It has been postulated that the control of thrombus formation on molecularly engineered surfaces is an important step in developing clinically durable small-diameter vascular prostheses. This has led to designing a membrane-mimetic assembly that contains physiological regulators of blood coagulation, thrombomodulin (TM) and heparin, to provide strategies for generating actively antithrombogenic surfaces. The membrane-mimetic construct contains polymeric phospholipid monolayer on an alkylated polyelectrolyte multilayer supported by planar substrate such as glass or silicone. When incorporated with TM, the model platform exhibited the biological function by catalyzing activation of protein C. Surface TM activity was extensively investigated at physiologic shear rates (50 sec-1 and 500 sec-1). Significantly, reaction rates become saturated at TM surface densities greater than or equal to ~ 800 fmole/cm2 due to due to a transport limitation. Based on the similar membrane-mimetic construct, a functional heparinized surface was designed as an alternative anticoagulant system. Immobilization of heparin onto membrane-mimetic surfaces was achieved through biotin-streptavidin binding specificity. Activity of surface heparin to facilitate thrombin inactivation was investigated at shear rates of 50 and 500 sec-1. Significantly, rate of thrombin decay becomes saturated when the surface coverage of heparin is higher than 4.4 pmole of heparin per cm2. We further investigated the effects of surface bound TM and heparin on tissue factor (TF) -induced thrombin generation in a flow model. Specifically, TF positioned over a 2 x 6 mm2 upstream region as a trigger for thrombin generation and TM and/or heparin positioned over the remaining downstream (34 x 6 mm2) portion of the test film. Compared to TF alone surface, thrombin generation was profoundly reduced in the presence of surface bound TM and/or heparin. Significantly, thrombin production was maximally inhibited more than 85% in the presence of TM and heparin, possibly due to anticoagulant synergism of both anticoagulants. We believe that current membrane-mimetic systems can potentially create actively antithrombogenic surfaces.

La réponse immune sous héparine (H) est associée à la synthèse d’anticorps (Ac) d’isotype IgG dirigés contre le facteur plaquettaire 4 (FP4) modifié par l’héparine. Ces anticorps se fixent par leur fragment Fc aux récepteurs FcγRIIa des plaquettes et induisent une forte activation plaquettaire. Les héparines de bas poids moléculaire sont constituées d’un mélange hétérogène d’oligosaccharides (OS) dont la structure varie en fonction de leur nombre de sucres et de groupements sulfates. Nous avons montré que seuls les OS ayant dix groupements sulfates ou plus, peuvent modifier le FP4 et permettre la fixation des Ac héparine-dépendants. La chirurgie cardiaque est associée à une forte activation plaquettaire et les patients sont exposés à de fortes concentrations d’héparine qui est neutralisée en fin d’intervention par le sulfate de protamine (SP). Alors que 30 à 50 % d’entre eux développent des Ac anti H/FP4 nous avons montré que 25% développent également des Ac dirigés contre les complexes H/SP et que ces Ac sont capables in vitro d’induire une activation plaquettaire. Le rôle de ces Ac in vivo reste cependant discuté. / The immune response under heparin (H) treatment is associated with IgG antibodies (Abs) synthesis against heparin-modified Platelet Factor 4 (PF4). These Abs bind FcγRIIa receptors via their Fc fragment and promote strong platelet activation. Low Molecular Weight Heparins are complex mixtures of polysaccharide fragments. These oligosaccharides (OS) have a variable structure due to variations in the type of sugar units and the number of sulphate groups. We demonstrated that OS longer than 10 saccharides and with a large number of sulphate groups are likely able to modify PF4 and allow the binding of heparin-dependent Abs. Cardiac surgery is associated with strong platelet activation and high doses of unfractionated heparin are administered to patients during surgery, and then neutralized with protamine sulfate (SP) at the end of the intervention. 30 to 50% of patients develop anti H/PF4 Abs, but we demonstrated that 25% do synthethized anti H/SP Abs able to activate platelets in vitro. The pathogenic role of these Abs to H/SP in vivo is controversial.

