Activation of the bradykinin-forming cascade on endothelial cells: a role for heat shock protein 90

Bradykinin is a major mediator of swelling in C1 inhibitor deficiency as well as the angioedema seen with ACE inhibitors and may contribute to bronchial hyper-reactivity in asthma. Formation of bradykinin occurs in the fluid phase and along cell surfaces requiring interaction of Factor XII, prekallikrein and high molecular weight kininogen (HK). The mechanism by which initiation occurs is uncertain. Recent data suggest that activation of the kinin-forming cascade can occur on the surface of endothelial cells, even in the absence of Factor XII. We demonstrate herein that during a 2-h incubation time, plasma deficient in either Factor XII or high molecular weight kininogen (HK) fail to activate kinin-forming cascade as compared to normal plasma. With more prolonged incubation, Factor XII deficient plasma gradually activates and HK deficient plasma does not. Our data support both Factor XII-dependent (rapid) and Factor XII-independent (slow) mechanisms; the latter may require a cell-derived protein (possibly protease) to activate prekallikrein in the presence of zinc ion and HK. To further define this cellular factor, we demonstrated that both cytosolic and membrane fractions from endothelial cells possessed the ability to catalyze prekallikrein conversion to kallikrein in the presence of HK and zinc ion. We purified this factor from cytosol by affinity chromatography employing corn trypsin inhibitor (CTI) as ligand. The fractions with peak activity were subjected to SDS-PAGE analysis, ligand blotted with biotinylated CTI, and positive bands were sequenced. Heat shock protein 90 (Hsp90) was identified as one of the proteins. Zinc-dependent activation of the prekallikrein-HK complex on endothelial cells was inhibited upon the addition of polyclonal antibody to Hsp90 in a dose-dependent manner. Although the mechanism by which Hsp90 activates the kinin-forming cascade is not yet clear, this protein represents the cellular contribution to the reaction and may become the dominant mechanism in pathologic circumstances in which Hsp90 is highly expressed or secreted.     

Interaction of factor XII and high molecular weight kininogen with cytokeratin 1 and gC1qR of vascular endothelial cells and with aggregated Abeta protein of Alzheimer's disease

High molecular weight kininogen (HK) attaches to endothelial cells at separate sites on the heavy and light chains by a process which requires 15-50 microM zinc. Previously identified binding proteins include gClqR, cytokeratin 1, and the urokinase plasminogen activator receptor (U-par), however, their relative contribution to binding are not yet clarified. We have purified the binding proteins by affinity chromatography, in the presence of zinc ion, and identified cytokeratin 1 and gC1qR by amino acid sequencing of an internal peptide and by immunoblot as heavy chain and light chain binding proteins, respectively. Antibody to cytokeratin 1 inhibited HK binding to endothelial cells by 30%, antibody to gClqR inhibited HK binding to endothelial cells by 72%, and a mixture of both inhibited binding by 86%. The binding and activation of the proteins of the kinin-forming cascade along the cell surface is zinc-dependent. Similarly, proteins of the plasma kinin-forming cascade can be activated by binding to aggregated A(beta) protein of Alzheimer's disease. Activation of the cascade using purified proteins or upon addition of Abeta to plasma requires aggregation of A(beta) and the reactions are zinc-dependent. In plasma, HK is cleaved and bradykinin is liberated. The data demonstrate that aggregated A(beta) can bind and activate proenzymes of the plasma kinin-forming cascade to release bradykinin and these reactions are dependent on zinc ion.     

Activation of the plasma kallikrein/kinin system on endothelial cell membranes

For more than three decades, it has been known that the plasma kallikrein/kinin system becomes activated when exposed to artificial, negatively charged surfaces. The existence of an encompassing in vivo, negatively charged surface capable of activation of the plasma kallikrein/kinin system has, however, never been convincingly demonstrated. In this report, we describe current knowledge on how the proteins of the plasma kallikrein/kinin system assemble to become activated on cell membranes. On endothelial cells, the activation of the plasma kallikrein/kinin system is not initiated by factor XII autoactivation as seen on artificial surfaces. On endothelial cells, prekallikrein is activated by an antipain sensitive protease. Prekallikrein activation is dependent on the presence of high molecular weight kininogen and an optimal free Zn2+ concentration. Kallikrein generated on the surface of endothelial cell is capable of activating factor XII. Further, kallikrein formed on endothelial cell membranes is capable of cleaving its receptor and native substrate, high molecular weight kininogen, liberating bradykinin and the HK PK complex from the endothelial cell surface. Endothelial cell-associated kallikrein also is capable of kinetically favorable pro-urokinase and, subsequent, plasminogen activation.     

