Browsing by Subject "Thrombosis"

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Thrombosis, or pathological blood clot formation, is intimately associated with cardiovascular disease and is the leading cause of morbidity and mortality in the western world. Antithrombotics are commonly prescribed as prophylactic medications or as rapid onset anticoagulants in acute care clinical settings. Although a number of antithrombotics are clinically available, their use is limited by immunogenicity, toxicity, and inability to be controlled with an antidote in the event of hemorrhage. Therefore, new antithrombotics that are effective, yet can be rapidly controlled are urgently needed.

Aptamers are oligonucleotides that form complex secondary and tertiary structures based on intramolecular base pairing and nucleic acid folding that allows them to bind to molecular targets with high affinity and specificity. Aptamers can be isolated that bind to proteins, such as clotting proteins, and modulate protein function. However, unlike most currently used antithrombotics, aptamers can be directly controlled with an antidote and therefore represent a safer class of therapeutic agents.

To generate a novel anticoagulant, we developed an aptamer-antidote pair against prothrombin. Prothrombin is a blood protein that plays an essential role in clot formation. I truncated, optimized, and studied the mechanism of an aptamer that can bind to prothrombin and inhibit prothrombin function, thereby severely impeding clot formation. Moreover, to increase the safety profile of this anticoagulant aptamer, I developed an antidote that can quickly reverse aptamer function and restore normal clotting. This aptamer and antidote pair is the first antidote reversible anticoagulant that targets prothrombin and may prove to be a valuable clinical anticoagulant.

A number of anticoagulants are in development, and a wide debate regarding the optimal protein target for anticoagulation is underway. We have previously generated anticoagulant aptamers to human coagulation factor VII, factor IX, factor X, and prothrombin. I compared the effects of these four anticoagulant aptamers to determine their impact on thrombin generation and clot formation. Each aptamer exerts its own unique effect on thrombin generation/clot formation, depending on the role that its protein target plays in coagulation. These studies provide valuable data regarding target validation and the anticoagulant effects of different therapeutic aptamers.

Robust anticoagulation is required during acute clinical surgical procedures to treat thrombosis. Currently used anticoagulants have several untoward side effects and most are not antidote controllable. I tested the effects of combining two anticoagulant aptamers to assess potential drug synergy. Several combinations of two anticoagulant aptamers were synergistic and severely impaired blood clot formation. One specific pair of aptamers that targeted factor X (FX) and prothrombin in combination was extremely potent and could keep blood fluid in an ex vivo model of extracorporeal circulation. Additionally, this pair of aptamers could be functionally modulated with two different types of antidotes. In conjunction with antidote reversal, this strategy of combining aptamer anticoagulants may prove useful in a variety of highly prothrombotic acute clinical settings.

Finally, to explore the potential of aptamers to regulate platelet function, I isolated and characterized an aptamer toward platelet glycoprotein VI. Glycoprotein VI is a platelet surface receptor that plays a key role in platelet activation and platelet plug formation. I isolated several aptamers that bind to glycoprotein VI, and show that the lead aptamer binds to platelets with high affinity and causes platelet activation and aggregation. This aptamer could potentially be further developed for topical administration to manage bleeding, or for biomarker detection of soluble glycoprotein VI in patient plasma.

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The number one cause of death in the industrialized world, atherosclerosis, can be treated through a variety of methods: angioplasty, stenting, vein graft bypass, synthetic grafts, and maybe one day tissue engineering vessels (TEBVs). The long term goal that motivated this research is the delivery of umbilical cord blood derived endothelial progenitor cells (CB-EPCs) to damaged arteries and possibly reducing the rate of re-occlusion by re-establishing a healthy, functional, intact endothelium. The proposed research tested the following hypotheses: (1) Mild trypsinization methods produces strong endothelial cell (EC) adhesion strength, (2) CB-EPCs are functionally similar to native ECs (specifically human aortic endothelial cells (HAECs)) and exhibit similar anti-thrombotic and anti-inflammatory behavior compared to HAECs, (3) CB-EPCs are capable of adhering to smooth muscle cells (SMCs) and extracellular matrix (ECM) proteins under flow conditions, (4) CB-EPCs can be used to prevent thrombosis in mice that have undergone vein bypass grafts through re-endothelialization of the vessel, and (5) CB-EPCs are capable of proliferating under flow conditions. In order to produce supraphysiological adhesion strengths of HAECs or CB-EPCs, the cells must be detached using 0.025% trypsin for 5 minutes prior to adhesion to adsorbed ECM proteins or SMCs. CB-EPCs have a high proliferation rate and express similar levels of important anti-thrombotic genes and inflammatory proteins compared to HAECs. CB-EPCs and HAECs produce similar levels of nitric oxide and alignment in the direction of flow when exposed to laminar shear stress for at least 24 hours. CB-EPCs are capable of adhering to many different substrates under flow conditions. The adhesion of CB-EPCs with response to shear stress appears to be biphasic and increases with shear stress up to 0.75 dyn/cm2 and then decreases above this value. CB-EPC adhesion is much greater than HAECs and EPCs isolated from peripheral blood (PB-EPCs) of healthy individuals, which can be related to their higher expression level of adhesion integrin α5β1 and their smaller size. When seeded onto FN coated plastic, CB-EPCs proliferated under flow conditions and had a much shorter doubling time than PB-EPCs and HAECs. Proliferation of CB-EPCs and HAECs on SMCs was limited. Further, Cb-EPCs formed network-like structures except when growth factors were removed and a shear stress of at least 5 dyne/cm2 was applied. To assess whether CP-EPCs could promote vessel repair in vivo, human CB-EPCs were injected into SCID mice that received a carotid interpositional vein grafts, resulting in 100% patency. In contrast, only 2 of the 8 saline injected mice had a patent vein graft 2 weeks post surgery. We found that CB-EPC injected mice had roughly 55% endothelialization compared to less than 20% for the patent saline controls, with CB-EPCs making up approximately 33% of this coverage. These results suggest that CB-EPCs could be used as a therapeutic method to prevent vessel re-occlusion in patients undergoing treatment for atherosclerosis.

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.

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.