Made available in DSpace on 2014-12-17T14:03:25Z (GMT). No. of bitstreams: 1 AdrianaSB.pdf: 1664966 bytes, checksum: 33b28b577c6c69f20823f4e4a84e7540 (MD5) Previous issue date: 2008-06-13 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The occurrence of bioactive compounds in marine organisms comes awaking the interest of the pharmaceutical industry. Heparin, a sulfated polysaccharide which presence was already identified in several marine invertebrates, is very attractive due its remarkable functional versatility. Besides to intervene in blood coagulation, this molecule has a great anti-inflammatory potential. However, its strong anticoagulant activity difficult the clinical exploitation of its anti-inflammatory properties. Thus, the aims of this work were to evaluate the effect of a heparin-like compound (heparinoid), isolated from the cephalotorax of the Litopenaeus vannamei shrimp, on the inflammatory response, hemostasia and synthesis of antithrombotic heparan sulfate by endothelial cells, besides studying some aspects concerning its structure. The purified heparinoid was structurally characterized following an analytical boarding, involving electrophoresis and chromatography. The structural analysis have shown that this compound possess a high content of glucuronic acid residues and disulfated disaccharide units. In contrast to mammalian heparin, the heparinoid was incapable to stimulate the synthesis of heparan sulfate by endothelial cells in the tested concentrations, beyond to show reduced anticoagulant activity and hemorrhagic effect. In a model of acute inflammation, the compound isolated from the shrimp reduced more than 50% of the cellular infiltration. Besides reduce the activity of MMP-9 and proMMP-2 of the peritoneal lavage of inflamed animals, the heparinoid also reduced the activity of MMP-9 secreted by activated human leukocytes. These results demonstrate the potential of heparinoid from L. vannamei to intervene in the inflammatory response. For possessing reduced anticoagulant activity and hemorrhagic effect, this compound can serve as a structural model to direct the development of more specific therapeutical agents to the treatment of inflammatory diseases / A ocorr?ncia de compostos bioativos em representantes da biodiversidade marinha vem despertando o interesse da ind?stria farmac?utica. Heparina, um polissacar?deo sulfatado cuja presen?a j? foi identificada em v?rios invertebrados marinhos, destaca-se por sua extraordin?ria versatilidade funcional. Al?m de interferir na coagula??o sangu?nea, essa mol?cula possui grande potencial antiinflamat?rio. No entanto, sua forte atividade anticoagulante dificulta o aproveitamento cl?nico de sua propriedade antiinflamat?ria, o que estimula a pesquisa por an?logos de heparina com efeitos colaterais reduzidos. Diante disso, este trabalho teve por objetivos avaliar o efeito de um composto semelhante ? heparina (heparin?ide), isolado do cefalot?rax do camar?o Litopenaeus vannamei, sobre a resposta inflamat?ria, hemostasia e s?ntese de heparam sulfato antitromb?tico pelas c?lulas endoteliais, al?m de estudar alguns aspectos relevantes a cerca de sua estrutura. O heparin?ide purificado foi estruturalmente caracterizado seguindo uma abordagem anal?tica, envolvendo eletroforeses e cromatografias. As an?lises estruturais mostraram que esse composto possui um elevado conte?do de res?duos de ?cido glucur?nico e de dissacar?deos dissulfatados. Ao contr?rio da heparina de mam?feros, o heparin?ide foi incapaz de estimular a s?ntese de heparam sulfato pelas c?lulas endoteliais nas concentra??es testadas, al?m de apresentar atividade anticoagulante in vitro e efeito hemorr?gico reduzidos. Em um modelo de inflama??o aguda, o composto isolado do camar?o reduziu mais de 50% da infiltra??o celular. Al?m de reduzir a atividade de MMP-9 e proMMP-2 no lavado peritoneal dos animais submetidos ao modelo de inflama??o, o heparin?ide tamb?m reduziu a atividade de MMP-9 secretada por leuc?citos humanos ativados. Esses resultados demonstram o potencial do heparin?ide de L. vannamei em interferir em v?rios eventos da resposta inflamat?ria. Por possuir atividade anticoagulante e efeito hemorr?gico reduzidos, esse composto pode servir como um modelo estrutural para direcionar o desenvolvimento de agentes terap?uticos mais espec?ficos para o tratamento de doen?as inflamat?rias

