Alpha 2-plasmin inhibitor inactivation by human granulocyte elastase

Plasminogen-binding human alpha 2-plasmin inhibitor is converted by human granulocyte elastase into its non-plasminogen-binding and finally into the inactive form of the inhibitor. This degradation of the plasmin inhibitor, described earlier as "spontaneously" occurring conversion, is shown in dodecyl sulfate polyacrylamide gel electrophoresis, in two-dimensional immunoelectrophoresis and by measuring the kinetics of plasmin inhibition. Experiments in the presence of normal human plasma required unphysiologically high concentrations of elastase to inactivate alpha 2-plasmin inhibitor, suggesting a role of elastase in this type of indirect fibrinolysis in a microenvironment only and not in systemic events.     

Endotoxin-mediated pulmonary endothelial cell injury

Infusion of endotoxin into sheep results in physiological and structural damage to the pulmonary endothelium. It is uncertain whether complement activation and granulocyte sequestration in the pulmonary microcirculation and the ensuing granulocyte migration into the interstitium seen with endotoxemia contribute to the endothelial damage. We have shown that infusion of complement-activated plasma into sheep, although causing the same degree of granulocyte sequestration in the lungs, results in only modest and transient endothelial damage. In addition, migration or chemotaxis of granulocytes across the endothelial layer of intimal explants is not accompanied by either structural evidence of endothelial damage or a detectable increase in vascular permeability. Such studies indicate that neither complement/granulocyte activation nor granulocyte migration across a vessel wall is entirely responsible for the severe endothelial damage seen with endotoxin. In vitro studies of bovine pulmonary endothelial monolayers indicate that endotoxin can cause direct damage to the endothelium; the damage is dose-dependent and more severe in the presence of serum. Structural studies show endothelial cell retraction, pyknosis, and sloughing. Prostacyclin production and lactic dehydrogenase release are increased, as are permeability to small solutes and hydraulic conductance across the endothelium. It seems that endotoxin can cause a direct injury to pulmonary endothelium but complement and granulocyte activation may enhance the damage.     

Characterization of the proteins of isolated human platelet alpha-granules. Evidence for a separate alpha-granule-pool of the glycoproteins IIb and IIIa

The protein composition of a well-defined alpha-granule preparation isolated from human platelets has been studied. Crossed immunoelectrophoresis against polyspecific platelet antibodies revealed more than 20 immunoprecipitates. The glycoprotein IIb-IIIa complex represented a major antigen in the Triton X-100-solubilized alpha-granule preparation and cross-reacted with the corresponding platelet membrane antigen. Furthermore, after lactoperoxidase-catalyzed 125I-iodination of whole platelets it was not labelled, in contrast to its membrane-located counterpart. This indicates an intracellular location of glycoproteins IIb and IIIa, probably as constituents of the alpha-granules. Fibrinogen, platelet factor 4, albumin, factor VIII-related antigen and the main granule glycoprotein (thrombinsensitive protein, thrombospondin) were identified in the alpha-granule preparation by the crossed immunoelectrophoresis technique. Crossed affinity immunoelectrophoresis using lectins revealed the presence of at least seven glycoproteins, and six sialoglycoproteins were identified by their altered electrophoretic mobility after neuraminidase treatment. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of reduced samples of the alpha-granules revealed at least 15 Coomassie Brilliant Blue-staining polypeptide bands, one of which comigrated with myosin heavy chain. No prominent band was observed in the actin region. Five glycopolypeptide bands were observed after periodic acid-Schiff staining. The dominant three represented the main granule glycoprotein, glycoprotein IIb and glycoprotein IIIa, respectively. More glycoproteins seem to be present in the alpha-granules than was previously recognized.     

Identity of human granulocyte kininogenase and plasminogen activator

Preparative isoelectrofocusing used for fractionating the whole human granulocyte lysate serine proteinases revealed multiple forms of elastase, cathepsin G, kininogenase, human granulocytes plasminogen activator (pI 6.2-10.75). Kinetic characteristics of their substrate specificity were also obtained. It is shown that serine kininogenase of human granulocytes is not identical with elastase as it had been supposed before, it is of trypsin-like nature and is identical with plasminogen activator of these cells. The results obtained reveal new aspects in comprehension of the role of the granulocyte plasminogen activator in development of the inflammatory reaction. It is found that acid-stable proteinase inhibitors formed from blood plasma inter-alpha-inhibitor of trypsin, have an inhibitory effect on the granulocyte plasminogen activator, that supports an assumption on the anti-inflammatory function of these inhibitors.     

