The thesis is focused on a determination of heparin and chondroitin sulfate, using flow injection analysis with spectrofluorimetric detection. The determination is based on the interaction of negatively charged heparin, chondroitin sulfate resp., with a cationic dye (azure B or phenosafranine) which is manifested by the decrease in fluorescence intensity of the dye in its emission maximum. The optimal conditions for the determination in static mode were found, and calibration dependencies were measured. The conditions of FIA were optimized and following parameters were established: the volume of dispensed sample of 100 ml, the length of the reaction coil 60 cm, the flow rate 0.7 ml min-1 , the concentration of azure B 1.6×10-5 mol dm-3 , the concentration of phenosafranine 3.5×10-5 mol dm-3 . For the determination of heparin using azure B it was found: LOD = 0.023 IU ml-1 , LOQ = 0.186 IU ml-1 , and linear dynamic range 0.19-1.43 IU ml-1 . For the determination of heparin using phenosafranine it was found: LOD = 0.102 IU ml-1 , LOQ = 0.192 IU ml-1 , and linear dynamic range 0.19-1.79 IU ml-1 . For the determination of chondroitin sulfate using azure B it was found: LOD = 0.58 mg dm-3 , LOQ = 2.37 mg dm-3 , and linear dynamic range 2.37-8.32 mg dm-3 . The developed determination was applied to the...

Les Thrombopénies Induites par l’Héparine (TIH) sont une complication sévère des traitements par l’héparine dues à des IgG qui ciblent le facteur plaquettaire 4 modifié par l’héparine (FP4/H) et induisent une activation cellulaire via FcγRIIA, conduisant à des complications thrombotiques. Nous avons caractérisé 5B9, IgG1 monoclonale chimérique anti-FP4/H mimant parfaitement les anticorps de TIH et qui est donc un excellent outil pour étudier la physiopathologie des TIH. La pathogénicité des anticorps (Ac) de TIH implique leur fixation aux FcγR. Nous avons montré que le clivage de la région charnière des IgG de TIH par IdeS inhibe ces interactions IgG-FcγR et supprime la pathogénicité des Ac. Nous avons aussi construit un Antibody-Drug Conjugate (ADC) antithrombotique, en bioconjuguant le tirofiban (inhibiteur de l’agrégation plaquettaire) et 5B9 déglycosylé grâce à un linker clivable par la thrombine, protéase générée en excès lors d’une TIH. / Heparin Induced Thrombocytopenia (HIT) is a rare but severe complication of heparin treatments. HIT is due to IgG antibodies specific to platelet factor 4 modified by heparin (PF4/H), which activate blood cells, (especially platelets) after binding to FcγRIIA, this process explaining frequent thrombotic complications. We characterized 5B9, a chimeric IgG1 targeting PF4/H and which fully mimics human HIT antibodies. Therefore, 5B9 is a perfect tool for studying the physiopathology of HIT. IgG antibodies to PF4/H are pathogenic by interacting with FcγR. In this regard, we showed that cleavage by IdeS, a bacterial protease, of the hinge of anti-PF4/H IgG, fully suppressed their pathogenicity. Furthermore, we designed an antithrombotic Antibody-Drug Conjugate that combined tirofiban, a GPIIbIIIa inhibitor with deglycosylated 5B9 using a thrombin cleavable linker.

