The role of the kallikrein-kinin system, matrix metalloproteinases, and tissue inhibitors of metalloproteinases in the early restenosis of covered stents in the femoropopliteal arterial segment.

OBJECTIVE: The purpose of this study was to investigate the roles of the kallikrein-kinin system and matrix metalloproteinases (MMPs) in the development of arterial restenosis attributable to intimal hyperplasia in the femoropopliteal arteries. METHODS: This report describes a single-center prospective study of 27 patients with peripheral artery disease who required percutaneous transluminal angioplasty and stenting of the femoropopliteal segment using covered stent grafts. The blood concentrations of total and kininogen fractions were evaluated using immunoenzymatic methods. Plasma kallikrein was evaluated by the colorimetric method. Tissue kallikrein was evaluated by the spectrophotometric method. The activity of kininase II was measured by fluorometric analysis. Quantification of MMPs was performed by zymography, and tissue inhibitors of metalloproteinases were measured by enzyme-linked immunosorbent assay. RESULTS: Four (15%) of the treated patients developed restenosis at the 6-month follow-up evaluation. These patients had significantly lower levels of high-molecular-weight kininogens (24 hours; P < .05) and low-molecular-weight kininogens (before, P < .05; 24 hours, P < .01; 6 months, P < .05) and lower levels of tissue inhibitor of metalloproteinases-2 (6 months; P < .05) than the patients without restenosis. The activity levels of plasma and tissue kallikrein, kininase II, and MMPs did not differ significantly between the patients with and without restenosis. CONCLUSIONS: This study demonstrates an involvement of the kallikrein-kinin system in in-stent restenosis, although we could not confirm the participation of metalloproteinases in the restenosis process.

Kinins and cytokines in plasma and cerebrospinal fluid of patients with neuropsychiatric lupus.

OBJECTIVE: To evaluate the kinin system components and selected cytokines in plasma and cerebrospinal fluid (CSF) of patients with neuropsychiatric lupus (NPL). METHODS: We studied 29 women with active NPL and 29 healthy women matched to patients for age. Low (LKg) and high molecular weight kininogen (HKg) and cytokine concentrations [interleukin 1beta (IL-1beta), IL-6, IL-8, IL-10, and tumor necrosis factor-a (TNF-a)] were determined by ELISA. The activities of tissue kallikrein, plasma prekallikrein, and kininase II were assayed by their action on selective substrates. RESULTS: Compared to controls, patients with NPL presented increased plasma and CSF levels of LKg, HKg, and prekallikrein, increased activity of tissue kallikrein and kininase II, and increased levels of IL-6, IL-10, and TNF-a (p < 0.001 each comparison). IL-1beta levels were increased in patient plasma (p < 0.001), whereas plasma IL-8 levels did not differ from controls. IL-1beta and IL-8 were not detected in CSF of patients or controls. CONCLUSION: The increased levels of kininogen fractions, kallikreins, and kininase II in patient plasma and CSF indicate overactivity of the kinin system, suggesting intense kinin production. Since kinins may induce the production of proinflammatory cytokines including IL-1beta, IL-6, and TNF-a, these findings support the participation of kinins and cytokines in the acute manifestations of NPL. Most of the variables evaluated in patients' CSF increased proportionally in relation to plasma levels. In contrast, the activity of tissue kallikrein in patient CSF increased out of proportion to plasma levels, appearing to be locally synthesized in response to brain involvement.

The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin.

The influence of the hyaluronan-binding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The pro-cofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the N-terminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5H and D6H are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system.

Whale high-molecular-weight and low-molecular-weight kininogens.

The expression of high-molecular-weight and low-molecular-weight kininogen mRNAs in the whale liver was examined by reverse transcription-polymerase chain reaction. The nucleotide sequences of the high-molecular-weight and low-molecular-weight kininogen cDNAs were analyzed and deduced to the amino acid sequences. The high-molecular-weight kininogen composed of 609 amino acid residues with 18 signal peptides possessed the consensus sequences of the cysteine protease inhibitor domains I and II, the bradykinin domain, the histidine-rich region, and the prekallikrein-binding region. Except for the histidine-rich region, the overall homologies with bovine, human, and rat high-molecular-weight kininogens were 81%, 76%, and 62%, respectively. The low-molecular-weight kininogen is composed of 408 amino acid residues. The nucleotide sequence down to C(1200) as well as the amino acid sequence till Ile(382) is identical to that of the high-molecular-weight kininogen. The remaining low-molecular-weight kininogen-specific carboxy-terminal portion possessed an amino acid sequence similar to that of the land mammals. The overall homologies with bovine, human, and rat low-molecular-weight kininogens were 82%, 79%, and 64%, respectively. The amino acid sequences of both whale high-molecular-weight and low-molecular-weight kininogens are most similar to those of the bovine among the land mammals analyzed so far. An incubation of dolphin/whale plasma with human plasma kallikrein, or with bovine trypsin, in the presence of carboxypeptidase inhibitors generated bradykinin antigen as well as the spasmogenic activity to the estrous rat uterus. The amount of bradykinin released by the latter enzyme was almost double of the former, indicating that the dolphin/whale plasma contained similar concentrations of low-molecular-weight and high-molecular-weight kininogens.

