In order to develop blood biocompatible materials a heparin surface and a phosphorylcholine (PC) functionalized polymer surface were characterized using photoelectron spectroscopy (PES). The formation of the heparin surface was studied by quartz crystal microbalance with dissipation monitoring (QCM-D). This heparin surface consists of heparin conjugates deposited on a conditioning layer, applied once or twice. The PC functionalized polymer, poly(trimethylene carbonate), was linked to a silicon substrate through 3-amino- propyltrimethoxysilane (APTMS), also studied using PES. Synchrotron radiation based PES showed that the thicker heparin film resulted in complete coverage of the substrate, while the thinner did not. This could explain the difference in blood biocompatibility between the two films, as observed by others. It was also found that the heparin chains bend down towards the substrate (under vacuum). For the thinner heparin film the modifications, resulting from extensive irradiation of the sample, were studied with synchrotron radiation based PES. This was done at a pressure of about 10-7 mbar and in 0.5 mbar water vapor. It was found that the modification is slower under water vapor than at low pressures and that the damaged film incorporates water upon exposure. The heparin coating was found to be stable and wear resistant enough to still be present on artificial heart valves after three weeks testing in circulating plasma. It then had about the same antithrombin uptake as a non-tested surface. The film was, however, partly destroyed by the durability test and plasma proteins were deposited. The PC functionalized, APTMS linked polymer was found to be much shorter than could be expected from random reactions. One plausible explanation is an interaction between the PC group and the silane surface, favoring aminolysis close to the PC group. This is consistent with our finding that the PC group bends down towards the surface.

Antithrombin (AT) is one of the most important anticoagulant factors in the blood, and its effects are increased by the interaction with glycosaminoglycans, especially heparin. AT appears in two additional variants, other than the native form, and those variants have antiangiogenic properties and also bind to heparin. AT is found in two distinct isoforms (alfa, beta) where the difference lie in the degree of glycosylation. This project has shown interesting results regarding the dependence of calcium ions on the binding between heparin and antithrombin. The results show that the beta-isoform increases its affinity for heparin in the presence of calcium in contrast to the alfa-isoform, which shows a decrease in the heparin affinity under the same conditions. This project has also given results that after further investigation and development could be used for an improved set-up of the immobilisation of AT variants in a surface plasmon resonance system. The results show that immobilisation of a protein in the reference channel gives a better shielding effect between the negatively charged heparin molecules and the negatively charged dextran matrix. Furthermore a more significant difference was seen between the two heparin moieties used during binding affinity studies, especially for native AT.

<p>Antithrombin (AT) is one of the most important anticoagulant factors in the blood, and its effects are increased by the interaction with glycosaminoglycans, especially heparin. AT appears in two additional variants, other than the native form, and those variants have antiangiogenic properties and also bind to heparin. AT is found in two distinct isoforms (alfa, beta) where the difference lie in the degree of glycosylation. This project has shown interesting results regarding the dependence of calcium ions on the binding between heparin and antithrombin. The results show that the beta-isoform increases its affinity for heparin in the presence of calcium in contrast to the alfa-isoform, which shows a decrease in the heparin affinity under the same conditions. This project has also given results that after further investigation and development could be used for an improved set-up of the immobilisation of AT variants in a surface plasmon resonance system. The results show that immobilisation of a protein in the reference channel gives a better shielding effect between the negatively charged heparin molecules and the negatively charged dextran matrix. Furthermore a more significant difference was seen between the two heparin moieties used during binding affinity studies, especially for native AT.</p>

<p>Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the embyronal tumor neuroblastoma, where differentiation is a validated treatment strategy, remain unclear. The neuroblastoma tumor stroma is thought to suppress neuroblast growth via release of soluble differentiating factors. Here we identify critical components of the differentiating stroma secretome and describe preclinical testing of a novel therapeutic strategy based on their mechanism of action.</p><p>Expression of heparan sulfate proteoglycans (HSPGs), including T&#946;RIII, GPC1, GPC3, SDC3, and SDC4, is decreased in neuroblastoma, high in the stroma, and suppresses tumor growth. High expression of T&#946;RIII, GPC1, and SDC3 is associated with improved patient prognosis. HSPGs signal via heparan sulfate binding to FGFR1 and FGF2, which leads to phosphorylation of FGFR1 and Erk MAPK, and upregulation of the transcription factor inhibitor of DNA binding 1 (Id1). Surface expression and treatment with soluble HSPGs promotes neuroblast differentiation via this signaling complex. Expression of individual HSPGs positively correlates with Id1 expression in neuroblastoma patient samples and multivariate regression demonstrates that expression of HSPGs as a group positively correlates with Id1 expression, underscoring the clinical relevance of this pathway. HSPGs also enhance differentiation from FGF2 released by the stroma and FGF2 is identified as a potential serum prognostic biomarker in neuroblastoma patients. </p><p>The anticoagulant heparin has similar differentiating effects to HSPGs, decreasing neuroblast proliferation and reducing tumor growth while extending survival in an orthotopic xenograft model of neuroblastoma. Dissection of individual sulfation sites identifies 2-O-, 3-O-de-sulfated heparin (ODSH) as a differentiating agent that suppresses orthotopic xenograft growth and metastasis in two models while avoiding anticoagulation. These studies form the preclinical rationale for a multicenter clinical trial currently being proposed.</p><p>In conclusion, these studies translate mechanistic insights in neuroblast HSPG function to identify heparins as differentiating agents for clinical development in neuroblastoma, while demonstrating that tumor stroma biology can inform design of targeted molecular therapeutics.</p> / Dissertation

No description available.

