Tissue plasminogen activator activity in human aqueous humor.

PURPOSE: To determine the levels of free plasminogen activator activity in human aqueous humor and to identify the type of activity (i.e., tissue-type t-PA or urokinase-type u-PA) that is responsible. METHODS: Aqueous humor was obtained by a simplified pipette paracentesis before cataract surgery in 31 subjects, ages 57 to 93 years. Levels of plasminogen activator activity were determined using a modified 17-hour specific amidolytic assay. The type of plasminogen activator was investigated in selected samples based on its dependence on soluble fibrin, inhibition by amiloride, and specific antibody blocking. Activity-antigen ratios were compared in seven samples. RESULTS: Plasminogen activator (PA) activity was present in all samples tested. PA activity ranged widely between 0.54 and 26.7 mIU/ml, with a mean value of 10.8 +/- 8.1 mIU/ml. Soluble fibrin, a known stimulator of tissue-type plasminogen activator (t-PA), was required in the assay system. Its absence decreased the measured activity by more than 90%. Amiloride, a known inhibitor of urokinase-type PA, had little or no effect in selected samples tested. The activity was blocked by anti-human t-PA antibodies but not by antibodies against human u-PA, further defining the type of PA responsible for the detected activity. t-PA antigen levels showed less variation among individuals than did activity levels. Antigen-activity ratios ranged between 89 and 552. CONCLUSION: Plasminogen activator activity is present in the human aqueous humor in measurable quantities. The type of PA activity present is almost exclusively t-PA. t-PA activity varies more widely than antigen, as is the case in plasma.

Increased basal levels of free plasminogen activator activity found in human aqueous humor.

PURPOSE: To quantify the basal free t-PA activity in human aqueous humor. METHODS: Human aqueous humor obtained by simplified pipette paracentesis at cataract surgery was tested for free t-PA activity in a revised 15-hour amidolytic assay using a t-PA standard curve (n = 15). Total antigenic levels of PAI-1, the principal PA inhibitor, were determined using an ELISA kit. The available PAI-1 activity was tested indirectly using anti-human PAI-1 antibody blocking before t-PA activity assay (n = 11). Plasminogen activator type was determined by anti-human t-PA and urokinase (u-PA) antibody blocking before activity assay (n = 7). RESULTS: Free PA activity ranged widely (0.072 to 0.47 IU/ml; mean, 0.20 +/- 0.10 IU/ml) and was almost completed inactivated (> or = 89%) by antibody against human t-PA but not by the u-PA antibody. PAI-1 total antigen also ranged widely between (0.25 to 8.0 ng/ml; mean, 2.25 +/- 2.54 ng/ml). However, pretreatment of samples with PAI-1 antibody or by acidification (pH 3.2) to inactivate inhibitors did not increase t-PA activity levels. CONCLUSIONS: A basal-free t-PA activity, which is predominantly t-PA, is present in human aqueous humor at approximately 20 times higher levels than previously found with an earlier assay. This activity is estimated to represent roughly 10% of total released t-PA antigen. PAI-1 is present in aqueous humor at levels considerably lower than reported values for plasma in a predominantly PA-complexed or inactive form.

Adenosine diphosphate stimulates the endothelial release of tissue-type plasminogen activator but not von Willebrand factor from isolated-perfused rat hind limbs.

The acute release of tissue-type plasminogen activator (t-PA) activity and of von Willebrand Factor (vWF) antigen from vascular endothelium was studied ex vivo using the rat hindquarter isolated perfusion model. The release of these proteins has been reported to occur simultaneously in response to a variety of endothelial cell agonists including bradykinin, thrombin and platelet activating factor. It has therefore been suggested that similar endothelial cell pathways and mechanisms are involved in the release of t-PA and vWF in vivo. This paper shows that the releases of t-PA and of vWF are not always closely linked and may depend on the agonist utilized to effect release. In our hands, the bradykinin-induced release of these proteins appears to be essentially identical, but this is not true for adenosine diphosphate (ADP). Rather, ADP is capable of causing the acute release of t-PA without the simultaneous release of vWF in the ex vivo rat hindquarter model. This indicates that the bradykinin- and ADP-induced pathways for t-PA release are probably distinct and that the releases of t-PA and vWF are not as closely linked as previously believed.

