syndrome) had a higher prevalence of aβ2GPI and/or anti-II than those from the miscellaneous group. In the univariate analysis, a significant association was shown between the presence of aβ2GPI-IgG (OR 3.2; 95% Cl 1.5-6.6) and previous VT, but not AT. Anti-II were related to VT but the multivariate analysis showed that aβ2GPI-IgG are the only independent risk factor for VT (OR 3.0; 95% Cl 1.3-6.2). The presence of aβ2GPI-IgM correlates well with a history of pregnancy loss (OR 2.6; 95% Cl 1.1-6.1). The coagulation tests profile showed that the clotting assays were more prolonged in patients having aCL, a(β2GPI or anti-II. But a higher prevalence of abnormal results was only found for the dilute Russell viper venom time in patients with VT, as compared to those without thrombosis (94.4% vs. 58.7%, p <0.02). The measurement of aβ2GPI of both isotypes could help to identify aPL-positive patients with a higher risk for thrombosis and pregnancy loss, although this association should be confirmed by prospective studies.
Absence of Inhibitors in Previously Untreated Patients with Severe Haemophilia A after Exposure to a Single Intermediate Purity Factor VIII Product
Authors: Yee T T , Williams M D , Hill F G  H , Lee C A , Pasi K J
Published: July 2018

Use of high purity and recombinant factor VIII (FVIII) concentrates has been thought to be associated with an increased incidence of FVIII inhibitors in patients with severe haemophilia A. Comparison with comparable historical control groups has suggested that the true incidence of inhibitors in patients with severe haemophilia A was ~20-25%, similar to the incidence seen with new high purity and recombinant FVIII products.

We have conducted a study of inhibitor development in a cohort of 37 boys with severe haemophilia A (VIII: C <2 u/dl) exposed only to a single FVIII concentrate (BPL 8Y) with no previous blood or blood product exposure. This factor VIII concentrate is an intermediate purity product with a specific activity of ~2 IU/mg protein and contains well preserved von Willebrand factor multimers. It is manufactured by conventional fractionation technologies and terminally dry heat treated at 80° C for 72 h.
Synergistic Cofactor Function of Factor V and Protein S to Activated Protein C in the Inactivation of the Factor Villa – Factor IXa Complex
Authors: Shea Lei , He Xuhua , Dahlbäck Björn
Published: July 2018

Human factor V has been shown not only to be a precursor to procoagulant factor Va but also to express anticoagulant properties. Thus, factor V was recently found to potentiate the effect of protein S as cofactor to activated protein C (APC) in the inactivation of the factor VIIIa-factor IXa complex. The purpose of this study was to determine whether the APC-cofactor function of factor V was also expressed in the bovine protein C system and to elucidate the molecular background for the species specificity of APC. For this purpose, the effects of protein S and factor V on APC-mediated inactivation of factor VIIIa were studied using purified APC, protein S and factor V of human and bovine.origin. The factor VIIIa investigated here was part of a Xase complex (i.e. factor IXa, factor VIIIa, phospholipid and calcium) and the APC-mediated inhibition of factor VIIIa was monitored by the ability of the Xase complex to activate factor X. Synergistic APC-cofactor function of factor V and protein S was demonstrated in the bovine system. The effect of bovine APC was potentiated by bovine protein S but not by human protein S, whereas both human or bovine protein S stimulated the function of human APC. Factor V did not express species specificity in its APC-cofactor activity even though bovine factor V was more potent than its human counterpart. Recombinant human/bovine protein S chimeras were used to demonstrate that the thrombin sensitive region and first epidermal growth factor-like module of protein S determine the species specificity of the APC-protein S interaction. In conclusion, both human and bovine factor V were found to express APC-cofactor activity which depends on the presence of protein S. The species specificity of APC was shown to be caused by the interaction between APC and protein S.
Isolation and Characterization of Mouse Coagulation Factor X – Biophysical and Enzymological Properties
Authors: James H L , Larson P J , Chao Y B , Yan S B , Kim D J
Published: July 2018

