<p>Thrombosis is associated with the occlusion of a blood vessel and can be triggered by a number of types of injury, such as the rupture of an atherosclerotic plaque on the artery wall, changes in blood composition, or blood stasis. The resulting thrombosis can cause major diseases such as myocardial infarction, stroke, and venous thromboembolic disorders that, collectively, account for the most common cause of death in the developed world. Anticoagulants are used to treat and prevent these thrombotic diseases in a number of clinical and surgical settings. Although commonly prescribed, currently approved anticoagulants have a major limitation of severe drug-induced bleeding that contributes to the high levels of morbidity and mortality associated with use. The "holy grail" for antithrombotic therapy is to identify a drug that inhibits thrombus formation without promoting bleeding. Understanding the differences between thrombosis and hemostasis in the vascular system is critical to developing these safe and effective anticoagulants, as this depends on striking the correct balance between inhibiting thrombus formation (efficacy) and reducing the risk of severe bleeding (safety). While it is commonly thought that the same factors play a similar role in hemostasis and thrombosis, recent evidence points to differing functions for FXI and FXII in each of these settings. Importantly, these factors seem to contribute to pathological thrombus formation without being involved in normal hemostasis.</p><p> The overall goal of this project was to evaluate the inhibition of the intrinsic pathway of coagulation as a potential anticoagulant strategy utilizing the aptamer platform. Aptamers are short, highly structured nucleic acids that act as antagonists by binding to large surface areas on their target protein and thus tend to inhibit protein-protein interactions. High affinity binding aptamers have been isolated that specifically target a diverse range of proteins, including transcription factors, proteases, viral proteins, and growth factors, as well as other coagulation factors. As synthetic molecules, aptamers have a small molecular weight, are highly amenable to modifications that can control their bioavailability, and have not been found to elicit an immune response, thus making them ideal drug candidates. Importantly, aptamers can be rapidly and effectively reversed with either a sequence specific antidote that recognizes the primary sequence of the aptamer or a universal antidote that binds to their backbone and reverses all aptamer activity independent of sequence. This ability lends itself well to their therapeutic application in coagulation, as rapid reversal of a drug upon the onset of bleeding is a key property for increasing the safety of this class of drugs.</p><p> Aptamers targeting FXI/FXIa and FXII/FXIIa were isolated in two separate SELEX (systematic evolution of ligands by exponential enrichment) procedures: the FXII aptamer was isolated in a convergent SELEX approach and the FXIa aptamer was isolated from a purified protein selection. In both processes, 2'fluoropyrimindine modified RNA with a 40-nucleotide random region was incubated with either the plasma proteome (in initial rounds of the convergent SELEX) or the purified protein target (FXII or FXIa). The nucleic acids that did not bind to the target were separated from those that bound, and these molecules were then amplified to generate an enriched pool with increased binding affinity for the target. This process was repeated under increasingly stringent conditions to isolate the aptamer that bound with the highest affinity to the purified target protein. Utilizing biochemical and in vitro coagulation assays, specific, high-affinity binding and functional anticoagulant aptamers were identified for both protein targets, and the mechanism of anticoagulation was ascertained for each aptamer. </p><p> Overall, both aptamers bound to an exosite on their target protein that was able to inhibit downstream activation of the next protein in the coagulation cascade. In order to specifically examine aptamer effects on several parameters of thrombin generation, a new assay was developed and fully characterized using aptamer anticoagulants targeting other coagulation factors. Aptamer inhibition of both FXI and FXII was able to decrease thrombin generation in human plasma. However, limited cross-reactivity in other animal species by both aptamers hindered our ability to assess aptamer inhibition in an in vivo setting. Moving forward, screening aptamers against a larger selection of animal plasmas will hopefully allow us to identify an animal species in which we can analyze aptamer inhibition of the intrinsic pathway for effectiveness and safety in inhibiting thrombosis. The further characterization and use of these aptamers in plasma and blood based settings will allow us to study the diverging functions of the intrinsic pathway in thrombosis and hemostasis.</p><p> A critical need exists for safe and effective anticoagulants to treat and prevent numerous thrombotic procedures and diseases. An ideal anticoagulant is one that strikes the correct balance between inhibiting thrombus formation and reducing drug-induced bleeding. Inhibition or depletion of factors XI and XII of the intrinsic pathway of coagulation have shown reduced thrombus formation without interruption of normal hemostasis in several models of thrombosis. By developing novel RNA aptamer anticoagulants to these factors, we have set the stage for evaluating the net therapeutic benefit of intrinsic pathway inhibition to effectively control coagulation, manage thrombosis, and improve patient outcome. As well as developing a safe anticoagulation, these agents can lead to important biological discoveries concerning the fundamental difference between hemostasis and thrombosis.</p> / Dissertation