A trombocitopenia induzida por heparina (TIH) é uma síndrome imunohematológica mediada por um anticorpo que causa ativação plaquetária na presença de heparina, induz à agregação plaquetária e pode estar associada a graves e paradoxais complicações trombóticas e morte. A freqüência de TIH nos pacientes que recebem heparina por mais de cinco dias é de 1% a 5%, e está relacionada a vários fatores. Este é um estudo pioneiro no Brasil, que objetivou avaliar aqui a freqüência de TIH nos pacientes em uso de heparina, a relação ao gênero, ao tipo de heparina e a associação do genótipo da FcRIIa de receptores plaquetários. Foram selecionados 278 pacientes das Unidades de Terapia Intensiva e Unidades Coronariana do InCor-HCFMUSP, que receberam anticoagulação por heparina não fracionada (HNF) e/ou heparina de baixo peso molecular (HBPM), por pelo menos 5 dias, e excluídas as possíveis causas conhecidas de trombocitopenia. Foi realizada a contagem plaquetária pré e pós terapia com heparina, e o teste de detecção do anticorpo anti-fator 4 plaquetário/heparina (ID-PaGIA, DiaMed; e Asserachrom®-HPIA, Stago). O estudo da genotipagem da FcRIIa de receptores plaquetários foi realizado pelo método de digestão com enzima de restrição alelo específica. A freqüência de TIH encontrada foi de 6 (2,2%), e a freqüência de trombocitopenia com a presença do anticorpo anti-fator 4 plaquetário foi de 24,3%. A análise do gênero do paciente não demonstrou correlação com a TIH, nem com a trombocitopenia e nem com o anticorpo anti-fator 4 plaquetário. As mulheres apresentaram mais trombose do que os homens. A trombocitopenia ocorreu com maior freqüência nos pacientes que utilizaram os dois tipos de heparina (HNF-HBPM) e, com menor freqüência, nos que utilizaram apenas HBPM. O genótipo da FcRIIa de receptores plaquetários não apresentou relação com o gênero, nem com a TIH. Este estudo determinou a freqüência de TIH em uma população brasileira com uso de heparina e auxiliou no diagnostico. O melhor teste para detectar o anticorpo anti-fator 4 plaquetário/heparina, na presença de trombocitopenia e trombose, foi o teste de imunoaglutinação ID-PaGIA (DiaMed). A utilização dos dois tipos de heparina promoveu uma maior freqüência de trombocitopenia. Porém, novos estudos precisam confirmar as relações entre o tipo de heparina, com a trombocitopenia e com a TIH / Heparin induced thrombocytopenia (HIT) is an immune-hematologic syndrome mediated by a heparin dependent antibody that causes platelet activation, platelet aggregation, and can be associated with thrombosis and death. HIT occurs in about 1-5% of patients receiving heparin therapy up to 5 days or more. Many factors influence on the frequency of HIT. This is a pioneer Brazilian study to determine the frequency of HIT on patients under heparin therapy, and the relationship of HIT with gender, heparin type and the FcRIIa platelet receptor genotype. 278 patients from the Intensive Care Unit and Cardiac Care Unit at InCor-HCFMUSP treated with Unfractionated Heparin (UFH) and/or Low Molecular Weight Heparin (LMWH) for 5 or more days were studied. Known causes of thrombocytopenia were excluded. Platelet count was monitored pre and post heparin therapy. All selected patients were tested for detection of anti-heparin/PF4 antibody (ID-PaGIA, DiaMed; and Asserachrom®-HPIA, Stago). HIT frequency found was 6 (2,2%) and the frequency of thrombocytopenia (determined by a decrease in the platelet count below 50%, after the introduction of heparin therapy) and positive anti-heparin/PF4 antibody test was 24,3%. Patients gender was not related to TIH, neither to thrombocytopenia nor to the presence of antiheparin/ PF4 antibody. Thrombosis events were more frequent in women than in men. Thrombocytopenia, related to the type of heparin, was more frequent in patients that had used both types of heparin and less frequent in those that used only LMWH. FcRIIa platelet receptor genotype was associated with neither HIT nor with gender. This study has provided the frequency of HIT in a Brazilian patient population under heparin therapy and auxiliary in the HIT diagnosis. The ID-PaGIA (DiaMed) was shown to be the best test to correlate the presence of anti-heparin/PF4 antibody to thrombocytopenia and thrombosis event. The use of both heparin types promotes more thrombocytopenia. New studies are needed to confirm the relationship between heparin type and thrombocytopenia with HIT

