Maria Kouyoumdjian

The recognition site for hepatic clearance of plasma kallikrein is on its heavy chain and is latent on prokallikrein

We partially purified the glycoproteins prokallikrein and kallikrein from rat plasma. The purification of rat plasma kallikrein may result in two forms: an intact form (alpha, M(r) 84-87 kDa) and a partially degraded form (beta, M(r) 46-51 kDa). The alpha-form is composed of a heavy chain (M(r) 50 kDa) and a light chain (M(r) 34-37 kDa) linked by a disulfide bond. The catalytic site is found on the light chain. The beta-form has a partially degraded heavy chain (M(r) 28 kDa). Using a preparation of exsanguinated and perfused rat liver, we verified that rat plasma prokallikrein is not activated by the liver and that neither the proenzyme nor the light chain is removed by the organ. Both forms (alpha and beta) of the active enzyme are similarly removed from the perfusate. We also observed that the clearance of plasma kallikrein is temperature-dependent, and not affected by substances that inhibit binding to galactosyl-, mannosyl-, fucosyl- or phosphomannosyl-specific lectins, but inhibited by beta-galactosides. We suggest that: (a) the binding site to hepatocytes is latent on prokallikrein and is located on its heavy chain, more specifically on the 28-kDa fragment still present in the beta form of the active enzyme and (b) plasma kallikrein is recognized by an S-type lectin.

Hepatic clearance of tissue‐type plasminogen activator and plasma kallikrein in experimental liver fibrosis

Abstract: We have previously shown that tissue‐type plasminogen activator (tPA) and rat plasma kallikrein (RPK) share a common, but not unique, pathway for liver clearance.

Participation of a galectin-dependent mechanism in the hepatic clearance of tissue-type plasminogen activator and plasma kallikrein

Tissue-type plasminogen activator (tPA) is a serine protease that plays a central role in the fibrinolytic system, activating plasminogen to plasmin, which degrades the fibrin that is present in blood clots. tPA has proven to be a potent drug in thrombolytic therapy, however, its use is limited due to the rapid clearance from circulation by active hepatic uptake [1]. The uptake mechanism for tPA in the liver involves endothelial and parenchymal cells [2]. A carbohydrate recognition system for tPA in sinusoidal endothelial cells and tPA binding to asialoglycoprotein receptor in parenchymal cells were described [3]. Uptake by sinusoidal endothelial cells involves an interaction between the carbohydrate group in the kringle1 domain and the mannose receptor. A minor involvement of fucose residue has also been suggested in the recognition of tPA by these cells [4,5]. On the other hand, uptake by parenchymal liver cells is mediated mainly by LDL receptor-related protein (LRP) [6,7], a well-known multiligand receptor that also mediates the clearance of a2Macroglobulin(a2M)-proteinases complexes [8]. We have shown that the hepatic clearance rate of complexes such as a2M-kallikrein [9] and a2M-trypsin [10] is decreased compared to the free enzyme. The 39-kDa receptor-associated protein (RAP) is a receptor antagonist that inhibits ligand interactions with the receptors that belong to the low-density lipoprotein receptor gene family. RAP can also function intracellularly as a molecular chaperone for LRP and can regulate its ligand binding activity along the secretory pathway. In addition, RAP also plays an important role in receptor folding [11]. Recently, Camani et al. [12] showed that in some cells the presence of functional LRP is not sufficient for efficient tPA degradation, suggesting that tPA degradation requires a co-receptor. Secretion of tPA is potently stimulated by bradykinin, a nonapeptide released from hydrolysis of high-molecularweight kininogen by kallikrein [13], linking the fibrinolytic and kallikrein–kinin systems. We have already shown that tPA and RPK compete for a common, but not unique, pathway for hepatic clearance [14]. In addition, a plasma kallikrein-dependent plasminogen cascade is required for adipocyte differentiation [15]. Plasma kallikrein circulates in the plasma as its zymogen, prekallikrein. After activation, plasma kallikrein is involved in different biological processes including the pathogenesis of inflammatory reaction, blood flow control, blood pressure control and intrinsic coagulation and fibrinolytic systems [16]. Recently, Akita et al. [17] showed that after partial hepatectomy an excessive amount of TNF-a, the major initiator of hepatic regeneration, may trigger the generation of TGF-h via enhancement of surface plasma kallikrein activity on stellate cells. The proteolytic activity of plasma kallikrein is modulated by plasma inhibitors and its concentration by liver clearance. Rat plasma prekallikrein and the light chain of rat plasma kallikrein (RPK) are not cleared by the isolated and exsanguinated rat liver. The binding site of RPK to hepatic cells is

