bluuebunny/crossref_metadata_2025_split huggingface dataset:
Habutobin Splits the Arg16-Gly17 Bond in the Aa Chain of Rabbit Fibrinogen
Authors: Kinjoh Kiyohiko , Kosugi Tadayoshi , Nakamura Mariko , Hanashiro Kazuhiko , Sunagawa Masanori , Tokeshi Yoshihiro , Eguchi Yukinori 
Published: July 2018

SummaryWe reported previously that habutobin, a thrombin-like enzyme from Trimeresurus flavoviridis venom, clotted only rabbit fibrinogen, whereas human, monkey, bovine, dog, rat and guinea-pig fibrinogens were unaffected. In the present study, we investigated the cleavage site of the rabbit Aα chain by habutobin. The fibrinopeptide released by habutobin was identical to the fibrinopeptide A released by thrombin, and its amino acid sequence corresponded to Aα 1-16 of rabbit fibrinogen. It was clarified therefore that habutobin cleaves the Arg16-Gly17 bond in the Aa chain of rabbit fibrinogen.
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

Summaryvon 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

SummaryDepolymerized 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

SummaryPlatelet-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

SummaryAlthough 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

SummaryThe 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

SummaryUse 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

SummaryHuman 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

SummaryMouse 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

SummaryPlatelet 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

SummaryThrombomodulin 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

SummaryAntiphospholipid-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

SummaryIn 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.