Activation of the plasma contact system and hemodynamic changes after graft revascularization in liver transplantation.

In this study, the relation between activation of the plasma contact system and hemodynamic changes during orthotopic liver transplantation was evaluated. Nineteen consecutive courses of OLT in 17 adult patients were investigated. Veno-venous bypass was used in all patients. Blood samples were drawn through all phases of the procedure, and analyzed for the following parameters using functional techniques (chromogenic peptide substrate assays): plasma kallikrein (KK), prekallikrein, functional plasma kallikrein inhibition, C1 inhibitor, and alpha 2-macroglobulin. Plasma high molecular weight kininogen (HK) degradation was evaluated using the immunoblotting technique. An abrupt rise in KK activities occurred within 1 min after portal reperfusion of the liver graft (7-16 U/L, P < 0.05). Simultaneously, proteolytic breakdown of HK was seen. The elevated KK activities were maintained the next 1 1/2 hr. Ten min after graft reperfusion, a significant increase in cardiac output compared with the anhepatic phase (7.2-12.4 L/min, P < 0.05) was found. At the same time, systemic vascular resistance fell significantly (817-408 dynes x sec/cm-5, P < 0.05). The increase in plasma KK activities accompanied by simultaneous degradation of HK seen immediately after reperfusion of the liver graft may be due to contact activation as recipient blood contacts with the underlying basement membrane of injured sinusoidal endothelium in the transplanted liver. We suggest that hemodynamic changes associated with the postreperfusion syndrome seen after revascularization of the liver in OLT could at least be caused in part by bradykinin release due to contact activation.

Polymorphonuclear elastase in human colorectal carcinoma.

proteinases are required for invasion through the extracellular matrix (ECM) during tumor invasion and metastasis. Polymorphonuclear (PMN) elastase can degrade ECM components and modulate other proteinases. In the present study PMN elastase activity was found in tissue extracts from 8 of 15 human colorectal carcinomas. Immunoreactive PMN elastase was demonstrated in all carcinoma biopsies with particular enrichment at the tumor-host interface. Immunofluorescence staining localized immunoreactive PMN elastase mainly to neutrophile granulocytes. The human colon carcinoma cell lines Caco-2 and HT-29 did not express PMN elastase. An interaction between tumor cells and elastase producing leukocytes is suggested.

Antigen levels of urokinase plasminogen activator and its receptor at the tumor-host interface of colorectal adenocarcinomas are related to tumor aggressiveness.

The distributions of urokinase and tissue plasminogen activators (uPA, tPA), uPA receptor (uPAR), and plasminogen activator inhibitors (PAI-1, PAI-2) were studied immunohistochemically in two subsets of colorectal adenocarcinomas with low and high aggressiveness, respectively: nine Dukes' stage A tumors with additional other good prognostic markers and 13 Duke's stage C tumors with also other poor prognostic markers (referred to as Dukes' stage A and Dukes' stage C tumors). The results showed that these components of the tissue destructive plasminogen activation system were accumulated at the invading front of the tumors. Both tumor groups showed accumulations of uPA, uPAR, and PAI-1 at the tumor-host interface compared with the location within the tumor epithelium and the adjacent normal mucosa and muscularis propria (all P < .05). However, the uPA level at the tumor-host interface in the Dukes' stage C tumors was twice the level in the Dukes' stage A tumors (P < .05). The uPAR level was also significantly higher in the Dukes' stage C tumors (P < .05), whereas the PAI-1 level was not significantly higher. This may indicate that uPA in more aggressive tumors exceeds the inhibitory capacity represented by PAIs, resulting in enhanced tissue destructive potential that promotes tumor invasion. uPA and uPAR antigen levels and the uPA/PAI-1 ratio at the tumor-host interface appeared to be related to tumor aggressiveness in colorectal cancer.

Location of plasminogen activator (PA) and PA inhibitor in human colorectal adenocarcinomas.

Both the coagulation and fibrinolytic cascades generate proteolytic enzymes which appear to be essential for tumor invasion. In the present investigation adenocarcinomas and normal colon from 14 patients with colorectal cancer were studied by immunohistochemistry. The most striking observation was an enrichment of plasminogen activator inhibitor in the tumor tissue, whereas no such immunoreactivity was detected in the biopsies of normal colon. The tumor-host interface was characterized by a massive accumulation of inflammatory cells, macrophages and T lymphocytes. In this area fibrin(ogen) immunoreactivity as a sign of local activation of the coagulation cascade was also seen. The transition zone between the tumor and normal tissue was furthermore characterized by a marked enrichment of urokinase plasminogen activator immunoreactivity. The study strongly indicates that proteases and inhibitors of the fibrinolytic system may be of great importance in tumor invasion.

