Intrinsic structural and conformational mechanisms regulate the functional specificity of the coagulation system. The study of these structure-function relationships is important for understanding the strategies used in the management of clinical thrombosis. Previous studies have shown that the central enzyme in clotting, thrombin, is sequestered inside of a clot, and protected from the natural downregulator antithrombin (AT). This is problematic for anticoagulants like heparin which depend on AT. Subsequently, it was found that the key upstream propagator of thrombin, the prothrombinase enzyme complex, is also resistant to the AT-heparin. Our data show that further upstream of prothrombinase, the intrinsic tenase is only moderately protected, while there is no protection at the level of the initiator complex, extrinsic tenase. This protection phenomenon possibly reflects steric and allosteric mechanisms that ensure maximal activation of the coagulation system once a threshold stimulus is achieved. These mechanisms likely evolved as a result of conformational rearrangement, as evidenced by the proteolytic activation of thrombin activity following proteolysis of prothrombin. Indeed, subtle differences in the structural interaction of ligands with the active site can lead to substantial differences in enzyme activity. The binding of rivaroxaban and apixaban to factor Xa is nearly identical; both interact with the active site with comparable affinity. Despite this, a 3-fold faster rate of the rivaroxaban on-rate yields significantly greater prolongation of the prothrombin time (PT) and activated partial thromboplastin time (aPTT), global tests of coagulation. These small differences in ligand interaction also have allosteric consequences. Structural differences between the direct thrombin inhibitors dabigatran and argatroban yield divergent exosite-mediated thrombin binding to physiologic ligands like yA-fibrin, y'-fibrin, factor Va, and factor VIII, interactions that govern clot-mediated protection from AT inhibition, and the various functions of thrombin. These divergent effects were robust and ligand-dependent, suggesting conserved energetic scaffolds within the thrombin molecule that govern allosteric changes throughout the molecule. Because proteolysis of prothrombin yields significant allosteric and structural rearrangement that capacitates the active site for substrate recognition amd catalytic ability, we investigated the role of Ser195, a key residue in the thrombin catalytic triad in also regulating thrombin allostery. Site directed mutagenesis of Ser195 to Ala yielded a significant increase in the flexibility of the entire thrombin molecule, as evidenced by increased potency of dabigatran and argatroban in terms of their capacity to modulate exosite binding through the active site, and increased interexosite cooperative and competitive allostery. Together, these studies represent an advance in our understanding of the consequences of both small molecule ligation of coagulation proteases, as well as the consequences of subtle structural modification for overall allosteric function. / Thesis / Doctor of Philosophy (PhD)

The extracellular matrix (ECM) is the highly hydrated, protein- and glycosaminoglycan- (GAG) based cell environment that provides cell-instructive cues like the mechanical stabilization of the cells and transmission of biochemical and physical signals. To biochemically and mechanically mimic the ECM, hydrogels with the highly negatively charged GAG heparin in interplay with a stabilizing polymer network are of high interest in biomaterial engineering. The application as cell-instructive materials allows for controlling transport processes of signaling molecules within the matrices, cell growth and differentiation behavior, and cellular fate decisions. In particular, heparin-based biomaterials enable targeted sequestration of signaling molecules on the one hand, but also sustained delivery of them with a lower necessary amount to be used, in contrast to the discontinuous application of solutes. In addition, heparin-based biomaterials can protect the loaded cargo from enzymatic degradation and conformational changes.[1]–[3] The affinity to signaling molecules as key feature provides the potential for applications in wound healing and tissue regeneration. Synthetic sulfonated polymers (SSPs) as synthetic heparin analogs can address multiple drawbacks of native heparin, such as its heterogeneous chemical structure and the potential risk of viral contamination from the animal isolation source.