A trombose venosa é a principal complicação da microcirurgia vascular e a intervenção precoce é necessária para o salvamento dos retalhos, com índices de sucesso de apenas 50% das revisões cirúrgicas; trombose da microcirculação, produção de radicais livres de oxigênio (RLO) e edema são os elementos principais da lesão de isquemia/reperfusão (I/R), e o planejamento das terapias protetoras tem como objetivo amenizar estas alterações. Os fármacos antioxidantes, antiagregantes plaquetários e anticoagulantes são utilizados no controle da lesão de I/R em diferentes órgãos. Neste estudo, em modelo de amputação subtotal de membro posterior de rato submetido a isquemia global primária, foi testado o efeito protetor dos fármacos alopurinol, heparina, tirofiban ou vitamina C durante a isquemia secundaria pós congestão venosa. Foram operados 100 ratos, que apos isquemia global de 90 minutos, foram divididos em cinco grupos de 20 animais recebendo uma das respectivas drogas na veia femoral contra-lateral: 1ml de solução fisiológica 0,9% no grupo controle (GC), 1ml de alopurinol 45mg/kg no grupo experimental 1 (G1), 1ml de heparina 200UI/kg no grupo experimental 2 (G2), 1ml de tirofiban 50 ug /ml no grupo experimental 3 (G3) e 1 ml de vitamina C 100mg/kg no grupo experimental 4 (G4); o clampe foi então retirado do feixe vascular e se iniciou a reperfusão de 60 minutos; a colocação do clampe vascular apenas na veia femoral direita iniciou a congestão venosa (isquemia secundária) do membro por 90 minutos seguido de outra reperfusão de 60 minutos; O músculo gastrocnêmio foi dissecado e retirado para analise histológica e os animais sacrificados por injeção letal. Foram estudados a porcentagem de viabilidade celular muscular, o edema e o extravasamento de hemácias. A porcentagem de lesão celular do músculo do grupo controle foi 54,6% (±10,6), do G1 31,5% (±13,6), do G2 24,7% (±11,7), do G3 24,6% (±8,6) e do G4 21,3% (±8,6). Os grupos foram comparados por modelo de comparação múltiplas one way-ANOVA e post-hoc Tukey com significância de p < 0,05. A porcentagem de lesão celular foi menor para os grupos G1, G2, G3 e G4 quando comparados ao GC (p < 0,001), e quando comparados os grupos experimentais entre si, apenas o G4 (vitamina C) foi menor estatisticamente que G1(alopurinol) (p < 0,029). A utilização individual dos fármacos alopurinol, heparina ,tirofiban e vitamina C mostraram efeito protetor na congestão venosa secundaria a isquemia global primária, e a vitamina C foi mais efetiva nesta ação que o alopurinol quando comparados os antioxidantes entre si. Quando avaliado o edema, apenas os antioxidantes tiveram índices menores estatisticamente que o GC, enquanto que todos os fármacos diminuíram o extravasamento de hemácias comparados com o grupo controle (p < 0,001) / Venous thrombosis is the main complication of vascular microsurgery an early intervention is mandatory to rescue the flap, with a success rate of only 50% of surgical revisions; microcirculation thrombosis, oxygen free radicals production and edema are the main elements of ischemia/reperfusion (I/R) injury, and protective therapies aim to mitigate these changes. Antioxidants, antiplatelets and anticoagulants are used in different organs to control this injury. In this study, in a partial hind limb amputation model submitted to global ischemia, it was tested the protective effect of Allopurinol, Heparin, Tirofiban or Vitamin C during secondary ischemia after venous congestion. A hundred rats divided in five groups of 20 animals each were operated; after global ischemia of 90 minutes each group was injected into the contra lateral femoral vein one of the following solutions: 1 ml of saline solution NaCl 0,9% - control group (CG); 1ml of Allopurinol 45mg/kg - experimental group 1 (G1); 1ml of Heparin 200 UI/kg - experimental group 2 (G2); 1ml of Tirofiban 50 ug /ml - experimental group 3 (G3); 1ml of Vitamin C 100mg/kg - experimental group 4 (G4). Sixty minutes of limb reperfusion was performed, and a secondary period of limb ischemia started with the clamping of the femoral vein only (limb congestion) which lasted for 90 minutes (secondary ischemia). After that, the vein clamp was removed and a 60 minute reperfusion period was observed; at the end of the second reperfusion period, the right gastrocnemius muscle was removed and fixed in 10% formaldehyde, animals were euthanized with a lethal dose of Pentobarbital. Muscle fibers were scored as uninjured or injured based on the morphology of individual fibers; interstitial edema and bleeding were graded on a four-point scale. The control group had more damaged muscle cells 54.6±10.6% when compared to allopurinol 31.5±13,6%, heparin 24.7±11.7%, tirofiban 24.6±8.6% and Vitamin C 21.3±8.6% all reached statistical significance (p < 0.00 0.029). These comparisons were analysed using ANOVA and post-hoc Tukey. The single use of Allopurinol, Heparin, Tirofiban or Vitamin C showed a protective effect on venous congestion after global ischemia, and Vitamin C was more effective than Allopurinol when compared both antioxidants. When evaluating the edema, only the antioxidants had statistically lower rates than the CG, whilst all drugs reduced the extravasation of red blood cells compared with the control group (p < 0.001)