Cleavage of plasma high molecular weight kininogen in surgical ICU patients.

OBJECTIVE: To characterize kininogens in plasma from surgical patients in the intensive care unit (ICU). DESIGN: Prospective study. SETTING: Surgical ICU. PATIENTS: 35 patients aged 19-79 years, divided into two groups: sepsis (defined by standard criteria) and nonsepsis. MEASUREMENTS AND RESULTS: Studies of proteolytic degradation of H-kininogen showed degradation in both patient groups compared to healthy controls. Functional quantification of prekallikrein showed a reduction of prekallikrein in plasma from both patients groups. Functional quantification of kininogens by a cysteine proteinase inhibitor assay showed no significant differences between the patients and the controls. Immunological levels of H-kininogen and total kininogen were not significantly different from normal plasma. No differences could be detected between the two patient groups in any of the parameters studied. CONCLUSIONS: This study showed that contact activation took place in surgical ICU patients: partial kinin release and consumption of prekallikrein took place in vivo.

Characterization of kininogens in human malignant ascites.

Ascites from seven patients with advanced cancer were studied to characterize the kininogens. Immunological quantification of low molecular weight kininogen (L-kininogen) and high molecular weight kininogen (H-kininogen) by rocket immunoelectrophoresis showed values of 42% and 39%, respectively, compared to control plasma. Release of kinin from the ascites samples was assayed on an isolated rat uterus. The total kinin released from the kininogens was 39% of the value in control plasma, while release selectively from H-kininogen amounted to 25% of plasma. This indicates about 30% of the bradykinin in H-kininogen to be released in vivo in ascites, and points to kinins as possible mediators of the increased vascular permeability causing accumulation of ascites. The function of kininogens as cysteine protease inhibitors (CPIs) was assayed as well, indicating that both L- and H-kininogen function as cysteine protease inhibitors in human ascitic fluid. The proteolytic cleavage of H-kininogen in ascites was studied by polyacrylamide gel electrophoresis and subsequent immunoblotting. H-kininogen was extensively cleaved in ascites compared to control plasma, with large amounts present of a degraded form with Mr of 99 kDa. The bands observed compared well with those described in plasma, and are consistent with contact activation taking place in ascites.

Quantification of kininogens in plasma. A functional method based on the cysteine proteinase inhibitor activity.

We have previously reported on a microassay based on human kininogens as cysteine proteinase inhibitors (CPIs), which could quantify partially purified kininogens from different biological fluids (J Pharmacol Meth 26, 113-124, 1991). In the present study we describe a functional method that, when assuming a 1:1 stoichiometry between papain and kininogen, allows a direct measurement of the molar concentration of kininogens in plasma. The principle of the method is that the target enzyme papain is inhibited by kininogens present in added diluted plasma. The residual activity of papain, not inhibited in this reaction, subsequently hydrolyzes the added peptide substrate (S-2302), generating a yellow colour which is read in a microplate reader at 405 nm. Relating the test samples to a standard curve established from known concentrations of E-64 (a selective low molecular weight inhibitor of cysteine proteinases), we could quantify kininogens on a molar basis. A major problem when first applying this method to plasma, was the interference of alpha 2-macroglobulin, which inhibited papain and generated a complex able to split the chromogenic substrate. The interference of alpha 2-macroglobulin was eliminated by an initial acid treatment of plasma followed by dilution with a buffer containing methylamine. The specificity for kininogens in this assay is demonstrated by the following observations: Commercial pooled normal plasma contained 3.2 microM CPI activity, in good agreement with the expected molar concentration of kininogens. After gel filtration of a plasma sample with a CPI activity of 3.4 microM, two peaks with CPI activity were identified as H-kininogen (0.9 microM) and L-kininogen (2.5 microM), both in good accordance with expected concentrations of the two kininogens. Plasma deficient of kininogens had a minimal inhibitory capacity towards papain.

Interleukin-1 and collagenases around loosening total hip prostheses.