Leukocyte adherence and sequestration following hemorrhagic shock and total ischemia in rats.

The pathogenesis of generalized microvascular injury following hemorrhagic shock and total ischemia appears to be dependent on leukocytes interacting with the venular endothelium. The purpose of this study was to compare leukocyte adherence and sequestration following hemorrhagic shock with that of total ischemia in the small bowel mesentery of rats. Leukocyte adherence and sequestration was measured by direct visualization in vivo using intravital microscopy. In addition, sequestration was also quantitated by measuring tissue levels of myeloperoxidase, a marker of leukocyte infiltration. Mean arterial blood pressure was decreased to 40 mm Hg for 30 min (hemorrhagic shock group). In the total ischemia group, both the superior and inferior mesenteric arteries were clamped for 30 min followed by reperfusion. Hemorrhagic shock (9.4+/-1.5 cell/100 microm) and total ischemia (8.3+/-3 cell/100 microm) caused a statistically significant increases in leukocyte adherence 60 min postinsult as compared with controls (.9+/-1.5 cell/100 microm). However, the increase in leukocyte adherence appeared earlier and to a greater degree initially following total ischemia. Leukocyte sequestration as measured by intravital microscopy was significant only after total ischemia [(24.6+/-1.7 cell/(100 microm)2; p<.01] and not hemorrhagic shock [3.4+/-.6 cell/(100 microm)2] versus controls [2.2+/-.2 cell/(100 microm)2]. This difference in sequestration was also confirmed by tissue levels of myeloperoxidase. The results of this study suggest that the microvascular response following hemorrhagic shock is different than that of total ischemia, and caution is warranted when extrapolating the experimental results of one to the other.

Effect of LFA-1beta antibody on leukocyte adherence in response to hemorrhagic shock in rats.

The activation and adherence of leukocytes to the venular endothelium are critical steps in the pathogenesis of generalized microvascular injury following hemorrhagic shock. Previous studies have shown that the integrins CD11/CD18 play a significant role in this interaction. The purpose of this study is to examine the efficacy of anti-LFA-1beta, an antibody to CD11a/CD18, in attenuating leukocyte adherence before, during, and after hemorrhagic shock. Following a control period, blood was withdrawn to reduce the mean arterial pressure to 40 mm Hg for 30 min in urethane-anesthetized rats. Mesenteric venules in a transilluminated segment of the small intestines were examined to quantitate leukocyte adherence using intravital microscopy. In sham-operated rats (control), there was minimal to no leukocyte adherence throughout the experiment. Hemorrhagic shock resulted in significant leukocyte adherence during resuscitation (10.8 +/- 1.7 cells/100 microm, P < 0.01) when compared to control. Anti-LFA-1beta, when given before hemorrhagic shock, significantly attenuated leukocyte adherence during resuscitation (1.1 +/- 0.8, P < 0.01) when compared with hemorrhagic shock alone. This protective effect of anti-LFA-1beta on leukocyte adherence was even demonstrated when it was given during (1.6 +/- 0.3, P < 0.01) and 10 min after hemorrhagic shock (5.8 +/- 0.4, P < 0.05). These results suggest that anti-LFA-1beta may be of potential therapeutic benefit against microvascular injury caused by hemorrhagic shock.

The local effect of PAF on leukocyte adherence to small bowel mesenteric venules following intra-abdominal contamination.

We have previously demonstrated that intra-abdominal contamination increases neutrophil infiltration into the gastrointestinal tract. The purpose of our current study was twofold: 1) to determine if leukocyte adherence to the mesenteric microvasculature occurred by local peritoneal contamination or by systemic mechanisms; and 2) to assess the role of platelet activation factor (PAF) in this process. Rats underwent cecal ligation and puncture (CLP), and 4 h after the procedure we used intravital microscopy to visualize the mesenteric microcirculation. Cecal ligation and puncture increased leukocyte adherence (22.3+/-5.5 leukocytes/100 microm) vs. sham (2.3+/-0.9, P < 0.05). WEB-2086, a PAF receptor antagonist, prevented this increase (6.47+/-4.8, P < 0.05). To assess if leukocyte adherence was due to topical effects, we performed similar experiments with the small bowel exteriorized. In such cases, CLP did not increase leukocyte adherence (1.2+/-0.8 vs. 1.4+/-0.9). In addition, topical application of highly diluted fecal matter (1:1000) increased leukocyte adherence (4.8+/-1.2) vs. control (0.6+/-0.3, P < 0.05). Our study demonstrates that leukocyte adherence in the mesenteric microcirculation following intra-abdominal contamination is due to direct topical exposure to fecal matter, and it is mediated by PAF.