A trombose venosa profunda (TVP) é uma patologia grave de alta incidência mundial. Quando não diagnosticada precocemente e tratada adequadamente pode evoluir causando sérias complicações, como a embolia pulmonar e insuficiência venosa crônica, as quais são responsáveis por altas taxas de morbidade e mortalidade. Seu tratamento utiliza terapia com anticoagulantes pelas vias parenteral e oral (para manutenção) que estão associadas a prejuízos bem documentados limitando seu uso, além de resultar em baixa adesão do paciente ao tratamento. Os sistemas de liberação modificada de fármacos, tais como as micropartículas poliméricas, representam uma grande área em desenvolvimento, a qual tem recebido atenção de pesquisadores e indústrias de todo o mundo e recebido investimentos crescentes nas últimas três décadas. As micropartículas poliméricas possuem grande estabilidade, capacidade industrial e possibilitam ajustes para alcançar o perfil de liberação adequado e/ou o direcionamento para determinado sítio de ação. O estudo teve início com o desenvolvimento e validação do método analítico para a quantificação da enoxaparina sódica. A turbidimetria foi a técnica de escolha, pois os resultados utilizando CLAE não foram satisfatórios. Este estudo teve como objetivo a obtenção e caracterização físico-química de um sistema de liberação microparticulado para veiculação de uma heparina fracionada (HF), a enoxaparina sódica, muito utilizada no tratamento da TVP, visando um aumento da biodisponibilidade do fármaco com controle da sua biodistribuição. As micropartículas contendo a enoxaparina sódica foram preparadas utilizando o copolímero dos ácidos lático e glicólico (50:50) (PLGA), biodegradável, através do método da dupla emulsificação/ evaporação do solvente. As partículas obtidas foram caracterizadas pela técnica de microscopia eletrônica de varredura (MEV) e apresentaram forma esférica com superfície lisa e regular. As análises do tamanho e distribuição dos tamanhos de partícula foram realizadas por dispersão de luz laser e apresentaram perfil monomodal para a maioria das formulações. O perfil de liberação in vitro do fármaco encapsulado foi avaliado por 35 dias e apresentou cinética de liberação de pseudo ordem zero, modelo de Higuchi (1961), indicando que a difusão foi o principal mecanismo de liberação. A velocidade de degradação das micropartículas é, através da difusão do fármaco, um parâmetro muito importante e determinante da liberação in vivo. / Deep vein thrombosis (DVT) is a severe disease with high incidence worldwide. When it is not early diagnosed and properly treated it can develop and to cause serious complications, such as pulmonary embolism and chronic venous insufficiency, which are responsible for high morbidity and mortality rates. The treatment of DVT is accomplished with parenteral and oral (for maintenance) anticoagulants. They are associated to damage well documented that limit their use resulting in poor adherence of patients to treatment. Drug delivery systems, such as polymeric microparticles, represent a significant development area. It has received attention of researchers and industries around the world and increased investments in last three decades. The polymeric microparticles have great stability, industrial capacity and they allow adjustments to achieve the suitable release profile and / or direction for a particular site of action. The study started with development and validation from the analytical method to quantification of enoxaparin sodium. Turbidimetric technique was used because the results by HPLC were not satisfactory. The aim of this work was the preparation and physical-chemical characterization of a microparticle release system for delivery of a fractionated heparin (FH), enoxaparin sodium, widely used to the treatment of DVT to increase the drug bioavailability and control their biodistribution. The microparticles containing enoxaparin sodium were prepared from a biodegradable polymer poly (lactic-co-glycolic acid) (50:50) (PLGA) using double emulsification / evaporation of the solvent method. The particles obtained were characterized by scanning electron microscopy technique (SEM) and showed spherical shape with smooth and regular surface. The analysis of the size and distribution of particle sizes were performed by scattering of laser light and showed unimodal profile for the most of formulations. In vitro drug release profile from the microparticles was evaluated in 35 days showing pseudo zero order kinetics, Higuchi model (1961). This indicated that main mechanism of drug release was diffusion.