Introduction: Thrombotic events are among the most serious complications of sepsis and also the most frequent causes of morbidity and mortality in patients with sepsis. Currently, the administration of low molecular weight heparins (LMWH) is recommended in patients with severe sepsis for prophylaxis of these complications. However, this prophylaxis often fails. Objectives of the study: One of the objectives of our study was to examine changes in haemostasis in relation to the inflammatory response during 15 days of severe sepsis. The next objective was to determine whether a prophylactic inhibition of F Xa in the range from 0.2 to 0.4 IU/mL is achieved in these patients, if they receive the recommended prophylaxis with LMWH. We also recorded the dynamics of changes in the F Xa inhibition during the entire study period. Moreover, we tried to identify the factors that may affect the antithrombotic efficacy of the subcutaneously administered enoxaparin. Patient population and methods: A total of 35 ICU patients meeting the criteria of severe sepsis were enrolled in the study. Only 16 of these patients could be followed throughout the entire 15-day period. Patients were treated according to the current guidelines, including LMWH prophylaxis; enoxaparin (40 mg sc per day) was used in this study....

(v AJ) The aim of this diploma thesis was namely to find out whether the saline solution, intended for the continuous flushing of arterial catheters, is as effective in preventing arterial blockage as compared to saline solution with heparin. In this diploma thesis I also deal with the comparison of material consumption by using both methods of flushing arterial sets and the financial costs associated with them. Patients with diagnosis of sepsis or septic shock were included in the study. Pseudorandomization was used to group 52 patients into either experimental and control group. The testing was performed using a non-parametric Wilcoxon two-assay test and Fisher precision test. Based on the results of my study, regard to the occurrence of complications (catheter closure) an arterial catheter flushing by saline solution is as effective as flushing by a saline solution with heparin. My research also confirmed that the use of saline solution as a flushing solution is less costly (regarding material) and therefore it is advisable to use saline solution for flushing arterial catheters. Based on my study, saline solution is now used as a flushing solution of arterial catheters at an anesthesiology-resuscitation department, where only a saline solution with heparin was used for the flushing. Using saline...

Articular cartilage is a highly specialized, hierarchically organized tissue covering the articular surfaces of diarthrodial joints that absorbs and distributes forces upon mechanical loading and enables low-friction movement between opposing bone ends. Despite a strong resilience towards mechanical stress, once damaged cartilage is generally not regenerated due to a limited repair potential of the residing cells (chondrocytes) and the local absence of vascularized blood vessels and nerves. Eventually, this may lead to osteoarthritis, a chronic degenerative disorder of the synovial joints which has a strongly growing prevalence worldwide. Modern regenerative therapies that aim to rebuild cartilage tissue in vivo and in vitro using chondrocyte- and stem cell-based methods are still not able to produce tissue constructs with desired biomechanical properties and organization for long-term repair. Therefore, cartilage tissue engineering seeks for new ways to solve these problems. In this regard, the application of hydrogel-based scaffolding materials as artificial matrix environments to support the chondrogenesis of embedded cells and the implementation of appropriate biofabrication techniques that help to reconstitute the zonal structure of articular cartilage are considered as promising strategies for sophisticated cartilage regeneration approaches. In this thesis, a modular starPEG-heparin hydrogel platform as cell-instructive hydrogel scaffold was used in combination with a custom-designed 3D inkjet bioprinting method with the intention to develop a printable 3D in vitro culture system that promotes the chondrogenic differentiation of human mesenchymal stromal cells (hMSC) in printed cell-laden hydrogels with layered architectures in order to fabricate cartilage-like tissue constructs with hierarchical organization. Firstly, the successful bioprinting of horizontally and vertically structured, cell-free and -laden hydrogel scaffolds that exhibit layer thicknesses in the range of the superficial zone, the thinnest articular cartilage layer is demonstrated. The long-term integrity of the printed constructs and the cellular functionality of the plotted cells that generally had a high viability after the printing process are shown by a successful PDGF-BB-mediated hMSC migration assay in a printed multilayered hydrogel construct over a culture period of 4 weeks. Secondly, when the established printing procedures were applied for the chondrogenic differentiation of hMSCs, it was found that the printed cell-laden constructs showed a limited potential for in vitro chondrogenesis as indicated by a weaker immunostaining for cartilage-specific markers compared to casted hydrogel controls. In order to increase the post-printing cell density to tackle the limited printable cell concentration which was regarded as the primary reason for the impaired performance of the printed scaffolds, different conditions with varying culture medium and hydrogel compositions were tested to stimulate 3D cell proliferation. However, a significant 3D cell number increase could not be achieved which ultimately resulted in shifting the further focus to casted hMSC-laden starPEG-heparin hydrogels. Thirdly, the chondrogenic differentiation of hMSCs in casted hydrogels proved to be successful which was indicated by a uniform deposition of cartilage-specific ECM molecules comparable with the outcomes of scaffold-free MSC micromass cultures used as reference system. However, the quantitative analysis of biochemical and physical properties of the engineered hydrogel constructs yielded still significant lower values in relation to native articular cartilage tissue. Fourthly, in order to improve these properties and to enhance the chondrogenesis in starPEGheparin hydrogels, a dualistic strategy was followed. In the first part, specific externally supplied stimulatory cues including a triple growth factor supply strategy and macromolecular crowding were applied. As second part, intrinsic properties of the modular hydrogel system such as the crosslinking degree, the enzymatic degradability and the heparin content were systematically and independently altered. It was found that while the external cues showed no supportive benefits for the chondrogenic differentiation, the reduction of the heparin content in the hydrogel proved to be a key trigger that resulted in a significantly increased cartilage-like ECM deposition and gel stiffness of engineered constructs with low and no heparin content. In conclusion, this work yielded important experiences with regards to the application of inkjet bioprinting for hMSC-based cartilage tissue engineering approaches. Furthermore, the obtained data provided valuable insights into the interaction of MSCs and a surrounding hydrogel-based microenvironment that can be used for the further development of chondrosupportive scaffolding materials which may facilitate the fabrication of cartilage-like tissue constructs.

