Avaliação da relação entre o polimorfismo C677T no gene para MTHFR e a concentração plasmática de homocisteína na doença arterial coronariana / Evaluation of MTHFR C677T gene polymorphism and homocysteine level in coronary atherosclerotic disease / Evaluation of MTHFR C677T gene polymorphism and homocysteine level in coronary atherosclerotic disease

OBJETIVO: O objetivo deste trabalho é determinar a prevalência do polimorfismo C677T do gene metilenotetraidrofolato redutase (MTHFR) e associá-la com a concentração plasmática de homocisteína plasmática na doença arterial coronariana (DAC). MÉTODOS: Foram avaliados 93 pacientes com DAC documentada, atendidos no Hospital Universitário Oswaldo Cruz (Recife, PE, Brasil), e 108 controles sem a doença. Foram determinados os perfis lipídicos de pacientes e controles. As concentrações plasmáticas de homocisteína e folato foram determinadas por HPLC e quimioluminescência, respectivamente. A genotipagem foi realizada por RFLP/PCR. RESULTADOS: Os grupos de pacientes e controles foram homogêneos quanto aos perfis genéticos do polimorfismo investigado. Nos pacientes, as concentrações plasmáticas médias de homocisteina (11,7 ± 4,4 æmol/L) e de folato (6,22 ± 3,0 ng/dL) foram estatisticamente diferentes daquelas observadas nos controles (8,84 ± 3,2 æmol/L e 7,69 ± 3,1 ng/dL, respectivamente), ao nível de significância de 0,05. Entretanto, não houve correlação entre concentração plasmática de homocisteína e folato nos pacientes (r= -0,202). Não foi observada associação entre a homozigosidade 677TT para MTHFR e a concentração plasmática de homocisteína sérica (p= 0,634). A comparação dos casos e controles que apresentaram simultaneamente alta concentração plasmática de homocisteína e baixa concentração de folato, resultou numa razão de chance superior à de cada variável analisada independentemente (RC= 11,9; IC 95 por cento= 4,16-34,42, p< 0,01). CONCLUSÕES: A mutação C677T não parece ser um fator genético importante capaz de explicar a hiperhomocisteinemia moderada observada nos pacientes com DAC. Outros fatores, ambientais e genéticos, devem ser investigados.

OBJECTIVE: The aim of this study is to determine the prevalence of C677T methylenetetrahydrofolate reductase (MTHFR) polymorphism and correlate it with plasma homocysteine levels in coronary artery disease (CAD). METHODS: Ninety-three patients with documented CAD from Hospital Universitário Oswaldo Cruz (Recife, PE, Brazil) and 108 healthy controls were evaluated. Homocysteine and folate levels were determined by HPLC and chemoluminescence, respectively, and lipid profile was considered. Genotyping was done by RFLP/PCR. RESULTS: The groups were homogeneous for the C677T polymorphisms. The homocysteine level in cases (11.7 æmol/L) was statistically different from that observed in controls (8.84 æmol/L, p< 0.05). It was also observed that 72 percent of the patients had homocysteine values above12 æmol/L while the control group presented only 32 percent in this range. There was no relationship between homozigosity for the C677T polymorphism and the homocysteine level (p= 0.634). We noticed statistical differences between folate levels from patients and controls (6.22 and 7.69 ng/dL, p< 0.05, respectively). However, there was no correlation between homocysteine and folate concentrations in the entire group (r= -0.202). Comparing cases and controls, the odds ratio (OR) when homocysteine is high and folate is low was OR= 11.9; CI 95 percent= 4.16-34.42, p< 0.01. CONCLUSION: A lack of correlation between C677T mutation and homocysteine level suggests that environmental factors and others genetic factors seem to exert more influence on homocysteine level in this population.

[Evaluation of MTHFR C677T gene polymorphism and homocysteine level in coronary atherosclerotic disease]. / Avaliação da relação entre o polimorfismo C677T no gene para MTHFR e a concentração plasmática de homocisteína na doença arterial coronariana.

