Hypotensive effect of the active fragment derived from factor XII is mediated by an activation of the plasma kallikrein-kinin system.

Plasma protein fraction (PPF) contaminated by factor XII active fragment (XIIf) may cause hypotensive reactions when infused to patients. This study was planned to assess in conscious normotensive rats whether the blood pressure response to the factor XIIf is mediated by an activation of the plasma kallikrein-kinin system or by stimulation of prostaglandin synthesis. To test whether the factor XIIf-induced blood pressure fall is due partially to an enhanced generation of vasodilating prostaglandins, the blood pressure effect of XIIf (1 microgram i.v.) was investigated 15 min after treatment with indomethacin (5 mg i.v.), an inhibitor of cyclo-oxygenase. Factor XIIf reduced mean blood pressure similarly in indomethacin- and vehicle-treated rats (-23 +/- 4 mmHg, n = 5, and -23 +/- 5 mmHg, n = 4, respectively). Other rats received factor XIIf 15 min after depletion of circulating prekallikrein by the administration of dextran sulfate. Thirty minutes after a 0.25 mg i.v. dose of this agent, plasma prekallikrein activity averaged 0.12 +/- 0.015 mumol/min/ml (n = 6) as compared to 2.48 +/- 0.31 mumol/min/ml in control rats (n = 4, P less than .001). Factor XIIf decreased mean blood pressure by only 4 +/- 2 mm Hg in rats pretreated with dextran sulfate. Thus, it was possible to blunt the acute hypotensive effect of factor XIIf by depleting circulating prekallikrein, but not by inhibiting prostaglandin production. This strongly suggests that the blood pressure effects of factor XIIf is mediated by a stimulation of the plasma kallikrein-kinin system.

Involvement of the kallikrein-kinin system in the antihypertensive effect of the angiotensin converting enzyme inhibitors.

1. Studies were performed in normal subjects and in rats to assess the effect of angiotensin converting enzyme (ACE) inhibition on the kallikrein-kinin system. As ACE is identical to kininase II, one of the enzymes physiologically involved in bradykinin degradation, bradykinin may be expected to accumulate during ACE inhibition. 2. A competitive antagonist of bradykinin was used to explore in unanaesthetized rats the contribution of circulating bradykinin to blood pressure control under ACE inhibition. 3. No evidence was found for a role of this vasodilating peptide in the blood pressure lowering effect of acute ACE inhibition. 4. The plasma activity of carboxypeptidase N (= kininase I), another pathway of bradykinin degradation, remained intact during a 1 week course of treatment with an ACE inhibitor in normal subjects. This therefore indicates that bradykinin formed during ACE inhibition can still be metabolized.

La dénervation rénale unilatérale et le système kallikréine-bradykinine rénal chez le rat [Unilateral renal denervation and the renal kallikrein-bradykinin system in the rat].

AIM: The antihypertensive effect of renal denervation in hypertensive patients is partially explained by increased tubular natriuresis. To study the possible contribution of the kallikrein-kinin system (KKS) to this natriuretic effect in rats, we measured kallikrein activity (KA) and bradykinin concentrations (BK) in plasma and tissues. METHODS: To measure KA, we adapted and validated an enzymatic assay that cleaves para-nitroaniline (pNA) from the tripeptide H-D-Pro-Phe-Arg-pNA. The coefficients of variation (CV) within- and between-assays were less than 8% for plasma and tissue KA (plasma n=6 and 13; tissue n=4). Linear results for serially diluted samples confirmed the assay specificity. Tissue BK determinations were based on an established assay for plasma BK: tissue was homogenized and kinins extracted in ethanol, and BK was isolated by high-performance (HPLC) liquid chromatography and quantitated by radioimmunassay. Within- and between-assay CV for plasma BK were 18% (n=8 and n=35, respectively) and for BK in various tissues less than 16% (n=5-8). RESULTS: In male Wistar rats (n=3), plasma BK was 8.2±6.6 fmol/mL (mean±SD), and tissue BK (fmol/g) in 14 tested organs varied between brain (14±3) and submaxillary gland (521±315). Six days after left-sided unilateral renal denervation, left renal tissue BK (89±9) was not different from right renal BK (75±23). Similarly, KA was comparable in the two kidneys (left 18.0±1.5, right 15.8±1.4μkat/g). CONCLUSION: Any possible effect of unilateral renal denervation on the kidney's KKS would have to be bilateral.

Chronic bradykinin receptor blockade modulates neonatal renal function.