OBJECTIVES: The present study was carried out to assess the expression of IL-1 in periprosthetic tissue and to relate it to local collagenase levels. METHODS: Interface tissue between the implant and bone was obtained from hip revision operations and was compared to healthy joint capsular tissue. Membrane bound IL-1 alpha was stained using the avidin-biotin-peroxidase complex method and was quantified using morphometry (VIDAS image analysis system). Soluble IL-1 beta was measured by ELISA. IL-1 levels were compared to collagenase activities in the same tissue as assessed by the degradation of triple helical type I collagen monomers analyzed by SDS-PAGE/laser densitometry. RESULTS: The IL-1 alpha staining index was high in interface tissue (7.25 +/- 1.28, p < 0.01) compared to normal joint capsular tissue (0.50 +/- 0.04). Similarly, IL-1 beta levels (ng/ml; ELISA) were high in such tissue (139.0 +/- 71.9 vs 0 +/- 0, p < 0.02). Total collagenase was higher in the interface (26.3 x 10(-6) +/- 7.57 x 10(-6) IU/l, p < 0.01) than in control tissue (0.55 x 10(-6) +/- 0.44 x 10(-6) IU/l). IL-1 alpha (r = -0.04, p > 0.05) and IL-1 beta (r = 0.25, p > 0.05) did not correlate with collagenase activity in interface tissue. CONCLUSION: Both IL-1 (alpha and beta) and collagenase are up-regulated around loose THR, suggesting that they may play a role in loosening. However, the correlations between IL-1 and collagenase were not significant, indicating, that IL-1 is probably not the only cytokine involved in the regulation of collagenase activity in vivo. IL-1 may also contribute to loosening by its osteoclast activating properties.

Cysteine proteinase inhibitors reduce malignant melanoma cell invasion in vitro.

Proteolytic enzymes are believed to be necessary for tumor cell invasion. We have studied the effects of the cysteine proteinase inhibitor E-64 and the serine and cysteine proteinase inhibitor leupeptin, on the ability of human malignant melanoma cells (LOX) to pass through an artificial basement membrane. Transwell chambers containing filters coated with the reconstituted basement membrane, Matrigel, were used. Nontoxic concentrations of the proteinase inhibitors reduced the invasion of LOX cells through Matrigel. E-64 (250 mumol/l) by 27% and leupepetin (250 micrograms/ml) by 46%. The proteinase inhibitors did not alter the growth rate of the tumor cells, their motility through uncoated filters, or their attachment to the Matrigel coated wells. Our results indicate that cysteine proteinases are involved in the degradation of basement membranes and thus contribute to the invasion of malignant melanoma cells.

Expression and release of plasminogen activators, their inhibitors and receptor by human tumor cell lines.

Plasminogen activators (PAs) and their inhibitors (PAIs) can be produced by tumor cells and surrounding inflammatory cells and fibroblasts. The present study evaluate both the expression and release of PAs (uPA and tPA) and PAIs (PAI-1 and PAI-2) from cultured cells, and also the expression of uPA receptor (uPAR). Immunocytochemistry showed that PAs, PAIs and uPAR were present to different extents on the surface of colon carcinoma cells (Caco-2, HT-29), malignant melanoma cells (LOX) and normal fibroblasts. uPA immunoreactivity was intermediate in Caco-2, HT-29 and LOX and weak in the fibroblasts. tPA immunoreactivity was intermediate in Caco-2 and LOX and weak in HT-29 and fibroblasts. PAI-1 and PAI-2 immunoreactivities were absent in HT-29, weak in Caco-2 and strong in fibroblasts. In LOX the immunoreactivity was intermediate for PAI-1 and strong for PAI-2. uPAR immunoreactivity was weak in Caco-2, HT-29 and LOX and negative in fibroblasts. ELISAs on conditioned medium detected that the colon carcinoma cells Caco-2 and HT-29 did not release any PAs or PAIs. LOX released tPA (median 9 ng/million cells at 72 hours), PAI-1 (1050 ng/million cells) and PAI-2 (245 ng/million cells), and fibroblasts released uPA (1 ng/million cells) and PAI-1 (910 ng/million cells). These results show that both tumor cells and fibroblasts express tissue destructive enzymes, PAs and PAIs, whereas only the tumor cells express the uPAR required for focalization and regulation of PA activity at the cell surface. The melanoma cells LOX and fibroblasts also released PAs and PAIs, in contrast to the colon carcinoma cells Caco-2 and HT-29.

Activation of the contact system in ascites from patients with gastrointestinal cancer.

Our observations indicates that the plasma contact system is activated in ascites from patients with gastrointestinal cancer: Factor XII is activated, plasma kallikrein is present in complex with the protease inhibitor alpha 2-macroglobulin, and the plasma kallikrein substrate high molecular weight kininogen, is highly degraded. Contact activation seems to take place in spite of a high level of inhibition. Activation of the contact system generates mediators, which may play a role in the accumulation of ascites.

Activation of the plasma kallikrein-kinin system in respiratory distress syndrome.