Mechanism of kinin release from human low-molecular-mass-kininogen by the synergistic action of human plasma kallikrein and leukocyte elastase

We have investigated the kinin release from human L-kininogen, a poor substrate for plasma kallikrein, by the synergistic action of human PMN elastase and plasma kallikrein. Although PMN elastase alone failed to generate kinin activity from L-kininogen, combination of PMN elastase with plasma kallikrein was found to be effective for the generation of kinin activity from L-kininogen. Two kinds of kinin, bradykinin and Met-Lys-bradykinin, were found to be released from L-kininogen by the synergistic action of PMN elastase and plasma kallikrein. Pretreatment of L-kininogen with PMN elastase facilitated the kinin release by plasma kallikrein, whereas pretreatment of L-kininogen with plasma kallikrein did not allow kinin release by the action of PMN elastase. These results suggested that PMN elastase would act firstly on L-kininogen to form a kinin containing fragment, from which kinin is released by the action of plasma kallikrein. The kinin-containing fragment was isolated by gel filtration and high-performance liquid chromatography of the elastase digest of L-kininogen. The amino-acid analysis and N-terminal amino-acid sequence analysis revealed that the kinin-containing fragment consisted of 26 amino-acid residues and is formed by cleavage of an Ile-Ser and a Ser-His bond of L-kininogen.     

Pig IαI appears unmodified in plasma in case of endotoxin-induced disseminated intravascular coagulation

The unrestricted activity of leukocyte proteinases is thought to contribute to the degradation of plasma proteins and thus amplify the coagulation disorders occurring in septic shock. Inter-α-inhibitor (IαI) is a plasma protein particularly susceptible to their action. Therefore we investigated its behavior in a procine model of endotoxin shock which reproduces the coagulation changes observed in human sepsis. We did not detect any qualitative or quantitative modification of porcine IαI in plasmas collected from pigs after endotoxin infusion. To explain these data. IαI was incubated with polymorphonuclear neutrophils (PMN) stimulated by FMLP in the presence of cytochalasin B. We found that, unlike human PMN, procine cells were unable to proteolyze IαI. Moreover, in the incubation medium of pig PMN, triggered either by FMLP or PMA, no measurable elastase activity was evidenced. Therefore, we urge to better take into account species differences in functional responses of PMN, to explain the experimental results obtained in animal models of septic shock.     

Quantification of granulocyte elastase inhibitors in human mixed saliva and in pure parotid secretion

The predominant inhibitors of granulocyte elastase in plasma (alpha 1-proteinase inhibitor and alpha 2-macroglobulin) together with antileukoproteinase were quantified in parotid secretion and mixed saliva. Antileukoproteinase was the only inhibitor found in parotid saliva and was present in a concentration about 30 times the serum level, suggesting a local production. In mixed saliva, antileukoproteinase accounted for more than 70% of the molar concentration of the granulocyte elastase inhibitors studied. alpha 1-Proteinase inhibitor was measurable in about 1/3 of the specimens of mixed saliva. In parotid secretion, antileukoproteinase was present only as a free, active inhibitor. In mixed saliva about 15% of antileukoproteinase was in complex with granulocyte elastase, while the remaining amount of 85% was inhibitorily active. This suggests that antileukoproteinase has a biological function in a local defence mechanism directed towards the effects of granulocyte elastase in the oral cavity and salivary glands.     

A fibronectin-binding glycoprotein from human platelet membranes.

Fibronectin ('cold-insoluble globulin') has been suggested as a possible mediator of platelet adhesion. A fibronectin-binding protein as partially purified from washed solubilized human platelet membranes by affinity chromatography on fibronectin-Sepharose. The isolated protein migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr (relative molecular mass) of approx. 125 000 under reducing conditions. The protein migrated as a dimer in non-reduced gels. The purified protein did not react with immunoglobulins against fibrinogen or fibronectin when tested in crossed immunoelectrophoresis or electroimmunoassay. The protein and purified fibronectin formed a complex that had a significantly faster mobility in crossed immunoelectrophoresis than did native fibronectin. The presence of heparin in the binding-protein-fibronectin mixture resulted in an even faster mobility of the complex, whereas the mobility of native fibronectin was unaffected. Crossed affinoimmunoelectrophoresis of the complex using different lectins suggested that the binding protein is a glycoprotein containing N-acetylglucosamine residues. The complex, but not purified fibronectin, bound to phenyl-Sepharose on crossed hydrophobic-interaction immunoelectrophoresis. The results strongly suggest the presence of a fibronectin-binding glycoprotein in the platelet membrane.     

Role of nitric oxide in the regulation of glucose kinetics in response to endotoxin in dogs.

