scholarly journals Inactivation of α2-Macroglobulin by Activated Human Polymorphonuclear Leukocytes

1994 ◽  
Vol 3 (2) ◽  
pp. 117-123 ◽  
Author(s):  
G. Deby-Dupont ◽  
J.-L. Croisier ◽  
G. Camus ◽  
D. Brumioul ◽  
M. Mathy-Hartert ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Proteolytic Activity ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Hypochlorous Acid ◽  
Myeloperoxidase Activity ◽  
Trypsin Activity ◽  
Α2 Macroglobulin ◽  
Human Polymorphonuclear Leukocytes

The proteolytic activity of trypsin releases the dye Remazol Brilliant Blue from its high molecular weight substrate, the skin powder (Hide Powder Azure, Sigma), with an increase in absorbance at 595 nm. Active α2- macroglobulin (80 μg/ml) totally inhibits the proteolytic activity of trypsin (14 μg/ml) by trapping this protease. But after a 20 min incubation of α2-macroglobulin at 37°C with 2 × 106human polymorphonuclear leukocytes activated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (10−7M) and cytochalasin B (10−8M), 100% of trypsin activity was recovered, indicating a total inactivation of α2-macroglobuHn. Incubation with granulocyte myeloperoxidase also inactivates α2-macroglobulin. Hypochlorous acid, a by-product of myeloperoxidase activity, at a concentration of 10−7M also inactivates α2-macroglobulin, which indicates that an important cause of α2-macroglobulin inactivation by activated polymorphonuclear leukocytes could be the activity of myeloperoxidase.

The Contact Phase of Blood Coagulation in Diabetes Mellitus and in Patients with Vasculopathy

1984 ◽  
Vol 52 (03) ◽  
pp. 221-223 ◽  
Author(s):  
M Christe ◽  
P Gattlen ◽  
J Fritschi ◽  
B Lämmle ◽  
W Berger ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Molecular Weight ◽  
Blood Coagulation ◽  
High Molecular Weight ◽  
Negative Correlation ◽  
Factor Xii ◽  
C1 Inhibitor ◽  
High Molecular Weight Kininogen ◽  
Contact Phase ◽  
Α2 Macroglobulin

SummaryThe contact phase has been studied in diabetics and patients with macroangiopathy. Factor XII and high molecular weight kininogen (HMWK) are normal. C1-inhibitor and also α2-macroglobulin are significantly elevated in diabetics with complications, for α1-macroglobulin especially in patients with nephropathy, 137.5% ± 36.0 (p <0.001). C1-inhibitor is also increased in vasculopathy without diabetes 113.2 ± 22.1 (p <0.01).Prekallikrein (PK) is increased in all patients’ groups (Table 2) as compared to normals. PK is particularly high (134% ± 32) in 5 diabetics without macroangiopathy but with sensomotor neuropathy. This difference is remarkable because of the older age of diabetics and the negative correlation of PK with age in normals.

Gene Expression of the High Molecular Weight Proteinase Inhibitor α2-Macroglobulin

1992 ◽  
Vol 373 (2) ◽  
pp. 509-516 ◽  
Author(s):  
EVA KRAUSE ◽  
URSULA WEGENKA ◽  
GARSTEN MÖLLER ◽  
FRIEDEMANN HORN ◽  
PETER C. HEINRICH
Keyword(s):  
Gene Expression ◽  
Molecular Weight ◽  
High Molecular Weight ◽  
Proteinase Inhibitor ◽  
Α2 Macroglobulin

Three noncontiguous peptides comprise binding sites on high-molecular-weight kininogen to neutrophils

1998 ◽  
Vol 275 (1) ◽  
pp. H145-H150 ◽  
Author(s):  
Mohammad M. H. Khan ◽  
Satya P. Kunapuli ◽  
Yingzhang Lin ◽  
Abraham Majluf-Cruz ◽  
Raul A. Dela Cadena ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Binding Site ◽  
High Molecular Weight ◽  
Binding Sites ◽  
Polymorphonuclear Leukocytes ◽  
High Molecular Weight Kininogen ◽  
Rich Region ◽  
Putative Receptor ◽  
Exon 9 ◽  
Stimulation Of