Components of the plasma kallikrein-kinin and fibrinolytic systems together with antithrombin III were measured the first days postpartum in 13 premature babies with severe respiratory distress syndrome (RDS). Seven of the patients received a single dose of porcine surfactant (Curosurf) as rescue treatment. Nine premature babies without lung disease or any other complicating disease served as controls. There were no differences in prekallikrein values between surfactant treated and non-treated RDS babies during the first 4 d postpartum. The controls had, however, significantly higher prekallikrein values than the RDS babies already at the first day of age (mean +/- SD 32 +/- 8% in controls versus 22 +/- 6 and 21.5 +/- 5% in the treated and nontreated RDS groups, respectively). Plasma kallikrein activities did not differ between RDS and control patients. Plasma kallikrein inhibition values, which increased steadily in all groups, were lower in the RDS babies treated with surfactant than in controls at d 2 and 4. The degree of degradation of plasma high molecular weight kininogen was measured in RDS patients treated with surfactant and was significantly higher when compared with controls at d 1, demonstrating an increased proteolysis of kininogen to kinin early in RDS. There were no differences in plasminogen and plasmin values between RDS and control babies. This study shows that the plasma kallikrein-kinin system is activated in RDS. This system as well as the fibrinolytic system does not seem to be influenced by rescue instillation of a single dose of porcine surfactant into the lungs of premature babies with RDS.

Bradykinin release from high molecular weight kininogen in surgical ICU patients.

The purpose of this prospective study was to characterize kininogens in plasma from surgical ICU patients. Thirty-five patients, ages 19-79 years, were divided into 2 groups: sepsis (defined by standard criteria) and nonsepsis. Studies of proteolytic degradation of H-kininogen showed degradation in both patient groups compared to healthy controls. Functional quantification of prekallikrein showed a reduction of prekallikrein in plasma from both patient groups. Functional quantification of kininogens by a CPI (cysteine proteinase inhibitor) assay showed no significant differences between the patients and the controls. Immunological levels of H-kininogen and total kininogen were not significantly different from normal plasma. No differences could be detected between the two patient groups in any of the parameters studied. In conclusion, this study supported contact activation taking place in surgical ICU patients, a partial kinin release and a consumption of prekallikrein has taken place in vivo.

[The role of proteases in the growth, invasion and spread of cancer cells]. / Proteasers betydning for cancercellers vekst, invasivitet og spredning.

Cancer cells show a greater capability than normal cells do to break down proteins in the surrounding tissue. This tissue destruction involving proteolytic enzymes is probably essential for the invasion and metastatic spread of malignant cells. The process takes place through an interplay of proteolytic enzyme systems where plasmin-mediated proteolysis plays an important role. Plasminogen activator activity and receptors for plasminogen activators have been discovered in tumors, mainly in areas with invasive growth and tissue degradation. Patients with malignant diseases often demonstrate abnormalities in their blood coagulation, including hyperaggregability of platelets. Experimental research has shown that therapy with antiplatelet drugs, and prophylaxis with protease inhibitors, can limit spread of tumors.

The contact system in human malignant and benign ascites.

The plasma contact system was studied in ascites and plasma from patients with gastrointestinal cancer and patients with liver failure. Our study demonstrates the presence of factor XII, factor XI, and prekallikrein and their main inhibitors in ascites and plasma from both patient groups. Both factor XII-like and plasma kallikrein-like activities were detected in the malignant ascites. The kallikrein-like activity in malignant ascites was found in complex with alpha 2-macroglobulin. In plasma samples from the patients functional values of factor XII and prekallikrein were decreased compared to controls. In benign ascites the proenzyme levels were significantly lower than in malignant ascites. Functional inhibition values in ascites and plasma from patients were unexpectedly high. Our findings indicate that the plasma contact system is activated in the ascites from cancer patients. Activation of the contact system generates vasoactive mediators, which may play a role in the accumulation of malignant ascites.

The fibrinolytic system in human ascites.

BACKGROUND: We recently reported that the contact and kallikrein-kinin systems are activated in malignancy-related ascites. We have now studied the fibrinolytic system in ascites and plasma from patients with gastrointestinal cancer(n = 14) and non-malignant liver disease (n = 18). METHODS AND RESULTS: Enzyme immunoassays (EIAs) showed that urokinase and tissue plasminogen activators (uPA, tPA) and PA inhibitors (PAI-1, PAI-2) were present in ascites from both patient groups and that tPA was the predominant PA. uPA, tPA, and PAI-1, were detected in plasma from patients and controls. These EIA findings were supported by zymography studies. Functional assays showed considerable generation of plasmin-like activity and low plasminogen and antiplasmin values in malignancy-related ascites. The plasmin/antiplasmin and tPA/PAI-1 ratios were particularly high in malignancy-related ascites as compared with non-malignant ascites. Plasma from the liver disease patients showed a higher tPA/PAI-1 ratio and, thus a higher potential for plasminogen activation than plasma from cancer patients and controls. Patient plasma showed low values of plasmin-like activity, antiplasmin, and plasminogen. CONCLUSION: Our findings show that the fibrinolytic system is activated in malignancy-related ascites.