<p>Cardiopulmonary bypass (CPB) is associated with inflammatory response and activation of coagulation. Heparin coating of the CPB circuit is shown to improve the biocompatibility of the surface. The biological effects of a new heparin surface, the Corline Heparin Surface (CHS), prepared according to a new principle, have been studied. </p><p>The CHS used during coronary artery bypass grafting with CPB in sixty patients prevented adhesion of cells to the extracorporeal device. The activation of inflammation, coagulation, and fibrinolysis was significantly reduced by the use of CHS. Both a reduced and an increased dose of systemic heparin in combination with the heparin-coated surface resulted in more activation of inflammation and coagulation. </p><p>Photoelectron spectroscopy studies of the molecular structure of the CHS demonstrated that a single layer of the heparin surface, equivalent to what was used in the <i>in vivo</i> studies, did not completely cover the substrate surface. Additional layer of immobilized heparin has resulted in a complete coverage. We examined the biological effects, i.e. activation of inflammation and coagulation, by CHS in one and two layers in an <i>in vitro</i>-study. The data from this study clearly demonstrated that a uniform surface coating of the CHS results in only minor activation of coagulation, inflammation and cell activation. </p><p>Monocytes do not normally express tissue factor (TF), initiator of the coagulation <i>in vivo</i>, but can be induced upon adhesion to artificial surfaces. TF is receptor for coagulation factor VIIa (FVIIa) and binding subsequently leads to formation of thrombin. Other biological effects beyond coagulation, as inflammation and angiogenesis, has recently been associated with the formation of TF·FVIIa. The TF∙FVIIa signal transduction induced an increased sensitivity to PDGF-BB-stimulated migration and an increased production of IL-8 and TNF-α in monocytes. These could be important mechanisms for continued recruitment of cells to sites of inflammation. </p>

<p>Heparan sulfate (HS) is a ubiquitous polysaccharide on the cell surface and in the extracellular matrix. HS is an important actor in the regulation of cell signaling, especially in the developing embryo. In combination with cell culture and biochemical experiments, <i>in vivo</i> studies of genetically modified animals have pointed out the sulfation pattern of HS as highly important for binding of ligands, their receptors and other signaling modulators.</p><p>The sulfation pattern of an HS chain is gained by several modifying steps, performed by multiple enzymes during biosynthesis in the Golgi apparatus. By alterations of sulfation pattern, and the amount of sulfate groups, a cell can regulate the binding properties of its HS to different molecules. The most highly sulfated form of HS is called heparin, and can only be found intracellularly in mast cells.</p><p>This thesis describes the phenotypes and the alterations in HS/heparin biosynthesis of two genetically modified mouse strains deficient in N-deacetylase/N-sulfotransferase-1 (NDST1) and -2 (NDST2) respectively. We have found NDST1 to be important for correct sulfation of HS and that NDST2 is crucial in heparin biosynthesis. NDST2 deficient mice completely lack heparin and therefore have a severe mast cell phenotype. NDST1 deficient mice produce undersulfated HS and show several developmental disturbances. Some NDST1 embryos die in utero while the rest die neonatally due to breathing difficulties. Defect brain, eye and skeletal development has also been observed while some organs, such as the liver, appear to be largely unaffected. Several phenotypes are similar to defects seen in other mouse strains with impaired fibroblast growth factor and bone morphogenetic protein signaling, among others. This suggests the phenotypes of NDST1 deficient embryos to be of a multi factorial origin, in complete accordance to the many signaling pathways HS is suggested to modulate.</p>

Cardiopulmonary bypass (CPB) is associated with inflammatory response and activation of coagulation. Heparin coating of the CPB circuit is shown to improve the biocompatibility of the surface. The biological effects of a new heparin surface, the Corline Heparin Surface (CHS), prepared according to a new principle, have been studied. The CHS used during coronary artery bypass grafting with CPB in sixty patients prevented adhesion of cells to the extracorporeal device. The activation of inflammation, coagulation, and fibrinolysis was significantly reduced by the use of CHS. Both a reduced and an increased dose of systemic heparin in combination with the heparin-coated surface resulted in more activation of inflammation and coagulation. Photoelectron spectroscopy studies of the molecular structure of the CHS demonstrated that a single layer of the heparin surface, equivalent to what was used in the in vivo studies, did not completely cover the substrate surface. Additional layer of immobilized heparin has resulted in a complete coverage. We examined the biological effects, i.e. activation of inflammation and coagulation, by CHS in one and two layers in an in vitro-study. The data from this study clearly demonstrated that a uniform surface coating of the CHS results in only minor activation of coagulation, inflammation and cell activation. Monocytes do not normally express tissue factor (TF), initiator of the coagulation in vivo, but can be induced upon adhesion to artificial surfaces. TF is receptor for coagulation factor VIIa (FVIIa) and binding subsequently leads to formation of thrombin. Other biological effects beyond coagulation, as inflammation and angiogenesis, has recently been associated with the formation of TF·FVIIa. The TF∙FVIIa signal transduction induced an increased sensitivity to PDGF-BB-stimulated migration and an increased production of IL-8 and TNF-α in monocytes. These could be important mechanisms for continued recruitment of cells to sites of inflammation.