L-selectin: mechanisms and physiological significance of ectodomain cleavage.

L-selectin is a cell adhesion molecule consisting of a large, highly glycosylated, extracellular domain, a single spanning transmembrane domain and a small cytoplasmic tail. It is expressed on most leukocytes and is involved in their rolling on inflamed vascular endothelium prior to firm adhesion and transmigration. It is also required for the constitutive trafficking of lymphocytes through secondary lymphoid organs. Like most adhesion molecules, L-selectin function is regulated by a variety of mechanisms including gene transcription, post-translational modifications, association with the actin cytoskeleton, and topographic distribution. In addition, it is rapidly downregulated by proteolytic cleavage near the cell surface by ADAM-17 (TACE) and at least one other "sheddase". This process of "ectodomain shedding" results in the release of most of the extracellular portion of L-selectin from the cell surface while retaining the cytoplasmic, transmembrane, and eleven amino acids of the extracellular domain on the cell. This review will examine the mechanism(s) of L-selectin ectodomain shedding and discuss the physiological implications.

Analysis of gamma-carboxyglutamic acid by reverse phase HPLC of its phenylthiocarbamyl derivative.

A method is presented for analysis of gamma-carboxyglutamic acid based on its derivatization with phenylisothiocyanate and reverse phase HPLC analysis of the resulting phenylthiocarbamyl derivative. Proteins were hydrolyzed with sodium hydroxide and the hydrolysates were desalted on Dowex 50 eluted with ammonium hydroxide. The resulting amino acid mixtures were derivatized with phenylisothiocyanate and the phenylthiocarbamyl derivatives were separated under isocratic conditions on either C18 or C8 reverse phase columns using 0.14 M Tris, 0.05% triethylamine, titrated to pH 7.5 with glacial acetic acid, plus 2% acetonitrile, and detected by absorbance at 254 nm. The method is linear over the range from 10 to 1000 pmol of gamma-carboxyglutamic acid and the limit of detection is near 2 pmol. The utility of the method was verified for analysis of purified prothrombin yielding a value of 10.3 mol of gamma-carboxyglutamic acid per mole in agreement with sequence data. No gamma-carboxyglutamate was detectable for acid-hydrolyzed samples of prothrombin, nor in acid- or base-hydrolyzed samples of bovine serum albumin. Application of this method failed to corroborate the reported presence of gamma-carboxyglutamate in a putative mitochondrial gamma-carboxyglutamate-containing calcium-binding protein. The method was also tested for determination of beta-carboxyaspartate, beta-hydroxyaspartate, phosphoserine, phosphothreonine, and phosphotyrosine in an attempt to identify an unknown material which appeared in preparations of the mitochondrial protein.

Vitamin K1 2,3-epoxide and quinone reduction: mechanism and inhibition.

The chemical and enzymatic pathways of vitamin K1 epoxide and quinone reduction have been investigated. The reduction of the epoxide by thiols is known to involve a thiol-adduct and a hydroxy vitamin K enolate intermediate which eliminates water to yield the quinone. Sodium borohydride treatment resulted in carbonyl reduction generating relatively stable compounds that did not proceed to quinone in the presence of base. NAD(P)H:quinone oxidoreductase (DT-diaphorase, E.C. 1.6.99.2) reduction of vitamin K to the hydroquinone was a significant process in intact microsomes, but 1/5th the rate of the dithiothreitol (DTT)-dependent reduction. No evidence was found for DT-diaphorase catalyzed reduction of vitamin K1 epoxide, nor was it capable of mediating transfer of electrons from NADH to the microsomal epoxide reducing enzyme. Purified diaphorase reduced detergent- solubilized vitamin K1 10(-5) as rapidly as it reduced dichlorophenylindophenol (DCPIP). Reduction of 10 microM vitamin K1 by 200 microM NADH was not inhibited by 10 microM dicoumarol, whereas DCPIP reduction was fully inhibited. In contrast to vitamin K3 (menadione), vitamin K1 (phylloquinone) did not stimulate microsomal NADPH consumption in the presence or absence of dicoumarol. DTT-dependent vitamin K epoxide reduction and vitamin K reduction were shown to be mutually inhibitory reactions, suggesting that both occur at the same enzymatic site. On this basis, a mechanism for reduction of the quinone by thiols is proposed. Both the DTT-dependent reduction of vitamin K1 epoxide and quinone, and the reduction of DCPIP by purified DT-diaphorase were inhibited by dicoumarol, warfarin, lapachol, and sulphaquinoxaline.

