bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heterogeneous Recognition of beta 2-glycoprotein I by Antibodies from Antiphospholipid Syndrome Patients
Authors: Guerin J. , Sim R. , Yu B-B. , Ferluga J. , Feighery C. , Jackson J. 
Published: March 2018

SummaryBeta 2-glycoprotein I plays a pivotal role in the binding of antiphospholipid antibodies to phospholipid in patients with antiphospholipid syndrome. In this study the nature of the epitopes on beta 2-glycoprotein I (β2-GPI) recognised by sera from antiphospholipid syndrome (APS) patients (n = 15) was investigated and compared to rabbit polyclonal and mouse monoclonal anti-β2-GPI antibodies. β2-GPI was only recognised when bound to a high affinity binding support. The antigenic epitope on β2-GPI recognised by all APS patients was also dependent on disulphide bond integrity. Digestion of β2-GPI with elastase rapidly destroyed the epitope(s) on β2-GPI recognised by antibodies in 91% of APS patients. The main cleavage occurred at tryptophan316-lysine317 in the fifth domain. Digestion with staphylococcal V8 protease resulted in a 50% reduction in antibody binding in 81% of patients and the cleavage sites mainly involved the first domain of the molecule. There was considerable variability in the recognition of six different species of β2-GPI by serum from APS patients. The epitopes on β2-GPI bound by APS sera appear conformationally determined in all patients but are quite heterogeneous in the regions of β2-GPI that are recognised.
Multi-Cellular Activation In Vivo by Endotoxin in Humans – Limited Protection by Adenosine Infusion
Authors: Li Nailin , Soop Anne , Sollevi Alf , Hjemdahl Paul 
Published: March 2018

SummaryThe influence of adenosine infusion (40 µg/kg/min for 4 h) on inflammatory and hemostatic parameters was investigated in healthy males without (n = 10) or with (n = 11) intravenous endotoxin injection (4 ng/kg). Without endotoxin, adenosine elevated circulating leukocytes and circulating platelet-leukocyte aggregates. Endotoxin activated platelets and leukocytes in vivo. Platelet activation was seen as slightly increased platelet P-selectin expression, decreased platelet counts, and elevated plasma soluble P-selectin (from 39.6 ± 3.4 to 68.9 ± 6.6 ng/ml; P <0.01). Leukocyte activation was evidenced by increased CD11b expression (from MFI of 0.54 ± 0.02 to 2.21 ± 0.17; P <0.01) and plasma elastase levels (from 25.3 ± 2.5 to 169.3 ± 22.5 ng/ml; P <0.01). Endotoxin also enhanced platelet and leukocyte responsiveness to in vitro stimulation. Endotoxin induced von Willebrand factor secretion (from 92 ± 8 units to 265 ± 19 units at 4 h; P <0.001) and enhanced thrombin generation in vivo. Endotoxin induced leukocytosis and thus increased circulating platelet-leukocyte, mainly platelet-neutrophil, aggregates. Adenosine caused slight attenuation of platelet reactivity to agonist stimulation, enhanced the endotoxin-induced leukocytosis, and detained more platelet-leukocyte aggregates in circulation, but did not attenuate endotoxin-induced neutrophil elastase secretion, von Willebrand factor secretion, or thrombin generation. Thus, endotoxemia induces multi-cellular activation in vivo. Adenosine inhibits leukocyte adhesion and extravasation, and mildly attenuates platelet responsiveness and soluble P-selectin release. Adenosine has the potential of becoming a therapeutic antiinflammatory drug, but an optimal treatment strategy needs to be developed.
Functional Analyses of Patient-derived IgG Monoclonal Anticardiolipin Antibodies Using In Vivo Thrombosis and In Vivo Microcirculation Models
Authors: Liu Xiaowei , Espinola Ricardo , Olee Tsawei , Zhu Min , Harris Nigel , Chen Pojen , Pierangeli Silvia 
Published: March 2018

