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- Chen, Daxin, et al.
(författare)
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Regulated inhibition of coagulation by porcine endothelial cells expressing P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins
- 1999
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Ingår i: Transplantation. - : Ovid Technologies (Wolters Kluwer Health). - 0041-1337. ; 68:6, s. 832-839
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Tidskriftsartikel (refereegranskat)abstract
- Background. Thrombotic vascular occlusion resulting in infarction occurs during hyperacute rejection of allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation. A similar process is also found in disorders of diverse etiology including atherosclerosis, vasculitis, and disseminated intravascular coagulation. Methods. We have previously constructed two membrane-tethered anticoagulant fusion proteins based on human tissue factor pathway inhibitor and the leech anticoagulant hirudin and demonstrated their functional efficacy in vitro. These constructs have now been modified by the addition of a P-selectin sequence to the cytoplasmic tail to localize them in Weibel-palade bodies. They have been transfected into Weibel-palade body-positive endothelial cells isolated from the inferior vena cava of normal pigs. Results. In resting endothelial cells, fusion protein expression colocalized with P-selectin and was confined to Weibel-palade bodies. These cells had a procoagulant phenotype in recalcified human plasma. However, after activation with phorbol ester the anticoagulant proteins were rapidly relocated to the cell surface where they specifically inhibited the clotting of human plasma. Conclusions. Novel anticoagulant molecules may prove useful therapeutic agents for gene therapy in thrombotic disease and postangioplasty or for transgenic expression in animals whose organs may be used for clinical xenotransplantation. Expression in vascular endothelial cells may be regulated by inclusion of P-selectin cytoplasmic sequence, to restrict cell surface expression to activated endothelium.
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- Riesbeck, Kristian, et al.
(författare)
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Expression of hirudin fusion proteins in mammalian cells : A strategy for prevention of intravascular thrombosis
- 1998
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Ingår i: Circulation. - 0009-7322. ; 98:24, s. 2744-2752
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Tidskriftsartikel (refereegranskat)abstract
- Background - Intravascular thrombosis occurs in disorders of diverse pathogeneses, including allograft and xenograft rejection. In this in vitro study, we describe an approach for tethering the specific thrombin inhibitor hirudin to plasma membranes as part of a genetic strategy for regulating intravascular coagulation. Methods and Results - An HLA class I leader sequence was fused with hirudin linked to domains 3 and 4 of human CD4 and intracytoplasmic sequence from either CD4 or human P-selectin. The constructs were transfected into mouse fibroblasts, Chinese hamster ovary (CHO)-K1 cells, immortalized porcine endothelial cells (IPECs), and a pituitary secretory cell line (D16/16). Thrombin binding to the hirudin fusion proteins expressed on fibroblasts and CHO-K1 cells could be blocked by an anti- hirudin monoclonal antibody and by pretreatment of thrombin with either the synthetic tripeptide thrombin inhibitor PPACK or native hirudin. Hirudin expression significantly modified the procoagulant phenotype of IPECs in human plasma, leading to prolongation of clotting times. Hirudin-CD4-P- selectin fusion proteins accumulated in adrenocorticotropic hormone- containing granules in D16/16 cells, with no cell surface expression except on activation with phorbol ester, when hirudin relocated to the outer membrane. Conclusions - Hirudin fusion proteins were expressed on mammalian cells, where they reduced local thrombin levels and inhibited fibrin formation. Regulated expression was achieved on activated cells by use of the cytoplasmic sequence from P-selectin. In vivo, these fusion proteins may prove useful transgenic or gene therapy agents for preventing intravascular thrombosis.
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- Riesbeck, Kristian, et al.
