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.

An intracranial aneurysm is a local dilation of an artery in the cerebral circulation. While the etiology of intracranial aneurysms is unknown, they likely result from a combination of factors including the weakening and degeneration of the collagen fibers and the internal elastic lamina comprising the arterial wall, as well as hemodynamic-associated stress resulting from blood pulsation inside the aneurysm sac. Intracranial aneurysm rupture leads to a devastating sequela, as 50% of patients die. In the U.S. alone there are approximately 30,000 cases of subarachnoid hemorrhage annually, a prevalence which has pushed practitioners to aggressively treat the aneurysm disease. Traditionally, intracranial aneurysms were managed with open craniotomy and microsurgical clipping; however, these treatment modalities carry relatively high morbidity and mortality depending upon the aneurysm location and surgical experience. In 2002 the International Subarachnoid Hemorrhage Aneurysm Trial established the superiority of the endovascular coiling of intracranial aneurysms compared to microsurgical clipping. This trial led to a paradigm shift in treating intracranial aneurysms with marked use of intracranial stenting, including devices used to assist endovascular coiling and stand-alone flow diverting devices. However, the placement of intracranial devices in the cerebral circulation mandates the adjunctive application of dual anti-platelet pharmaceuticals to minimize thromboembolic events, despite being associated with increased patient risk. This dissertation proposes a novel multilayer, nanometer-scale coating technology suitable for commercially available intracranial stents and flow diverting devices to minimize the use of dual anti-platelet therapy in the elective setting and expand the use of intracranial devices in the acute setting of ruptured intracranial aneurysms. A combination of qualitative and quantitative chemical characterization techniques was used to assess the composition, uniformity, and thickness of each coating layer on commercially available flow diverting devices; overall the coating was found to be relatively uniform and conformal to the device wires. Furthermore, in-vitro and in-vivo testing on commercially available intracranial devices suggest some hemocompatible and antithrombotic properties. Finally, the proposed coating technology can be modified for use as a platform for the attachment of FDA-approved molecules. With further optimization and testing this technology has the potential to minimize the adjunctive use of dual-antiplatelet therapy in the endovascular treatment of intracranial aneurysms.

<p>The aim of this study was to examine the expression of Thrombomodulin, Tissue Factor, Tissue Factor Pathway Inhibitor and Endothelial Protein C Receptor in placenta throughout the three phases of the third trimester in the normal placenta and in IUGR placenta from full term.</p><p>Twenty-five normal placenta samples and twenty-five IUGR placenta samples were obtained and each sample was stained by immunohistochemistry using monoclonal antibodies. Each antibody was optimised for antigen retrieval method and for optimal dilution, before been applied to the test tissue.</p><p>The results showed that each of the antibodies mentioned was expressed in normal placenta and in IUGR placenta.</p><p>No significant difference could be established concerning the expression of each antibody mentioned between normal and IUGR placenta.</p>

<p>Synthetic small diameter vascular grafts fail clinically due to thrombosis and intimal hyperplasia. The attachment of endothelial cells (ECs) onto the inner lumen of synthetic small diameter vascular grafts can improve graft patency; however, significant challenges remain that prevent wide clinical adoption. These issues include difficulties in the autologous sourcing of ECs, the lack of attachment, growth and retention of the layer of ECs to the graft lumen, and the maintenance of an anti-thrombotic and anti-inflammatory profile by the layer of ECs. </p><p>This dissertation describes the isolation, characterization, and use of endothelial progenitor cells (EPCs) to improve the performance of small diameter vascular grafts. First, EPC isolation efficiency and expression of critical EC markers was compared between young healthy volunteers and patients with documented coronary artery disease (CAD). EPCs were isolated and expanded from patients with CAD and had a similar phenotype to EPCs isolated from healthy donors, and a control population of human aortic ECs. Second, we assessed the ability to enhance the anti-thrombotic activity of patient derived EPCs through the over expression of thrombomodulin (TM). In vitro testing showed TM-transfected EPCs had significantly increased production of key anti-thrombotic molecules, reduced platelet adhesion, and extended clotting times over untransfected EPCs. Finally, native and TM-transfected EPCs were seeded onto small diameter vascular grafts and tested for their ability to improve graft performance. EPCs sodded onto the lumen of small diameter ePTFE vascular grafts had strong adhesion and remained adherent during graft clamping and exposure to flow. TM-transfected EPCs improved graft anti-thrombotic performance significantly over bare grafts and grafts seeded with native EPCs. Based on these promising in vitro results, grafts were implanted bilaterally into the femoral arteries of athymic rats. Bare grafts and grafts with air removed clotted and had only 25% patency at 7 days. In contrast, graft sodded with native EPCs or TM-transfected EPCs had 87% and 89% respective patency rates. High patency rates continued with 28 day implant testing with EPC sodded grafts (88% Native; 75% TM). There were no significant differences in patency rates at 7 or 28 days between native and TM-transfected grafts. These in vivo data suggest patient blood-derived EPCs can be used to improve the performance of small diameter vascular grafts.</p> / Dissertation

The aim of this study was to examine the expression of Thrombomodulin, Tissue Factor, Tissue Factor Pathway Inhibitor and Endothelial Protein C Receptor in placenta throughout the three phases of the third trimester in the normal placenta and in IUGR placenta from full term. Twenty-five normal placenta samples and twenty-five IUGR placenta samples were obtained and each sample was stained by immunohistochemistry using monoclonal antibodies. Each antibody was optimised for antigen retrieval method and for optimal dilution, before been applied to the test tissue. The results showed that each of the antibodies mentioned was expressed in normal placenta and in IUGR placenta. No significant difference could be established concerning the expression of each antibody mentioned between normal and IUGR placenta.

