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by Chan Po Kwok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 132-140). / Abstract --- p.i / Acknowledgement --- p.iii / Publication --- p.iv / Abbreviations --- p.v / Table of Contents --- p.vi / Chapter Chapter 1 --- Introduction / Chapter Section a --- Blood Coagulation --- p.1 / Chapter Section b --- The role of factor XII --- p.4 / Chapter Section c --- Genomic organisation of the human factor XII gene --- p.8 / Chapter Section d --- Protein structure of the human factor XII --- p.12 / Chapter Section e --- Mutations in factor XII --- p.16 / Chapter Section f --- Methods to detect mutations --- p.25 / Chapter Section g --- About the patient with factor XII deficiency --- p.29 / Chapter Section h --- Strategies used in this study --- p.30 / Chapter Chapter 2 --- Methods and Materials / Chapter Section a --- Genomic DNA extraction --- p.33 / Chapter Section b --- Polymerase chain reaction amplification --- p.34 / Chapter Section c --- Cycle sequencing --- p.35 / Chapter Section d --- Restriction enzyme digestion and cloning of PCR products --- p.36 / Chapter Section e --- Reverse transcription and polymerase chain reaction of factor XII ectopic transcript --- p.36 / Chapter Section f --- Subcloning of 95-bp novel fragment --- p.38 / Chapter Section g --- Gel mobility shift assay --- p.39 / Chapter Chapter 3 --- Results / Chapter Section a --- Analysis of the catalytic region of the human factor XII --- p.41 / Chapter Section b --- Ectopic transcript of factor XII in peripheral blood lymphocytes --- p.65 / Chapter Section c --- Analysis of 3'end untranslated region of factor XII gene --- p.70 / Chapter Section d --- Analysis of 5' flanking region of factor XII gene --- p.75 / Chapter Section e --- Analysis of intron B --- p.94 / Chapter Section f --- Analysis of 5'-b PCR product --- p.98 / Chapter Chapter 4 --- Discussions / Chapter Section a --- Mutations in the coding sequence of factor XII gene --- p.107 / Chapter Section b --- Ectopic transcript of factor XII in lymphocytes --- p.113 / Chapter Section c --- The 3'untranslated region of factor XII gene --- p.116 / Chapter Section d --- The 5'flanking region of factor XII gene --- p.117 / Chapter Section e --- Other possible explanations of undetectable factor XII mRNA --- p.120 / Chapter Section f --- The 95-bp novel sequence in ´5ة flanking region --- p.123 / Chapter Section g --- Technical difficulties --- p.126 / Chapter Section h --- Future study --- p.130 / Chapter Chapter 5 --- References --- p.132 / List of Tables --- p.137 / List of Figures --- p.138

A human liver cDNA library was screened by colony hybridization with two mixtures of synthetic oligodeoxyribonucleotides as probes. These oligonucleotides encoded regions of β-factor Xlla as predicted from the amino acid sequence. Four positive clones were isolated that contained DNA coding for most of factor XII mRNA. A second human liver cDNA library was screened by colony hybridization with ³²P-labeled cDNA clones obtained from the first screen and two identical clones were isolated. DNA sequence analysis of these overlapping clones showed that they contained DNA coding for the signal peptide sequence, the complete amino acid sequence of plasma factor XII, a TGA stop codon, a 3' untranslated region of 150 nucleotides, and a poly A⁺ tail. The cDNA sequence predicts that plasma factor XII consists of 596 amino acid residues. Within the predicted amino acid sequence of factor XII, were identified three peptide bonds that are cleaved by kallikrein during the formation of β-factor Xlla. Comparison of the structure of factor XII with other proteins revealed extensive sequence identity with regions of tissue-type plasminogen activator (the epidermal growth factor-like region and the kringle region) and fibronectin (type I and type II homologies). As the type II region of fibronectin contains a collagen-binding site, the homologous region in factor XII may be responsible for the binding of factor XII to collagen. The carboxyl-terminal region of factor XII shares considerable amino acid sequence homology with other serine proteases including trypsin and many clotting factors. A human genomic phage library was screened by using a human factor XII cDNA as ahybridization probe. Two overlapping phage clones were isolated which contain the entire human factor XII gene. DNA sequence and restriction enzyme analysis of the clones indicate that the gene is approximately 12 kbp in size and is comprised of 13 introns and 14 exons. Exons 3 through 14 are contained in a genomic region of only 4.2 kbp with introns ranging in size from 80 to 554 bp. The multiple regions found in the coding sequence of FXII that are homologous to putative domains in fibronectin and tissue-type plasminogen activator are contained on separate exons in the factor XII gene. The intron/exon gene organization is similar to the serine protease gene family of plasminogen activators and not to the clotting factor family. Analysis of the 5' flanking region of the gene shows that it does not contain the typical TATA and CAAT sequences found in other genes. This is consistent with the finding that transcription of the gene is initiated at multiple start sites. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

O angioedema hereditário (AEH) é uma doença autossômica dominante, caracterizada por episódios recorrentes de edema subcutâneo, do trato gastrointestinal e das vias aéreas superiores. Em sua forma clássica, o AEH é causado por deficiência do inibidor de C1 (C1- INH) e está associado a mutações no gene SERPING1. Recentemente, foi descrito o AEH com inibidor de C1 normal, resultante de mutações no gene F12, que codifica o Fator XII da coagulação (FXII), denominado AEH-FXII; e de mutações nos genes PLG e ANGPT1, que codificam Plasminogênio e Angiopoietina-1, caracterizando os tipos AEH-PLG e AEHANGPT1. O AEH com inibidor de C1 normal não cursa com diminuição de C1-INH quantitativo e/ou funcional, ou de C4, sendo o diagnóstico realizado pela presença de sintomas clínicos sugestivos e história familiar. Os objetivos do presente estudo foram: avaliar a discriminação alélica como método alternativo ao sequenciamento por método de Sanger, para o diagnóstico de AEH com inibidor de C1 normal por mutação c.983C>A no gene F12; determinar se a discriminação alélica seria uma técnica de melhor custo-benefício; e investigar outras mutações previamente descritas no exon 9 do gene F12, e nos genes PLG e ANGPT1 em pacientes com suspeita clínica de AEH com inibidor de C1 normal, e negativos para mutação c.983C>A em F12. Cento e oitenta e quatro indivíduos incluindo 51 pacientes- índice com suspeita clínica de AEH com inibidor de C1 normal, e seus familiares, foram previamente investigados por sequenciamento de Sanger. Esses indivíduos foram investigados por discriminação alélica, e a concordância entre os resultados para a mutação c.983C>A foi verificada por estatística Kappa. Custos e tempo de execução entre os dois métodos foram avaliados. Casos-índice negativos para a mutação c.983C>A no gene F12 foram avaliados para outras mutações no exon 9 do gene F12 e para mutações previamente descritas c.988A>G e c.807G>T em PLG e ANGPT1, respectivamente, usando sequenciamento por método de Sanger. Mutação no gene F12 foi identificada em 96 indivíduos (24 pacientes- índice e 72 familiares). Em todos esses pacientes foi encontrada a mutação c.983C>A. 88 indivíduos foram negativos para esta mutação. Os resultados foram 100% concordantes entre os dois métodos. Setenta e um dos 96 pacientes positivos para mutação c.983C>A eram do sexo feminino; 78,9% e 56% eram pacientes sintomáticos do sexo feminino e masculino, respectivamente. A discriminação alélica apresentou custo e tempo de execução 79,7% e 82,3% menores em comparação ao sequenciamento por Sanger, respectivamente. Outras mutações no gene F12, e mutações em PLG e ANGPT1 não foram encontradas. Entre os pacientes negativos para mutações associadas ao AEH com inibidor de C1 normal, 11 foram diagnosticados como AEH-desconhecido e 16 como angioedema idiopático adquirido não histaminérgico. Os resultados do presente estudo nos permitiram construir um algoritmo para diagnóstico de pacientes com AEH com inibidor de C1 normal. Em áreas onde a mutação c.983C>A em F12 é predominante entre pacientes com AEH-FXII, a discriminação alélica pode ser um método adequado para screening inicial. Em pacientes com resultados negativos, sequenciamento do exon 9 de F12 pelo método de Sanger estaria indicado. Pacientes remanescentes com resultados negativos seriam genotipados para mutações previamente descritas em PLG e ANGPT1. Estudos futuros em outros locais serão necessários para estabelecer se a discriminação alélica apresentaria melhor custo-benefício, com potencial para mais acessibilidade ao diagnóstico em pacientes portadores da doença no Brasil / Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of edema of subcutaneous tissue, gastrointestinal tract, and upper airways. In its classical form, HAE is caused by deficiency of C1 inhibitor (C1-INH) and it is associated with mutations in the SERPING1 gene. Recently, HAE with normal C1 inhibitor has been described, resulting from mutations in F12 gene, which encodes coagulation factor XII (FXII), designated as FXII-HAE; and from mutations in PLG and ANGPT1 genes, which encode Plasminogen and Angiopoietin-1, characterizing PLG-HAE and ANGPT1-HAE types. HAE with normal C1-INH does not present with decrease in quantitative and/or functional levels of C1-INH or C4, and diagnosis is carried out by presence of suggestive clinical symptoms and family history. Diagnosis can only be confirmed by genetic sequencing. The objectives of the present study were: to evaluate the allelic discrimination as an alternative method to sequencing by Sanger method for the diagnosis of HAE with normal C1-INH due to mutation c.983C>A in F12 gene; to determine whether allelic discrimination would be a better cost-effective technique; and to investigate presence of other mutations previously described in exon 9 of F12 gene and in PLG and ANGPT1 genes in patients with clinical suspicion of HAE with normal C1-INH who were negative for c.983C>A mutation in F12. One hundred and eighty-four individuals including 51 index patients with clinical suspicion of HAE with normal C1-INH and their relatives were previously investigated by Sanger sequencing. These individuals were investigated for allelic discrimination, and concordance of the results for the c.983C>A mutation was verified by Kappa statistics. Costs and execution time of the two methods were evaluated. Negative index cases for c.983C>A mutation in F12 gene were evaluated for other mutations in exon 9 of the F12 gene and for mutations previously described c.988A>G and c.807G>T in PLG and ANGPT1, respectively, using sequencing by Sanger method. Mutation in F12 gene was identified in 96 individuals (24 index patients and 72 relatives). In all these patients, the c.983C>A mutation was found. 88 subjects were negative for this mutation. The results were 100% concordant between the two methods. Seventy-one of the 96 patients positive for the c.983C>A mutation were female; 78.9% and 56% were symptomatic female and male patients, respectively. The allelic discrimination presented cost and execution time 79.7% and 82.3% lower in comparison to sequencing by Sanger, respectively. Other mutations in F12 gene, and mutations in PLG and ANGPT1 were not found. Among patients who were negative for mutations associated to HAE with normal C1-INH, 11 were diagnosed as unknown HAE and 16 as idiopathic nonhistaminergic acquired angioedema. The results of present study allowed us to construct an algorithm for diagnosis of patients with HAE with normal C1-INH. In areas where the c.983C>A mutation in F12 is predominant among patients with FXII-HAE, allelic discrimination may be a suitable method for initial screening. In patients with negative results, sequencing of F12 exon 9 by the Sanger method would be indicated. Remaining patients with negative results would be genotyped for mutations previously described in PLG and ANGPT1. Future studies at other sites will be needed to establish whether allelic discrimination would be more cost-effective, with potential for more accessibility to diagnosis in patients with the disease in Brazil

Activated coagulation Factor XII (FXIIa) elevates blood pressure (BP) acutely by stimulating adrenomedullary catecholamine (CA) release in Brown Norway (BN) bioassay rats. These effects are absent in kininogen-deficient BN Katholiek (BNK) bioassay rats, indicating that these FXIIa-induced responses require an intact kallikrein-kinin system (KKS). In three hypertensive anephric pediatric patients, ΔFXIIa concentrations tracked peri-dialytic ΔBP. We hypothesized that FXIIa exerts a vasoconstrictor pro-hypertensive action, via the KKS, particularly evident in chronic renal failure (CRF). In CRF patients (n=11) receiving conventional hemodialysis, mean plasma FXIIa concentrations were 3-fold (p<0.05) greater than in healthy controls. Although conversion from conventional to nocturnal hemodialysis did not change mean FXIIa concentrations there was intra-session variation within individuals, such that ΔFXIIa concentrations correlated with changes in mean arterial pressure (MAP, r=0.66, p=0.026) and total peripheral resistance (TPR, r=0.75, p=0.007). In normotensive BN rats, FXIIa infusion (85 ng/min/kg for 60 mins) increased MAP (10±1 mmHg), TPR (0.5±0.1 Units), and CA, whilst left-ventricular end-diastolic volume (LVEDV) and heart rate decreased (all p<0.05). After adrenalectomy, FXIIa infusion decreased MAP (5±1 mmHg), did not raise CA or induce sustained vasoconstriction, and caused a greater fall in LVEDV (all p<0.05). In the 5/6 nephrectomized (NX) rodent CRF model, MAP and TPR were significantly greater in BN NX (n=16) than in BNK NX (n=15) (147±4 vs. 133±2 mmHg, 2.8±0.2 vs. 2.3±0.2 Units; all p<0.05). Plasma FXIIa measured using our semi-quantitive ELISA was 3-fold higher in both BN NX and BNK NX than in controls (p<0.01), but only correlated with MAP (r=0.48, p=0.01) in the BN NX. Plasma CA were elevated in the BN NX (p<0.05) but not in BNK NX. Infusion of a specific FXIIa inhibitor into BN NX decreased MAP (-12 mmHg) and TPR (-0.5 Units) proportionally to baseline FXIIa (ΔMAP: r=-0.72, p=0.002; ΔTPR: r=-0.57, p=0.021), and plasma CA fell by 40-67% (all p<0.05). No such changes occurred in the BNK NX. In summary, a significant component of the hypertension of CRF can be attributed to FXIIa-induced vasoconstriction mediated via the KKS and stimulated CA release. In normal rats, FXIIa appears also to directly or indirectly decrease preload and heart rate.

Activated coagulation Factor XII (FXIIa) elevates blood pressure (BP) acutely by stimulating adrenomedullary catecholamine (CA) release in Brown Norway (BN) bioassay rats. These effects are absent in kininogen-deficient BN Katholiek (BNK) bioassay rats, indicating that these FXIIa-induced responses require an intact kallikrein-kinin system (KKS). In three hypertensive anephric pediatric patients, ΔFXIIa concentrations tracked peri-dialytic ΔBP. We hypothesized that FXIIa exerts a vasoconstrictor pro-hypertensive action, via the KKS, particularly evident in chronic renal failure (CRF). In CRF patients (n=11) receiving conventional hemodialysis, mean plasma FXIIa concentrations were 3-fold (p<0.05) greater than in healthy controls. Although conversion from conventional to nocturnal hemodialysis did not change mean FXIIa concentrations there was intra-session variation within individuals, such that ΔFXIIa concentrations correlated with changes in mean arterial pressure (MAP, r=0.66, p=0.026) and total peripheral resistance (TPR, r=0.75, p=0.007). In normotensive BN rats, FXIIa infusion (85 ng/min/kg for 60 mins) increased MAP (10±1 mmHg), TPR (0.5±0.1 Units), and CA, whilst left-ventricular end-diastolic volume (LVEDV) and heart rate decreased (all p<0.05). After adrenalectomy, FXIIa infusion decreased MAP (5±1 mmHg), did not raise CA or induce sustained vasoconstriction, and caused a greater fall in LVEDV (all p<0.05). In the 5/6 nephrectomized (NX) rodent CRF model, MAP and TPR were significantly greater in BN NX (n=16) than in BNK NX (n=15) (147±4 vs. 133±2 mmHg, 2.8±0.2 vs. 2.3±0.2 Units; all p<0.05). Plasma FXIIa measured using our semi-quantitive ELISA was 3-fold higher in both BN NX and BNK NX than in controls (p<0.01), but only correlated with MAP (r=0.48, p=0.01) in the BN NX. Plasma CA were elevated in the BN NX (p<0.05) but not in BNK NX. Infusion of a specific FXIIa inhibitor into BN NX decreased MAP (-12 mmHg) and TPR (-0.5 Units) proportionally to baseline FXIIa (ΔMAP: r=-0.72, p=0.002; ΔTPR: r=-0.57, p=0.021), and plasma CA fell by 40-67% (all p<0.05). No such changes occurred in the BNK NX. In summary, a significant component of the hypertension of CRF can be attributed to FXIIa-induced vasoconstriction mediated via the KKS and stimulated CA release. In normal rats, FXIIa appears also to directly or indirectly decrease preload and heart rate.

The physiological role of the plasma contact system still remains a partial enigma. The aim of the presented work was to expand our understanding of the plasma contact system, focusing on its physiological activation and function, principally from a hemostatic perspective. It also explored contact system activation under pathological conditions. We found that when human platelets become activated in blood, plasma proteins of the contact system bind to platelets and initiate contact activation. The platelet-triggered contact activation contributed to clot formation by shortening the clotting time and enhancing clot stability. We demonstrated that the regulation of contact activation elicited by activated platelets differed from the previously described contact activation elicited by negatively charged material surfaces. Platelet-triggered contact activation and activation propelled by clotting blood were found to be regulated by antithrombin, whereas material-induced activation was regulated by C1 inhibitor. We also showed that the fibrin fibers that are formed during the clot process further enhance and propagate the contact activation initially induced by activated platelets. Fibrin not only activated factor XII but also seemed to increase the affinity of antithrombin for the proteases of the contact system, leading to the generation of contact enzyme-antithrombin complexes during clot formation. To determine whether the contact system might be involved in the inflammation and vascular disease associated with systemic lupus erythematosus (SLE), we analyzed plasma samples from SLE patients. These patients were found to have altered levels of contact enzyme-serpin complexes, supporting the concept that the contact system may be involved in the pathophysiology of SLE. The contact enzyme-antithrombin complexes were clearly linked to platelet activation in vivo. Altered levels of both FXIIa-antithrombin and FXIIa-C1 inhibitor were found to be correlated with previous vascular disease and may therefore be potential biomarkers for assessing the risk of thrombotic events in SLE patients. These findings add to our knowledge of how the plasma contact system is activated and functions in vivo and will help us to understand the role of the contact system, not only in hemostasis but also in vascular disease and thrombotic conditions.

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)

Histidine-rich glycoprotein (HRG) is a protein present in plasma at ~ 2 μM, but whose physiologic function is unclear. HRG is a multi-domain protein that contains a unique histidine-rich core that interacts with zinc and hydrogen ions to modulate ligand binding. Due to its modular structure and capacity to sense local changes in zinc and pH, HRG binds several ligands including complement proteins, phospholipids, DNA, fibrin(ogen), heparin, factor (F) XIIa and plasmin. Thus, it is hypothesized that HRG functions as an accessory or adapter protein that bridges different ligands together. Despite the array of ligands and potential involvement in immunity, angiogenesis, coagulation and fibrinolysis, no clear role for HRG has emerged. Congenital HRG deficiency in humans has been associated with a variable phenotype; some investigators report increased susceptibility to thrombosis while others do not. However, studies in HRG-deficient mice reveal that HRG attenuates coagulation. Coagulation is initiated via the intrinsic (or contact) and extrinsic (or tissue factor) pathways and culminates in the generation of thrombin. Thrombin catalyzes the conversion of fibrinogen into a fibrin meshwork that reinforces the platelet plug at sites of vascular injury. There are two circulating isoforms of fibrinogen that differ with respect to their γ-chains. Bulk fibrinogen is composed of a pair of γA-chains, and is designated γA/γA-fibrinogen, whereas a minor variant contains a γA-chain and a γʹ-chain, and is designated γA/γʹ-fibrinogen. The γʹ-chain contains an anionic 20-amino acid residue extension at its COOH-terminus, which provides an accessory binding site for thrombin. Thrombin possesses an anion binding pocket termed exosite II that flanks the active site and mediates its interaction with the γʹ-chain of fibrinogen. Exosite II is an evolutionary feature that is unique to thrombin, as this region is not observed on the prototypic serine protease trypsin or on other defibrinogenating enzymes from snake venom such as batroxobin. Although the physiologic function of the thrombin-γʹ-chain interaction is unclear, it is proposed that this interaction modulates thrombin’s activity when it is bound to fibrin clots. Consistent with this, we show that γA/γʹ-fibrin attenuates thrombin’s capacity to promote clot expansion compared with thrombin bound to γA/γA-fibrin clots, thereby demonstrating that γA/γʹ-fibrin attenuates thrombin’s activity. In the presence of physiologic concentrations of zinc, HRG binds the γʹ-chain of fibrino(gen) and competes with thrombin for binding, thereby suggesting that HRG is a unique modulator of thrombin activity on fibrin clots. Platelets store zinc and HRG in their α-granules and release both components when they undergo activation at sites of injury, which localizes HRG in the vicinity of fibrin-bound thrombin. Consistent with the role of HRG in modulating coagulation, we also show that HRG attenuates contact activation of coagulation, but has no impact on clotting initiated by the extrinsic pathway. The intrinsic pathway is initiated when FXII is activated by polyanions such as RNA and DNA, which are released into the blood after cellular activation, injury or death. FXIIa activates FXI, thereby propagating coagulation and leading to thrombin generation and fibrin formation. Recently, studies using rodent, rabbit and non-human primate models of thrombosis have shown that knock down of FXII or FXI with antisense oligonucleotides or blocking FXIIa or FXIa activity with inhibitors attenuates thrombosis, while having a minimal impact on hemostasis. With increasing evidence that the intrinsic pathway plays an important role in thrombosis, FXII and FXI have emerged as prominent targets for new anticoagulants. However, little is known about how the intrinsic pathway is regulated, so as to prevent uncontrolled clotting. HRG attenuates the intrinsic pathway by binding both FXIIa and the contact activators, RNA and DNA. By binding nucleic acids, HRG is localized to the site of contact activation, where it is poised to inhibit FXIIa. HRG binds to an allosteric region on FXIIa and attenuates its capacity to feedback activate FXII and to activate FXI, thereby inhibiting the initiating steps of contact activation. In addition, HRG attenuates the cofactor role of RNA and DNA in thrombin activation of FXI, which is an important feedback step. With the capacity to modulate multiple steps in the intrinsic pathway, HRG likely serves as a dynamic regulator of contact activation. We tested our hypothesis