OBJECTIVE: The aim of this study is to determine the prevalence of C677T methylenetetrahydrofolate reductase (MTHFR) polymorphism and correlate it with plasma homocysteine levels in coronary artery disease (CAD). METHODS: Ninety-three patients with documented CAD from Hospital Universitário Oswaldo Cruz (Recife, PE, Brazil) and 108 healthy controls were evaluated. Homocysteine and folate levels were determined by HPLC and chemoluminescence, respectively, and lipid profile was considered. Genotyping was done by RFLP/PCR. RESULTS: The groups were homogeneous for the C677T polymorphisms. The homocysteine level in cases (11.7 micromol/L) was statistically different from that observed in controls (8.84 micromol/L, p< 0.05). It was also observed that 72% of the patients had homocysteine values above 12 micromol/L while the control group presented only 32% in this range. There was no relationship between homozygosity for the C677T polymorphism and the homocysteine level (p= 0.634). We noticed statistical differences between folate levels from patients and controls (6.22 and 7.69 ng/dL, p< 0.05, respectively). However, there was no correlation between homocysteine and folate concentrations in the entire group (r= -0.202). Comparing cases and controls, the odds ratio (OR) when homocysteine is high and folate is low was OR= 11.9; CI 95%= 4.16-34.42, p< 0.01. CONCLUSION: A lack of correlation between C677T mutation and homocysteine level suggests that environmental factors and others genetic factors seem to exert more influence on homocysteine level in this population.

Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI.

A Bauhinia variegata trypsin inhibitor (BvTI) cDNA fragment was cloned into the pCANTAB5E phagemid. The clone pAS 1.1.3 presented a cDNA fragment of 733 bp, including the coding region for a mature BvTI protein comprising 175 amino acid residues. The deduced amino acid sequence for BvTI confirmed it as a member of the Kunitz-type plant serine proteinase inhibitor family. The BvTI cDNA fragment encoding the mature form was cloned into the expression vector, pET-14b, and ex-pressed in E. coli BL21 (DE3) pLysS in an active form. In addition, a BvTI mutant form, r(mut)BvTI, with a Pro residue as the fifth amino acid in place of Leu, was produced. The recombinant proteins, rBvTI and r(mut)BvTI, were purified on a trypsin-Sepharose column, yielding 29 and 1.44 mg/l of active protein, respectively, and showed protein bands of approximately 21.5 kDa by SDS-PAGE. Trypsin inhibition activity was comparable for rBvTI (Ki=4 nM) and r(mut)BvTI (Ki=6 nM). Our data suggest that the Leu to Pro substitution at the fifth amino-terminal residue was not crucial for proteinase inhibition.

Vitamin E prevents cell death induced by mild oxidative stress in chicken skeletal muscle cells.

Apoptosis and necrosis are two forms of cell death that can occur in response to various agents and oxidative damage. In addition to necrosis, apoptosis contributes to muscle fiber loss in various muscular dystrophies as well participates in the exudative diathesis in chicken, pathology caused by dietary deficiency of vitamin E and selenium, which affects muscle tissue. We have used chicken skeletal muscle cells and bovine fibroblasts to study molecular events involved in the cell death induced by oxidative stress and apoptotic agents. The effect of vitamin E on cell death induced by oxidants was also investigated. Treatment of cells with anti-Fas antibody (50 to 400 ng/mL), staurosporine (0.1 to 100 microM) and TNF-alpha (10 and 50 ng/mL) resulted in a little loss of Trypan blue exclusion ability. Those stimuli conducted cells to apoptosis detected by an enhancement in caspase activity upon fluorogenic substrates but this activity was not fully blocked by the caspase inhibitor Z-VAD-fmk. Oxidative stress induced by menadione (10, 100 and 250 muM) promoted a significant reduction in cell viability (10%, 20% and 35% for fibroblasts; 20%, 30% and 75% for muscle cells, respectively) and caused an increase in caspase activity and DNA fragmentation. H2O2 also promoted apoptosis verified by caspase activation and DNA fragmentation, but in higher doses induced necrosis. Vitamin E protected cells from death induced by low doses of oxidants. Although it was ineffective in reducing caspase activity in fibroblasts, this vitamin diminished the enzyme activity in muscle cells. These data suggested that oxidative stress could activate apoptotic mechanisms; however the mode of cell death will depend on the intensity and duration of the stimulus, and on the antioxidant status of the cells.

Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies.

Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase ( K i(app) 5.3 nM), porcine pancreatic elastase ( K i(app) 40 nM), cathepsin G ( K i(app) 160 nM) and the cysteine proteinases cruzipain ( K i(app) 1.2 nM), cruzain ( K i(app) 0.3 nM) and cathepsin L ( K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein ( K i(app) 2.4 nM) and plasmin ( K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the beta-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.

The BIR domain of IAP-like protein 2 is conformationally unstable: implications for caspase inhibition.

Several IAP (inhibitor of apoptosis) proteins regulate cell fate decisions, and the X-linked IAP (XIAP) does so in part by inhibiting caspases, proteases that execute the apoptotic pathway. A tissue-specific homologue of XIAP, known as ILP2 (IAP-like protein 2), has previously been implicated in the control of apoptosis in the testis by direct inhibition of caspase 9. In examining this protein we found that the putative caspase 9 interaction domain is a surprisingly weak inhibitor and is also conformationally unstable. Comparison with the equivalent domain in XIAP demonstrated that the instability is due to the lack of a linker segment N-terminal to the inhibitory BIR (baculovirus IAP repeat) domain. Fusion of a 9-residue linker from XIAP to the N-terminus of ILP2 restored tight caspase 9 inhibition, dramatically increased conformational stability and allowed crystallization of the ILP2 BIR domain in a form strikingly similar to the XIAP third BIR domain. We conclude that ILP2 is an unstable protein, and cannot inhibit caspase 9 in a physiological way on its own. We speculate that ILP2 requires assistance from unidentified cellular factors to be an effective inhibitor of apoptosis in vivo.

A proteinase inhibitor from Caesalpinia echinata (pau-brasil) seeds for plasma kallikrein, plasmin and factor XIIa.

Caesalpinia echinata is a tree belonging to the Leguminosae family. The red color of the trunk, looking like burning wood ('brasa' in Portuguese), is the origin of the name Brazil. Seeds of leguminous plants contain high amounts of serine proteinase inhibitors that can affect different biological processes. Here we show that a protein isolated from seeds of C. echinata is able to inhibit enzymes that participate in blood coagulation and fibrinolysis. This inhibitor (CeKI) was purified to homogeneity by ion exchange and reversed-phase chromatography. SDS-PAGE indicated a single polypeptide chain with a molecular mass of 20 kDa. CeKI inhibits human plasma kallikrein ( K i =3.1 nM), plasmin ( K i =0.18 nM), factor XIIa ( K i =0.18 nM), trypsin ( K i =21.5 nM) and factor Xa ( K i =0.49 mM). CeKI inhibited kinin release from highmolecular- mass kininogen by kallikrein in vitro . The N-terminal sequence, determined by automatic Edman degradation, identified the inhibitor as a member of the Kunitz family. The secondary structure, determined by circular dichroism, is mainly a random coil followed by beta-sheet structure. The action of CeKI on enzymes of the blood-clotting intrinsic pathway was confirmed by prolongation of the activated partial thromboplastin time.

Plasma prekallikrein/kallikrein processing by lysosomal cysteine proteases.

Plasma kallikrein plays a role in coagulation, fibrinolysis and inflammation. Cathepsins B and L participate in (patho)physiological processes such as peptide antigen processing, tissue remodeling events, protein turnover in cells, hormone processing and tumor invasion. The present work analyzes the processing of prekallikrein/kallikrein by lysosomal cathepsins. Prekallikrein is not hydrolyzed by catB, and catL generates an inactive fragment of prekallikrein. Both kallikrein chains are hydrolyzed by catL and the light chain is mainly hydrolyzed by catB; kallikrein activity is lower after incubation with catL compared to catB. Our data suggest that the plasma kallikrein/ kinin system can be controlled by cathepsins.