SummaryAntiphospholipid antibodies (aPL) have been associated with thrombosis and pregnancy losses in patients diagnosed with antiphospholipid syndrome (APS) and enhance thrombus formation in vivo in mice, but the mechanism of thrombosis by aPL is not completely understood. It has been proposed that aPL may affect endothelial cell (EC) function and/or induce their activation, transforming their anticoagulant surface into procoagulant, thus predisposing to thrombosis. It has been proposed that aPL may affect EC cell function and/or induce their activation, transforming their anticoagulant surface into procoagulant, thus predisposing to thrombosis. This study proposes to test the hypotheses that some IgG anticardiolipins (IgG aCL) with thrombogenic properties in mice, exert their effects through activation of endothelium. We studied seven patient-derived monoclonal aCL for their thrombogenic properties in an in vivo pinch-induced thrombosis model, and their functional activities in activating EC by analyzing in vivo leukocyte adhesion to endothelium in microcirculation in venules in exposed murine cremaster muscle and in vitro adhesion molecule expression in cultured EC. The binding of the monoclonal aCL to EC was also tested. In addition to the previous identified thrombogenic IS2, four of the five new more IgG monoclonal aCL (from two patients) were found to be thrombogenic. Of these five thrombogenic aCL, three caused more in vivo leukocyte adhesion to EC in microcirculation, as compared to that induced by the H2 control human monoclonal IgG, and enhanced expression of adhesion molecules (particularly VCAM-1) on cultured EC. These data show that about 2/3 patientderived IgG monoclonal aCL are thrombogenic and suggest that some thrombogenic IgG aCL exert their effects through activating EC.
Abbreviations: aCL, anticardiolipin antibodies; aPL, antiphospholipid antibodies; APS, antiphospholipid syndrome; CL, cardiolipin; dRVVT, dilute Russell’s viper venom time; EC, endothelial cells; ELISA, enzyme-linked immunosorbent assay; GP, glycoprotein; h, hour; HUVEC, human umbilicalvein endothelial cells; ICAM, intercellular adhesion molecule; KCT, kaolin clotting time; LA, lupus anticoagulant; PBS, phosphate-buffered saline; PL, phospholipid; sq, square; TBS, Tris-buffered saline; VCAM, vascular cell adhesion molecule; PMBC, peripheral mononuclear blood cells
Protein S Thr103Asn Mutation Associated with Type II Deficiency Reproduced In Vitro and Functionally Characterised
Authors: Giri Tusar , de Frutos Pablo , Dahlbäck Björn 
Published: March 2018

SummaryProtein S functions as a cofactor to activated protein C (APC) in the degradation of FVa and FVIIIa. In protein S, the thrombin sensitive region (TSR) and the first EGF-like domain are important for expression of the APC cofactor activity. A naturally occurring Thr103Asn (T103N) mutation in the first EGF-like domain of protein S has been associated with functional (type II) protein S deficiency. To elucidate the functional consequences of the T103N mutation, recombinant protein S mutant was expressed in mammalian cells and functionally characterised. The expression level of protein S T103N from transiently transfected COS 1 cells was equal to that of wild type protein S. The mutant protein S and wild type protein S were also expressed in 293 cells after stable transfection, and the recombinant proteins purified. In APTT-and PT-based coagulation assays, the mutant protein demonstrated approximately 50% lower anticoagulant activity as compared to wild type protein S. The functional defect was further investigated in FVa-and FVIIIa-degradation assays. The functional defect of mutant protein S was attenuated at increasing concentrations of APC. The results demonstrate the region around residue 103 of protein S to be of functional importance, possibly through a direct interaction with APC.
Single Founder Mutation (W380G) in Type II Protein C Deficiency in Finland
Authors: Kuismanen Kristiina , Holopainen Päivi , Vahtera Elina , Rasi Vesa , Krusius Tom , Levo Antti , Partanen Jukka 
Published: March 2018

SummaryThe present study investigated the genetic basis for type II protein C deficiency in Finland, where this form has an unusually high incidence. We demonstrated that, first, a single novel mutation W380G in the protein C gene (PROC) explained 25/26 index patients, estimated to represent two thirds of all families with type II deficiency in Finland. Second, extended chromosomal conservation, i. e. a specific haplotype, around the W380G mutation was indicated in unrelated patients. Third, a local geographical origin for the W380G mutation was suggested by genealogical data. These results are in contrast to the heterogeneity in type II protein C deficiency elsewhere, but closely parallel disorders of the Finnish disease heritage. The high frequency of the type II disease can be explained by founder effect and subsequent enrichment of a single mutation in Finland. The present study also provided a simple means for genetic diagnosis of this disease and the genetic test can be included in the routine screenings in this population.
Characterization of the Murine Coagulation Factor X Promoter
Authors: Wilberding Julie , Castellino Francis 
Published: March 2018