Subcutaneous anticoagulant medications like Heparin and Low-Molecular Weight Heparin are injections that readily cause bruising, pain, induration, and hematoma formation at the injection site. It is known that these adverse reactions can be correlated to the technique used to administer these medications; however, there is no established technique that reduces bruising, pain, induration, and hematoma formation at the site. Currently, the only protocol for subcutaneous Heparin and Low-Molecular Weight Heparin is that it is to be administered subcutaneously in the abdomen and when using a prefilled syringe, the air bubble should not be removed. The purpose of this study was to identify current nursing practice for the administration of these medications and to compare the results to researched techniques that resulted in less adverse site reactions. A total of 33 participants were recruited. The survey targeted six researched techniques found, after a comprehensive literature review, to have reduced site adverse effects associated with subcutaneous Heparin and Low-Molecular Weight Heparin. After completing the survey, it was found that current practice does not reflect techniques researched to reduce bruising, pain, induration, and hematoma formation at the site. In fact, very few completed one of the six research techniques that were questioned, which included: a two minute application of a cold compress/pack before and/or after the injection, an injection duration lasting 30 seconds, slow removal of the needle over five seconds, application of pressure after the injection for a minimum of 30 seconds, use of a hot pack/compress after the injection, and the use of a3 mL syringe. It was also found that there were inconsistencies in techniques that have been previously established as current protocol for these medications.

<p>Prothrombinase is composed of a proteinase, factor Xa (Xa), its cofactor Va (Va), Ca²⁺ and a zymogen, prothrombin (II), assembled on a phospholipid surface. During coagulation, prothrombinase accelerates II to thrombin conversion; but during anticoagulation, it protects the proteinase from inhibition by antithrombin (AT) ± unfractionated heparin (UFH). Although the degree of Xa protection by prothrombinase varies according to the reports in literature, moderate to significant protective effects have been consistently reported by most investigators. To overcome the limitations of UFH, our laboratory has developed a covalent complex of AT and UFH (ATH) with superior anticoagulant responses. To further understand the mechanisms of enhanced anticoagulant activity of ATH, we proceeded to study inhibition of the prothrombinase complex<em> </em>on synthetic vesicles, activated platelets and red blood cells (RBCs). Using discontinuous inhibition assays, we determined the rate of inhibition of prothrombinase-complexed Xa compared to control Xa. With synthetic vesicles, Xa was protected from inhibition by AT+UFH when in prothrombinase, while only a mild protective effect was observed with ATH. Omission of various components of the prothrombinase led to a reduction in Xa protection for AT+UFH. However, an increased Xa protection against ATH was observed when II was omitted from the prothrombinase. In comparison to the synthetic vesicle system, activated platelets showed a similar trend for protection of Xa in reactions involving prothrombinase ± components, while no protection of Xa was observed for ATH reactions. Alternatively, RBCs showed differences relative to vesicles in that increased protection of Xa occurred with omission of II and Va for AT+UFH, whereas omission of Va increased protection against ATH inhibition. In addition, ATH had improved inhibition of thrombin generation, fibrin formation and plasma coagulation compared to AT+UFH. Studies of fluorescently labelled Xa and inhibitors detailed binding interactions with prothrombinase subunits. Overall, the results suggest that a covalent linkage between AT and heparin improves inactivation of prothrombinase complexed-Xa leading to down-regulation of prothrombinase function.</p> / Doctor of Philosophy (Medical Science)