High molecular weight kininogen as substrate for cathepsin B.

We investigated the influence of pH and divalent cations (Zn2+, Mg2+ and Ca2+) on high molecular weight kininogen processing by cathepsin B. At pH 6.3, high molecular weight kininogen is hydrolyzed by cathepsin B at three sites generating fragments of 80, 60 and 40 kDa. Cathepsin B has kininogenase activity at this pH which is improved in the absence of divalent cations. At pH 7.35, high molecular weight kininogen is slightly cleaved by cathepsin B into fragments of 60 kDa, and cathepsin B kininogenase activity is impaired. Our results suggest that high molecular weight kininogen is a substrate for cathepsin B under pathophysiological conditions.

Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities.

A small combinatorial library of LDTI mutants (5.2 x 10(4)) restricted to the P1-P4' positions of the reactive site was displayed on the pCANTAB 5E phagemid, and LDTI fusion phages were produced and selected for potent neutrophil elastase and plasmin inhibitors. Strong fusion phage binders were analyzed by ELISA on enzyme-coated microtiter plates and the positive phages had their DNA sequenced. The LDTI variants: 29E (K8A, I9A, L10F, and K11F) and 19E (K8A, K11Q, and P12Y) for elastase and 2Pl (K11W and P12N), 8Pl (I9V, K11W, and P12E), and 10Pl (I9T, K11L, and P12L) for plasmin were produced with a Saccharomyces cerevisiae expression system. New strong elastase and plasmin inhibitors were 29E and 2Pl, respectively. LDTI-29E was a potent and specific neutrophil elastase inhibitor K(i) =0.5 nM), affecting no other tested enzymes. LDTI-2Pl was the strongest plasmin inhibitor ( K(i) =1.7nM) in the LDTI mutant library. This approach allowed selection of new specific serine proteinase inhibitors for neutrophil elastase and plasmin (a thrombin inhibitor variant was previously described), from a unique template molecule, LDTI, a Kazal type one domain inhibitor, by only 2-4 amino acid replacements. Our data validate this small LDTI combinatorial library as a tool to generate specific serine proteinase inhibitors suitable for drug design and enzyme-inhibitor interaction studies.

Action of Bauhinia bauhinioides synthetic peptides on serine proteinases.

The kallikrein inhibitor found in Bauhinia bauhinioides seeds (BbKI) differs from classical Kunitz plant inhibitors in the lack of disulfide bridges in its structure [Biochim. Biophys. Acta 1477 (2000) 64-74]. In this study, we examined whether structural properties may be involved in inhibitory specificity and, if so, whether those properties might be useful tools in designing compounds that interfere with enzyme activity. Peptides structurally related to the BbKI (RPGLPVRFESPLRINIIKE-NH(2)) reactive site were synthesized by solid-phase method and assayed for serine proteinase activity. The peptides RPGLPVRFESPLRINIIKE-NH(2), RPGLPVRFESPL-NH(2), and GLPVRFES-NH(2) were efficient tissue kallikrein inhibitors, with I(50) values of 0.54 microM, 0.87 microM, and 0.5mM, respectively. The lasting inhibitory effect was observed in incubation periods of up to 120 min. None of the studied peptides interfere with the activity of thrombin, factor Xa or trypsin, although the native protein BbKI is a potent trypsin inhibitor.

Effect of plant Kunitz inhibitors from Bauhinia bauhinioides and Bauhinia rufa on pulmonary edema caused by activated neutrophils.

Mediators released from polymorphonuclear neutrophils, in particular elastase, are known to induce acute edematous lung injury. In this study we show that the pulmonary edema in isolated perfused rabbit lungs caused by activated neutrophils via release of elastase is significantly decreased by the Kunitz-type Inhibitor BbCI (10(-5) M) from Bauhinia bauhinoides to the same degree as by eglin C (10(-5) M) from Hirudo medicinalis, which was used as a reference. The highly homologous proteinase inhibitor BrPI (10(-5) M) from Bauhinia rufa, however, did not reduce edema formation. The major difference between these inhibitors is the much higher Ki value of BrPI (Ki = 38 nM) for elastase compared to BbCI (Ki = 5.3 nM) and eglin C (Ki = 0.2 nM), respectively. Elastase liberation from activated PMNs was not influenced by the inhibitors. Our results indicate that BbCI can be a useful tool to study the role of neutrophil elastase in pathophysiological processes.

Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles.

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes.

A prothrombin activator from Bothrops erythromelas (jararaca-da-seca) snake venom: characterization and molecular cloning.

A novel prothrombin activator enzyme, which we have named 'berythractivase', was isolated from Bothrops erythromelas (jararaca-da-seca) snake venom. Berythractivase was purified by a single cation-exchange-chromatography step on a Resource S (Amersham Biosciences) column. The overall purification (31-fold) indicates that berythractivase comprises about 5% of the crude venom. It is a single-chain protein with a molecular mass of 78 kDa. SDS/PAGE of prothrombin after activation by berythractivase showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin, independent of the prothrombinase complex. Chelating agents, such as EDTA and o -phenanthroline, rapidly inhibited the enzymic activity of berythractivase, like a typical metalloproteinase. Human fibrinogen A alpha-chain was slowly digested only after longer incubation with berythractivase, and no effect on the beta- or gamma-chains was observed. Berythractivase was also capable of triggering endothelial proinflammatory and procoagulant cell responses. von Willebrand factor was released, and the surface expression of both intracellular adhesion molecule-1 and E-selectin was up-regulated by berythractivase in cultured human umbilical-vein endothelial cells. The complete berythractivase cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 2330 bp and encodes a preproprotein with significant sequence similarity to many other mature metalloproteinases reported from snake venoms. Berythractivase contains metalloproteinase, desintegrin-like and cysteine-rich domains. However, berythractivase did not elicit any haemorrhagic response. These results show that, although the primary structure of berythractivase is related to that of snake-venom haemorrhagic metalloproteinases and functionally similar to group A prothrombin activators, it is a prothrombin activator devoid of haemorrhagic activity. This is a feature not observed for most of the snake venom metalloproteinases, including the group A prothrombin activators.

Glycosaminoglycans affect the action of human plasma kallikrein on kininogen hydrolysis and inflammation.

Human plasma kallikrein (huPK) is a serine proteinase involved in many biological processes including those of the kallikrein-kinin system. The action of huPK on kininogen results in bradykinin (BK) release, a potent mediator of inflammatory responses. BK generation may be influenced by several agents, and the aim of this work was to investigate the effect of glycosaminoglycans (GAGs) on human high-molecular-weight kininogen (HK) hydrolysis by huPK and on inflammation. huPK was pre-incubated in the absence and presence of different GAGs, followed by the addition of kininogen. Bradykinin released at different times was measured by radioimmunoassay, and KM and kcat were calculated. Tuna and bovine dermatan sulfates, the most potent GAGs studied, reduced by 80% and 68%, respectively, the catalytic efficiency of huPK (control = 4. x 10(4) M(-1) s(-1) in BK release. The effect of bovine dermatan sulfate (BDS) on inflammatory response was studied in rat paw edema induced by carrageenin and hourly determined (1-4 h) by plethysmography. BDS significantly reduced the inflammatory response in the first and second hours of measurements (24% and 28%, respectively), p < 0.05. GAGs were shown to reduce bradykinin release "in vitro" and in an inflammation model. This reduction may play a role in the control or maintenance of some pathological and physiological processes.

BmTI antigens induce a bovine protective immune response against Boophilus microplus tick.

Boophilus microplus trypsin inhibitors (BmTIs) present in larvae were preliminarily characterized as active proteins, approximately 10-18 kDa, by SDS-PAGE. BmTIs showed trypsin inhibitory activity on reverse zymography containing gelatin (0.03%) and also inhibited others serine proteinases (human neutrophil elastase and human plasma kallikrein). Bos indicus, Nelore breed calves, previously sensitized with BmTIs and challenged with tick larvae (20,000 larvae/animal), showed 72.8% efficacy to interfere in tick development with 69.7% and 71.3% reduction of both tick number and egg weight, respectively. Cattle BmTls antiserum titer was approximately 1:8000. The maximum level of BmTls antibody production was detected 40 days after the first immunization by ELISA. Our preliminary results suggest that B. microplus serine proteinase inhibitors may play a role in the tick larvae fixation and feeding processes. Therefore, the development of antibodies against BmTIs might impair the normal parasitism.