Recent data indicate that bradykinin participates in the regulation of neonatal glomerular function and also acts as a growth regulator during renal development. The aim of the present study was to investigate the involvement of bradykinin in the maturation of renal function. Bradykinin beta2-receptors of newborn rabbits were inhibited for 4 days by Hoe 140. The animals were treated with 300 microg/kg s.c. Hoe 140 (group Hoe, n = 8) or 0.9% NaCl (group control, n = 8) twice daily. Clearance studies were performed in anesthetized rabbits at the age of 8-9 days. Bradykinin receptor blockade did not impair kidney growth, as demonstrated by similar kidney weights in the two groups, nor did it influence blood pressure. Renal blood flow was higher, while renal vascular resistance and filtration fraction were lower in Hoe 140-treated rabbits. No difference in glomerular filtration rate was observed. The unexpectedly higher renal perfusion observed in group Hoe cannot be explained by the blockade of the known vasodilator and trophic effect of bradykinin. Our results indicate that in intact kallikrein-kinin system is necessary for the normal functional development of the kidney.

The role of bradykinin B(1) and B(2) receptors for secondary brain damage after traumatic brain injury in mice.

Inflammatory mechanisms are known to contribute to the pathophysiology of traumatic brain injury (TBI). Since bradykinin is one of the first mediators activated during inflammation, we investigated the role of bradykinin and its receptors in posttraumatic secondary brain damage. We subjected wild-type (WT), B(1)-, and B(2)-receptor-knockout mice to controlled cortical impact (CCI) and analyzed tissue bradykinin as well as kinin receptor mRNA and protein expression up to 48 h thereafter. Brain edema, contusion volume, and functional outcome were assessed 24 h and 7 days after CCI. Tissue bradykinin was maximally increased 2 h after trauma (P<0.01 versus sham). Kinin B(1) receptor mRNA was upregulated up to four-fold 24 h after CCI. Immunohistochemistry showed that B(1) and B(2) receptors were expressed in the brain and were significantly upregulated in the traumatic penumbra 1 to 24 h after CCI. B(2)R(-/-) mice had significantly less brain edema (-51% versus WT, 24 h; P<0.001), smaller contusion volumes ( approximately 50% versus WT 24 h and 7 d after CCI; P<0.05), and better functional outcome 7 days after TBI as compared with WT mice (P<0.05). The present results show that bradykinin and its B(2) receptors play a causal role for brain edema formation and cell death after TBI.

Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P'1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.

New human kallikrein 14: enzymatic characterization and inhibitors development.