SummaryFactor X (FX) is a vitamin K-dependent serine protease zymogen that functions in both the extrinsic and intrinsic pathways of blood coagulation. In this study, the 5’-flanking region of the murine FX gene was analyzed to determine those elements that govern its transcriptional activity and regulation. Consistent with other TATA-less promoters, murine FX contains two start sites of transcription, at bp −5 and −21 relative to the ATG translational initiation codon. The mRNA of FX was found in a number of tissues, including the liver, stomach, intestine, kidney, ovary, testes, spleen, skeletal muscle, and lung. Using DNase I footprinting, three areas of protection have been identified in the proximal 287 bp of the promoter, spanning bp −28 to −218. Further examination of this region revealed transcription factor binding sites for NF-Y, HNF-4, and a GATA factor. Electrophoretic mobility shift analysis (EMSA) confirmed the identities of NF-Y, HNF-4, and GATA-4, all of which were found by transient transfection analyses in HepG2 cells to influence the activity of the promoter. Ablation of the NF-Y site was most dramatic, reducing activity to 10% of that of the wild-type construct. Deletion of the HNF-4 site led to an activity of 25% of wild-type, and a GATA-4 mutation reduced activity to 63% of wild-type values. This investigation revealed the identity of the factors bound at the proximal promoter of the FX gene, and the relative importance of each. This is the first report of a member of the GATA family of transcription factors being important in the regulation of a coagulation-based gene.
Monitoring Hypocoagulant Conditions in Rat Plasma: Factors Determining the Endogenous Thrombin Potential of Tissue Factor-Activated Plasma
Authors: Nieuwenhuys Cécile M. , Feijge Marion A. , Béguin Suzette , Heemskerk Johan W. 
Published: March 2018

SummaryAutomated human plasma, continuous monitoring of the formation and inactivation of thrombin during the coagulation process provides an adequate way to detect hypo- and hypercoagulant conditions. Here, we describe an analogous procedure to determine the endogenous thrombin potential (ETP), i. e. the free thrombin concentration-time integral, of coagulating rat plasma. When activated with tissue factor, the ETP of plasma from Wistar rats was comparable to the ETP of human plasma, in spite of a relatively short half-life time of free thrombin in rat plasma. The ETP was highly sensitive to heparin as well as to administration of vitamin K antagonist or feeding of the animals with a vitamin K-deficient diet. In plasma that was activated under sub-optimal conditions (reduced levels of tissue factor or vitamin K-dependent coagulation factors), the ETP increased with the rate of thrombin formation in the first minutes of the coagulation process. Since both parameters are dependent of the prothrombin concentration, it appears that this level plays an important role in determining both the initial and total activity of the coagulation system. Thus, automated measurement of free thrombin during the coagulation process of rat plasma allows a detailed analysis of hypocoagulability in this animal model.
Immunoassays for the Quantitation of Porcine PAI-1 Antigen and Activity in Biological Fluid Samples
Authors: Leng Henry M. , Brouwers Els , Knockaert Isabelle , Declerck Paul 
Published: March 2018

SummaryTwo monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) for the quantitation of porcine plasminogen activator inhibitor-1 (PAI-1) antigen and activity in plasma were constructed and validated. The intra-assay, interassay, and interdilution coefficients of variation were 4.3, 13, and 8%, respectively, for the antigen ELISA and 5, 16, and 11% for the activity assay. Assay recoveries, in the antigen ELISA, of either latent or active recombinant porcine PAI-1 (10 and 50 ng/ml) added to plasma were 86 ± 9% and 92 ± 22%, respectively, for the latent form and 89 ± 9% and 87 ± 7% for the active form (mean ± SD, n = 3 to 4). In the immunofunctional assay, recoveries for the same concentrations of active PAI-1 were 108 ± 16% and 92 ± 21%, respectively. In male porcine plasma the level of PAI-1 antigen was 31 ± 11 ng/ ml and the activity, 34 ± 16 ng/ml (mean ± SD, n = 10). In female plasma PAI-1 antigen levels were 20 ± 5.2 ng/ml and the PAI-1 activity 42 ± 17 ng/ml (n = 13). A linear correlation was found between PAI-1 antigen and activity levels in male (r = 0.60) and female (r = 0.70) plasma. Immunodepletion resulted in a decrease of >95% of the original PAI-1 antigen or activity levels. Incubation of plasma samples at 37° C for 16 h resulted in a significant decrease (70 to 85%) of PAI-1 activity. Under these conditions (37° C, 16 h) PAI-1 antigen levels remained unchanged in males whereas the response of the female samples in the PAI-1 antigen assay increased two-fold.In lysed platelet-rich plasma males had 990 ± 470 ng/ml antigen and 160 ± 80 ng/ml activity and females, 920 ± 500 ng/ml antigen and 150 ± 98 ng/ml activity corresponding to 2.1 ± 0.77 fg PAI-1 antigen per platelet. Only 16% of PAI-1 released from platelets was found to be active. Linear correlations between PAI-1 antigen and activity were found for both males (r = 0.61) and females (r = 0.67).The assays are both sensitive and specific and may, therefore, aid the elucidation of the pathophysiological role of PAI-1 in swine experimental models of atherosclerosis and other thrombotic disorders.
Long-term Clinical Follow-up in 265 Patients with Deep Venous Thrombosis Initially Treated with either Unfractionated Heparin or Dalteparin: A Retrospective Analysis
Authors: Åberg W. , Lockner D. , Paul C. , Holmström M. 
Published: March 2018