Characterization of a Kunitz trypsin inhibitor with one disulfide bridge purified from Swartzia pickellii.

Swartzia pickellii is a Leguminosae that belongs to the Caesalpinioideae sub-family the Swartzia pickellii Trypsin Inhibitor (SWTI), a serine proteinase inhibitor was isolated from its seeds. SWTI is a single polypeptide chain protein and it's structure has 174 amino acid residues, it homologous to other Kunitz plant inhibitors, however shows some major differences: it contains only one disulfide bridge, instead two which are usually found in plant Kunitz inhibitors, and the SWTI reactive site does not contain the usual Arg or Lys residues at the putative reactive site (position 65). A glycosylation site was detected at Asn38 with 1188 kDa carbohydrate portion. The primary structure micro heterogeneity was found combining the sequence determination and mass spectrometry. Three forms of SWTI were actually defined: two glycosylated forms a 20,204 kDa (Arg 165) and 20,185 kDa (His 165) and one deglycosylated form 19,016 kDa (Arg 165), all of them contain a Met residue at position 130.

Proteínas anticoagulantes das glândulas salivares da sanguessuga Haementeria depressa

Sanguessugas possuem substâncias em sua saliva que são capazes de desequilibrar o sistema hemostático de suas presas tornando o sangue incoagulável. Foi feita a purificação da hementerina ( substância fibrinogenolítica ) e do lefaxin ( inibidor de fator Xa). O mecanismo de ação e peptídeos internos das duas proteínas foi determinado. A hementerina é uma metaloprotainase de cadeia única de 80 kDa, capaz de degradar o fibrinogênio e a fibrina. Essa atividade é maior quando o plasminogênio é adicionado. No entanto nenhuma atividade tipo t-PA ou pro-UK foi descoberta em sistema específico purificado. O N- terminal da hementerina apresenta 80% de homologia com a hementina, outra fibrinogenase purificada da Haementeria ghilianii. O lefaxin ( inibidor de FXa) é uma proteína de cadeia única de 30 kDa, pl 5,7 e possui o N-terminal bloqueado. Inibe o fator Xa por um mecanismo do tipo lento " slow" e apresenta um IC 50 de 50 pM para 1 nM de fator Xa humanoou bovino. Ele é especifico para o fator Xa e é capaz de inibir a formação da trombina no complexo protombinase. A sequência dos peptídeos internos não revelou homologia com a antistasina , inibidor de fator Xa da Haementeria officinalis. Foi construida uma biblioteca de cDNA para o complexo salivar de H. depressa.

Leeches posses salivary substances wich are able to disequilibrate the hemostatic system thus rendering the blood unclottable . Purification of hementerin (fibrinogenolytic substance) and of a lefaxin ( a FXa inhibitor ) was undertaken. The mecanism of action and the internal peptides of both proteins were determined. Purified hementerin is a single chain metaloproteinase of 80 kD, able do degrade fibrinogen and fibrin . This activity is higher on plasma or when plasminogen is added. However, no t-PA or Pro-UK like activity is disclosed in a purified specific system. The hementerin N- terminal has 80% homology with hementerin, another fibrinogenolytic component purified from Haementeria ghilianii. The lefaxin ( Fxa inhibitor) is a single chain protein of 30 kD, pl 5.7 with N-terminal bloqued. The inhibition is of the slow type and presents an IC 50 of 50 pM for 1 nM of human or bovine Fxa. It is specific for FXa and is able to inhibit thrombin formation from the prothrombinase complex. The internal peptide sequence does not show homology with antistasin ( another FXa inhibitor from H. officinalis). A cDNA library was constructed for the "salivary complex".