Effect of WEB 2086 on leukocyte adherence in response to hemorrhagic shock in rats.

BACKGROUND: The pathogenesis of generalized microvascular injury after hemorrhagic shock is known to involve the generation of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine [PAF]). The release of PAF is manifested in several ways, including by increased vascular permeability, altered vascular reactivity, and increased leukocyte adherence to the endothelium. WEB 2086 is a PAF antagonist that has been shown experimentally to improve survival after hemorrhagic shock. The purpose of this study was to examine the efficacy of WEB 2086 in attenuating leukocyte adherence before, during, and after hemorrhagic shock. METHODS: After a control period, blood was withdrawn to reduce the mean arterial pressure to 40 mm Hg for 30 minutes in urethane-anesthetized rats. Mesenteric venules in a transilluminated segment of the small bowel were examined to quantitate leukocyte adherence using intravital microscopy. RESULTS: In sham-operated rats (control), there was minimal to no leukocyte adherence throughout the experiment. Hemorrhagic shock resulted in a significant increase in leukocyte adherence postshock during resuscitation (10.9 +/- 1.8 cells/100 microm, p < 0.01) when compared with controls. WEB 2086, when given before shock, significantly attenuated leukocyte adherence (0.1 +/- 0.08 cells/100 microm, p < 0.01) when compared with hemorrhagic shock alone. This effect of WEB 2086 on adherence could be demonstrated even when it was given during (3.5 +/- 0.9 cells/100 microm, p < 0.01) and 10 minutes into (5.8 +/- 1.1 cells/100 microm, p < 0.05) hemorrhagic shock. CONCLUSION: Our findings suggest that WEB 2086 may be of therapeutic benefit against the microvascular damage sustained after hemorrhagic shock.

Endothelial cells prevent accumulation of lipid hydroperoxides in low-density lipoprotein.

A variety of cell types, including endothelial cells, oxidize low-density lipoprotein (LDL). To investigate the mechanisms by which endothelial cells modulate LDL oxidation states, endothelial cell cultures were incubated with LDL (240 mg cholesterol/dL) for 24 hours in M199 supplemented with fetal bovine serum (FBS, 16.7%). These conditions were not toxic to endothelial cells over the time frame of the study. Changes in LDL oxidation were monitored by measuring thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxide (LOOH), and conjugated dienes (A234nm). LDL medium incubated in the absence of endothelial cells contained higher TBARS than did LDL medium incubated with endothelial cells (0.35 +/- 0.08 versus 0.23 +/- 0.08 nmol MDA/mg, respectively). LOOHs were higher in LDL medium incubated without endothelial cells than in LDL medium incubated with endothelial cells (6.8 +/- 4.4 versus 0.49 +/- 0.89 nmol/mg, respectively). Conjugated diene formation, based on changes in absorbance at 234 nm, increased to a greater extent in LDL medium incubated in the absence of endothelial cells than when endothelial cells were present. To increase oxidative stress on the endothelial cell cultures, increasing concentrations of Cu2+ (0 to 4 mumol/L) were added to LDL medium. Endothelial cells prevented LOOH accumulation until the concentration of Cu2+ exceeded 0.75 mumol/L. At 1.5 and 4 mumol/L Cu2+, endothelial cells enhanced LOOH formation nearly 3 and 2.5 times the LOOH values in the corresponding medium incubated in the absence of endothelial cells. This loss of protective function however, was not permanent. Endothelial cells, preincubated for 24 hours with Cu(2+)-containing LDL medium, were still able to prevent LOOH accumulation in fresh LDL medium. Endothelial cells prevented LOOH accumulation even when exposed to LDL medium that contained low concentrations of LOOHs (< 22 nmol/mg). However, endothelial cells accelerated the accumulation of LOOHs in LDL when exposed to LDL medium that contained slightly higher concentrations of preexisting LOOHs (approximately equal to 33 nmol/mg). These data indicate that endothelial cells have a limited capacity for preventing LOOH formation and that small increases in LOOHs may play a critical role in enhancing the potential of endothelial cells for oxidative modification of LDL.