The purpose of the present in vivo study was to determine the role of nitric oxide (NO) in the regulation of glucose metabolism in response to endotoxin by blocking NO synthesis with N(G)-monomethyl-L-arginine (L-NMMA). In five dogs, the appearance and disappearance rates of glucose (by infusion of [6,6-(2)H(2)]glucose), plasma glucose concentration, and plasma hormone concentrations were measured on five different occasions: saline infusion, endotoxin alone (E coli, 1.0 microg/kg i.v.), and endotoxin administration plus three different doses of primed, continuous infusion of L-NMMA. Endotoxin increased rate of appearance of glucose from 13.7 +/- 1.6 to 23.6 +/- 3.3 micromol x kg(-1) x min(-1) (P < 0.05), rate of disappearance of glucose from 13.9 +/- 1.1 to 24.8 +/- 3.1 micromol x kg(-1) x min(-1) (P < 0.001), plasma lactate from 0.5 +/- 0.1 to 1.7 +/- 0.1 mmol/l (P < 0.01), and counterregulatory hormone concentrations. L-NMMA did not affect the rise in rate of appearance and disappearance of glucose, plasma lactate, or the counterregulatory hormone response to endoxin. Plasma glucose levels were not affected by endotoxin with or without L-NMMA. In conclusion, in vivo inhibition of NO synthesis by high doses of L-NMMA does not affect glucose metabolism in response to endotoxin, indicating that NO is not a major mediator of glucose metabolism during endotoxemia in dogs.     

A mouse partially protective glycoprotein antigen inStreptococcus agalactiae serotype Ia

The immunological properties of a glycoprotein antigen (antigen 2) ofStreptococcus agalactiae serotype Ia were investigated. A specific antiserum was prepared by immunizing rabbits with antigen 2 immunoprecipitates excised from Crossed immunoelectrophoresis (Crossed IEP) gels. This antiserum produced a single peak representing antigen 2 when reacted with a Triton X-100 sonicate of heat-killed whole serotype Ia cells in Crossed IEP analysis. With polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and subsequent immunoelectroblotting, three strongly reacting polypeptides were detected at 60, 56, and 35 kilo-Daltons. Many faintly reacting polypeptides were detected between 67 and 30 kilodalton. The specific anti-antigen 2 serum used in Crossed immunoisoelectric focusing (XIEF) detected three immunoprecipitates, two with a pI of 8.4 and one with a pI of 6.7. Identification of the antigens detected in XIEF with the polypeptides detected by immunoelectroblotting was not attempted. The specific anti-antigen 2 serum partially protected mice against lethal serotype Ia infection.     

Vascular effects, formation, clearance and release of endothelin peptides in the pig

The formation, release, clearance and vascular effects of endothelin (ET)-like immunoreactivity (-LI) was studied in the pig in vivo. Intravenous infusion of ET-1, 2 and 3 (20 pmol/kg/min for 20 min) increased vascular resistance in the kidney, spleen and skeletal muscle. The most pronounced effects were evoked by ET-1 which caused increases in renal, splenic and skeletal muscle vascular resistance of 554, 528 and 38%, respectively, and a threshold response was observed at 80 pmol/l ET-LI in arterial plasma. During the infusion a large portion of arterial plasma, ET-LI was cleared over the kidney, spleen and skeletal muscle, whereby the most pronounced clearance was observed for ET-1 (73–93%). The ET-1 precursor Big-ET (1–39) given in a similar dose produced only a slight increase in renal vascular resistance (by 20%) and was cleared only over the kidney and not over the spleen or skeletal muscle. Using an ET-1 specific antiserum it was found that plasma ET-1 levels increased 11-fold during the infusion of Big-ET, indicating formation of ET-1 from Big-ET. The half-lives of circulating ET-1, 2 and 3 were 1.3–2.1 min and of Big-ET 8.9 min. Induction of asphyxia for 2 min increased the overflow of ET-LI from the spleen, suggesting local release, and caused splenic vasoconstriction. During i.v. administration of endotoxin for 4 h, arterial plasma ET-LI increased 7-fold and renal and splenic vasoconstrictor responses developed that correlated significantly with the arterial plasma ET-LI. Furthermore, a local release of ET-LI in the spleen was observed during endotoxin administration. Chromatographic characterization of the ET-LI in plasma during endotoxin administration revealed presence of ET-1 and Big-ET. It is concluded that there exists specificity both concerning the vasoconstrictor effects and removal from the circulation of ET peptides, both mechanisms being most prominent for ET-1 in the kidney. Furthermore ET-1 seems to be formed from circulating Big-ET and release of ET-LI can be detected during situations like asphyxia and sepsis.     