Effect of Neurotropin on the activation of the plasma kallikrein-kinin system.

Bradykinin (BK), an important mediator of allergic reactions and pain induction, is released by the activation of the plasma kallikrein-kinin (K-K) cascade. Neurotropin is a biological material obtained from inflamed rabbit skin inoculated with vaccinia virus and is widely used clinically in Japan as an effective agent for these disorders. Since its mechanism of action is not clearly known, we have investigated the effects of Neurotropin on the human plasma K-K system. In dextran sulfate-activated plasma, Neurotropin inhibited the formation of BK, the cleavage of high molecular weight kininogen (HK) and the formation of kallikrein-C1 inhibitor and activated coagulation factor XII (FXIIa)-C1 inhibitor complexes. Experiments using purified enzyme of the K-K cascade indicated that Neurotropin inhibited surface-mediated activation of coagulation factor XII (FXII) and the activation of prekallikrein by FXIIa. Neurotropin also inhibited the binding of FXII and HK to the activating surface. These data suggest that the ameliorating effects of Neurotropin in allergic disorders and pain syndromes may be related to this ability to inhibit activation of the K-K cascade and consequently the formation of BK.     

The fibrinolytic system during short-term treatment with tenoxicam

The effect of 20 mg tenoxicam once daily for 7 days on various components of the fibrinolytic system was studied in 10 healthy volunteers. Plasma plasminogen, antithrombin 3, and prekallikrein decreased significantly while plasma plasminogen activator inhibitor increased significantly. The medication did not affect fibrin plate lysis area or the plasma level of plasminogen activator, alpha-2-antiplasmin, alpha-2-macroglobulin, C1 inactivator or Factor XII. It is suggested that these changes may be caused by interference with hepatic enzyme systems. The reduction in plasma prekallikrein may indicate that tenoxicam exerts its anti-inflammatory effect by more than one mechanism.     

Assembly and activation of the plasma kallikrein/kinin system: a new interpretation

Understanding the importance and physiologic activity of the plasma kallikrein/kinin system (KKS) has been thwarted by the absence of an inclusive theory for its assembly and activation. The contact activation hypothesis describes the assembly and activation of this system in test tubes and disease states, but not under physiologic circumstances. Recent investigations have indicated a new cohesive hypothesis for understanding physiologic activation of this system. Prekallikrein (PK) and factor XI (FXI) through high molecular weight kininogen (HK) assemble on a co-localized, multiprotein receptor complex on endothelial cells that consists of at least cytokeratin 1 (CKI), gClqR, and urokinase plasminogen activator receptor (muPAR). When assembled on these proteins, prekallikrein becomes activated to kallikrein by the membrane-expressed enzyme prolylcarboxypeptidase (PRCP). Formed kallikrein then activates factor XII (FXII) for amplification of its activation and single chain urokinase. The plasma kallikrein/kinin system may serve as a physiologic counterbalance to the plasma renin angiotensin system (RAS) by lowering blood pressure and preventing thrombosis. Insights into the integrated role of these two systems may afford the development of novel therapeutic drugs to manage hypertension and thrombosis.     

Activation of factor XII in rat plasma: protection by benzamidine of the cofactor function of high molecular weight kininogen

Factor XII has been assayed as kaolin-activated prekallikrein activator in rat citrated plasma pretreated with acetone (Briseid et al. 1978 & 1979; Briseid & Berstad 1979). In the present work benzamidine added during blood collection increased the extent of activation by a factor of 6. Rat high molecular weight kininogen (HMWK) added to acetone-treated citrated plasma likewise increased the activation, providing evidence of the protection by benzamidine of the cofactor function of HMWK. All cofactor capacity was retained after the removal of the kinin part of HMWK. Experiments carried out with plasminogen-free plasma showed that plasmin could hardly be the the factor responsible for the destruction of HMWK. The stoichiometric factor XII concentration-effect curve obtained by diluting acetone-treated rat plasma with acetone-treated human factor XII deficient plasma showed that factor XII is present in functional excess, the concentration of HMWK deciding the extent of activation. By diluting acetone-treated rat plasma with buffer, HMWK concentration-effect curves were obtained which were approximately linear over a range of 0.03-0.40 microgram (bradykinin equivalents) per ml kaolin incubate. No further activation of factor XII was obtained at 0.80 microgram/ml.     