(författare)
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Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface
- 1997
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Ingår i: Thrombosis and Haemostasis. - 0340-6245. ; 78:6, s. 1488-1494
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Tidskriftsartikel (refereegranskat)abstract
- Tissue factor pathway inhibitor (TFPI) is one of the main regulators of the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third Kunitz domain were fused with domains three and four and the carboxy-terminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expression using monoclonal antibodies directed against the first two TFPI Kunitz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition of FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition, TFPI-CD4-expressing cells, preincubated with FXa, specifically bound human TF-FVIIa complexes as revealed with an anti-human TF polyclonal antibody. No functional difference was observed between full length or truncated TFPI-CD4. These results demonstrate that functionally intact TFPI can be tethered to the cell surface. Genetic manipulation of, for example, endothelial cells leading to the stable expression of TFPI may inhibit the development of coronary artery heart disease following cardiac allotransplantation, and may inhibit thrombosis in the context of xenotransplantation.
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- Sandborg, Michael, 1961-, et al.
(författare)
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Demonstration of correlations between clinical and physical image quality measures in chest and lumbar spine screen-film radiography
- 2001
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Ingår i: British Journal of Radiology. - : British Institute of Radiology. - 0007-1285 .- 1748-880X. ; 74:882, s. 520-528
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Tidskriftsartikel (refereegranskat)abstract
- The ability to predict clinical image quality from physical measures is useful for optimization in diagnostic radiology. In this work, clinical and physical assessments of image quality are compared and correlations between the two are derived. Clinical assessment has been made by a group of expert radiologists who evaluated fulfilment of the European image criteria for chest and lumbar spine radiography using two scoring methods: image criteria score (ICS) and visual grading analysis score (VGAS). Physical image quality measures were calculated using a Monte Carlo simulation model of the complete imaging system. This model includes a voxelized male anatomy and was used to calculate contrast and signal-to-noise ratio of various important anatomical details and measures of dynamic range. Correlations between the physical image quality measures on the one hand and the ICS and VGAS on the other were sought. 16 chest and 4 lumbar spine imaging system configurations were compared in frontal projection. A statistically significant correlation with clinical image quality was found in chest posteroanterior radiography for the contrast of blood vessels in the retrocardiac area and a measure of useful dynamic range. In lumbar spine anteroposterior radiography, a similar significant correlation with clinical image quality was found between the contrast and signal-to-noise ratio of the trabecular structures in the L1-L5 vertebrae. The significant correlation shows that clinical image quality can, at least in some cases, be predicted from appropriate measures of physical image quality.
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- Steen, Mårten, et al.
(författare)
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Functional characterization of Factor V-Ile359Thr, a novel mutation associated with thrombosis.
- 2004
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 103:9, s. 3381-3387
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Tidskriftsartikel (refereegranskat)abstract
- A missense mutation, FV-IIe359Thr (FV Liverpool), associated with thrombosis has recently been described. This mutation creates an additional potential N-linked glycosylation site (Asn-X-Ser/Thr) in factor V (FV) at Asn357 that could interfere with secretion and/or protein interactions. To investigate the molecular pathology of FV-IIe359Thr, the mutation was created by site-directed mutagenesis and expressed together with other mutations that could help explain the phenotype (FV-Arg306GIn/IIe359Thr/Arg679GIn, FV-IIe359Thr/Arg506GIn/Arg679GIn, and FV-Asn357GIn/IIe359Thr). The FV-IIe359Thr was secreted normally and had full procoagulant activity. Western blot analysis showed that FV-IIe359Thr migrated more slowly, while the FV-Asn357GIn/IIe359Thr was indistinguishable from FV-wild type (FV-WT), indicating that FV-IIe359Thr was expressed with an additional carbohydrate chain. Activated protein C (APC)-mediated inactivation in an FVa degradation assay showed that the IIe359Thr mutation significantly reduced the cleavage at Arg306 both in the presence and absence of protein S, whereas the cleavage at Arg506 was unaffected. When tested in an FVIIIa degradation assay, the FV-IIe359Thr variant exhibited equally low APC cofactor activity as FV Leiden (FVArg506GIn). In conclusion, the IIe359Thr mutation appears to affect anticoagulation by 2 mechanisms, impeding the APCmediated down-regulation of the FVa molecule and additionally being a poor APC cofactor for the down-regulation of FVIIIa. These findings explain the association of the FV-IIe359Thr mutation with thrombosis. (C) 2004 by The American Society of Hematology.
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