Heparan sulfate (HS) is a ubiquitous polysaccharide on the cell surface and in the extracellular matrix. HS is an important actor in the regulation of cell signaling, especially in the developing embryo. In combination with cell culture and biochemical experiments, in vivo studies of genetically modified animals have pointed out the sulfation pattern of HS as highly important for binding of ligands, their receptors and other signaling modulators. The sulfation pattern of an HS chain is gained by several modifying steps, performed by multiple enzymes during biosynthesis in the Golgi apparatus. By alterations of sulfation pattern, and the amount of sulfate groups, a cell can regulate the binding properties of its HS to different molecules. The most highly sulfated form of HS is called heparin, and can only be found intracellularly in mast cells. This thesis describes the phenotypes and the alterations in HS/heparin biosynthesis of two genetically modified mouse strains deficient in N-deacetylase/N-sulfotransferase-1 (NDST1) and -2 (NDST2) respectively. We have found NDST1 to be important for correct sulfation of HS and that NDST2 is crucial in heparin biosynthesis. NDST2 deficient mice completely lack heparin and therefore have a severe mast cell phenotype. NDST1 deficient mice produce undersulfated HS and show several developmental disturbances. Some NDST1 embryos die in utero while the rest die neonatally due to breathing difficulties. Defect brain, eye and skeletal development has also been observed while some organs, such as the liver, appear to be largely unaffected. Several phenotypes are similar to defects seen in other mouse strains with impaired fibroblast growth factor and bone morphogenetic protein signaling, among others. This suggests the phenotypes of NDST1 deficient embryos to be of a multi factorial origin, in complete accordance to the many signaling pathways HS is suggested to modulate.

Δύο από τα σημαντικότερα δομικά και λειτουργικά βιομόρια του εξωκυττάριου χώρου είναι το κολλαγόνο και οι γλυκοζαμινογλυκάνες (GAGs), ανιοντικοί πολυσακχαρίτες που αποτελούν το βασικό δομικό συστατικό των πρωτεογλυκανών. Οι κύριοι τύποι γλυκοζαμινογλυκανών είναι η θειική χονδροϊτίνη, η θειική δερματάνη, η ηπαρίνη, η θειική ηπαράνη, η θειική κερατάνη και το υαλουρονικό οξύ. Το κολλαγόνο τύπου Ι είναι η πιο άφθονη πρωτεΐνη στους ιστούς των θηλαστικών. Το κολλαγόνο τύπου ΙΙ αποτελεί το κύριο συστατικό του εξωκυττάριου χώρου του αρθρικού χόνδρου και άλλων ιστών. Τα παραπάνω μακρομόρια είναι υπεύθυνα για τη ρύθμιση διαφόρων διεργασιών των κυττάρων τόσο σε φυσιολογικές όσο και σε παθολογικές καταστάσεις, όπως παθήσεις των αρθρώσεων και νεοπλασματικές ασθένειες. Αντικείμενο της παρούσας εργασίας αποτέλεσε η ανάπτυξη μιας μεθοδολογίας για τον προσδιορισμό των αλληλεπιδράσεων μεταξύ γλυκοζαμινογλυκανών και των δύο τύπων κολλαγόνου, η οποία θα συνεισφέρει στη βαθύτερη κατανόηση της βιολογικής τους λειτουργίας. Μερικές από τις τεχνικές που έχουν χρησιμοποιηθεί για το συγκεκριμένο σκοπό είναι η χρωματογραφία συγγένειας, η ηλεκτροφόρηση και η φασματοσκοπία φθορισμού. Η φασματοσκοπία πυρηνικού μαγνητικού συντονισμού (NMR), η περίθλαση ακτίνων-Χ και ο κυκλικός διχρωισμός (Circular Dichroism, CD) προσφέρουν δομικές πληροφορίες για τις αλλαγές στη διαμόρφωση και τα σημεία πρόσδεσης