Dextran-induced anaphylactoid reaction in man: altered reactivity of high molecular weight kininogen

Normal levels of factor XII and of high and low molecular weight kininogens (HMWK and LMWK) were registered in plasma specimens from 5 individuals who had developed anaphylactoid reactions upon injection of dextran during surgery (dextran reactors, DR). Factor XII was assayed as prekallikrein activator (PKA) activated with kaolin at 0 degrees, and kininogen fractions were estimated through the release of kinin caused by plasma kallikrein or hog pancreas kallikrein (HPK). Subnormal levels of factor XII apparently present in plasma from one DR, and after affinity chromatography on a lysine-Sepharose column also in plasma from another DR, were normalized by addition of plasma deficient in factor XII or by addition of purified HMWK. Treatment of plasma from DR with acetone (25% v/v) induced a conversion of HMWK into a state which was non-functional as a cofactor in the surface-dependent activation of factor XII, and the passage of plasma from DR through a lysine-Sepharose column altered the HMWK present to a substance that released kinin only very slowly by incubation with HPK. It is concluded that the treatments mentioned will favour the activation in plasma from DR of a factor that will cause the conversion of HMWK. Previous experiments with rat plasma demonstrated that plasmin and also a plasmin-like factor without affinity for lysine-Sepharose were able to destroy the capacity of HMWK to function as a cofactor in the surface-dependent activation of factor XII, without a corresponding release of kinin.     

Acetylcysteine in Rats: Inhibition of Activation of Prekallikrein and Factor XII - Protection against Dextran-Induced Blood Pressure Fall

Abstract: Acetylcysteine (AC) injected intravenously into rats (200 mg/kg) had no effect on blood pressure, but significantly inhibited dextran-induced (40 mg/kg) blood pressure fall. Injection of AC also reversibly blocked the activation of prekallikrein (PK) normally obtained in plasma incubated with acetone. Kallikrein was assayed as plasminogen activator, S-2302 amidase and BAEe esterase. Also the activation of factor XII to factor XIIa, assayed as prekallikrein activator, was strongly inhibited in AC-treated rats. It is suggested that the partial blockade of dextran-induced shock is correlated with an inhibition of activation of PK and factor XII. Previous experiments had demonstrated an extensive, but reversible in vitro inhibition of human plasma kallikrein by AC. In view of such data it is concluded that the present results obtained with AC in rats are probably due to an inhibition of plasma kallikrein and its activation of factor XII.     

Acetylcysteine in rats: inhibition of activation of prekallikrein and factor XII--protection against dextran-induced blood pressure fall

Acetylcysteine (AC) injected intravenously into rats (200 mg/kg) had no effect on blood pressure, but significantly inhibited dextran-induced (40 mg/kg) blood pressure fall. Injection of AC also reversibly blocked the activation of prekallikrein (PK) normally obtained in plasma incubated with acetone. Kallikrein was assayed as plasminogen activator, S-2302 amidase and BAEe esterase. Also the activation of factor XII to factor XIIa, assayed as prekallikrein activator, was strongly inhibited in AC-treated rats. It is suggested that the partial blockade of dextran-induced shock is correlated with an inhibition of activation of PK and factor XII. Previous experiments had demonstrated an extensive, but reversible in vitro inhibition of human plasma kallikrein by AC. In view of such data it is concluded that the present results obtained with AC in rats are probably due to an inhibition of plasma kallikrein and its activation of factor XII.     

Fine mapping of the H-kininogen binding site in plasma prekallikrein apple domain 2

Plasma prekallikrein (PPK), the zymogen of the contact phase protease plasma kallikrein, forms a non-covalent complex with its substrate H-kininogen (HK). HK binds to cell surface proteoglycans, indirectly anchoring this bradykinin-generating protease to endothelial cells. The heavy chain of PPK consisting of four apple domains designated A1 to A4. Previous studies indicated that a major HK binding site on PPK is within the A2 domain, with additional contributions to binding provided by the N-terminal portion of Al and the central part of A4. To precisely map the relevant binding segments in A2, we employed a monoclonal anti-PPK antibody (PKH6) that binds to A2 and blocks HK-PPK complex formation with an apparent IC50 of 8 nM. Using recombinant A2 C-terminal deletion mutants, we mapped the target epitope of PKH6 to the N-terminal portion of A2, residues 92-153. C-terminal deletion of A2 to residue 145 resulted in a loss of PKH6 binding, as did proteolytic cleavage of A2 at Lys140-Arg141. A comparison of HK binding to various A2 deletion mutants revealed that the major HK binding site is localized to residues 145-153 in the central portion of A2, where it overlaps with the PKH6 epitope. This sequence is conserved in the A2 domain of the related protease factor XI, explaining the unusual strong cross-reactivity of PHK6 with factor XI, as well as the similar HK-binding characteristics of PPK and factor XI.     