[4],[5] Due to a large number of molecular design opportunities in particular the degree of sulfation, sulfate volume concentration, sulfate or sulfonate nature, distance of the sulf(on)ate from the backbone, and hydrophobicity of the polymers, biochemical processes may be controlled in a targeted manner. The chemical possibilities for forming a hydrogel network based on SSPs are far more diverse with synthetic, freely designable polymers to achieve a targeted structure and chemical nature of the network. Here, the aim was to introduce a library of SSPs to replace heparin in fully synthetic hydrogels capable of modulating cell-instructive cues such as soluble factor signaling, adhesiveness, and growth behavior of integrated cells. Accordingly, a library of systematically varied SSPs differing in degree of sulfation, sulfate or sulfonate conjugation, hydrophobicity, and sulf(on)ate distance to the backbone have been synthesized from by polymer analog reaction of various sulf(on)ated amines with a polyacrylate (15 kDa, sodium salt) as the polymeric backbone. The polymers have been thoroughly characterized by proton nuclear magnetic resonance (1H-NMR), Fourier-transform infrared spectroscopy (FTIR), asymmetric flow field flow fractionation (AF4) coupled light scattering analysis, and microscale thermophoresis (MST) for their molecular composition, stability in aqueous solution, conformation, and interaction with a chosen signal molecule. The affinity of the very stable coiled polymers under physiological conditions to signaling molecules depends mainly on the degree of sulfation, sulfate or sulfonate nature, and hydrophobicity. The SSPs are crosslinked with 4-arm star-shaped poly(ethylene glycol) (starPEG) either directly to form amide-crosslinked hydrogels or by pre-functionalization via Michael-type addition to prepare cell-instructive hydrogels, each with graded mechanical properties. The affinity of these hydrogels for various signaling molecules can be quantified compared to heparin-based ones and attributed to the influence of the degree of sulfation, sulfate volume concentration, sulfate or sulfonate nature, and hydrophobicity. The potential of SSPs in functional 3D tissue cultures could be confirmed by renal morphogenesis and neural network formation in the corresponding hydrogels by collaborators. Further on, the synthesis procedure of hydrogel precursors has been transferred to fully automated procedures. Because standardized production of cell-instructive hydrogels at low compositional and batch-to-batch variation and material compliance can benefit from high-throughput synthesis and liquid handling robots. An automated multistage workflow was developed to synthesize hydrogel precursors, carry out hydrogel formation, and execute cell culture experiments with cells embedded in the hydrogels. The protocol combines two robotic liquid handling systems and a microscope for automated sample imaging and cell analysis. The customized heparin and SSP maleimidation procedures, including temperature-regulated synthesis, purification, and aliquotation, were implemented on a customized liquid-handling robot. The resulting hydrogel precursors were analyzed for their maleimide conjugation efficiency and purity by 1H-NMR and conductivity measurements and for their hydrogel formation ability. This automated synthesis can ensure the quality and production of good manufacturing practice (GMP)-compliant hydrogel materials. Automated SSP hydrogel preparation, cell culture, and analysis can further promote combinatorial approaches to biomedical applications of cell-instructive materials. References [1] Lohmann, N.; Schirmer, L.; Atallah, P.; Wandel, E.; Ferrer, R. A.; Werner, C et al. Glycosaminoglycan-Based Hydrogels Capture Inflammatory Chemokines and Rescue Defective Wound Healing in Mice. Sci. Transl. Med. 2017, 9 (386), 1–12. [2] Schirmer, L.; Atallah, P.; Werner, C.; Freudenberg, U. StarPEG-Heparin Hydrogels to Protect and Sustainably Deliver IL-4. Adv. Healthc. Mater. 2016, 5 (24), 3157–3164. [3] Liang, Y.; Kiick, K. L. Heparin-Functionalized Polymeric Biomaterials in Tissue Engineering and Drug Delivery Applications. Acta Biomater. 2014, 10 (4), 1588–1600. [4] Blossom, D. B.; Kallen, A. J.; Patel, P. R.; Elward, A.; Robinson, L.; Gao, G. et al. Outbreak of Adverse Reactions Associated with Contaminated Heparin. N. Engl. J. Med. 2008, 359 (25), 2674–2684. [5] Hirsh, J.; Dalen, J. E.; Anderson, D. R.; Poller, L.; Bussey, H.; Ansell, J. et al. Oral Anticoagulants. Chest 1998, 114 (5), 445S-469S.