The plasminogen activation system is upregulated in loosening of total hip prostheses.

Interface tissues and pseudocapsules from loose total hip replacements were removed during revision of 11 cases and were investigated for the plasminogen activation system and IL-1beta. Control samples of synovium were taken during knee arthroscopy (n 8), and from the hip joint during primary total hip replacement (n 5). The concentrations of urokinase plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and interleukin 1beta were all found to be significantly different in interfaces and in pseudocapsules, compared to controls. Immunohistochemistry disclosed localization in periprosthetic tissues of uPA, uPA-receptor and tPA in macrophages with phagocytosed metal, polyethylene, cement particles or accompanying pieces of necrotic bone. PAI-1 staining was present in the neighboring areas that stained for uPA or tPA, but PAI-1 staining was also found overlapping and outside these areas. These findings suggest a role for the uPA/uPA- receptor and PAI-1 in activation and focalization of extracellular matrix degradation in periprosthetic tissues. The expression of the plasminogen activation system by macrophages containing phagocytosed material suggests undegradable microdebris as a possible initiating and perpetuating stimulus for a proteolytic activation cascade, which may contribute to loosening of the prosthesis.

Differential diagnosis of human ascites: inhibitors of the contact system and total proteins.

To assess their accuracies as markers for malignancy, we assayed alpha 2-macroglobulin, C1-inhibitor, alpha 1-protease inhibitor, and total proteins in ascites and plasma from patients with gastrointestinal cancer (n = 15) and non-malignant liver disease (n = 13), using functional and immunologic assays. For all inhibitors and total proteins determined in ascites, the values in the cancer group were significantly higher than the corresponding values in the group with non-malignant liver disease. The diagnostic accuracy for differentiating malignancy-related from non-malignant ascites was 93% for a alpha 1-protease inhibitor value > or = 50% of the pool plasma value and 90% for alpha 2-macroglobulin > or = 16%, C1-inhibitor > or = 40% (all functional assays), and total proteins > or = 20 g/l (biuret). In conclusion, functional assays for alpha 2-macroglobulin, C1-inhibitor, and alpha 1-protease inhibitor and determination of total proteins in ascites appeared to be very informative tests for the differential diagnosis of ascites. The test for alpha 1-protease inhibitor gave higher specificity (92% versus 77%) and likelihood ratio for a positive test (12 versus 4) compared with the other tests.

Potentiative effects of neutral proteinases in an inflamed lung: relationship of neutrophil procollagenase (proMMP-8) to plasmin, cathepsin G and tryptase in bronchiectasis in vivo.

We attempted to study the possible relationships between neutrophil-type procollagenase/pro-matrix metalloproteinase (MMP-8) and the serine proteinases plasmin, cathepsin G and tryptase in bronchiectasis. The presence of the plasmin/plasminogen system and plasmin-, cathepsin G- and tryptase-like activities were compared to the activity of endogenously activated MMP-8 in bronchoalveolar lavage (BAL) fluid in 38 bronchiectasis patients and in 14 healthy controls by means of immunohistochemistry, Western-blot and substrate-based functional assays. In contrast to cathepsin G- and tryptase-like activities, the plasmin/plasminogen activator system in BAL fluid was observed to have a relatively weak activation stage and no correlation with disease severity. Neither plasmin-like activities nor concentrations of plasminogen activators from the bronchiectatic patients differed significantly from the values of healthy controls. Immunolocation of plasminogen activator inhibitor-1 showed a marked, but not significant, increase in bronchiectatic lung as compared to controls. In contrast to cathepsin G- and tryptase-like activities, with their strong and significant correlation with endogenously activated collagenase (r=0.9; p=0.0001; and r=0.6; p=0.03, respectively), no correlations were observed between plasmin-like and endogenously activated collagenase (r=0.3; p=0.2) in bronchiectasis. These findings suggest that cathepsin G- and tryptase-like activities may act as potent pro-matrix metalloproteinase-8 activators in patients with bronchiectasis, whereas the plasminogen activator/plasmin cascade was shown to be down-regulated.