Mouse factor X was highly purified from plasma using barium ion precipitation and chromatography on anion-exchange and heparin- agarose affinity chromatography columns. Intact and reduced patterns of mouse factor X in SDS-PAGE were similar to those of human factor X. The specific absorption E 1%/1 cm at 280 nm of mouse factor X was found to be 11.2. Content of carbohydrate moieties of mouse factor X, determined to be 10% by weight, differs both quantitatively and quantitatively from that of human factor X, while the γ-carboxy- glutamic acid (Gla) and β-hydroxy-aspartic acid (β-OH-Asp) content were essentially the same as for human factor X. The amino-terminal amino acid sequences of the light and heavy chains of mouse factor X separated by SDS-PAGE were ANSFF--FKK and SVALXTSDSE, respectively. Underlined residues are non-identical with those of human factor X. Clotting time-based assays using human factor X- deficient plasma as substrate exhibited the following apparent extents of activation of factor X in mouse plasma, using human plasma as the standard: 195% (intrinsic); 200% (extrinsic); and 190% (RVV-X). Using the purified proteins in the same assay systems, the following apparent activation of mouse factor X was demonstrated, compared with human factor X: 195% (intrinsic); 27% (extrinsic); and 41% (RVV-X). These activity profiles suggest that the human extrinsic coagulation pathway functions less efficiently than the corresponding mouse pathway in the activation of mouse factor X. Furthermore, mouse brain thromboplastin satisfactorily replaced rabbit brain thromboplastin in extrinsic activation of factor X in mouse plasma, but not of human plasma or purified mouse or human factor X, in line with studies by others suggesting that human factor Vila poorly activates factor X in the presence of mouse tissue factor. While fully RVV-X-activated mouse factor X activated human prothrombin at a rate equal to about 117% of that for human factor X, it hydrolyzed the synthetic substrate, S-2222, at a rate of only about 18% of that for human factor X. These results are expected to be useful in making the mouse suitable for study of the mammalian blood coagulation pathways.
Platelet Adhesion to Fibrinogen, Fibrin Monomer, and Fibrin Protofibrils in Flowing Blood - The Effect of Fibrinogen Immobilization and Fibrin Formation
Authors: Jiroušková Markéta , Dyr Jan Evangelista , Suttnar Jiří , Holada Karel , Trnková Bohuslava
Published: July 2018

Platelet fibrin(ogen) adhesive interactions were investigated in whole citrated blood using the rectangular perfusion chamber at wall shear rates of 300 and 1600s1 with regard to the amount and structure of immobilized protein. Only single platelets adhered to adsorbed fibrinogen at both low and high surface fibrinogen concentrations and at 1600 s1 almost no adhesion was observed. When using spray-immobilized protein, platelet adhesion was significantly higher than to ad sorbed protein. Conversion of adsorbed fibrinogen to fibrin monomer resulted in the formation of pronounced platelets aggregates and with the elevation of wall shear rate 50% decrease of adhesion took place. Degree of platelet adhesion to fibrin monomer was significantly influenced by immobilized protein concentration at both shear rates. However, the morphology (small and dense platelet aggregates) and extent of platelets adhered to fibrin pentamer was nearly the same at both shear rates. Starting with surface-bound fibrinogen and alternating addition of thrombin and fibrinogen fibrin pentamer was prepared using the stepwise synthesis. This methodology is based on the observation that at low concentration immobilized fibrin monomer binds fibrinogen in 1:1 molar ratio. The gradually formed fibrin of a defined size and composition can be a useful tool in the further understanding of the role of fibrin architecture in the pathophysiology of thrombosis.
A Novel Thrombomodulin Gene Mutation in a Patient Suffering from Sagittal Sinus Thrombosis
Authors: Norlund Lena , Zöller Bengt , Öhlin Ann-Kristin
Published: July 2018

Thrombomodulin is an endothelial cell membrane glycoprotein that promotes protein C activation. It has been clearly demonstrated that the anticoagulant functions of the protein C system are important in the prevention of thromboembolic disease. Patients with protein C or protein S deficiency and/or resistance to activated protein C (APC resistance) are at higher risk for developing thromboembolic disease. The first mutation in the thrombomodulin gene was discovered in an American patient suffering from pulmonary embolism at the age of 45 (Öhlin and Marlar 1995). Here we report a case of sagittal sinus thrombosis in a 42-year-old Swedish woman. She was found to carry a heterozygous point mutation changing G127 to A, predicting an Ala25 to a Thr change in the mature thrombomodulin protein. This mutation was also found in her 16-year-old daughter, who so far has not suffered from any thrombotic events. The patient had no other detectable prothrombotic genetic defects associated with the coagulation system. This case supports the hypothesis of an association between mutations in the thrombomodulin gene and venous thrombosis.
Thrombin Generation Measured Ex Vivo Following Microvasculor Injury Is Increased in SLE Patients with Antiphospholipid-protein Antibodies
Authors: Musiał Jacek , Swadźba Jakub , Jankowski Miłosz , Grzywacz Marek , Bazan-Socha Stanisława , Szczeklik Andrzej
Published: July 2018