Fate of bradykinin on the rat liver when administered by the venous or arterial route

Background and Aim: Bradykinin (BK) infused into the portal vein elicits a hypertensive response via the B2 receptor (B2R) and is efficiently hydrolyzed by the liver. Our purpose was to characterize the mechanism of interaction between BK and the liver.

Thimet oligopeptidase EC 3.4.24.15 is a major liver kininase

Bradykinin (BK) is a potent hepato-portal hypertensive agent although it is efficiently inactivated by the liver. The organ converts angiotensin I to AII, but at a much slower rate than it inactivates BK. We had previously identified EC 3.4.24.15 as an hepatic bradykinin inactivating endopeptidase that hydrolyzes BK at the F5-F6 bond. The aim of this study was to determine the relative importance of BIE, as compared to other kininases, in normal, cirrhotic or inflamed rat livers, as well as in samples of human liver. Using specific substrates and inhibitors we showed that: 1) purified BIE preparation hydrolyzed BK and a BK analogue (BK-Q) with similar efficacy; BK-Q was functionally active since it caused an increase in hepato-portal pressure, as did BK itself. 2) BK degradation in rat serum was performed by ACE since BIE and prolylendopeptidase (PEP) activities were negligible. 3) normal rat liver homogenate contained a large amount of BIE activity which was eliminated by a specific EC 3.4.24.15 inhibitor; ACE and PEP activities were negligible. 4) There was no difference (p>0.05) in BIE activity in the liver homogenates from rats with normal, inflamed or cirrhotic livers. 5) BIE activity was efficiently removed from livers (normal, inflamed or cirrhotic) that were perfused with TritonX-100.6) Human liver had an similar enzymatic pattern although ACE activity was detected. We concluded that in normal, inflamed or cirrhotic rat livers, as well as in the human liver, the bradykinin inactivating endopeptidase (EC 3.4.24.15), and not ACE, is the major hepatic kininase.

Anicteric cholangiopathy in schistosomiasis patients

UNLABELLED We previously reported that in anicteric patients with the isolated form of schistosomiasis (without co-morbidities) an ursodeoxycholic acid-sensitive increase in serum gamma-glutamyltransferase activity (gammaGT) occurs. We now describe the presence of cholangiopathy in these patients. METHODS Sixteen adult anicteric patients with the isolated form of schistosomiasis mansoni were carefully selected: nine with increased gammaGT and seven with normal gammaGT. High sensitive C-reactive protein (CRP), to exclude inflammatory status, hyaluronic acid (HA), and other laboratory parameters were determined. The ultrasonographic study measured spleen length, portal vein and splenic vein diameters, and the portal flow. Magnetic resonance cholangiopancreatography (MRCP) images were interpreted by a blind observer. MRCP was deemed abnormal when focal narrowing and/or paucity of second and third order biliary branches and/or irregularities in the contours of biliary pathways were identified. RESULTS Both groups (normal and elevated gammaGT) have preserved hepatic function tests (HA, serum albumin, prothrombin time) and clinical significant portal hypertension (low platelet count and ultrasonographic parameters). MRCP was abnormal in all patients with elevated gammaGT but in only 3 of the 7 patients with normal gammaGT (p=0.003). CONCLUSION Magnetic resonance cholangiopancreatography characterized a cholangiopatic disorder in anicteric patients with the isolated form of schistosomiasis, even preceding laboratory test alterations.

Kallikrein-kinin system in hepatic experimental models.