Complex formation between plasminogen activator inhibitor 1 and vitronectin in purified systems and in plasma

Functionally active PAI-1 is bound to a discrete binding or carrier protein in plasma, which was recently identified as vitronectin. In the present study, the interaction between PAI-1 and vitronectin has been studied in purified systems and in plasma by agarose gel electrophesis using non-denaturing conditions and by crossed immunoelectrophoresis using an antiserum produced towards purified PAI-1/vitronectin complex. Both methods revealed a clearly distinguishable complex with electrophoretic mobility in between the parent molecules. Virtually all of the purified vitronectin, which did not contain any appreciable amounts of polymerized material, and almost all of the vitronectin in plasma, had the capacity to form a complex with PAI-1. The results suggested a stoichiometry of 1:1 as the most likely ratio between the two molecules in the complex. In contrast to functionally active PAI-1, latent or chloramine T-inactivated PAI-1 did not form such a complex with vitronectin.     

Complex formation between plasminogen activator inhibitor 1 and vitronectin in purified systems and in plasma.

Functionally active PAI-1 is bound to a discrete binding or carrier protein in plasma, which was recently identified as vitronectin. In the present study, the interaction between PAI-1 and vitronectin has been studied in purified systems and in plasma by agarose gel electrophoresis using non-denaturing conditions and by crossed immunoelectrophoresis using an antiserum produced towards purified PAI-1/vitronectin complex. Both methods revealed a clearly distinguishable complex with electrophoretic mobility in between the parent molecules. Virtually all of the purified vitronectin, which did not contain any appreciable amounts of polymerized material, and almost all of the vitronectin in plasma, had the capacity to form a complex with PAI-1. The results suggested a stoichiometry of 1:1 as the most likely ratio between the two molecules in the complex. In contrast to functionally active PAI-1, latent or chloramine T-inactivated PAI-1 did not form such a complex with vitronectin.     

Purification and characterization of Aleutian disease virus

Virus was isolated from infected mink organs by a combination of tissue homogenization, fluorocarbon extraction and ultracentrifugation. The final preparation was analysed by crossed immunoelectrophoresis and electronmicroscopy. Virions had a capsid diameter of 22 nm. Preparative agarose electrophoresis separated virions from contaminating ferritin. Crossed immunoelectrophoresis of virus gave a single precipitate with sera from infected mink. Crossed immunoelectrophoretic analysis with intermediate gels showed that a part of the virus preparation was complexed with antibody. Serum from a certain mink was found to contain precipitating antibody to (poly)nucleotid. Virus and virus-antibody complexes were found to focus at pH 4.0-4.4 in isoelectric focusing. In SDS-polyacrylamide gel-electrophoresis the main virus protein was found to have a molecular weight of 69000. This study gives further support to the classification of aleutian disease virus as a parvovirus.     

Diethylcarbamazine on pulmonary vascular response to endotoxin in awake sheep

Diethylcarbamazine (DEC) is an inhibitor of lipoxygenase, with protective effects in several experimental models of anaphylaxis and lung dysfunction. The hypothesis of this study was that DEC would alter the pulmonary response to endotoxin infusion, especially the prolonged pulmonary hypertension, leukopenia, hypoxemia, and high flow of protein-rich lung lymph. We prepared sheep for chronic measurements of hemodynamics and collection of lung lymph. In paired studies we gave six sheep endotoxin (0.5 micrograms/kg iv) either with or without DEC. DEC was given (80-100 mg/kg iv) over 30 min followed by a continuous infusion at 1 mg X kg-1 X min-1. Endotoxin was given after the loading infusion of DEC, and variables were monitored for 4 h. The response to endotoxin was characterized by pulmonary hypertension, leukopenia, hypoxemia, and elevations of thromboxane B2 and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha). Lymph flow and protein content reflected hemodynamic and permeability changes in the pulmonary circulation. DEC did not significantly modify the response to endotoxin by any measured variable, including pulmonary arterial and left atrial pressures, cardiac output, lymph flow and protein content, alveolar-to-arterial PO2 difference, blood leukocyte count, and lymph thromboxane B2 and 6-keto-PGF1 alpha. We could not find evidence of release of leukotriene C4/D4 by radioimmunoassay in lung lymph after endotoxin infusion with or without DEC treatment. We conclude that lipoxygenase products of arachidonic acid may not be a major component of the pulmonary vascular response to endotoxin.     