Antiphospholipid-protein antibodies (APA) include lupus-type anticoagulant (LA) and antibodies recognizing complexes of anionic phospholipids (e.g. cardiolipin) and proteins (e.g. prothrombin and (β2-glycoprotein I). The presence of APA is associated with an increased risk of both arterial and venous thrombosis. However, the pathogenic mechanism leading to thrombosis in patients with APA remains unclear. We studied 32 patients with systemic lupus erythematosus (SLE) who were divided into two groups depending on the presence (n = 19) or absence (n = 13) of APA. Healthy volunteers (n = 12) matched by age and sex served as controls. In all subjects LA and IgG class anticardiolipin antibodies (ACA) were determined. Thrombin generation was monitored ex vivo measuring fibrinopeptide A (FPA) and prothrombin fragment F1 + 2 (F1 + 2) in blood emerging from a skin microvasculature injury, collected at 30 second intervals. In subjects with antiphospholipid antibodies mean FPA and F1 + 2 concentrations were signiF1cantly higher at most blood sampling times than in controls. In some SLE patients with APA the process of thrombin generation was clearly disturbed and very high concentrations of F1brinopeptide A were detected already in the F1rst samples collected. Two minutes after skin incision SLE patients without APA produced slightly more FPA, but not F1 + 2, as compared to healthy subjects. Mathematical model applied to analyze the thrombin generation kinetics revealed that APA patients generated signiF1cantly greater amounts of thrombin than healthy controls (p = 0.02 for either marker). In contrast, in the same patients generation of thrombin in recalciF1ed plasma in vitro was delayed pointing to the role of endothelium in the phenomenon studied. In summary, these data show for the F1rst time that in SLE patients with antiphospholipid-protein antibodies thrombin generation after small blood vessel injury is markedly increased. Enhanced thrombin generation might explain thrombotic tendency observed in these patients.
Risk Factors for Pregnancy Associated Venous Thromboembolism
Authors: McColl Mark D , Ramsay Jane E , Tait R C , Walker Isobel D , McCall Frances , Conkie J A , Carty M J , Greer I A
Published: July 2018

In an attempt to reduce the incidence of pregnancy associated venous thromboembolism (PA-VTE), some researchers have advocated screening of all women for the factor VLeiden mutation during early pregnancy. We have conducted a large retrospective study (over 72,000 deliveries) to determine if this would be useful. Sixty-two objectively confirmed venous thrombotic events (51 DVT, 11 PE) were recorded at two maternity units in the UK. The incidence of DVT was 0.71 per 1000 deliveries (95% CI 0.5-0.9) with 0.50 occurring in the antenatal period (95% CI 0.34-0.66) and 0.21 in the puerperium (95% CI 0.11-0.31). The incidence of PE was 0.15 per 1000 deliveries (95% CI 0.06-0.24), 0.07 antenatal (95% CI 0.01-0.13) and 0.08 in the puerperium (95% CI 0.02-0.14). Of these 62,50 attended for follow-up and thrombophilia screening. 28% of all episodes of PA-VTE had no CIinical risk factor for thrombosis or an identifiable thrombophilic abnormality. Deficiency of antithrombin was identified in 12% of individuals (95% CI 3-21) and the factor VLeiden mutation in 8% (95% CI 0.5-15.5). Based on estimates of the prevalence of the factor VLeiden mutation in the population, we estimate that the thrombotic risk for a woman during pregnancy or the puerperium with the defect is approximately 1 in 400-500. This figure would not lend support to the idea of random screening for the mutation in early pregnancy.
Bleeding Classification in Clinical Trials: Observer Variability and Clinical Relevance
Authors: Graafsma Yvonne P , Prins Martin H , Lensing Anthonie W A , de Haan Rob J , Huisman Menno V , Büller Harry R
Published: July 2018

To evaluate the bleeding classification in a recent trial on venous thrombosis treatment, a selection of reported bleeding episodes was adjudicated twice by an independent committee and graded by the treating physician and independent clinical experts on the clinical severity and impact on the patient’s life.

The kappa values for the dichotomy major bleeding versus minor or no bleeding were 0.79 (95% CI, 0.57-1.0) for the agreement between the two members of the adjudication committee and 0.77 (95% CI, 0.52-1.0) for the agreement between both adjudication sessions. The kappa values for the dichotomy major or minor bleeding versus no bleeding were 0.42 and 0.44. The weighted kappa values for the agreement between the treating physician and the independent experts were 0.76 for the Clinical severity and 0.79 for the impact on the patient’s life (95% CI, 0.63-0.88 and 0.70-0.89). The association between the adjudication result expressed as major bleeding or minor or no bleeding and the Clinical grading by the treating physician resulted in an ROC curve with an area under the curve of 0.98 for the Clinical severity and 0.99 for the impact on the patient’s life. The dichotomy major or minor bleeding versus no bleeding resulted in areas under the curve of 0.70 and 0.66.

