Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 181 pages. Includes bibliographical references.

Distúrbios de hemostasia são uma das principais manifestações clínicas observadas nos acidentes por serpentes do gênero Bothrops. Tendo em vista a importância da ativação de fatores da cascata de coagulação no desenvolvimento da patologia no envenenamento, o presente trabalho descreve o isolamento e a caracterização bioquímica e funcional de uma metaloprotease capaz de induzir a ativação de fatores de coagulação, a partir da peçonha de Bothrops moojeni. A metaloprotease foi isolada por três etapas cromatográficas utilizando colunas de exclusão molecular (Sephacryl S-200), interação hidrofóbica (Phenyl Sepharose) e troca aniônica (ES 502N). A protease isolada, denominada moojenactivase, é uma glicoproteína com massa molecular de aproximadamente 89 kDa e ponto isoelétrico de 4,92, sendo composta por três cadeias com massas de 66; 17 e 14 kDa, ligadas por pontes dissulfeto. A determinação da sequência de aminoácidos por espectrometria de massas evidenciou grande identidade sequencial com outras metaloproteases, indicando a presença dos domínios metaloprotease, desintegrina-like e lectinas-like e classificando-a como uma protease da classe PIIId. Funcionalmente, a moojenactivase foi capaz de induzir a cogulação de plasma humano pela ativação dos fatores II (protrombina) e X da cascata de coagulação, gerando -trombina e fator X ativado, respectivamente. A protease apresentou atividade fibrinogenolítica, especialmente sobre a cadeia da molécula de fibrinogênio, porém não foi capaz de induzir a formação do coágulo de fibrina pela ativação deste. A moojenactivase foi parcialmente inibida quando incubada em condições de pH entre 3,5 e 5,0 e em pH 9,0, além de temperaturas acima de 60ºC, bem como na presença de ions Cu2+, além dos inibidores EDTA, SDS, DTT e soro anti-ofídico crotalico/botrópico. A protease induziu agregação plaquetária e não apresentou atividades fibrinolítica e hemorrágica. Células mononucleares de sangue periférico (PBMC) tratadas com a protease foram capazes de produzir TNF- assim como expressar fator tecidual (Fator III da coagulação) na forma ativa, fazendo com que essas células apresentassem caráter procoagulante. Com o objetivo avaliar os efeitos nos parâmetros hematológicos in vivo, a moojenactivase foi administrada em ratos (3g/Kg) onde foi observado que a protease foi capaz de prolongar o tempo de sangramento dos animais e induzir a diminuição do número de plaquetas sanguíneas, caracterizando um quadro de trombocitopenia. Ainda, o plasma dos animais administrados com a moojenactivase apresentaram valores elevados do tempo de protrombina e tempo de tromboplastina parcialmente ativada, assim como redução na concentração de fibrinogênio. Na análise dos parâmetros da série branca, foi observado aumento leucocitário na circulação, com predominância de neutrófilos até 3h após a administração, indicando a instalação de um quadro inflamatório. Com relação à análise da série vermelha, a moojenactivase não foi capaz de alterar nenhum dos parâmetros estudados. Os resultados obtidos no presente trabalho mostram, pela primeira vez, o isolamento de uma metaloprotease da classe P-IIId da peçonha de Bothrops moojeni capaz de atuar sobre diferentes ii eventos do processo hemostático, sendo essa ação prócoagulante responsável pelo quadro de incoagulabilidade sanguínea em animais. Os dados gerados podem auxiliar no entendimento dos distúrbios de coagulação em pacientes envolvidos em acidentes por serpentes da espécie Bothrops moojeni, levando ao melhor direcionamento na terapia anti-ofídica. Ainda, a função da moojenactivase sobre componentes biológicos credencia a molécula para uma possível aplicação biotecnológica em processos que envolvem o sistema hemostático. / Haemostasis disorders are a major clinical manifestation induced by Bothrops snake envenomations. Considering the relevance of the activation of coagulation factors during the envenomation pathophysiology, the present work describes, for the first time, the isolation and functional and biochemical characterization of a coagulation factor activator metalloprotease from Bothrops moojeni snake venom. The protease was purified by three chromatographic procedures using size exclusion (Sephacryl S-200), hydrophobic interaction (Phenyl Sepharose) and anion exchange (ES 502N) chromatographies. The isolated protease, named moojenactivase, is a glycoprotein with molecular mass of approximately 89 kDa by SDS-PAGE, and composed of 66 kDa, 17 kDa and 14 kDa disulfide linked chains, with pI of 4,92. The amino acid sequence determination of tryptic peptides from moojenactivase by mass spectrometry presented fragments with high identity to snake venom metalloproteases, confirming the presence of the metalloprotease, disintegrin-like and lectin-like domains, which allowed its classification as a PIIId class snake venom metalloprotease. Regarding its functional properties, the protease was capable to induce human plasma coagulation by inducing activation of coagulation factors II and X, forming-thrombin and factor X activated, respectively. Also, moojenactivase presented fibrinogenolitic activity, by cleaving preferentially -chain of fibrinogen, however was not capable to induce the formation of fibrin clot from fibrinogen. The enzyme stability was assessed and showed that moojenactivase presented a reduced functional activity when preincubated in pH values ranging from 3,5 to 5,0 and at pH 9,0, and in temperature conditions over 60ºC. Cu2+ ions and inhibitors such as EDTA, SDS, DTT and crotalic/bothropic antiophidian serum reduced the protease activity. Moojenactivase induced platelet aggregation, but no fibrinolytic and haemorrhage activities. In order to evaluate the stimulation of peripheral blood mononuclear cells (PBMC), cells were treated with the protease and we observed the release of proinflammatory cytokine TNF- and expression of active Tissue Factor (coagulant factor III), inducing a procoagulant state on PBMC. In order to evaluate in vivo haematological effects, the protease (3 g/Kg) was administered in rat (i.v.) and was observed that moojenactivase induced a prolonged bleeding time and reduced platelet counting (indicating a thrombocytopenia state). Moreover, the evaluation of the hemostasis parameters was assessed by the the prothrombin time and activated partial thromboplastin time assays and showed a prolonged clot time on both tests, and also a decrease in fibrinogen plasma levels. The leukogram analysis showed an increase in the circulating leukocyte number up to 3 hours after moojenactivase administration, composed predominantly of neutrophils. However, parameters envolving red cells shows that the protease do not affect. The results obtained in the present work show, for the first time, the isolation of a PIIId class metalloprotease from Bothrops moojeni snake venom involved on the activation of several hemostatic events, inducing a pro coagulant activity and leading to blood unclottable state in experimental animals. These data can assit in understanding coagulation disturbs in iv patients involved in Bothrops moojeni envenomation and leading to a better anti ophidic therapy guidance. Moreover, moojenactivase functional activities accredits this protease as a possible molecular instrument applied on biotechnological prospect related to the hemostasis.

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)

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