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<p>Unfractionated heparin (UFH) is used as an adjunct during thrombolytic therapy. However, its use is associated with many clinical limitations, such as the inability to inhibit fibrin-bound coagulation factors, increasing the potential for sustained procoagulant activity. We have developed a covalent conjugate of antithrombin (AT) and heparin (ATH) with superior anticoagulant properties to those of UFH. Some advantages of ATH include enhanced inhibition of surface-bound enzymes and its ability to reduce the overall size and mass of clots <em>in vivo</em>. However, the potential interactions of UFH or ATH with the components of the fibrinolytic pathway are not well understood. Therefore, our study utilized discontinuous second order rate constant (<em>k₂</em>) assays to determine rates of inhibition of plasmin (Pn) in the presence or absence of fibrin by AT+UFH <em>vs.</em> ATH. In addition, we monitored the rates of Pn generation in a system comprised of preformed fibrin clots with the aim of evaluating the inhibitory effect of AT+UFH or ATH in this more native system. The <em>k₂</em>values for the inhibition of Pn without fibrin were 5.74x10⁶±0.278x10⁶ and 6.39x10⁶±0.588x10⁶ for AT+UFH and ATH, respectively (p=0.36). In the presence of fibrin, the <em>k₂</em>values decreased to 1.45x10⁶±0.0971x10⁶ for AT+UFH and 3.07x10⁶±0.192x10⁶for ATH (<em>p</em></p> / Master of Science (MS)

Proper formation of the heart is a critical developmental event which requires strict regulation of coordinated cardial cell adhesion, alignment, and migration. The simple, tube-like heart of the fruit fly, Drosophila melanogaster, has proven to be an attractive system in which to study the regulatory pathways which control cardiogenesis. This is mainly due to its strikingly similarity to the vertebrate heart during early embryogenesis. In addition, many genes identified in association with congenital heart disease in humans have homologues in Drosophila, suggesting that this model organism has great potential to contribute to cardiovascular research. The extracellular matrix protein encoded by slit is a ligand for the receptors Robo, and Robo2 (lea). Recently, a third receptor for Slit has been identified as the heparin sulfate proteoglycan Syndecan. The main objective of this thesis was to use time lapse confocal imaging in order to develop further understanding of the mechanisms which result in heart assembly defects in slit, robo, lea, and syndecan mutants. We also aimed to gain a better understanding of the role of Syndecan within the Slit-Robo pathway and elucidate its relative contribution to development of the mature heart. In mutants homozygous for slit, as well as mutants doubly heterozygous for robo and lea, cardial cell alignment, adhesion, and synchronized migration were disrupted. The heart phenotype of syndecan homozygous mutants was similar that of slit and robo, lea, however the migration speed of cells to the midline did not seem to be affected. Based on our findings, we hypothesize that Slit may have Syndecan-dependent and Syndecan-independent functions in the heart. / Thesis / Master of Science (MSc)

Background: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by a lowered platelet count (50% from baseline) 4-10 days after heparin exposure. Autoantibodies specific for platelet factor 4 (PF4) bind PF4 and heparin complexes (PF4/H) and activate platelets through the FcgammaRIIA receptor. Severe cases of HIT can result in thrombotic complications including deep vein thrombosis, pulmonary embolism, and death. Pathogenic class-switched antibodies against PF4/H (IgG) are detectable in circulation as early as five days post-heparin exposure and peak at 14 days. The timeline and class of antibody found in HIT patients suggest that there must be pre-existing immunity against PF4/H. Thus, B cells producing anti-PF4/H antibodies must exist prior to heparin exposure. Cardiac surgery patients are disproportionately prone to anti-PF4/H seroconversion (up to 70%) and thus are utilized in this study as a model patient group. Research objective: The objective of this study is to determine whether the preoperative immune profile is associated with postoperative anti-PF4/H antibody production in a cohort of patients undergoing cardiac pulmonary bypass (CPB) surgery. Materials and methods: To characterize the preoperative immune profile, we used 1) a peripheral blood mononuclear cell (PBMC) enzyme linked immunospot (ELISPOT) assay to measure the prevalence of preoperative anti-PF4/H specific antibody secreting cells (ASC) and 2) a PF4/H-dependant enzyme immunoassay (EIA) to measure the anti-PF4/H antibodies produced by PBMCs in vitro. To characterize postoperative anti-PF4/H seroconversion in CPB patients, we used a PF4/H dependent EIA to measure in vivo levels of anti-PF4/H antibodies produced postoperatively. We