In conCIusion, the applied criteria for major bleeding are reproducible and Clinically relevant. The criteria for minor bleeding are not reproducible and are less associated with the observed Clinical relevance.
Ultrasensitive Fluorogenic Substrates for Serine Proteases
Authors: Butenas Saulius , DiLorenzo Maria E , Mann Kenneth G
Published: July 2018

Selective, sensitive assays for the quantitation of serine proteases involved in coagulation and fibrinolysis have been developed employing fluorogenic substrates containing a 6-amino-1-naphthalenesulfonamide leaving group (PNS-substrates). Over one hundred substrates were evaluated for hydrolysis by the serine proteases of blood coagulation and fibrinolysis, and substrate structure-efficiency correlations were examined. PNS-substrates which contain Lys in the P1 position are specific for Lys-plasmin and are either not hydrolyzed or hydrolyzed at a relatively low rate by factor Xa, thrombin, or urokinase-type plasminogen activator (uPA). These substrates allow quantitation of Lys-plasmin at concentrations as low as 1 pM. Eighteen of over 90 substrates tested for factor XIa are hydrolyzed by this enzyme at a relatively high rate reaching a kcat value of 170 s-1 and allowing quantitation of factor XIa at 10 fM. Eighteen of almost 90 PNS-substrates tested display high specificity for thrombin, some exceeding that for factor Xa by > 10,000-fold and > 100-fold for activated protein C (APC). Seven of these substrates have a over 100 s-1 and three of them have a KM below 1 μM. They allow the quantitation of thrombin at concentrations as low as 20 fM. For APC, uPA and the factor Vila/tissue factor complex, quantitation is feasible at 1 pM concentration. For factor Xa and factor VIIa the limits are 0.4 pM and 40 pM respectively. The PNS-substrates presented in this study may be employed for the development of direct and sensitive serine protease assays.
Effects of Plasma Kallikrein Specific Inhibitor and Active-site Blocked Factor VIIa on the Pulmonary Vascular Injury Induced by Endotoxin in Rats
Authors: Uchiba Mitsuhiro , Okajima Kenji , Murakami Kazunori , Okabe Hiroaki , Okamoto Shosuke , Okada Yoshio
Published: July 2018

The acute respiratory distress syndrome (ARDS) is a serious complication of sepsis. To evaluate the role of the coagulation system in the pathogenesis of ARDS in sepsis, we examined the effects of the administration of a synthetic plasma kallikrein specific inhibitor (PKSI) and of active-site blocked factor VIIa (DEGR-VIIa) on the pulmonary vascular injury induced by E. coli endotoxin (ET) in rats. Administration of PKSI prevented the pulmonary vascular injury induced by ET as well as pulmonary histological changes in animals administered ET, but it did not affect the intravascular coagulation. The opposite effect was seen with DEGR-VIIa, which prevented the intravascular coagulation but not the pulmonary vascular injury. PKSI did not inhibit the activation of the complement system induced by ET leading to the activation of neutrophils.

Findings suggest that PKSI may prevent the pulmonary vascular injury induced by ET by inhibiting kallikrein, which activates the neutrophils. The intrinsic pathway of coagulation may be more important than the extrinsic pathway in the pulmonary vascular injury produced byET.
A Fast and Robust Dual-label Nonradioactive Oligonucleotide Ligation Assay for Detection of Factor V Leiden
Authors: Chakravarty Anders , Hansen Torben Stiig , Hørder Mogens , Kristensen Søren Risom
Published: July 2018

Activated protein C resistance is in almost all cases caused by the factor V Leiden mutation (FV:R506Q). Due to the high prevalence and clinical significance of the mutation reliable methods suited for processing large sets of samples are in demand. We here present the oligonucleotide ligation assay (OLA) with lanthanide labeled oligonucleotides for the detection of FV Leiden. The assay is based on time resolved fluorescence measurement of lanthanide labeled oligonucleotides (DELFIA: Delayed Enhanced Lanthanide Fluorescence Immuno Assay) and on the specificity of T-4 DNA Ligase to join two adjacent oligonucleotides only when the two are complementary to the PCR template at the ligation junction. The Europium/Samarium fluorescence pattern is specific for each of the three genotypes (G/G, G/A, A/A) and clearly separates the three genotypes. By using a wildtype probe (Samarium labeled) and a mutant-specific probe (Europium labeled) simultaneously an internal control of the assay is included in each reaction. The assay is simple to perform, can be partly automated and is ideal for processing large sets of samples.
The Regulation of Platelet Aggregation In Vitro by Interleukin-1 β and Tumor Necrosis Factor-α: Changes in Pregnancy and in Pre-eclampsia
Authors: Bar J , Zosmer A , Hod M , Elder M G , Sullivan M H F
Published: July 2018