μεταξύ γλυκοζαμινογλυκανών και πρωτεϊνών. Θερμοδυναμικές πληροφορίες για τις αλληλεπιδράσεις πρωτεϊνών-γλυκοζαμινογλυκανών αντλούνται από τη θερμιδομετρία ισόθερμης τιτλοδότησης (Isothermal Titration Calorimetry, ITC), ενώ με την τεχνική της διέγερσης επιφανειακών πλασμονίων (Surface Plasmon Resonance, SPR) μελετώνται η σταθερά σύνδεσης και η σταθερά διάστασης της αλληλεπίδρασης σε πραγματικό χρόνο. Το κυριότερο μειονέκτημα των παραπάνω τεχνικών είναι το ότι δεν προσφέρουν πληροφορίες για χημικούς δεσμούς, ενώ ο χρόνος ανάλυσης είναι μεγάλος και απαιτούνται μεγάλες ποσότητες δειγμάτων. Η τεχνική που χρησιμοποιήθηκε ήταν εκείνη της φασματοσκοπίας micro-Raman, μια μη καταστρεπτική τεχνική, η οποία προσφέρει πληροφορίες για τη χημική δομή του εξεταζόμενου δείγματος, ενώ παράλληλα είναι γρήγορη και ακριβής. Παρασκευάστηκαν δύο είδη μιγμάτων γλυκοζαμινογλυκανών με κολλαγόνο. Στην πρώτη περίπτωση, κολλαγόνο τύπου Ι ή τύπου ΙΙ εμβαπτίστηκε σε διάλυμα θειικής χονδροϊτίνης, ηπαρίνης ή μίγμα τους που παρασκευάστηκε με αναλογία όγκων 1:1. Στη δεύτερη περίπτωση, μίγματα των δύο ουσιών προέκυψαν με ανάμιξη ίσων ποσοτήτων των δύο ουσιών. Τα παραπάνω μίγματα μελετήθηκαν με φασματοσκοπία Raman και με την τεχνική της Διαφορικής Θερμιδομετρίας Σάρωσης (Differential Scanning Calorimetry, DSC) και συγκρίθηκαν με τα φάσματα των προτύπων ουσιών. Κάθε ουσία έχει ένα χαρακτηριστικό φάσμα Raman, η ερμηνεία του οποίου οδήγησε στην ταυτοποίηση χαρακτηριστικών ομάδων των μορίων, όπως οι δεσμοί C-OH, οι θειικές ομάδες (O-SO3-, N-SO3-), η Ν-ακετυλομάδα, οι δεσμοί C=O και οι δεσμοί C-Ν. Οι φασματικές περιοχές που παρουσιάζουν τα πιο έντονα χαρακτηριστικά στα φάσματα Raman των μιγμάτων GAG-κολλαγόνου είναι οι εξής: 800-920 cm-1, 900-1000 cm-1 και 980-1170 cm-1. Όσον αφορά στην τελευταία φασματική περιοχή, παρατηρήθηκε σημαντική μετατόπιση της χαρακτηριστικής κορυφής της δόνησης έκτασης των θειομάδων προς χαμηλότερους κυματάριθμους (από τους 1070 cm-1 περίπου στους 1062-1064 cm-1) και η εμφάνιση μιας κορυφής στους 1072 cm-1, σε σχέση με τα αντίστοιχα φάσματα των προτύπων ουσιών, στα φάσματα όλων των μιγμάτων που μελετήθηκαν. Η μετατόπιση της συγκεκριμένης κορυφής αποτελεί ένδειξη αλληλεπίδρασης μεταξύ των δύο ουσιών και καταδεικνύει το σημαντικό ρόλο των θειομάδων των γλυκοζαμινογλυκανών στις αλληλεπιδράσεις τους με τη συγκεκριμένη πρωτεΐνη. Τα αποτελέσματα της φασματοσκοπίας Raman βρίσκονται σε συμφωνία με εκείνα που προκύπτουν από την τεχνική της Διαφορικής Θερμιδομετρίας Σάρωσης (DSC), καθώς τα θερμογραφήματα DSC των μιγμάτων θειικής χονδροϊτίνης-κολλαγόνου τύπου Ι είναι διαφορετικά από εκείνο του μίγματος που προέκυψε από την ανάμιξη των δύο συστατικών, υποδεικνύοντας την ύπαρξη αλληλεπίδρασης μεταξύ των δύο ουσιών. Με την τεχνική της φασματοσκοπίας Raman διαπιστώθηκε ότι το κολλαγόνο τύπου Ι έδειξε μεγαλύτερη «χημική προτίμηση» προς την ηπαρίνη σε σχέση με τη θειική χονδροϊτίνη, ενώ το κολλαγόνο τύπου ΙΙ προτίμησε να αλληλεπιδράσει με τη θειική χονδροϊτίνη. / Collagen and glycosaminoglycans (GAGs) co-exist as major constituents of the extracellular matrix (ECM) in a variety of tissues. Collagen type I is the most abundant protein in the human body, whereas another important type of collagen is type II, which forms the extracellular matrix of cartilage and other tissues. Glycosaminoglycans are