also utilize a functional assay, 14C-serotonin release assay (SRA) to determine if seroconverting patients produced platelet activating antibody. Results: All patients were able to produce anti-PF4/H spots in the ELISPOT; however, this did not correlate with the titer of antibody production in vitro nor did it correlate with antibody production in the postoperative period. Instead, we found that pre-operative in vitro anti-PF4/H IgM production was associated with post-operative IgG anti-PF4/H seroconversion (Spearman’s r=0.39, P=0.018). We observed that 92.1% of CPB patients produced PF4/H antibody at postoperative week 3 with some combination of IgA, IgG, and IgM. Of the anti-PF4/H seropositive patients, 26% developed platelet activating antibody and were found seropositive when the SRA was supplemented with PF4 instead of heparin, while 15.7% were seropositive in the original SRA. It was noted that 4 of 10 patients that caused the most robust platelet activation were also seropositive for anti-PF4/H IgA antibody. Lastly, throughout this serosurveillance study, several patients that demonstrated unique immunological features are presented in this study as case studies. Specifically, we report the preoperative, surgical, clinical and postoperative characteristics for 3 patients of interest: 1) in a preoperative setting, a CPB patient’s PBMC were able to be activated and produce anti-PF4/H IgG antibody in vitro, 2) the second patient had platelet-activating antibodies in circulation prior to intraoperative heparin challenge and early post-surgery 3) the third patient who developed probable HIT. Conclusions: Based on our findings, we conclude that preoperative PF4/H ELISPOTs were unable to predict post-operative production of anti PF4/H antibodies. However, preoperative in vitro production of anti-PF4/H IgM may be associated with postoperative production of anti-PF4/IgG antibody and should be investigated further as this may help to elucidate the mechanisms for anti-PF4/H production related to HIT. / Thesis / Master of Science (MSc)

Heparin is an anticoagulant widely administered to patients undergoing major orthopedic or cardiac surgery. Though heparin is effective at preventing thrombosis, it is paradoxically associated with the development of heparin-induced thrombocytopenia (HIT). HIT is strongly associated with thrombotic complications and is an adverse drug reaction that occurs when heparin binds to the self-protein, platelet factor 4 (PF4) and forms immunogenic multimolecular complexes. As a result, anti-PF4/heparin antibodies are formed, which bind to these complexes, and can cross-linking Fc receptors on platelets and monocytes causing intense platelet activation, thrombocytopenia, and thrombosis. Patients who receive heparin frequently form antibodies against these PF4/heparin complexes; however, most of these antibodies do not cause HIT. Over-diagnosis of HIT is common due to the detection of clinically insignificant non-pathogenic anti-PF4/heparin antibodies. Current enzyme immunoassays (EIAs) cannot distinguish between pathogenic and non-pathogenic anti-PF4/heparin antibodies and will give a false positive result in the presence of the clinically insignificant non-pathogenic anti-PF4/heparin antibodies. Further functional testing is required to identify samples containing the pathogenic anti-PF4/heparin antibodies that will lead to HIT; however, these tests are not readily available in most centres, and delay timely diagnosis. There is little known about the differences between pathogenic and non-pathogenic HIT antibodies. The identification of antigenic determinations of pathogenic HIT antibodies binding to PF4 from this project will have direct implications for patient care. We will be able to accurately and rapidly identify “true” HIT patients from learning more about the pathogenic HIT antibody epitope. / Dissertation / Doctor of Science (PhD) / At least 30% of patients admitted into the hospital will be exposed to the anticoagulant, heparin. 1-3% of these patients develop heparin-induced thrombocytopenia (HIT): an adverse drug reaction. HIT is a major cause of morbidity and mortality in patients receiving heparin if not diagnosed and treated in a timely manner. HIT occurs when patients form antibodies against the platelet protein, platelet factor 4, in complex with heparin leading to an immune response. However, most heparin-exposed patients produce these antibodies but do not have HIT. Current rapid and available diagnostics tools cannot distinguish between antibodies that can or cannot cause the disease. To improve HIT diagnosis, we will identify the molecular differences between the antibodies that cause HIT and those that do not. From this, we can develop a new diagnostic assay that will be able to dictate whether the antibodies found in patients are specific for HIT.

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