Platelet activation occurs in early pregnancy in women at risk of developing pre-eclampsia. Cytokines have been implicated in the pathogenesis of pre-eclampsia, so we determined the effects of interleukin- 1β (IL-1β) and tumor necrosis factor-α (TNF-α) on the in vitro aggregation of human platelets. IL-1β increased aggregation of platelets from non-pregnant and pre-eclamptic women, and inhibited the aggregation of platelets from normal pregnant women. This latter effect was linked to a diminished P-selectin expression on ADP-stimulated whole blood platelets in normal pregnant women (p = 0.011). Platelet aggregation in response to ADP was found to be inhibited after preincubation with TNF-α in non-pregnant (38%, p = 0.01) and in normal pregnant women (54%, p = 0.001) and not affected in pre-eclamptic women. The inhibitory effects of TNF-α were mediated through the P75 receptor for TNF-α.
Sensitive Measurement of Thrombopoietin by a Monoclonal Antibody Based Sandwich Enzyme-linked Immunosorbent Assay
Authors: Folman Claudia C , von dem Borne Albert E G K , Rensink Irma H J A M , Gerritsen Winald , van der Schoot C Ellen , de Haas Masja , Aarden Lucien
Published: July 2018

In this report a sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of plasma thrombopoietin (Tpo) is described that is solely based on monoclonal antibodies (MoAbs).

The assay has an intra and inter-assay variance of 5-7% and 7-13%, respectively. Native and recombinant human Tpo (rhTpo) were recognized equally well, no cross reactivity with other cytokines was found and rhTpo added to plasma and serum was completely recovered. With the ELISA, Tpo concentrations in EDTA-anticoagulated plasma of all controls (n = 193) could be determined, since the limit of detection (2 ± 0.8 A.U./ml, mean ± sd) was lower than the concentration found in controls (11 ± 8 A.U./ml, mean ± sd; 2.5th-97.5th percentile: 4-32 A.U./ml). Tpo levels in serum were on average 3.4 times higher than in plasma.

We showed in vivo that Tpo is bound by platelets, as in thrombocytopenic patients (n = 5) a platelet transfusion immediately led to a drop in plasma Tpo level, whereas in patients receiving chemotherapy the induced thrombocytopenia was followed by a rise in plasma Tpo levels.

In summary, these results indicate that this ELISA is a reliable tool for Tpo measurements and is applicable for large scale studies.
The Importance of Enzyme Inhibition Kinetics for the Effect of Thrombin Inhibitors in a Rat Model of Arterial Thrombosis
Authors: Elg Margareta , Gustafsson David , Deinum Johanna
Published: July 2018

The relation between the antithrombotic effect in vivo, and the inhibition constant (K
i) and the association rate constant (k
on) in vitro was investigated for eight different thrombin inhibitors. The carotid arteries of anaesthetized rats were exposed to FeCl3 for 1 h, and the thrombus size was determined from the amount of incorporated 125I- fibrinogen. The thrombin inhibitors were given intravenously, and complete concentration- and/or dose-response curves were constructed. Despite a 50,000-fold difference between the k
i-values comparable plasma concentrations of hirudin and melagatran were needed (0.14 and 0.12 μmol 1-1, respectively) to obtain a 50% antithrombotic effect (IC50) in vivo. In contrast, there was a comparable in vitro (k
i-value) and in vivo (IC50) potency ratio for melagatran and inogatran, respectively. These results can be explained by the concentration of thrombin in the thrombus and improved inhibition by the low-molecular-weight compounds. For all eight thrombin inhibitors tested, there was an inverse relationship between k
on-values in vitro and the slope of the dose response curves in vivo. Inhibitors with k
on-values of <1 X 107 M-1 s-1 gave steep dose response curves with a Hill coefficient >1. The association time for inhibition of thrombin for slow-binding inhibitors will be too long to give effective antithrombotic effects at low plasma concentrations, but at increasing concentrations the association time will decrease, resulting in a steeper dose-response curve and thereby a more narrow therapeutic interval.
Free Protein S Deficiency Is a Risk Factor for Venous Thrombosis
Authors: Faioni Elena M , Valsecchi Carla , Palla Alessandra , Taioli Emanuela , Razzari Cristina , Mannucci Pier Mannuccio
Published: July 2018