Anticoagulants are the basis for treatment and prevention of thrombotic diseases. The currently available medicines are associated with a wide range of adverse reactions that mandates developing new anticoagulants. Several lines of evidence support the superiority of factor Xa (FXa) as a promising target to develop novel anticoagulants. This work focuses on the design of direct and indirect FXa inhibitors using an interdisciplinary approach. As indirect FXa inhibitors, a focused library of tetrasulfated N–arylacyl tetrahydroisoquinoline (THIQ) nonsaccharide allosteric antithrombin activators was designed, synthesized, and biochemically evaluated to establish their structure–activity relationship (SAR). An N–arylacyl THIQ analog having carboxylate at position–3, two sulfate groups at positions–5 and –8 of THIQ moiety, butanoyl linker, and two sulfate groups at positions–2 and –5 of the phenolic monocyclic moiety was identified as the most promising nonsaccharide antithrombin activator with KD of 1322 ± 237 μM and acceleration potential of 80–fold. Its biochemical profile indicates a strong possibility that it activates antithrombin by the pre–equilibrium pathway rather than the induced–fit mechanism utilized by heparin analogs. A similar interdisciplinary approach was exploited to design direct FXa inhibitors that possess high selectivity and are potentially orally bioavailable. Structurally, the designed direct FXa inhibitors are neutral THIQ dicarboxamides. THIQ dicarboxamide is a privileged structure with a semi–rigid character, a structural feature that potentially offers high selectivity for targeting FXa over other coagulation and digestive proteases. It can also be thought of as an amino acid–like structure, which affords accessibility to a large number of compounds using well established peptide chemistry. Mechanistically, the designed inhibitors were expected to bind to FXa in the active site and function as orthosteric inhibitors. These direct FXa active site inhibitors are also likely to inhibit clot–bound enzyme. Nearly 60 THIQ dicarboxamides were synthesized and biochemically evaluated. Through detailed SAR analysis, the most potent analog was designed and found to exhibit an IC50 of 270 nM (Ki = 135 nM), an improvement of more than 207–fold over the first inhibitor synthesized in the study. The most potent inhibitor displayed at least 1887–fold selectivity for FXa over other coagulation enzymes and a selectivity index of at least 279–fold over the digestive serine proteases. This analog doubled plasma clotting times at 17–20 μM, which are comparable to those of agents being currently studied in clinical trials. Overall, allosteric and orthosteric approaches led to the design of indirect and direct small molecule inhibitors of FXa based on the THIQ scaffold. This work introduces two promising molecules, a tetrasulfated N–arylacyl THIQ analog as a heparin mimetic and a neutral THIQ dicarboxamide as a potent, selective, and potentially bioavailable peptidomimetic, for further advanced medicinal chemistry studies.

Hypercoagulable states occur when the process of hemostasis is disturbed. It manifests as a deep vein thrombosis or a pulmonary embolism. It is also one of the most common cardiovascular diseases. There are two factors- genetic and acquired. Inherent factors involve coagulation inhibitor deficiency, prothrombin gene mutation and activated protein C resistance. Acquired factors include antiphospolipid syndrome, pregnancy, overweight etc. The analytic part of the thesis is focused on the effect of a low molecular weight heparin. It was shown in many theses that the LMWH could have a negative impact on a parturition complications or a week of parturition, a birth weight. The results of statistical analysis show that there is a coherence between the week of parturition and the type of parturition. What is more, the anticoagulation drug therapy shows a positive effect on the week of parturion. The data used in the thesis originate in laboratory information management system of Clinical Haematology ward in the hospital Nemocnice České Budějovice a.s.