SummaryThe primary objective of this retrospective study was to describe the frequency of a post-thrombotic syndrome in 265 patients previously treated for deep venous thrombosis (DVT). The secondary objectives were to document the frequency of recurrent venous thromboembolism (VTE) and mortality, especially from malignant disease. The patients were evaluated 5-14 years after inclusion in three randomized trials comparing continuous intravenous (i. v.) infusion of unfractionated heparin (UFH) (n = 85) with a low molecular weight heparin (LMWH), dalteparin (n = 180). The median post-thrombotic score at follow-up was 2 (range 0-8). In a multiple step-wise regression analysis the post-thrombotic score was significantly higher among patients with initial proximal DVT (p = 0,0001) as compared with those who had distal DVT. A recurrent venous thromboembolic event was diagnosed in 29,4% of the patients treated with dalteparin and in 23,5% of the patients treated with UFH (ns). A secondary risk factor for venous thromboembolism and a longer duration of treatment with oral anticoagulants (OAC) were significantly associated with a lower risk for recurrent VTE, whereas malignant disease diagnosed during follow-up was associated with a higher risk. During follow-up a total of 40,7% of patients had died. No difference in total mortality or mortality from malignant disease was demonstrated between the two drugs. In conclusion, a severe post-thrombotic syndrome occured relatively infrequent. considering the long observation period. Proximal DVT was significantly associated with a more severe post-thrombotic syndrome. After 14 years follow-up, no significant differences were observed in overall mortality, mortality from malignant disease or recurrent VTE between UFH- and dalteparin-treated patients. Malignant disease was a risk factor for recurrent VTE, the presence of a secondary risk factor and a longer duration of treatment with OAC decreased the risk for recurrent VTE.
Comparison of Three Methods to Assess Therapeutic Quality Control of Treatment with Vitamin K Antagonists
Authors: Prins Martin , Ken Redekop W. , Tijssen Jan , Heisterkamp Siem , Büller Harry , Hutten Barbara 
Published: March 2018

SummaryDuring treatment with vitamin K antagonists, International Normalized Ratios (INR) are determined periodically to maintain a therapeutic level of anticoagulation. We evaluated two existing methods for therapeutic quality control (linear interpolation and equidivision), with regard to their validity and reproducibility. In addition, we proposed and evaluated a (hybrid) method that takes into account potential effects of dosage modifications when INRs are far out of the target range. Validity was assessed by deleting intermediary INR results and estimating this INR based on the two surrounding INRs with each of the three methods. The estimated INRs were then compared with the observed INR.Reproducibility of time spent in an INR range was evaluated for each of the three methods by deleting at random increasing proportions of INRs and comparing these estimates with the situation without deletions. We found that estimates of time spent in INR categories obtained with equidivision were most reproducible, but least valid. The hybrid method showed slightly higher validity and reproducibility in comparison with linear interpolation. Since these differences were small, linear interpolation is preferable to the hybrid method, since the calculations involved are easier.
Allelic Discrimination of Factor V Leiden Using a 5’ Nuclease Assay
Authors: Schwaab R. , Hanfland P. , Hoernschemeyer D. , Happich D. 
Published: March 2018

SummaryThe G1691A (Leiden) mutation of the factor V gene is the most prevalent identified cause of venous thrombosis. Therefore, we developed a new genetic test using the TaqMan system. With this assay which combines PCR amplification and detection reaction in one closed tube, a cohort of 234 patients with a history of thrombosis was screened. In parallel, amplification products of the same patients were screened with a previously described test using endonuclease digestion of PCR products followed by gel electrophoresis. Identical results were obtained by both methods. Among cases, 122 (52%) individuals were homozygous normal, 99 (42%) were heterozygous affected and 13 (5.5%) showed homozygous pattern for the Factor V Leiden mutation. Thus, it could be demonstrated that the new TaqMan assay is a robust, rapid and automated method for high throughput application which avoids time consuming and difficult post-PCR steps.
The Biology of P-Selectin Glycoprotein Ligand-1: Its Role as a Selectin Counterreceptor in Leukocyte-Endothelial and Leukocyte-Platelet Interaction
Authors: Furie Barbara , Furie Bruce , Yang Jing 
Published: March 2018