Assembly, activation, and physiologic influence of the plasma kallikrein/kinin system

The plasma kallikrein/kinin system that consists of the proteins factor XII, prekallikrein, and high molecular weight kininogen was first recognized as a surface-activated coagulation system arising when blood or plasma interacts with artificial surfaces. Although surface-activated contact activation occurs in vivo when various negatively charged surfaces become exposed, including a developing platelet thrombus, a physiologic, non-injury mechanism for activation, regulation, and function of this system has been elusive. Recent investigations have shown that there is a physiologic pathway for assembly and activation of this system independent of factor XII. Gene deficient mice of the bradykinin B2 receptor and factor XII have been recognized to have reduced risk for arterial thrombosis. This plasma proteolytic system influences arterial thrombosis independent of influencing hemostasis. Thus, the plasma kallikrein/kinin system has two mechanisms for its activation: one that is dependent and another independent of factor XII. Better understanding of this system may lead to insight into mechanisms for arterial thrombosis, independent of hemostasis.     

Histidine-lysine peptides as carriers of nucleic acids

With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo.     

Increased percentages of tumor necrosis factor-alpha+/interferon-gamma+ T [corrected] lymphocytes and calprotectin+/tumor necrosis factor-alpha+ monocytes in patients with acute Kawasaki disease

In vivo exposure to microorganisms resident in the oral cavity is considered as a possible cause of Kawasaki disease (KD), and some epitopes derived from streptococci display homology with Factor H of Complement. Additionally, calprotectin, a major calcium binding protein released by neutrophils and activated monocytes, could be directly involved in endothelial damage occurring in KD. The aim of our study is to evaluate the percentages of IFN-gamma+ and/or TNF-alpha+ lymphocytes and double positive calprotectin/TNF-alpha monocytes (CD14+) after in vitro stimulation with streptococcal- and/or Factor H-derived peptides, in patients with acute KD. Peripheral Blood Mononuclear Cells (PBMCs) obtained from KD patients and febrile controls were stimulated in vitro with peptides. After culture, cells were collected, stained with fluorochrome-labelled monoclonal antibodies against CD3, CD14, calprotectin, IFN-gamma and TNF-alpha, and cytofluorimetric analyses were performed. Our results showed increased percentages of TNF-alpha+/IFN-gamma+ lymphocytes in KD patients in respect to controls when PBMCs were stimulated with streptococcal or Factor H-derived epitopes. In addition, also calprotectin+/TNF-alpha+ monocytes from KD patients were activated after PBMC in vitro stimulation. These findings lead us to speculate that some peptides, derived from oral streptococci and cross-reactive with the human Factor H of Complement, could induce lymphocyte and monocyte activation potentially involved in the pathogenesis of KD. Our results should be confirmed by further studies enrolling more patients and controls than those analyzed in our study.     

Activation of factor XII in acetone-treated human plasma: significance of the functional state of plasma kallikrein for the extent of activation

The kaolin-induced activation of factor XII (XII) to XIIa was studied in plasminogen-free human citrated plasma treated with acetone in the presence of benzamidine 7.5 mM. XIIa was assayed as prekallikrein (PK) activator. The significance of the concentrations of XII, PK and high molecular weight kininogen (HMrK) was examined using mixtures of normal plasma and plasma genetically deficient in these factors. At the high plasma dilution used (1 + 23 v/v in the kaolin incubate) a joint estimation of the factors was obtained. A reduction in amount of XII, PK or HMrK resulted in a correspondingly reduced yield of XIIa. Plasma kallikrein present was assayed as S-2302 amidase. The concentration of PK in XII-deficient plasma was normal, in HMrK-deficient plasma about 30% of normal. The activation of XII was studied in fresh plasma as well as in plasma stored for 3-6 months at -70 degrees, and the activation with acetone was carried out in the presence and in the absence of benzamidine, EDTA or purified HMrK. In previous work benzamidine was found to protect the cofactor function of purified HMrK in the assay system used, and EDTA was found to inhibit purified human plasma kallikrein assayed as plasminogen activator. The present results support the previous observations, and indicate that acetone treatment of fresh human plasma (benzamidine present) results in the activation of plasma kallikrein in a functional state that requires kinin-free, but otherwise native HMrK as a cofactor for the activation of XII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of serotonin antagonists on the dextran-induced anaphylactoid reaction in the rat