Role of circulating granulocytes in sheep lung injury produced by phorbol myristate acetate

Phorbol myristate acetate (PMA) and endotoxin cause pulmonary granulocyte sequestration and alteration in lung fluid and solute exchange in awake sheep that are felt to be analogous to the adult respiratory distress syndrome in humans. The basic hypothesis that PMA causes lung injury by activating circulating granulocytes has never been tested. The effects of infused PMA on lung mechanics and the cellular constituents of lung lymph have also not been reported. We therefore characterized the effects of intravenous PMA, 5 micrograms/kg, on lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, blood and lymph leukocyte counts, and plasma and lymph cyclooxygenase products of arachidonate metabolism in 10 awake sheep with normal granulocyte counts and after granulocyte depletion with hydroxyurea. PMA significantly altered lung mechanics from base line in both nongranulocyte depleted and granulocyte-depleted sheep. Dynamic compliance decreased by over 50% and resistance to airflow across the lungs increased over threefold acutely following PMA infusion in both sets of experiments. Changes in lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, and plasma and lymph arachidonate metabolites were not significantly affected by greater than 99% depletion of circulating granulocytes. We conclude that the lung injury caused by PMA in chronically instrumented awake sheep probably is not a result of activation of circulating granulocytes.     

Determination of the elastase activity of the leukocyte thermostable alpha-glycoprotein from human blood plasma

The possibility of isolation of specific human granulocyte antigen--leukocyte thermostable alpha-glycoprotein (LTG) from plasma was shown. The isolation of preparations containing LTG was carried out on CNBr-activated sepharose with immobilized soluble fraction of pus. Using immunochemical analysis with standard antisera, the absence of plasma and leukocyte proteins and the presence of LTG in the preparations obtained were demonstrated. Pus may comprise a protein component capable of binding LTG. Elastase activity of the preparations obtained was established. The identity of LTG and granulocyte elastase is suggested.     

Human inter-alpha-trypsin inhibitor. Limited proteolysis by trypsin, plasmin, kallikrein and granulocytic elastase and inhibitory properties of the cleavage products.

The acid-labile inter-alpha-trypsin inhibitor is cleaved enzymatically in vivo, liberating a smaller acid-stable inhibitor. The molar ratio of native inhibitor to this smaller inhibitor in plasma is significantly changed in some severe cases of inflammation and kidney injury. To clarify this observation on a molecular basis, the action of four different types of proteinases (trypsin, plasmin, kallikrein and granulocyte elastase) on the inter-alpha-trypsin inhibitor was studied. The initial rate of cleavage of the inter-alpha-trypsin inhibitor by a 1.3-fold molar excess of proteinase over inhibitor was found to be 4375 nM x min-1 with granulocyte elastase, 860 nM x min-1 with trypsin, 67 nM x min-1 with plasmin, and 0.3 nM X min-1 with kallikrein. Obviously, of the enzymes studied so far, the granulocyte elastase known to be released during severe inflammatory processes is by far the most potent proteinase in the transformation of the inter-alpha-trypsin inhibitor. The inter-alpha-trypsin inhibitor and its cleavage products inhibit bovine trypsin very strongly (Ki = 10(-9)--10(-11) M), porcine plasmin much less strongly, human plasmin very weakly and pancreatic kallikrein practically not at all.     

Glycoprotein Ib in the triton-insoluble (cytoskeletal) fraction of blood platelets

Glycoprotein Ib could be demonstrated in the Triton-insoluble (cytoskeletal) fraction of platelets prepared with EGTA by SDS-polyacrylamide gel electrophoresis and staining with the periodic acid Schiff's reagent. Crossed immunoelectrophoresis showed that glycoprotein Ib could be extracted from such Triton-insoluble residues when the extraction solution contained 1% Triton X-100 plus 5 mM CaCl₂, but not if it also contained leupeptin. This indicates that glycoprotein Ib was associated to structures in the cytoskeletal fraction in such a way that it could be extracted only after activation of a calcium-dependent protease, and degradation of the actin-binding protein was demonstrated. After crossed immunoelectrophoresis of platelet extracts prepared in the presence of leupeptin or EDTA, a glycoprotein Ib-related, rocket-shaped immunoprecipitate was seen originating from the application well. This was interpreted as being related to glycoprotein Ib associated to actin polymers which did not sediment at low-speed centrifugation. Incubation of platelets with ³²P as sodium phosphate led to incorporation of phosphatase-sensitive ³²P in all of the glycoprotein Ib-related immunoprecipitates except for that of glycocalicin. This support the idea that glycoprotein Ib traverses the plasma membrane and can be phosphorylated at the inner surface whereas glycocalicin represents the terminal part of the glycoprotein Ibα-chain exposed at the outer surface.