RESUME DESTINE A UN LARGE PUBLIC En biologie, si une découverte permet de répondre à quelques questions, en général elle en engendre beaucoup d'autres. C'est ce qui s'est produit récemment dans le monde des kallicréines. De la famille des protéases, protéines ayant la faculté de couper plus ou moins spécifiquement d'autres protéines pour exercer un rôle biologique, la famille des kallicréines humaines n'était composée que de 3 membres lors du siècle dernier. Parmi eux, une kallicréine mondialement utilisée pour détecter le cancer de la prostate, le PSA. En 2000, un chercheur de l'hôpital universitaire Mont Sinaï à Toronto, le Professeur Eleftherios Diamandis, a découvert la présence de 12 nouveaux gènes appartenant à cette famille, situés sur le même chromosome que les 3 premières kallicréines. Cette découverte majeure a placé les spécialistes des kallicréines face à une montagne d'interrogations car les fonctions de ces nouvelles protéases étaient totalement inconnues. La kallicréine humaine 14 (hK14) présente un intérêt particulier, car elle se retrouve associée à différents cancers, notamment les carcinomes ovariens et mammaires. Cette association ne répond cependant pas à la fonction de cette protéase. L'objectif de ce travail de thèse était donc de découvrir, dans un premier temps, la spécificité de cette nouvelle kallicréine, c'est-à-dire le type de coupure qu'elle engendre au niveau des protéines qu'elle cible. Utilisant une technologie de pointe qui exploite la propriété des bactériophages à se répliquer dans les bactéries à l'infini, des dizaines de millions de combinaisons protéiques aléatoires ont été présentées à hK14, qui a pu sélectionner celles qui lui étaient favorables pour la coupure. Cette technique qualitative porte le nom de Phage Display Substrate. Une fois la sélection réalisée, il fallait transférer ces séquences coupées ou substrats dans un système permettant de donner une valeur quantitative à l'efficacité de coupure. Pour cela nous avons développé une technologie qui permet d'évaluer cette efficacité en utilisant des protéines fluorescentes de méduse, modifiées génétiquement, dont l'excitation de la première (CFP : cyan fluorescent protein) par la lumière à une certaine longue d'onde permet le transfert d'énergie à la seconde (YFP : yellow fluorescent protein), via un substrat qui les lie. Pour que ce transfert d'énergie se produise, il faut que les deux protéines fluorescentes soient proches, comme c'est le cas lorsqu'elles sont liées par un substrat. La coupure de ce lien provoque un changement de transfert d'énergie qui est quantifiable en utilisant un spectrofluoromètre. Cette technologie permet donc de suivre la réaction d'hydrolyse (coupure) des protéases. Afin de poursuivre certaines expériences permettant de mieux comprendre la fonction biologique d'hK14 ainsi que son éventuelle implication dans le cancer, nous avons développé des inhibiteurs spécifiques d'hK14. Les séquences qui on été le plus efficacement coupées par hK14 ont été utilisées pour transformer deux types d'inhibiteurs classiques, qui circulent dans notre sang, en inhibiteurs d'hK14 hautement efficaces et spécifiques. Selon les résultats obtenus in vitro, ils pourront être évalués in vivo en tant que traitement potentiel contre le cancer. RESUME Les protéases sont des enzymes impliquées dans des processus physiologiques mais aussi parfois pathologiques. La famille des kallicréines tissulaires humaines représente le plus grand groupe de protéases humaines, dont plusieurs pourraient participer au développement de certaines maladies. D'autre part, ces protéases sont apparues comme des marqueurs de pathogénicité potentiels, notamment dans les cas de cancers hormono-dépendants. La kallicréine humaine 14 a été récemment découverte et son implication dans quelques maladies, particulièrement dans le cas de tumeurs, semble probable. En effet, son expression génique est augmentée au niveau des tissus cancéreux de la prostate et du sein et son expression protéique s'est révélée plus élevée dans le sérum de patientes atteintes d'un cancer du sein ou des ovaires. Cependant, comme c'est le cas pour la plupart des kallicréines, sa fonction est encore inconnue. Afin de mieux connaître son rôle biologique et/ou pathologique, nous avons décidé de caractériser son activité enzymatique. Nous avons tout d'abord mis au point un système de substrats entièrement biologique permettant d'étudier in vitro l'activité des protéases. Ce système est basé sur le phénomène de FRET, à savoir le transfert d'énergie de résonance fluorescente qui intervient entre deux molécules fluorescentes voisines si le spectre d'émission de la protéine donneuse chevauche le spectre d'excitation de la protéine receveuse. Nous avons fusionné de manière covalente une protéine fluorescente bleue (CFP) et une jaune (YFP) en les liant avec diverses séquences. Par clivage de la séquence de liaison, une perte du transfert d'énergie peut être mesurée par un spectrofluoromètre. Cette technologie représente un moyen facile de suivre la réaction d'hydrolyse des protéases. Les conditions optimales de production de ces substrats CFP-YFP ont été déterminées, de même que les paramètres pouvant éventuellement influencer le FRET. Ce système possède une grande résistance à la protéolyse non spécifique et est applicable à un grand nombre de protéase. Contrairement aux substrats fluorogéniques, il permet d'étudier les acides aminés se trouvant des deux côtés du site de clivage. Ce système étant entièrement biologique, il est le reflet des interactions protéine-protéine et représente un outil biologique facile, bon marché et rapide pour caractériser les protéases. Dans un premier temps, hK14 a été mise en présence d' une banque de haute diversité de pentapeptides aléatoires présentée à la surface de phages afin d'identifier des substrats spécifiques. Ensuite, le système CFP-YFP a été employé pour trier les peptides sélectionnés afin d'identifier les séquences de substrats les plus sensibles et spécifiques pour hK14. Nous avons montré, qu'en plus de sa prévisible activité de type trypsine, hK14 possède aussi une très surprenante activité de type chymotrypsine. Les séquences les plus sensibles ont été choisies pour cribler la banque de donnée Swissprot, permettant ainsi l'identification de 6 substrats protéiques humains potentiels pour hK14. Trois d'entre eux, la laminine α-5, le collagène IV et la matriline-4, qui sont des composants de la matrice extracellulaire, ont démontré une grande susceptibilité à l'hydrolyse par hK14. De plus, la séparation éléctrophorétique a montré que la dégradation de la laminine α-5 et de la matriline-4 par hK14 devait se produire aux sites identifiés par la technologie du phage display. Pour terminer, nous avons transformé, par mutagenèse dirigée, deux serpines (inhibiteurs de protéases de type sérine) connues, AAT et ACT (alpha anti-trypsine et alpha anti-chymotrypsine), qui inhibent un vaste éventail d'enzymes humaines en inhibiteurs d'hK14 hautement efficaces et spécifiques. Ces inhibiteurs pourront être utilisés d'une part pour poursuivre certaines expériences permettant de mieux comprendre l'implication d'hK14 dans des voies physiologiques ou dans le cancer et d'autre part pour les évaluer in vivo en tant que traitement potentiel contre le cancer. SUMMARY Proteases consist of enzymes involved in physiological events, but also, in case of dysregulation, in pathogenicity. The human tissue kallikrein family represents the largest human protease cluster and includes several members that either could participate in the course of certain diseases or emerged as potential biological markers, especially in hormone dependent cancers. The human kallikrein 14 has been recently discovered and suggested implications in some disorders, particularly in tumors since its gene expression is up-regulated in prostate and breast cancer tissues and its protein expression increased in the serum of patients with breast and ovarian cancers. However, like most kallikreins, its function remains unknown. To better understand hK14 biological and/or pathological role, we decided to characterize its enzymatic activity. First of all, we developped a biological system suitable for in vitro study of protease activity. This system is based on the so-called FRET phenomenon, that is the Fluorescence Resonance Energy Transfer that occurs between two nearby fluorescent proteins if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. We fused covalently a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) with diverses sequences. Upon cleavage of the linker sequence by protease, the loss of energy transfer can be measured by a spectrofluorometer allowing an easy following of hydrolysis reaction. The optimal conditions to produce in bacterial system these CFP-YFP substrates were determined as well as the parameters that could eventually influence the FRET. This system demonstrated a high degree of resistance to non-specific proteolysis and applicability to various conditions corresponding to a great number of existing proteases. Other avantages are the possibility to study the amino acids located both sides of the cleavage site as well as the interest to work in a full biological system reflecting protein-protein interaction. A phage substrate library with exhaustive diversity was used prior to CFP-substrate-YFP system to isolate specific human kallikrein 14 substrates. After that the CFP-YFP system was used to sort peptides and identify highly sensitive and specific substrate sequences for hK14. We showed that besides its predictable trypsin-like activity, hK14 also possesses a surprising chymotrypsin-like activity. The screening of the Swissprot database was achieved with the most sensitive sequences and allowed the identification of 6 potential human protein substrates for hK14. Three of them, laminin α-5, collagen IV and matrilin-4, which are components of the extracellular matrix were incubated with hK14, by which they were efficiently hydrolyzed. Moreover, electrophoretic separation revealed that degradation of laminin α-5 and matrilin-4 by hK14 generated fragments with identical molecular size than the predicted N-terminal fragments that would result from hK14 specific cleavage, proving the value of phage display substrate to identify potential substrates. Finally, with site-directed mutagenesis, we transformed two well-known serpins (serine protease inhibitors), AAT and ACT (alpha anti-trypsin and alpha anti-chymotrypsin), which inhibit a vast spectrum of human enzymes into highly efficient and specific hK14 inhibitors. These inhibitors will be used to pursue experiments that could help understand hK14 implication in physiological pathways as well as in cancer biology and also to perform their in vivo evalution as potential cancer treatment.

