Browsing by Subject "Thrombogenicity"

Creation of novel tools for biomedical applications is critical for the improvement of patient diagnostics and therapeutics. Two particularly important needs lie in (1) improved in vitro testing and increased performance of prosthetic vascular grafts and (2) purification methods for cells that do not compromise their utility. Progress in these areas is urgently needed and would facilitate the availability of higher quality devices and treatments that raise the quality of patient care. This work focused on developing new approaches toward that goal.

A tremendous and immediate need exists for high-performance small-diameter synthetic vascular grafts, as a fifth of the 500,000 annual coronary artery bypass grafting (CABG) patients lack suitable autologous vessels for revascularization. This problem has driven intense research and development of increasingly diverse prosthetics that could be viable alternatives in the years to come. Evaluating these designs in vitro offers high-throughput, low-cost screening for promising graft technologies ahead of more stringent vetting in vivo.

Offering a fresh take on assessing vascular graft thrombogenicity in vitro, the buildup of pressure upstream to a clot was used as a metric to quantify the physical interaction between the graft lumen and a maturing thrombus. A closed tubing system was devised and continuously monitored as clotting solutions of fibrin glue, platelet-rich plasma or whole blood were cured to varying maturities and then purged from small-diameter ePTFE grafts or Tygon graft mimics. This approach provided insight into how blood flow resistance is influenced by a number of clinically relevant factors, such as the level of vessel occlusion and the physical nature of the resident coagulum.

Endothelialization of synthetic vascular grafts yields viable alternatives to native vessels and can be accomplished non-invasively using late-outgrowth endothelial progenitor cells (LO-EPCs) isolated from peripheral blood. However, the time required to amass sufficient cells to prepare a graft with current methods is risky for waiting CABG patients. An ambitious approach conceived to significantly decrease this wait period involved developing affinity ligands selective for LO-EPCs that would enable their capture directly from the circulation to facilitate rapid amassment. An in vitro directed evolution strategy to generate aptamers, the nucleic acid analogs of antibodies, that specifically bind these cells was carried out with initially promising but ultimately unsuccessful results. While the particular strategy executed here did not prevail, the high value and impact LO-EPC aptamers would deliver merit revisiting this work with a revised strategy such as the one proposed in this document.

Purification of autologous and allogenic cells is essential for their use in a variety of therapeutic and basic research applications in addition to augmenting graft performance. However, the antibody stains conventionally used to selectively purify cells are permanent and their continued presence can elicit an immune response in vivo and compromise native cell behavior. To avoid these issues, a cell purification strategy was crafted utilizing aptamers and matched oligonucleotide antidotes that enabled reversible cell staining. The reversible stains were robust enough for cell purification via fluorescence-activated cell sorting (FACS) yet subsequently able to be removed with gentle heat treatment and antidote. Importantly, cell function that was compromised without antidote was rescued to match the native behavior of non-stained cells following purification and antidote treatment.

About one in four deaths in the United States are currently due to some form of heart disease [1]. Patients with cardiovascular disease usually require some form of perfusion-based surgical intervention at some point in their lives. To this end, biomaterials used in these applications elicit thrombus formation at some point after implantation. To date, accurately predicting the thrombogenecity of biomaterials remains difficult, and current testing methods are not comprehensive [2]. This is due to the fact that although blood clotting has been a topic of research for many decades, the interplay between the myriad of biochemical and physical reactions that occur in this process is not yet fully understood. One problem is the unpredictability of working with whole blood, as its precise contents vary from person to person, and its activity varies with handling methods [2]. Therefore, it would be interesting to use a bottom-up approach and build an in vitro assay with minimal and specific clotting components to determine differences in thrombogenicity between different blood-contacting surfaces that may be used on an implantable biomaterial.

By combining clotting factors Va, Xa, II, and fibrinogen and focusing on anti-thrombotic modulators antithrombin III and heparin, samples were tested to show a dose response in clotting times and fibrin clot formation. This was done by measuring turbidity in a plate reader, and by manually assessing the time at which the first fibrin strand could be pulled up with a pipette tip. R squared values of greater than 0.96 for all correlation plots demonstrated a strong correlation between manual and plate reading times in all cases. Clotting factor cocktails with thrombin and prothrombinase complex factors showed comparable clotting times at 1U, 0.5U, 0.25U, and 0.125U/mL of equivalent thrombin activity in plasma. A significant dose response to heparin sodium salt with antithrombin III and 0.1U/mL prothrombinase complex was demonstrated at 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 ug/mL of heparin sodium salt. These results show that it will be possible to construct specific microplate-based thrombogenicity assays with purified components of clotting.