negatively charged polysaccharides that occur as a structural component of proteoglycans and can be divided in four major groups: i) chondroitin sulfate and dermatan sulfate, ii) heparin and heparin sulfate, iii) keratan sulfate, and iv) hyaluronic acid. Both GAGs and collagen not only regulate a variety of cellular functions but they also seem to be involved in many pathological conditions, including cancer and joint diseases. Therefore, a more detailed investigation of the interactions between them will result in a deeper understanding of their biological function. Most common methods for identifying GAG-collagen interactions include affinity chromatography, affinity electrophoresis and fluorescence spectroscopy. Nuclear Magnetic Resonance (NMR), X-ray diffraction and Circular Dichroism (CD) provide structural data characterizing conformational changes and contact points between the interacting species. Using Isothermal Titration Calorimetry (ITC), information on the thermodynamics of glycosaminoglycan-protein interactions can be obtained. Surface Plasmon Resonance (SPR) allows the measurement of association and dissociation constants of glycosaminoglycan-protein interactions in real time. The major disadvantage of the techniques described above is the inability to identify specific chemical bonds. Other disadvantages are the long analysis time and that large amounts of the interacting substances are required. In the present work, Raman spectroscopy, a non-destructive, vibrational technique which yields information on the chemical composition of the specimen, was employed for the exploration of the interactions between collagen type I and type II and two glycosaminoglycans, chondroitin sulfate and heparin. Two sets of mixtures composed of glycosaminoglycans and each type of collagen were prepared: i) collagen type I or type II was immersed in aqueous solutions of chondroitin sulfate, heparin and a 1:1 mixture of both GAGs, and ii) GAG-collagen mixtures were obtained by blending suitable amounts of the two substances. Differential Scanning Calorimetry (DSC) was also applied on the latter mixtures. From the Raman spectra identification of vibrational frequencies of the functional groups of the above molecules, such as C-OH linkages, sulfate groups (O-SO3-, N-SO3-), N-acetyl group, carboxyl group and C-Ν linkages is possible. The prominent features arising from the Raman spectra of GAG-collagen interactions are found in the regions 800-920 cm-1, 900-1000 cm-1 and 980-1170 cm-1. Processing of the spectra of all GAG-collagen mixtures has revealed that a shift of the most characteristic vibration of chondroitin sulfate’s and heparin’s spectrum from 1070 cm-1 to 1062-1064 cm-1, while a vibration at approximately 1072 cm-1 emerges. The sulfate band shift is indicative of an interaction between collagen and glycosaminoglycans and depicts the important role of the sulfate group of glycosaminoglycans in the interactions with the protein. This observation was in accordance with the results from Differential Scanning Calorimetry (DSC), which demonstrated an interaction between collagen and chondroitin sulfate. A stronger preference of collagen type I to interact with heparin rather than chondroitin sulfate and of collagen type II to interact with chondroitin sulfate was also observed.