A recent study suggests that protein S deficiency is not a risk factor for venous thrombosis. Since this unexpected finding would have important clinical implications if confirmed, we performed a case-control study with the aim to determine the prevalence of protein S deficiency in patients with thrombosis and in healthy individuals taken from the general population and the relative risk of thrombosis in protein S-deficient patients. Free protein S concentration was measured in 327 consecutive patients with at least one venous thrombotic episode and in 317 age- and sex-matched control individuals. Different normal reference ranges were obtained and adopted for men and women. Protein S deficiency was found in 3.1% (95% Cl: 1.5-5.2) of patients and in 1.3% of controls (95% Cl: 0.3-2.8). Ten patients and 4 control subjects had protein S deficiency, which determined a relative risk of thrombosis (sex- and age-adjusted odds ratio) of 2.4 (95% Cl: 0.8-7.9). When men and women were analyzed separately, the risk was 5.0 (95% CI: 0.6-43.6) and 1.6 (95% Cl: 0.4-6.7) respectively. PS-deficient men had more thrombotic episodes than women and later in life. Multivariate analysis established that sex was an independent determinant of the number of episodes, as was age, while PS deficiency was not. However sex and PS deficiency status were both determinants of age at first thrombotic episode.
Prevalence of the Prothrombin Gene Variant (nt20210A) in Venous Thrombosis and Arterial Disease
Authors: Arruda Valder R , Annichino-Bizzacchi Joyce M , Gonçalves Marilda S , Costa Fernando F
Published: July 2018

The prothrombin gene variant resulting form a G→A transition at position 20210 has been described as a common genetic risk factor for venous thrombosis. However, the risk for developing arterial disease is unknown. In this investigation, we studied 116 patients with venous thrombosis and 71 with arterial disease, all of whom were compared with 295 controls. Additionally, we also investigated the distribution of the prothrombin alleles among African descendents and Amazonian Indians from Brazil. The prevalence of 0.7% for 20210A allele in the control group increased to 4.3% (P = 0.021) among patients with venous thrombosis. There was also a high prevalence of the mutated allele in a selected arterial disease group (5.7%) without hyperlipoproteinemia, hypertension, and diabetes mellitus when compared to the controls (P = 0.013). Heterozygotes for the allele 20210A were common among individuals of African descent (2%) and rare among Indians. These data support the hypothesis that the prothrombin variant is a risk factor for venous thrombosis and suggest that it may also be a risk factor for arterial disease.
Electrospray Ionisation Mass Spectrometry Facilitates Detection of Fibrinogen (Bβ 14 Arg → Cys) Mutation in a Family with Thrombosis
Authors: Brennan Stephen O , Hammonds Bruce , Spearing Ruth , George Peter M
Published: July 2018

We report the first direct detection of a fibrinogen mutation by electrospray ionisation mass spectrometry. The propositus, from a family with a history of thrombosis, came to attention after a pulmonary embolism subsequent to a spontaneous abortion. Prolonged thrombin (41 s) and reptilase times (26 s) together with an impairment of fibrinopeptide B release suggested a mutation at the thrombin cleavage site of the Bβ chain. Direct mass analysis of purified fibrin chains from a thrombin induced clot showed that 50% of the Bβ chains remained uncleaved. The measured mass of the mono sialo isoform of this uncleaved chain was 54150 Da, compared to a value of 54198 Da for normal Bp chains. This decrease of 48 Da in the intact protein is indicative of either a Bβ 14 Arg to Cys, or Arg to Leu substitution. Heterozygosity for the Bβ 14 Arg → Cys mutation was verified by PCR amplification and DNA sequence analysis.
Erythropoietin Administration Increases Production and Reactivity of Platelets in Dogs
Authors: Wolf Roman F , Peng Jinpeng , Friese Paul , Gilmore Laura S , Burstein Samuel A , Dale George L
Published: July 2018