All artificial organ systems and medical devices that operate in direct contact with blood elicit activation of coagulation and platelets, and their long-term use often necessitates antithrombotic therapies that carry significant cost and bleeding risk. Thrombomodulin (TM) is a major endogenous inhibitor of blood coagulation localized on the endothelial cell surface. The overall objective of this research is to develop clinically durable synthetic materials by incorporating TM as a solid-supported film to actively and sustainably attenuate thrombus formation at the blood-contacting interface. During the course of this research, we developed site-specific approaches to covalently attach TM on the luminal surface of commercial vascular grafts using bioorthogonal chemistry that was compatible with ethylene oxide sterilization. Notably, we demonstrated the superior efficacy of TM to reduce platelet deposition compared with commercial heparin modified grafts using a non-human primate model of acute graft thrombosis. Finally, we optimized a novel reversible chemistry to rapidly and repeatedly regenerate immobilized TM, with the potential to significantly extend the lifetime of biologically active films.

Thesis (Ph. D.)--Chemical Engineering, Georgia Institute of Technology, 2006. / Chaikof, Elliot, Committee Chair ; Hanson, Stephen, Committee Member ; Lollar, John "Pete", Committee Member ; Sambanis, Athanassios, Committee Member ; Yoganathan, Ajit, Committee Member.

Abstract Hypothermia deaths pose a difficult challenge from the medico-legal point of view because no specific traces are left on the cadaver to be examined post-mortem. The concentrations of urinary catecholamines, adrenaline and noradrenaline increase in various stressful situations including cold stress, and high levels have been considered to be suggestive of lethal hypothermia. There is, however, a need for a better hypothermia indicator. A potential candidate could be thrombomodulin (TM), an endothelially expressed protein whose plasma concentration has been shown to elevate in response to hypothermia. TM and catecholamine levels were studied in short-term cold exposure (human subjects, n = 7), in mild and severe hypothermia with or without rewarming (rats, n = 96) and in hypothermia deaths compared with deaths from cardiovascular diseases, traumas and other causes (autopsy cases, total n = 552). Myocardial thrombomodulin transcript expression was increased in severely hypothermic rats, but was lower in hypothermia deaths than in other causes. The circulating TM level was transiently reduced in severe hypothermia. The myocardial and urinary TM protein levels were reduced in lethal hypothermia compared with other causes of death. TM and catecholamine levels correlated significantly in blood and urine both in living subjects and post-mortem examination. In severely hypothermic rats, there was an inverse relationship between plasma adrenaline concentration and myocardial thrombomodulin transcript level. The results suggest that TM expression and secretion are altered by hypothermia, possibly linked to the actions of catecholamines. Analysing the post-mortem catecholamine and TM levels provides evidence of ante-mortem cold stress in suspected hypothermia deaths. Further studies should be conducted in order to reveal the exact mechanisms behind the regulation of TM on cell level. / Tiivistelmä Paleltumiskuolemat ovat oikeuslääketieteellisesti haastavia, koska vainajaan ei jää paleltumiseen viittaavia yksiselitteisiä löydöksiä. Virtsan katekoliamiinien, adrenaliinin ja noradrenaliinin, pitoisuudet kasvavat stressitilanteissa kuten kylmäaltistuksessa. Korkeita pitoisuuksia on pidetty paleltumiseen viittaavana tekijänä. Paremmalle paleltumista osoittavalle merkkiaineelle on kuitenkin selkeä tarve. Eräs mahdollinen merkkiaine voisi olla trombomoduliini (TM), joka on endoteelisolujen tuottama proteiini. Sen plasmapitoisuuden on aiemmin osoitettu nousevan alilämpöisyystilassa. TM- ja katekoliamiinitasoja tutkittiin lyhyessä kylmäaltistuksessa (koehenkilöt, n = 7) sekä lievässä ja vaikeassa alilämpöisyystilassa joko lämmityksen jälkeen tai ilman lämmitystä (rotat, n = 96). Lisäksi verrattiin paleltumisen, sydän- ja verisuonitautien, vammojen sekä muiden syiden aiheuttamia kuolemia (ruumiinavausaineisto, n = 552). Sydänlihaksen trombomoduliini-transkriptin taso oli kohonnut vaikeasti alilämpöisillä rotilla, mutta se oli matalampi paleltumiskuolemissa kuin muissa kuolemissa. Veren TM-taso oli hetkellisesti alentunut vaikeassa alilämpöisyystilassa. Sydänlihaksen ja virtsan TM-proteiinipitoisuudet olivat matalampia paleltumiskuolemissa kuin muissa kuolemansyissä. TM- ja katekoliamiinitasot korreloivat merkittävästi veressä ja virtsassa sekä koehenkilöillä ja -eläimillä että vainaja-aineistossa. Vaikeasti alilämpöisillä rotilla todettiin käänteinen suhde plasman adrenaliinipitoisuuden ja sydänlihaksen trombomoduliini-transkriptitason välillä. Tulosten perusteella alilämpöisyystila muuttaa TM:n ekspressiota ja erittymistä, mikä voi liittyä katekoliamiinien vaikutuksiin. Kuolemanjälkeisten TM- ja katekoliamiinitasojen määritys tuo lisänäyttöä kuolemaa edeltäneestä kylmävaikutuksesta epäiltäessä paleltumiskuolemaa. Lisätutkimuksia tarvitaan TM:n solutason säätelymekanismien selvittämiseksi.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23072号 / 医博第4699号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 浅野 雅秀, 教授 福田 和彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20267号 / 医博第4226号 / 新制||医||1021(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 坂井 義治, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM