A rapid, two-step procedure is described for the isolation of both "high molecular weight" (H-) and "low molecular weight" (L-) plasma kininogens from a single sample of plasma. Affinity chromatography on carboxymethyl-papain-Sepharose is used, together with high-resolution anion exchange chromatography.

Tryptases are trypsin-like enzymes found in mast cell granules. Although in vivo substrates have not been positively identified, tryptases cleave a limited number of potential physiological substrates in vitro, including high molecular weight kininogen (HMWK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) apparently exists as a tetramer with an M$\sb{\rm r}$ of 135-144 kDa by gel filtration, whereas SDS-PAGE yielded two bands of M$\sb{\rm r}$ 29 Kda and 33 Kda. Tryptases are resistant to inhibition by most natural trypsin inhibitors and display some affinity for heparin. The existence of tryptase isozymes has been implied from the cloning of two tryptase cDNAs from human lung tissue, but distinct isozymes have not been isolated and characterized. This knowledge gap has been filled by isolating and characterizing two electrophoretically different forms of human lung mast cell tryptase, designated high-HLT (high molecular weight HLT) and low-HLT (low molecular weight HLT). These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high M$\sb{\rm r}$ form eluting with 10 $\mu$M heparin and the low M$\sb{\rm r}$ form subsequently eluting with 1 M NaCl. Using HMWK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMWK at a single Arg residue, whereas low-HLT cleaved HMWK simultaneously at multiple sites. Both isozymes cleaved VIP at multiple sites, but differed with regard to the preferential site of cleavage. Low-HLT was, on an active site basis, 25 and 2 times more active than high-HLT on HMWK and VIP, respectively. In addition, gel filtration of the isozymes yielded M$\sb{\rm r}$s of 125 Kda for high-HLT and 28 kDa for low-HLT, indicating tetrameric and monomeric quaternary structures, respectively. Both isozymes were inhibited by human secretory leukocyte proteinase inhibitor (SLPI), but not by other trypsin inhibitors tested. This work provides the first evidence for the existence of distinct tryptase isozymes, with supposedly different in vivo functions, and identification of an inhibitor that may control tryptase activity in vivo.

Arteriogenese bezeichnet das adaptive Wachstum von präexistenten kollateralen Arterien. Im Falle eines Arterienverschlusses ist Arteriogenese der endogen effizienteste Kompensationsmechanismus, um das Hypoperfusionsgebiet mit ausreichend Blut zu versorgen (Biologischer Bypasses). In dieser Arbeit wurde das frühe Wachstum von Kollateralgefäßen im Gehirn im ersten Modell für zerebrale Arteriogenese, dem 3-VO Modell (3-vessel occlusion), in der Ratte charakterisiert. (I) Die Untersuchung am nicht-ischämischen 3-VO Hypoperfusionsmodell zeigten, dass 7 Tage nach 3-VO die Arteria cerebri posterior (PCA) signifikant im Diameter anwächst. Histologische Untersuchungen konnten ein vermehrtes Zellwachstum in der PCA und das Einwandern von Makrophagen in den perivaskulären Bereich (24 Stunden und 3 Tage post 3-VO) darstellen und eine Aktivierung des Endothels 3 Tage nach 3-VO wurde mittels Rasterelektronenmikroskopie identifizieren. (II) Für eine genaue Anaylse des globalen Genexpressionsprofils der zerebralen Arteriogense wurde die wachsende PCA selektiv aus dem Gehirn entnommen und ein Genexpressionsprofil für die frühe zerebrale Arteriogenese erstellt (164 Gene dereguliert). Eine Unteruschung von biologischen und molekularen Prozessen zeigte, dass eine Vielzahl der deregulierten Gene in Zellproliferation und Inflammation involviert sind. Die Expression der Protease-Inhibitoren Kininogen und TIMP-1 wurde als “Marker” der frühen Arteriogenese in der PCA lokalisiert werden. Zusammenfassend zeigt diese Arbeit erstmals eine Übersicht der biologischen Prozesse in der zerebralen Arteriogenese und eröffnet neue Ideen für eine mögliche therapeutische Strategie. / Arteriogenesis, the adaptive outward growth of pre-existing collateral arteries, is the most efficient endogenous rescue mechanisms in vertebrates against the occlusion of a major artery (biological bypass). Here, collateral growth was induced using the first model for cerebral arteriogenesis, the 3-vessel occlusion (3-VO) rat model. (I) 3-VO resulted in a significant diameter increase within 7 days in the posterior cerebral artery (PCA) and posterior communicating artery (Pcom), classifying the region of interest. Immunhistological staining demonstrated proliferative activation and macrophage invasion, already 24h post 3-VO within the PCA, confirming the arteriogenic phenotype. Furthermore, activation of the PCA endothelium was detected within 3 days post 3-VO by scanning electron microscopy. (II) For analysing the molecular mechanism of cerebral arteriogenesis, collateral tissue from the growing PCA was selectively isolated. Here, 24h post 3-VO 164 genes were detected to be significantly deregulated. Analysis of molecular annotations and networks associated with differentially expressed genes revealed that expression patterns contain gene transcripts predominantly involved in proliferation, inflammation, and migration. Early-phase cerebral arteriogenesis is characterized by protease inhibitor expression and showed that protease inhibitors TIMP-1 and kininogen are molecular markers of early-phase cerebral arteriogenesis. In summary, this work characterizes morphological features and genomic profiles of growing collaterals in the brain and develops novel ideas for a therapeutic stimulation of arteriogenesis.