Administration of erythropoietin (EPO) to adult dogs resulted in a dramatic increase in the number of thiazole orange-positive (TO+) platelets, also referred to as reticulated platelets. Pre-treatment level of TO+ platelets was 6.2 ± 0.5% (mean ± 1 SE; n = 5); following day 5 of treatment with 500 U EPO/kg/day, the percentage of TO+ platelets peaked at 16.8 ± 2.3% (n = 5; p <0.02). After cessation of the hormone, the number of TO+ platelets fell rapidly to below starting levels. Unexpectedly, there was a significant decline in total platelet count during EPO administration despite an increased level of TO+ platelets. To assess platelet reactivity, total platelets and TO+ platelets from EPO- treated dogs were analyzed for thrombin-responsiveness as quantitated by P-selectin expression on the cell surface; reactivity was expressed as a thrombin EC50, the thrombin concentration required to activate 50% of platelets. Both total and TO+ platelets were hyperreactive during EPO treatment when compared either to pre-treatment values or to control animals. Thrombin EC50 values for total and TO+ platelets on day 5 fell to 66.5 ± 5.4% (mean ± 1 SE; n = 5; p <0.02) and 62.2 ± 8.7% (n = 5; p <0.025), respectively, of pre-treatment levels. These data indicate that EPO not only promotes the synthesis of increased numbers of TO+ platelets in the dog but that these newly produced platelets are hyperreactive when compared to TO+ platelets from control animals.
Peptide-specific Antibodies as Pro\bes of the Topography of the Ca2+-binding Motifs in αIIbβ3
Authors: Niewiarowska Jolanta , Świderska Mariola , Majewski Tadeusz , Cierniewski Czesław S
Published: July 2018

Antibodies against synthetic peptides corresponding to four Ca2+- binding motifs of the αIIb subunit have been obtained and used as molecular probes to analyze the topography of the αIIbβ3 complex. The specificity of the antibodies has been characterized by ELISA and Western immunoblotting in terms of binding capacity and affinity to the isolated αIIbβ3 and its αIIb subunit. Our data suggest that: (a) all four Ca2+-binding motifs of the αIIb are partially exposed on the surface of the intact molecule and accessible to antipeptide antibodies. However, they are not in close vicinity to the ligand recognition domain since the antibodies do not produce complete inhibition of platelet aggregation, (b) The conformation of amino acid stretches which form the second Ca2+-binding motif of αIIb is particularly dependent upon the presence of cation, and this region undergoes significant conformational alterations upon Ca2+ expulsion.
Formation of a macroscopically occupied polariton state in a tunable open-access microcavity under resonant excitation
Authors: Giriunas L. , Li Feng , Sich M. , Cancellieri E. , Emmanuele R. P. A. , Trichet A. A. P. , Farrer I. , Ritchie D. A. , Smith J. M. , Whittaker D. M. , Skolnick M. S. , Krizhanovskii D. N.
Published: July 2018

We experimentally demonstrate the formation of a macroscopically occupied state (condensation) of exciton-polaritons in a tunable 3D open-access microcavity system under resonant excitation. We observe a high conversion efficiency of polaritons from the pumped state to the ground mode, leading to the population of the condensate 1.5 times greater than that of the driven state for a total number of polaritons of ∼110. Comparative numerical simulations show that coherent polariton-polariton scattering mechanisms alone cannot explain the observed experimental results and that other relaxation channels play an important role.
Interdot spin transfer dynamics in laterally coupled excited spin ensemble of high-density InGaAs quantum dots
Authors: Hiura Satoshi , Takeishi Kazuki , Urabe Masayuki , Itabashi Kodai , Takayama Junichi , Kiba Takayuki , Sueoka Kazuhisa , Murayama Akihiro
Published: July 2018

Interdot spin transfer dynamics is studied in a laterally coupled excited spin ensemble of high-density InGaAs quantum dots (QDs). We observe a rise time of the photoluminescence intensity of ∼100 ps and a simultaneous increase in the spin polarization of the excited spin ensemble, indicating spin injection from higher-energy levels in smaller QDs. Moreover, this coupled ensemble exhibits decay properties of the spin polarization that vary with the excited spin density. This phenomenon can be quantitatively understood by considering interdot spin transfer into lower-energy levels of the surrounding QDs, where the transfer rate depends on the degree of state filling of each QD level.
On the nonlinear solitary and shock waves in Maxwellian multicomponent space plasma
Authors: Sarma Ridip , Das Ganesh C. , Das Ranjan , Adhikary Nirab C.
Published: July 2018

Our objective is to study the nonlinear wave phenomena in a multicomponent plasma with Maxwellian electrons and ions. The main reason for this consideration is to exhibit the effects of dust charge fluctuations in plasma acoustic modes. Different aspects of nonlinear dynamics have been studied through the Korteweg-deVries (K-dV) equation (solitons) and the Burgers equation (shocks) derived by the reductive perturbation technique. A special method, known as the (G′/G) method, has been used for a solution. It predicts the different coherent features of various nonlinear waves successfully, and finally, their importance has been recognized in space plasmas. Evolutions have been shown with the input of appropriate typical plasma parameters to support our observations in space plasmas. All conclusions are in good agreement with the actual occurrences and could be of interest in furthering investigations in experiments and satellite observations in spaces. An important aspect of this exercise is a comparative study of K-dV and Burgers solutions in different plasma spaces.
Modelling and measurement of the electrostatic potential perturbation in consideration of the non-adiabatic electron response to trace tungsten concentration in a tokamak
Authors: Li Erzhong , Chen K. , Liu H. , Zhang L. , Gong X.
Published: July 2018