that HRG is a novel inhibitor of the intrinsic pathway in a murine model of FeCl3-induced arterial injury. HRG-deficient mice exhibit accelerated thrombosis compared with wild type controls, an effect that was abolished by repletion with human HRG. Therefore, these studies indicate that HRG deficiency induces a prothrombotic phenotype. Consistent with the role of HRG as a modulator of the intrinsic pathway, we show that thrombosis after the FeCl3-induced arterial injury is attenuated by administration of RNase, but not DNase, or by knock down of FXII, but not FVII. Therefore, these studies show that thrombosis in this model is induced by RNA and occurs in a FXII-dependent manner. Furthermore, blood loss after tail tip amputation is similar in HRG-deficient and wild type mice, demonstrating that HRG does not modulate hemostasis. Therefore, these studies suggest that HRG is a dynamic regulator of the intrinsic pathway, and acts as a molecular brake to limit procoagulant stimuli. The observations that HRG binds fibrin(ogen), FXIIa and nucleic acids and modulates the thrombin-γʹ-interaction and intrinsic pathway of coagulation, suggest that HRG is a key regulator of coagulation. HRG, the contact system and fibrin are also important in the innate immune response, demonstrating that the interaction of HRG with these factors may provide a unique link between coagulation and immunity. Since immune cells and the coagulation system contribute to both deep vein thrombosis and sepsis, further characterization of the role of HRG in these conditions will contribute to a better understanding of the pathophysiological role of HRG, and may identify novel therapeutic directions. / Thesis / Doctor of Philosophy (PhD)

Made available in DSpace on 2014-07-29T16:11:50Z (GMT). No. of bitstreams: 1 Dissertacao Laura Maia Mairink Nassau.pdf: 2613768 bytes, checksum: d2e4321ca6a3e7930e1a7f57b3099a11 (MD5) Previous issue date: 2010-09-15 / Thromboembolic disorders produce the majority of diseases affecting the heart, and are the third leading cause of death worldwide. Venous thromboembolism is a thromboembolic disorder that occurs in the venous circulation, is a disease of high prevalence and remains a major cause of morbidity and mortality in hospitalized patients undergoing major surgery, the fact that affect patients from the very young, disease free prior to the more elderly mean that it is a problem with major socioeconomic burden by as much as by absenteeism in hospital patients in productive age. This describes the planning and synthesis of intermediates for the collection of new derivatives guanidine drawn from the infestina-4, protein extracted from the stomach Barber Triatoma infestans, with potent inhibitory activity of factor XII. Factor XII acts of direct and indirect enzymatic cascades in four of our body and has an important contribution to the formation of pathological vascular thrombi. The previous study of molecular modeling justified based planning in the structural and the electrostatic infestin. Thus it is concluded that the synthesis of intermediates 25-28, were getting adequate yields of 88.0 and 92.0% for compounds 25 and 26 and 71.4 and 65.0% for compounds 27 and 28 respectively. / As desordens tromboembólicas geram a maioria das doenças que acometem o coração, e são a terceira causa de morte mundial. O tromboembolismo venoso é uma desordem tromboembólica que ocorre na circulação venosa, é uma enfermidade de alta prevalência e continua sendo uma causa importante de morbimortalidade em pacientes hospitalizados e submetidos a cirurgias de grande porte, o fato de afetar pacientes desde os mais jovens, sem doenças prévias, até os mais idosos fazem com que ela constitua um problema sócio-econômico com grandes ônus tanto pelas internações quanto pelo absenteísmo em pacientes em idade produtiva. Este trabalho descreve o planejamento e a síntese de intermediários para obtenção de novos derivados guanidínicos desenhados a partir da infestina-4, proteína extraída do estômago do barbeiro Triatoma infestans, com potente atividade inibitória do fator XII. O fator XII atua de forma direta e indireta em quatro cascatas enzimáticas do nosso organismo e possui importante contribuição na formação patológica de trombos vasculares. O estudo prévio de modelagem molecular justificou o planejamento baseado nas características estruturais e eletrostáticas da infestina. Desta forma, conclui-se que a síntese dos intermediários 25-28 foram adequadas obtendo rendimentos de 88,0 e 92,0% para os compostos 25 e 26 e de 71,4 e 65,0% para os compostos 27 e 28, respectivamente.