Recent studies suggest that factor (F) XII, which is dispensable for hemostasis, is important for thrombus stabilization and growth. Therefore, FXIIa inhibition may attenuate thrombosis without disrupting hemostasis. FXII activation is stimulated by polyanions such as polyphosphates released from activated platelets, and nucleic acids released by cells. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa with high affinity, inhibits FXII autoactivation and FXIIa-mediated activation of FXI, and attenuates ferric chloride-induced arterial thrombosis in mice. Thus, HRG has the capacity to downregulate the contact pathway in vitro and in vivo. This thesis aimed to identify the complementary binding domains of HRG and FXIIa, and to further explore the anticoagulants effects of HRG on FXIIa-mediated contact activation. We hypothesized that FXIIa binds to the zinc-binding histidine-rich region (HRR) of HRG and that HRG binds to the non-catalytic heavy chain of FXIIa to exert its anticoagulant activities on FXIIa-mediated contact activation. We have localized the complementary binding sites of HRG and FXIIa to be within the HRR domain of HRG and NH2-FNII-EGF1 (NFE) domains of FXIIa. Moreover, we show that the HRR binds to short chain polyphosphate with high affinity, suggesting a dynamic complex between HRG, FXIIa, and polyphosphate (polyP) on activated platelets. We provide evidence for two potential mechanisms through which HRG modulates the contact system. These include by 1) inhibiting FXIIa activity and 2) attenuating the procoagulant effect of polyanions, such as polyP on FXIIa-mediated reactions. Indeed, we show that the interaction of HRG with FXIIa and polyphosphate is predominantly mediated by the HRR domain and that HRR analogs have the capacity to recapitulate the anticoagulant effects of HRG in purified and plasma systems. Therefore, by modulating FXIIa-mediated contact pathway reactions, like HRG, HRR analogs may attenuate thrombosis without disrupting hemostasis. / Thesis / Doctor of Philosophy (Medical Science)

Recent studies suggest that factor (F) XII plays a key role in thrombus stabilization and growth but is dispensable for hemostasis. We have previously shown that histidine-rich glycoprotein (HRG), a protein present in platelets and plasma, binds FXIIa and inhibits FXII autoactivation and FXIIa-mediated activation of FXI, thereby downregulating thrombosis. HRG binds various ligands, including FXIIa, fibrin(ogen), nucleic acids and polyphosphate (polyP). Studies have shown that polyP, released from activated platelets, and artificial surfaces like catheters, can promote FXII activation. This suggests that HRG can downregulate the activation of the contact system. This thesis aims to determine the potential mechanisms by which HRG modulates platelet function and thrombosis induced by polyP or catheters. We show that HRG binds polyP with high affinity and inhibits the procoagulant, prothrombotic and cardiotoxic effects of polyP via at least two mechanisms. First, HRG binds polyP and neutralizes its procoagulant activities and cytotoxic effects. Second, HRG binds FXIIa and attenuates its capacity to promote autoactivation and activate FXI. Also, we identify that HRG serves as a molecular brake for the contact system by attenuating the procoagulant activity of FXIIa regardless of whether FXII activation is triggered systemically with polyP or occurs locally on the surface of catheters. Our studies have identified HRG as a novel ligand for platelet receptor GPIbα on resting platelets, and upon activation, it competes with fibrinogen for binding to GPIIb/IIIa integrin, thereby inhibiting platelet aggregation. These findings suggest that HRG may modulate coagulation as well as platelet function. Therefore, supplementation with HRG or HRG analogs may serve as a potential therapeutic option to attenuate polyP or catheter-induced thrombosis without perturbing hemostasis. / Dissertation / Doctor of Philosophy (PhD)

Medical devices, such as catheters and heart valves, are an important part of patient care. However, blood-contacting devices can activate the blood coagulation cascade to produce factor (f) Xa, the clotting enzyme that induces thrombin generation. By activating platelets and converting soluble fibrinogen to fibrin, thrombin leads to blood clot formation. Blood clots that form on medical devices create problems because they may foul the device and/or serve as a nidus for infection. In addition, clots can break off from the device, travel through the circulation and lodge in distant organs; a process known as embolization. This is particularly problematic with central venous catheters because clots that form on them can break off and lodge in pulmonary arteries, thereby producing a pulmonary embolism. Similarly, clots that form on heart valves can break off and lodge in cerebral arteries, thereby producing a stroke. Therefore, anticoagulants, blood thinning drugs, are frequently used to prevent clotting on medical devices. Conventional anticoagulants, such as heparin and warfarin, target multiple clotting factors. Heparin binds to antithrombin in plasma and accelerates the rate at which it inhibits fXa, thrombin and many other clotting enzymes. Warfarin, which is a vitamin K antagonist, attenuates thrombin generation by interfering with the synthesis of the vitamin