Trace tungsten ions are almost unavoidable in tokamaks with the tungsten as the first wall material. In high performance plasma, tungsten ions are easily concentrated in the core with a finite gradient along the radius. Prevention of the concentration is an important task for long-pulse H-mode operations. In this paper, it is shown that the tungsten concentration generates electrostatic perturbations due to the bulk electron drift with dissipation. That leads to a phase difference between the electron and the tungsten ion density perturbations. The resultant electrostatic potential extends away from the resonant surface of an internal mode which is characterized by the mode frequency chirping down. This work indicates that the electrostatic potential is mainly determined by the electron kinetics as well as the tungsten-bulk ion collisions, and that the impurity ion dynamics is efficiently influenced by the electron perturbations. Modification of the electron kinetics is a candidate to control the tungsten transport in experiments.
Saltwater transport through pristine and positively charged graphene membranes
Authors: Nguyen Chinh Thanh , Beskok Ali
Published: July 2018

Transport of saltwater through pristine and positively charged single-layer graphene nanoporous membranes is investigated using molecular dynamics simulations. Pressure-driven flows are induced by motion of specular reflecting boundaries at feed and permeate sides with constant speed. Unlike previous studies in the literature, this method induces a desired flow rate and calculates the resulting pressure difference in the reservoirs. Due to the hexagonal structure of graphene, the hydraulic diameters of nano-pores are used to correlate flow rate and pressure drop data. Simulations are performed for three different pore sizes and flow rates for the pristine and charged membrane cases. In order to create better statistical averages for salt rejection rates, ten different initial conditions of Na+ and Cl− distribution in the feed side are used for each simulation case. Using data from 180 distinct simulation cases and utilizing the Buckingham Pi theorem, we develop a functional relationship between the volumetric flow rate, pressure drop, pore diameter, and the dynamic viscosity of saltwater. A linear relationship between the volumetric flow rate and pressure drop is observed. For the same flow rate and pore size, charged membranes exhibit larger pressure drops. Graphene membranes with 9.90 Å pore diameter results in 100% salt rejection with 163.2 l/h cm2 water flux, requiring a pressure drop of 35.02 MPa.
Zeros of rational functions and solvable nonlinear evolution equations
Authors: Calogero Francesco
Published: July 2018

Recently a convenient technique to relate the time evolution of the N zeros of a time-dependent polynomial pNz;t of degree N in the (complex) variable z to the time evolution of its N coefficients has been exploited to identify large classes of dynamical systems solvable by algebraic operations. These models also include N-body problems that evolve in the complex plane (or, equivalently, in the real Cartesian plane) according to systems of nonlinearly-coupled equations of motion of Newtonian type (“accelerations equal forces”). Many of these models feature remarkable properties: for instance, they are Hamiltonian and integrable and/or multiply periodic or even isochronous (featuring completely periodic solutions with a fixed period largely independent of the initial data), or asymptotically isochronous (featuring isochrony only up to corrections vanishing in the remote future). In this paper, an analogous technique is introduced that focuses instead on the time evolution of the N zeros of an appropriate class of time-dependent rational functions RNz;t, thereby opening large vistas of new dynamical systems solvable by algebraic operations and featuring remarkable properties. A few examples are reported.
Scaling of spoke rotation frequency within a Penning discharge
Authors: Powis Andrew T. , Carlsson Johan A. , Kaganovich Igor D. , Raitses Yevgeny , Smolyakov Andrei
Published: July 2018

A rotating plasma spoke is shown to develop in two-dimensional full-sized kinetic simulations of a Penning discharge cross-section. Electron cross-field transport within the discharge is highly anomalous and correlates strongly with the spoke phase. Similarity between collisional and collisionless simulations demonstrates that ionization is not necessary for spoke formation. Parameter scans with discharge current Id, applied magnetic field strength B, and ion mass mi show that the spoke frequency scales with eErLn/mi, where Er is the radial electric field, Ln is the gradient length scale, and e is the fundamental charge. This scaling suggests that the spoke may develop as a non-linear phase of the collisionless Simon-Hoh instability.