Dextran injected intravenously into rats causes a rapid lowering of the extent of activation of factor XII and of the plasma levels of prekallikrein and plasminogen, and at the same time a profound fall in blood pressure. The effects on these dextran-induced reactions of three serotonin antagonistic lysergic acid derivatives were studied in the rat: bromolysergic acid diethylamide (BOL 148), methysergide, and ergotamine. BOL 148 was found to be the only effective inhibitor of the blood pressure fall, possessing an inhibitory effect over the dose range 1.0-4.0 mg/kg intravenously. The finding supports the assumption that part of the dextran-induced blood pressure fall is mediated by serotonin through D-receptors. All three serotonin antagonists showed a serotonin-like lowering effect on the activation of factor XII and on the level of prekallikrein in plasma (BOL 148 1.0-4.0 mg/kg; methysergide 0.10-0.60 mg/kg; ergotamine 0.10-0.60 mg/kg). The results with the serotonin antagonist alone and in combination with dextran provided evidence that the serotonin-mediated part of the blood pressure fall can not be secondary to the effect of dextran on the plasma parameters assayed.     

Effects of intravenous radiographic contrast media on the blood pressure and on factors of the contact activation system in the rat

Blood pressure (BP), plasma prekallikrein (PK), and the extent of activation of factor XII (XII-ACT) were studied after the intravenous injection into rats of dextran (Macrodex), the ionic radiographic contrast substance iodipamide (Biligrafin), or the non-ionic contrast substance iohexol (Omnipaque). After acetone activation plasma kallikrein was assayed as plasminogen activator, BAEe esterase or S-2302 amidase, and factor XIIa was assayed as kaolin-activated prekallikrein activator. Dextran induced a strong and lasting hypotension, preceded by significant lowerings in PK and XII-ACT. Iodipamide induced a rapid and dose dependent BP fall, no change in plasma PK, but a slightly reduced XII-ACT. Iohexol induced no significant alterations, neither in BP, nor in plasma parameters. Pretreatments of the rats with iodipamide abolished the dextran-induced reductions in PK and XII-ACT, and almost blocked the fall in BP. We conclude that the ionic contrast substance iodipamide is capable of blocking dextran shock in the rat by preventing an activation of the contact activating system in plasma.     

Plasma kallikrein localisation in human blood vessels

Immunoreactive plasma kallikrein/prekallikrein was detected in the endothelial cells and the smooth muscle cells of the arteries examined. The most intense overall immunolabelling of plasma kallikrein/prekallikrein was visualized in the medium to small size arteries. The endothelial cells of the pulmonary artery and the smooth muscle cells of the supracallosal artery showed the highest intensity of plasma kallikrein/prekallikrein labelling. The least defined labelling occurred in the tunica adventitia. The renal vein was the only blood vessel that showed no trace of immunoreactive plasma kallikrein/prekallikrein. The question arises as to the mechanisms that could be involved in the in vivo conversion of plasma kallikrein/prekallikrein into the active enzymatic molecule. The experiments indicate that a bacterial elastase cleaves the Arg371-Ile372 scissile bond within a disulphide bridge of the prekallikrein molecule. This is the bond that is cleaved also during activation of prekallikrein by trypsin-like proteinases. Functionally, the endogenous activation of plasma prekallikrein is of considerable importance, both in the regulation of blood flow and blood pressure and in the causation of septic shock. The incidental finding at histology, of patchy atheromatous disease in the coronary, vertebral and supracallosal arteries, assisted in elucidating the role of plasma kallikrein/prekallikrein in the commonest disease affecting human blood vessels. Intense labelling for plasma kallikrein was observed in the endothelial cells, foamy macrophages, inflammatory cells and fibroblasts within the thickened intima of the plaque as well as in smooth muscle cells of the underlying tunica media. The intense immunolabelling of plasma kallikrein/prekallikrein in these regions suggest that these may be induced by atheromatous disease.     

Kininogen binding to the surfaces of macrophages

Kinin generation may be initiated on the cell surfaces via a primary kininogen docking which has been characterized for endothelial cells, platelets, neutrophils, astrocytes and smooth muscle cells. In this work we describe the adsorption of biotin-labeled human kininogens by murine RAW 264.7 macrophages and human U-937 monocytes/macrophages. Both cell types strongly bound high molecular mass kininogen (HK) in a zinc-ion dependent manner with the dissociation constants of 9.1 nM and 3.3 nM, respectively, and the binding capacities of 46 fmol and 71 fmol per million of respective cells. The HK binding was quenched by 50% by antibodies against Mac-1, gC1qR and uPAR proteins indicating that these macrophage surface receptors are involved in the HK adsorption. A significant increase of HK binding was observed after cell activation with phorbol myristate acetate. Our results suggest that macrophages, similarly to neutrophils, may supply kininogens to the inflammatory foci to support the local kinin production at these sites.