Estudamos a ação das proteases bothropasina e Bothrops protease A, do veneno da serpente Bothrops jararaca, sobre o fibrinogênio (FBG), fibronectina (FN) e cininogênio (HK), como ferramenta para geração de peptídeos bioativos. As sequências primárias dos produtos de digestão foram identificadas por espectrometria de massas, com as buscas direcionadas por peptídeos em comum gerados pelas duas proteases. Foram encontradas oito sequências em comum provenientes do FBG e onze, da FN. Apenas a bothropasina clivou o HK, liberando desArg9BK. Foram sintetizados peptídeos derivados do FBG (FBG1-6) e da FN (FN1-4), além de des-Arg9-BK. Oito peptídeos apresentam potencial atividade antiangiogênica predita in silico. Observamos a inibição da elastase (28-20%) causada por FBG1-2-5-6. A melhor inibição da trombina foi de 17%, por FBG1. Contudo, a maioria dos peptídeos intensificou sua atividade. Por fim, este trabalho sugere que as proteases de veneno de serpentes podem ser usadas como ferramentas para processar componentes do plasma, visando à busca por peptídeos bioativos. / We studied the action of the proteases bothropasin and Bothrops protease A purified from the venom of snake Bothrops jararaca upon fibrinogen (FBG), fibronectin (FN) and kininogen (HK), as a tool to generate bioactive peptides. The primary sequences of the digestion products were identified by mass spectrometry and we focused the search for common peptides released by both proteases simultaneously. Sequences in common released by both proteases were found, being eight peptides from FBG, and 11 from FN. Only bothropasin was able to cleave HK releasing des-Arg9-BK. Peptides from fibrinogen (FBG1-6) and from fibronectin (FN1-4), as well as the des-Arg9-BK were synthetized. Eight peptides have potential antiangiogenic predicted in silico. We observed the inhibition of elastase (28-20%) caused by FBG1-2-5-6. The best inhibition of thrombin was 17% by FBG1. However, most of the peptides intensified its activity. Finally, this work suggests that the snake venom protease can be used as tools to process plasma components in order to search for bioactive peptides.

Estudamos a ação das proteases bothropasina e Bothrops protease A, do veneno da serpente Bothrops jararaca, sobre o fibrinogênio (FBG), fibronectina (FN) e cininogênio (HK), como ferramenta para geração de peptídeos bioativos. As sequências primárias dos produtos de digestão foram identificadas por espectrometria de massas, com as buscas direcionadas por peptídeos em comum gerados pelas duas proteases. Foram encontradas oito sequências em comum provenientes do FBG e onze, da FN. Apenas a bothropasina clivou o HK, liberando desArg9BK. Foram sintetizados peptídeos derivados do FBG (FBG1-6) e da FN (FN1-4), além de des-Arg9-BK. Oito peptídeos apresentam potencial atividade antiangiogênica predita in silico. Observamos a inibição da elastase (28-20%) causada por FBG1-2-5-6. A melhor inibição da trombina foi de 17%, por FBG1. Contudo, a maioria dos peptídeos intensificou sua atividade. Por fim, este trabalho sugere que as proteases de veneno de serpentes podem ser usadas como ferramentas para processar componentes do plasma, visando à busca por peptídeos bioativos. / We studied the action of the proteases bothropasin and Bothrops protease A purified from the venom of snake Bothrops jararaca upon fibrinogen (FBG), fibronectin (FN) and kininogen (HK), as a tool to generate bioactive peptides. The primary sequences of the digestion products were identified by mass spectrometry and we focused the search for common peptides released by both proteases simultaneously. Sequences in common released by both proteases were found, being eight peptides from FBG, and 11 from FN. Only bothropasin was able to cleave HK releasing des-Arg9-BK. Peptides from fibrinogen (FBG1-6) and from fibronectin (FN1-4), as well as the des-Arg9-BK were synthetized. Eight peptides have potential antiangiogenic predicted in silico. We observed the inhibition of elastase (28-20%) caused by FBG1-2-5-6. The best inhibition of thrombin was 17% by FBG1. However, most of the peptides intensified its activity. Finally, this work suggests that the snake venom protease can be used as tools to process plasma components in order to search for bioactive peptides.