K-dependent clotting factors, which include fX and prothrombin, the precursor of thrombin. In contrast to heparin and warfarin, more recent anticoagulants inhibit only a single clotting enzyme. For example, fondaparinux, a synthetic heparin fragment, only inhibits fXa and dabigatran, an oral thrombin inhibitor, only targets thrombin. Although effective for many indications, fondaparinux was less effective than heparin for preventing clotting on catheters in patients undergoing heart interventions and dabigatran was less effective than warfarin for preventing strokes in patients with mechanical heart valves. The failure of these new anticoagulants highlights the need for a better understanding into the drivers of clotting on medical devices. Therefore, the overall purpose of this thesis is to gain this understanding so that more rational approaches to its prevention can be identified. In the classical model of blood coagulation, clotting is triggered via two distinct pathways; the tissue factor (TF) pathway or extrinsic pathway and the contact pathway or intrinsic pathway; pathways which are initiated by fVIIa and fXIIa, respectively. The mechanism by which medical devices initiate clotting is uncertain. Platelet and complement activation and microparticle formation have been implicated, which would drive clotting via the TF pathway. Alternatively, medical devices can bind and activate fXII, thereby initiating the contact pathway. We hypothesized that medical devices trigger clotting via the contact pathway and induce the local generation of fXa and thrombin in concentrations that exceed the capacity of fondaparinux and dabigatran to inhibit them. To test this hypothesis, we used catheters as a prototypical medical device and we used a combination of in vitro and rabbit models. Several lines of evidence indicate that catheters initiate clotting via the contact pathway. First, catheter segments shortened the clotting time of human plasma, and this activity was attenuated in fXII- or fXI-deficient plasma, which are key components of the contact pathway, but not in fVII-deficient plasma, which is the critical component of the extrinsic pathway. Second, corn trypsin inhibitor (CTI), a potent and specific inhibitor of fXIIa, attenuates catheter thrombosis. Third, selective knockdown of fXII or fXI with antisense oligonucleotides attenuated catheter-induced thrombosis in rabbits, whereas knockdown of fVII had no effect. Therefore, these results revealed the importance of the contact pathway in device-associated thrombosis, and identified CTI or fXII or fXI knockdown as novel strategies for preventing this problem. Focusing on fXIIa as the root cause of medical device associated clotting, we coated catheters with CTI using a polyethylene glycol (PEG) spacer. In addition to unmodified catheters, other controls included catheters coated with albumin via a PEG spacer or catheters coated with PEG alone. Compared with unmodified catheters or with the other controls, CTI-coated catheters attenuated clotting in buffer or plasma systems and were resistant to occlusion in rabbits. These findings support the concept that catheter-induced clotting is driven via the contact pathway and identify CTI coating as a viable strategy for its prevention. We next set out to test the hypothesis that fondaparinux and dabigatran, which inhibit fXa and thrombin, respectively, are less effective than heparin, which inhibits multiple clotting enzymes. Fondaparinux and dabigatran were less effective than heparin at preventing catheter induced clotting and thrombin generation, respectively. Likewise, in a rabbit model of catheter thrombosis, fondaparinux was less effective than heparin and dabigatran was only effective when administered at doses that yielded plasma dabigatran levels similar to those found at peak in human given the drug; at trough levels, dabigatran was no better than placebo. Finally, we also showed synergy between heparin and either fondaparinux or dabigatran. Thus, when co-administered to rabbits in doses that on their own had no effect, the combination of fondaparinux or dabigatran plus heparin extended the time to catheter thrombosis. These findings support the hypothesis that when catheters trigger clotting via the contact pathway, fXa and thrombin are generated in concentrations that overwhelm the capacity of fondaparinux or dabigatran to inhibit them. Furthermore, the synergy between heparin and fondaparinux or dabigatran has clinical implications because it explains why supplemental heparin attenuated the risk of catheter thrombosis in patients treated with fondaparinux who underwent cardiac procedures and it identifies the potential role of supplemental heparin in dabigatran-treated patients who require such interventions. In summary, we have shown that catheters trigger clotting via the contact pathway and have identified CTI coating or fXII or fXI knockdown as viable strategies for prevention of this problem. In addition, for prevention of catheter thrombosis, we also have shown that heparin, which inhibits multiple coagulation enzymes, is more effective than fondaparinux or dabigatran, which only inhibit fXa or thrombin, respectively; findings consistent with the clinical observations. Moreover, the synergy that we observed between fondaparinux or dabigatran and heparin identifies supplemental heparin as strategy for preventing catheter thrombosis in patients receiving these drugs. Taken together, these studies provide insight into the mechanisms of catheter thrombosis and potential strategies for its prevention. / Thesis / Doctor of Philosophy (PhD)