High-molecular-weight kininogen preadsorbed to glass surface markedly reduces neutrophil adhesion.

Adsorbed proteins on biomaterial surfaces determine whether cells adhere, but rheological variables are also critical. Neutrophil adhesion under well-defined radial flow conditions was studied on glass preadsorbed with plasma proteins or plasma protein domain fragments. Fibrinogen, low-molecular-weight kininogen (LK), high-molecular-weight kininogen (HK), cleaved HK (HKa), and recombinant HK domains 3 and 5 (D3 and D5H) were used. The number of adherent cells on the HK and HKa surfaces was less than 10% that found on the fibrinogen absorbed surface. The degree of spreading was minimal and detachment of adherent neutrophils was observed. HK and HKa contain binding sites for both anionic surfaces and neutrophils in the same domain (D5H). When adsorbed to surfaces, HK and HKa did not have the neutrophil binding sites available and therefore exhibited an anti-adhesive effect. Although D5H contains anionic surface binding sites, its small molecular size required a higher number of adsorbed molecules to cover the surface before a significant decrease in cell adhesion was observed. Since LK and D3 do not possess specific anionic surface binding sites, the adsorption of these proteins on glass was very low compared to HK and HKa. Thus, extensive cell adhesion and spreading were observed on the surfaces partially covered with preadsorbed LK and D3.

Parallel reduction of plasma levels of high and low molecular weight kininogen in patients with cirrhosis.

Little is known about the regulation of high-molecular-weight-kininogen (HK) and low-molecular-weight-kininogen (LK) or the relationship of each to the degree of liver function impairment in patients with cirrhosis. In this study, we evaluated HK and LK quantitatively by a recently described particle concentration fluorescence immunoassay (PCFIA) and qualitatively by SDS PAGE and immunoblotting analyses in plasma from 33 patients with cirrhosis presenting various degrees of impairment of liver function. Thirty-three healthy subjects served as normal controls. Patients with cirrhosis had significantly lower plasma levels of HK (median 49 microg/ml [range 22-99 microg/ml]) and LK (58 microg/ml [15-100 microg/ml]) than normal subjects (HK 83 microg/ml [65-115 microg/ml]; LK 80 microg/ml [45-120 microg/ml]) (p<0.0001). The plasma concentrations of HK and LK were directly related to plasma levels of cholinesterase (P<0.0001) and albumin (P<0.0001 and P<0.001) and inversely to the Child-Pugh score (P<0.0001) and to prothrombin time ratio (P<0.0001) (reflecting the clinical and laboratory abnormalities in liver disease). Similar to normal individuals, in patients with cirrhosis, plasma HK and LK levels paralleled one another, suggesting that a coordinate regulation of those proteins persists in liver disease. SDS PAGE and immunoblotting analyses of kininogens in cirrhotic plasma showed a pattern similar to that observed in normal controls for LK (a single band at 66 kDa) with some lower molecular weight forms noted in cirrhotic plasma. A slight increase of cleavage of HK (a major band at 130 kDa and a faint but increased band at 107 kDa) was evident. The increased cleavage of HK was confirmed by the lower cleaved kininogen index (CKI), as compared to normal controls. These data suggest a defect in hepatic synthesis as well as increased destructive cleavage of both kininogens in plasma from patients with cirrhosis. The decrease of important regulatory proteins like kininogens may contribute to the imbalance in coagulation and fibrinolytic systems, which frequently occurs in cirrhotic patients.

Bradykinin release from high molecular weight kininogen and increase in plasma kallikrein-like activity following sensory stimulation by food in the rat.