Native LDL increases endothelial cell adhesiveness by inducing intercellular adhesion molecule-1.

Native LDL (n-LDL) increased human umbilical vein endothelial cell (EC) adherence of mononuclear cells. Such phenotypic changes suggest that n-LDL alters the usual expression of cell adhesion molecules to enhance the adhesive properties of the endothelium. To investigate n-LDL mechanisms governing adherence, ECs were exposed to n-LDL in concentrations up to 240 mg/dL for 2 and 4 days. n-LDL-treated ECs bound nearly threefold more phorbol myristate acetate (PMA)-stimulated U937 cells than control ECs but did not bind unstimulated U937 cells. Anti-cellular adhesion molecule-1 (ICAM-1) antibodies blocked PMA-stimulated U937 cell binding to control and n-LDL-treated ECs by more than 80%, suggesting that increases in ICAM-1 may be involved in this increased adherence. Although increases in PMA-stimulated U937 cell binding developed with respect to time and concentration, statistically significant increases were achieved only when n-LDL concentrations exceeded 180 mg cholesterol/dL at day 4. n-LDL increased endothelial adherence of freshly isolated human monocytes more than twofold and neutrophils by almost twofold. Fluorescent-linked immunoassays revealed that n-LDL increased ICAM-1 protein expression by twofold, which corresponded with increased ICAM-1 message levels. n-LDL also appeared to increase E-selectin and vascular cell adhesion molecule-1 message levels, but these changes did not translate into statistically significant differences in protein levels. Taken together, these data indicate that n-LDL increases ICAM-1 expression to enhance the adhesive properties of the endothelium. Such perturbations in EC function likely represent a proinflammatory response to protracted n-LDL exposure and one of the early steps in atherogenesis.

PAF and CD18 mediate neutrophil infiltration in upper gastrointestinal tract during intra-abdominal sepsis.

Neutrophil infiltration is a critical event in the development of multiple organ failure during sepsis. We hypothesized that platelet-activating factor (PAF) release contributes to neutrophil infiltration in the gastrointestinal tract during sepsis. In the first experiments we administered exogenous PAF (1.56, 6.25, 25, and 100 ng . kg-1 . min-1 for 30 min) to urethan-anesthetized Sprague-Dawley rats. PAF was administered alone or in combination with either the PAF antagonist WEB-2086 (250 microg . kg-1 . min-1), a monoclonal antibody (MAb) to CD18, or a MAb to intercellular adhesion molecule 1 (ICAM-1). In separate groups of rats, cecal ligation and incision (CLI) was performed to create intra-abdominal sepsis, which we hypothesized would stimulate the release of endogenous PAF. CLI was performed in rats given either saline, WEB-2086, anti-CD18, or anti-ICAM-1 MAb. After these experiments, tissue myeloperoxidase (MPO) levels were determined as a marker of neutrophil infiltration. Both exogenous PAF and CLI induced significant increases in MPO activity in the stomach and duodenum. These increases were significantly attenuated by WEB-2086, anti-CD18 MAb, and anti-ICAM-1 MAb in both PAF- and CLI-treated rats. These results suggest that both the inflammatory mediator PAF and the CD18 integrins play a major role in neutrophil infiltration in the upper gastrointestinal tract during sepsis.

Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion.

To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O2-) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O2- by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [3H]citrulline assay. N omega-Nitro-L-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O2- by > 300% but decreases LDL-treated EC (LDL-EC) release by > 95%. L-Arginine inhibits the release of O2- from LDL-ECs by > 95% but did not effect C-EC release of O2-. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O2- production by approximately 50% and 30%, respectively. Thus, n-LDL increases O2- and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO-), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO-, and tyrosine by approximately 50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs.(ABSTRACT TRUNCATED AT 250 WORDS)