The purpose of this brief review is to describe some characteristics of the kallikrein-kinin system (KKS) in the liver. The liver synthesizes kininogens and prekallikrein and the synthesis of both proteins is increased in rats during the acute phase reaction. It is also the main organ to clear tissue as well as plasma kallikrein from the circulation in normal and pathological conditions. Bradykinin (BK), yielded by the kallikrein-kinin system, is a potent arterial hypotensive peptide, but in the liver it induces a portal hypertensive response. The portal hypertensive action of bradykinin is mediated by B2 receptors located on sinusoidal cells of the periportal region and is followed by its hydrolysis by angiotensin-converting enzyme, which is primarily present in the perivenous (centrolobular) region.

Hepatic conversion of angiotensin I and the portal hypertensive response to angiotensin II in normal and regenerating liver

Background and Aim:  Angiotensin I (AI) and angiotensin II (AII) induce a portal hypertensive response (PHR) and the liver is able to convert AI into AII to trough the action of the angiotensin‐converting enzyme (ACE). Our purpose was to characterize angiotensin I liver conversion.

Hemodynamic and metabolic effects of angiotensin II on the liver

To ascertain the mechanism of interaction between angiotensins (AI and AII) and the liver, an angiotensin-converting enzyme inhibitor (captopril) and a receptor antagonist (losartan) were used. Monovascular or bivascular liver perfusion was used to assess both hemodynamic (portal and arterial hypertensive responses) and metabolic (glucose production and oxygen consumption) effects. Microphysiometry was used for isolated liver cell assays to assess AII or losartan membrane receptor-mediated interaction. Captopril abolishes portal hypertensive response (PHR) to AI but not the AII effect. AII infused via the portal pathway promotes calcium-dependent PHR but not a hypertensive response in the arterial pathway (AHR); when infused into the arterial pathway AII promotes calcium-dependent PHR and AHR. Losartan infused into the portal vein abolishes PHR to AII but not the metabolic response; when infused via both pathways it abolishes the hypertensive responses and inhibits the metabolic effects. Isolated liver cells specifically respond to AII. Sinusoidal cells, but not hepatocytes, respond to 10 nM losartan. We conclude that AI has to be converted to AII to produce PHR. Quiescent stellate cells interacts in vitro with AII and losartan. Hemodynamic responses to AII are losartan-dependent but metabolic responses are partially losartan-independent. AII hemodynamic actions are mainly presinusoidal.

Liver bradykinin-inactivating-endopeptidase is similar to the metalloendopeptidase (EC 3.4.24.15)

The bradykinin-inactivating-endopeptidase (BIE) removal from rat liver, by perfusing the organ with 0.05% Triton X-100, achieved its maximum at 10 min of perfusion and falls to 50% of the maximum in 30 min, a pattern similar to AST removal. Using an internally quenched fluorescent BK analogue (Abz-RPPGFSPFRQ-EDDnp) we further characterized this enzyme: it is activated by low concentrations of 2-mercaptoethanol, inhibited by p-hydroxymercuribenzoate, o-phenanthroline and EDTA, and is resistant to enalapril, E-64 and PMSF. These results suggest that BIE is a metalloendopeptidase containing a thiol group important for its activity. BIE also hydrolyses the peptides Abz-GGFLRRVQ-EDDnp, Abz-GPQGLAGQ-EDDnp, Abz-FRSVQ-EDDnp, and Abz-ARVRRANSFLQ-EDDnp. All these properties are very similar to those described or assayed by us for EC 3.4.24.15, isolated initially from rat testes and then from several organs of different animals. Both BIE and EC 3.4.24.15: hydrolyze the F5S6 bond of the BK fluorescent substrate; are efficiently inhibited by Orlowski specific inhibitor (CFP-AAF-pAB, Ki 4.4 x 10(-7) M and 1.25 x 10(-7) M, respectively); have the same electrophoretic mobility in SDS-PAGE (Mr 78,000); and are both recognized by three polyclonal antibodies raised against rat testes EC 3.4.24.15. In conclusion, BIE appears to be EC 3.4.24.15.