Endotoxin-induced hypotension in rats is not mediated by prekallikrein activation.

To test whether endotoxin decreases blood pressure acutely in rats by activating the plasma kinin-forming system, plasma kallikrein activity was determined in different experimental settings of endotoxemia. Conscious normotensive rats were infused for 45 min with endotoxin (LPS E. coli 0111:B4) at a dose (0.01 mg/min) which had no effect on blood pressure. Additional rats were infused with the vehicle of endotoxin. Plasma prekallikrein activity was measured at the end of the 45 min infusions. In other rats, a bolus intravenous injection of endotoxin (2 mg) was administered following the 45 min infusion of endotoxin or its vehicle. In these two latter groups of rats, plasma prekallikrein activity was determined 15 min after administration of the bolus dose of endotoxin. In rats pretreated with the endotoxin infusion, the bolus dose of endotoxin had no significant effect on blood pressure, whereas rats infused with the vehicle became and remained hypotensive up to the end of the experiment. There was however no significant difference in plasma prekallikrein activity within the different groups of rats. In another group of rats, dextran sulfate (0.25 mg i.v.), which activates factor XII and thereby the conversion of prekallikrein to kallikrein, induced a short-lasting fall in blood pressure. 15 min after administration of dextran sulfate, plasma prekallikrein activity was almost completely suppressed. These results obtained in unanesthetized rats strongly suggest that the blood pressure fall induced by E. coli endotoxin is not due to activation of prekallikrein and consequently of the kinin-forming system.