SummaryCell-cell interactions mediating leukocyte trafficking, thrombogene-sis and inflammation are crucial for the host defense mechanism. The selectin family of integral membrane proteins includes E-selectin, L-selectin and P-selectin. Selectins mediate tethering and rolling of leukocytes to the vessel wall at the site of inflammation. The counter-receptor for P-selectin and possibly the other selectins is P-selectin glycoprotein ligand-1 (PSGL-1). This review focuses on the properties and biology of PSGL-1.
APC-resistance Is a Risk Factor for Postoperative Thromboembolism in Elective Replacement of the Hip or Knee – A Prospective Study
Authors: Lundahl Tom , Nilsson Lennart , Andersson Christer , Lindahl Tomas 
Published: March 2018

SummaryPostoperative venous thromboembolic complications are commonly seen after total replacement of the hip or knee. Recently, an inherited defect with resistance to the anticoagulant activity of activated protein C (APC-resistance) has been detected. APC-resistance seems to be a common risk factor, especially in Sweden, and it increases the propensity for venous thrombosis. This study assesses the prevalence of APC-resistance in a general population and its clinical significance for patients undergoing surgery associated with a high risk of thromboembolic complications. In a prospective cohort study, we analysed for APC-resistance in 645 consecutive patients before elective replacement of the hip or knee at 3 hospitals in southern Sweden. Thromboprophylaxis with LMWH-heparin was given to all patients throughout the hospitalisation period. We recorded events of clinical thromboembolism for 3 months postoperatively. Venography, ultrasonography or pulmonary scintigraphy was requested by the clinicians according to the existing routines, i.e. only patients with symptoms of thromboembolism were examined. A thromboembolic complication was registered in 20 (3.1%) patients. Fifty per cent of the venous thrombi had a proximal location. Only 0.3% of the patients had verified pulmonary embolism. APC-resistance was found in 14.1% of the patients, of whom 9.9% had experienced postoperative thromboembolism compared with 2.0% of the patients without APC-resistance (p <0.0007). We conclude that APC-resistance is a frequent risk factor for symptomatic postoperative deep venous thrombosis with an estimated relative risk of 5.0 (95% confidence interval: from 1.9 to 12.9) in elective replacement of the hip or knee.
Crotalase, a Fibrinogen-Clotting Snake Venom Enzyme: Primary Structure and Evidence for a Fibrinogen Recognition Exosite Different from Thrombin
Authors: Henschen-Edman Agnes , Theodor Ida , Edwards Brian , Pirkle Hubert 
Published: March 2018

SummaryCrotalase, a fibrinogen-clotting enzyme isolated from the venom of Crotalus adamanteus, and its overlapping fragments were subjected to Edman degradation. The resulting amino acid sequence, VIGGDEC NINEHRFLVALYDYWSQLFLCGGTLINNEWVLTAAHCDRTHI LIYVGVHDRSVQFDKEQRRFPKEKYFFDCSNNFTKWDKDIM LIRLNKPVSYSEHIAPLSLPSSPPIVGSVCRAMGWGQTTSPQET LPDVPHCANINLLDYEVCRTAHPQFRLPATSRTLCAGVLEG GIDTCNRDSGGPLICNGQFQGIVFWGPDPCAQPDKPGLYTK VFDHLDWIQSIIAGEKTVNCP, is characteristic of a serine protein-ase. Comparison with thrombin, the physiological fibrinogen-clotting enzyme, showed that thrombin’s fibrinogen-recognition exosite (FRE) is poorly represented in crotalase. Hirudin, a FRE-dependent inhibitor, had no effect on crotalase. Spatial modeling of crotalase yielded a possible alternative fibrinogen-recognition site comprised of Arg 60F, Lys 85, Lys 87, and Arg 107 (underlined in the sequence above). Crotalase also lacks thrombin’s YPPW loop, as well as its functionally important ETW 146-148, and its heparin-binding site. The enzyme contains a single asparagine-linked glycosylation site, NFT, bearing neutral and amino sugars that account for 8.3% of the enzyme’s total molecular weight of 29,027. The calculated absorbance of crotalase at 280 nm, 1%, cm-1is 15.2.