Beta 2 glycoprotein I (β2GPI) is a major antigenic target in antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiological functions, as it displays both anticoagulant and procoagulant properties. Beta 2GPI may bind to FXI and serve as a regulator of FXI activation by thrombin. The possible interaction of β2GPI with thrombin is investigated using enzyme linked immunosorbent assays and surface plasmon resonance based studies. It is demonstrated for the first time that domain V of β2GPI is involved in direct binding to thrombin, and exosites I and II on thrombin take part in this interaction. It is also shown that cleavage of β2GPI at Lys317-Thr318 does not interrupt this binding. A quaternary complex is proposed on the surface of activated platelets in which β2GPI may colocalise with FXI and thrombin to regulate FXIa generation. The effect of anti-β2GPI monoclonal antibodies (mAbs) were investigated on this system using 8 anti-β2GPI mAbs directed against domain I. Anti-β2GPI Abs potentiate the suppressing activity of β2GPI on FXI activation by thrombin. Moreover, they restore the inhibitory effect of clipped β2GPI on this system. The current study demonstrates for the first time a novel biological consequence of thrombin interaction with β2GPI. The effect of β2GPI on thrombin inactivation by the serine protease inhibitor heparin cofactor II (HCII) is investigated using chromogenic assays, platelet aggregation studies, and the platelet release response. The current work shows that β2GPI protects thrombin from inactivation by HCII/Heparin. This ability is modulated by the cleavage of β2GPI. A ternary structure is proposed between β2GPI, thrombin and heparin which may limit the N-terminus of HCII to exosite I therefore inhibit thrombin inactivation by HCII. The effect of anti-β2GPI Abs is examined in this system using patient polyclonal IgGs and a murine anti-β2GPI mAb. Anti-β2GPI Abs potentiate the protective effect of β2GPI on thrombin inhibition by HCII/Heparin. In view of the importance of HCII in regulating thrombin activity within the arterial wall, disruption of this function by β2GPI/anti-β2GPI Ab complexes may be particularly relevant in arterial thrombosis in APS. Beta 2 glycoprotein I (β2GPI) is a major antigenic target in antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiological functions, as it displays both anticoagulant and procoagulant properties. Beta 2GPI may bind to FXI and serve as a regulator of FXI activation by thrombin. The possible interaction of β2GPI with thrombin is investigated using enzyme linked immunosorbent assays and surface plasmon resonance based studies. It is demonstrated for the first time that domain V of β2GPI is involved in direct binding to thrombin, and exosites I and II on thrombin take part in this interaction. It is also shown that cleavage of β2GPI at Lys317-Thr318 does not interrupt this binding. A quaternary complex is proposed on the surface of activated platelets in which β2GPI may colocalise with FXI and thrombin to regulate FXIa generation. The effect of anti-β2GPI monoclonal antibodies (mAbs) were investigated on this system using 8 anti-β2GPI mAbs directed against domain I. Anti-β2GPI Abs potentiate the suppressing activity of β2GPI on FXI activation by thrombin. Moreover, they restore the inhibitory effect of clipped β2GPI on this system. The current study demonstrates for the first time a novel biological consequence of thrombin interaction with β2GPI. The effect of β2GPI on thrombin inactivation by the serine protease inhibitor heparin cofactor II (HCII) is investigated using chromogenic assays, platelet aggregation studies, and the platelet release response. The current work shows that β2GPI protects thrombin from inactivation by HCII/Heparin. This ability is modulated by the cleavage of β2GPI. A ternary structure is proposed between β2GPI, thrombin and heparin which may limit the N-terminus of HCII to exosite I therefore inhibit thrombin inactivation by HCII. The effect of anti-β2GPI Abs is examined in this system using patient polyclonal IgGs and a murine anti-β2GPI mAb. Anti-β2GPI Abs potentiate the protective effect of β2GPI on thrombin inhibition by HCII/Heparin. In view of the importance of HCII in regulating thrombin activity within the arterial wall, disruption of this function by β2GPI/anti-β2GPI Ab complexes may be particularly relevant in arterial thrombosis in APS.

Zugl.: Aachen, Techn. Hochsch., Diss., 2004.

Techn. Universiẗat, Diss., 2005--München.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available online.