Cephalic stimulation by food elicits, among other responses, dilatation of mesenteric blood vessels preparatory for digestion. The possible participation of bradykinin (BK), a powerful endogenous vasodilator, in this response was studied in fasted rats prior and following stimulation by sight and scent of food (sensory stimulation, SS), actual ingestion being denied to the animals. BK content of plasma high (HK) and low molecular weight kininogen (LK) was determined by bioassay on the atropinized, antihistamine-treated isolated guinea-pig ileum following release by trypsin from heat/acid denatured plasma. BK corresponding to LK was estimated in plasma which prior to denaturation had been incubated with kaolin, a process which leads to quantitative release and inactivation of BK from HK, but does not affect LK. BK corresponding to (HK + LK) was determined in plasma not exposed to kaolin. BK contained in HK was the difference between BK of (HK + LK) and of BK of LK. Plasma and glandular kallikreins were estimated by fluorimetry, using specific synthetic substrates. A 40.6+/-4.0% decrease (P<0.001) of BK in HK occurred in rats after 90 s of SS; LK remained unaffected. Ten minutes of SS did not result in further change. Atropine inhibited the effect of SS. Return of HK to pretreatment levels occurred when, following 90s of SS, rats were allowed to rest for 60 min in the absence of food. Renewed capacity to respond to SS was then observed. Plasma kallikrein, but not glandular kallikrein, increased in plasma of rats after SS. Increased free BK was detected in the circulation of Enalapril-protected rats after SS. Electrical stimulation of the distal sector of the sectioned left abdominal vagus nerve of Nembutal-anesthetized fasted rats reproduced the effect of SS on HK. It is concluded that visuo-olfactory stimulation by food generates nerve impulses, possibly carried by the vagus nerve, which by activating plasma kallikrein lead to cleavage of circulatory HK and release of BK in the rat.

Low-salt diet downregulates plasma but not tissue kallikrein-kinin system.

The kallikrein-kinin system (KKS) is involved in the regulation of blood pressure and in the sodium and water excretion. In humans, the KKS is divided functionally into a plasma KKS (pKKS) generating the biologically active peptide bradykinin and into the tissue (glandular) KKS (tKKS) generating the active peptide kallidin. The objective of this study was to examine the effect of a low-NaCl diet on the concentration of both pKKS and tKKS in plasma and urine in 10 healthy volunteers. After a 4-day low-NaCl diet, the urinary sodium and chloride excretions had decreased from 234 to 21.2 mmol/24 h and from 198 to 14.6 mmol/24 h, respectively. The plasma levels of ANG I, aldosterone, and angiotensin converting enzyme (ACE) significantly increased from 50.4 to 82.8 pg/ml, from 129 to 315 pg/ml, and from 46.4 to 59.8 U/ml, respectively, demonstrating the physiological adjustment to the low-salt diet. In plasma, the levels of bradykinin and plasma kallikrein had significantly decreased from 13.7 to 7.57 pg/ml and 14.4 to 7.13 U/ml, respectively. However, the levels of high-molecular-weight kininogen (HMW kininogen) remain unchanged (101 vs. 112 microg/ml, not significant). Contrary to plasma kallikrein, the plasma levels of tissue kallikrein increased (0.345 vs. 0.500 U/ml; P < 0.01). The plasma kallidin levels, however, did not change (64.7 vs. 68.6 pg/ml, not significant). This can be explained by a simultaneous decrease in the plasma low-molecular-weight kininogen (LMW kininogen) levels (89.9 vs. 44.4 microg/ml; P < 0.05). As in plasma, we find increased urinary concentrations of renal (tissue) kallikrein (23.3 to 42.8 U/24 h; P < 0.05) that contrast with, and are presumably counterbalanced by, urinary LMW kininogen levels (77.0 vs. 51.8 microg/24 h; P < 0.05). Consequently, in urine low-NaCl diet caused no significant change in either bradykinin or kallidin (9.2 vs. 10.8 microg/24 h, and 10.9 vs. 10.3 microg/24 h). It is concluded that the stimulation of the renin-angiotensin system on a low-NaCl diet is associated with a decrease in pKKS (bradykinin and plasma kallikrein) but not in tissue and renal KKS. Although tissue kallikrein is increased, there is no change in kallidin, as LMW kininogen in plasma and urine is decreased. These data suggest a difference in the regulation of pKKS and tKKS by low-salt diet.

Evidence for a role of kallikrein-P6nin system in patients with shock after blunt trauma.

Bradykinin (BK) is activated via plasma and/or tissue kallikrein-kinin (K-K) system pathways during hypotension after blunt trauma. The precise role of the K-K system in human subjects has not been defined. We developed a new method for measuring levels of BK in the blood and examined the role of the K-K system in patients with shock after trauma. Eight patients were entered into this study. We measured the levels of a high-molecular-weight kininogen (HMWK), a low-molecular-weight kininogen (LMWK), BK, and (1-5)-BK in the blood of patients in an unstable state (Pre) and a stable state (Post). At Pre, the blood BK level was significantly elevated, the HMWK and LMWK levels were significantly lower, and the (1-5)-BK level was significantly higher than the respective levels at Post. Our data suggest a significant role for the K-K system in the pathogenesis of shock after blunt trauma. This newly developed method for determination of the activation of the plasma K-K system appears to be useful for determining the severity of a trauma.