The binding of high-molecular-weight kininogen (HK) to neutrophils (polymorphonuclear leukocytes, PMN) is required for the stimulation of aggregation and degranulation by human plasma kallikrein as well as the displacement of fibrinogen from this cell surface. The putative receptor for HK is the leukocyte integrin αMβ2, and domains 3 (D3) and 5 (D5) of HK form its binding site. To further map the binding sites on HK for PMN, we used D3 recombinant exon products and designed peptides from D3 and D5. In D3, a heptapeptide, Leu271-Ala277, from exon 7 product, and a peptide, Cys333-Cys352, from exon 9 product can inhibit binding of kininogen to PMN. Two contiguous peptides from D5 in the histidine-glycine-rich region, Gly442-Lys458and Phe459-Lys478, each inhibit the binding of HK to PMN. This study has thus delineated three noncontiguous surface-oriented sequences on HK, which together comprise all or most of the binding site for human PMN.

scholarly journals Ca2+-Induced Cleavage of Human Platelet Polypeptides Mediated by the Calcium Ionophore A-23187

1977 ◽  
Author(s):  
Milica Jakábová ◽  
David R. Phillips
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Proteolytic Activity ◽  
Platelet Activation ◽  
Human Platelet ◽  
Calcium Ionophore ◽  
Lactic Dehydrogenase ◽  
Actin Binding ◽  
A 23187 ◽  
Hydrolysis Of

The effect of calcium on human platelet polypeptides was investigated. When lysed platelets were incubated with mM Ca++, two major intracellular polypeptides (Mr = 255,000 and 230,000) were found to rapidly disappear. A similar phenomenon was also observed when intact platelets were treated with the calcium ionophore A-23187 in the presence of mM Ca++. Determinations of lactic dehydrogenase activity in supernatant fractions demonstrated that these losses occurred before platelet lysis. Investigations into the identity of the high molecular weight polypeptides revealed that one (Mr = 255,000) had similar properties to actin binding protein. The loss of the high molecular weight polypeptides was accompanied by formation of lower molecular weight polypeptides (Mr = 135,000, 93,000 and 48,000), indicating that Ca++ activates a polypeptide cleavage mechanism. The Ca++-activated polypeptide cleavages were rapid, with significant changes being observed within the first 0.5 min of incubation. An obvious explanation for these effects is. that there is Ca++-activated proteolytic activity within platelets. The Ca++-activated proteolytic activity was determined by the hydrolysis of the artificial substrate azocasein. We found that more than 90% of the proteolytic activity in lysed platelets was due to Ca++-activated proteases. These studies show that Ca++-activated proteases may play an important role in platelet activation.

scholarly journals Molecular analysis of myeloperoxidase deficiency shows heterogeneous patterns of the complete deficiency state manifested at the genomic, mRNA, and protein levels

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1317-1322 ◽  
Author(s):  
ME Selsted ◽  
CW Miller ◽  
MJ Novotny ◽  
WL Morris ◽  
HP Koeffler
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Hypochlorous Acid ◽  
Restriction Pattern ◽  
Northern Analysis ◽  
Molecular Analyses ◽  
Protein Levels ◽  
Blood Tests ◽  
Digestion Pattern ◽  
Human Polymorphonuclear Leukocytes ◽  
Unrelated Subject

Myeloperoxidase (MPO) is a glycosylated hemoprotein contained in the azurophil granules of human polymorphonuclear leukocytes (PMNs). MPO is thought to play a role in the oxidative antimicrobial activity of neutrophils by catalyzing the formation of hypochlorous acid, a potent microbicide, from hydrogen peroxide and chloride anions. Seven unrelated individuals with complete MPO deficiency, a relatively common heritable defect of neutrophils, were identified during routine blood tests. Molecular analyses were conducted to determine the level of the abnormality in these individuals. Western blot analysis showed that 6 of the 7 donors were devoid of immunoreactive MPO protein, while neutrophils from one individual contained only the 55-Kd subunit. Northern analysis of bone marrow RNA from one MPO-deficient donor showed the presence of the normal-sized 3.3-kb transcript indicating that the defect in MPO biosynthesis in this case was posttranscriptional. Southern analysis of four MPO-deficient donors showed a normal Bgl II digestion pattern, whereas an abnormal restriction pattern was observed in a fifth individual. Although the Bgl II pattern was similar to that observed in an unrelated subject described by Nauseef (Blood 73:290, 1989), our study strongly suggests that the point mutation does not reflect a polymorphism. Taken together, these analyses show the existence of diverse abnormalities associated with MPO-deficiency that may be detected at the level of the MPO polypeptide, mRNA, and gene.

scholarly journals Role of microtubule assembly in lysosomal enzyme secretion from human polymorphonuclear leukocytes. A reevaluation.

1977 ◽  
Vol 73 (1) ◽  
pp. 242-256 ◽  
Author(s):  
S Hoffstein ◽  
I M Goldstein ◽  
G Weissmann
Keyword(s):  
Plasma Membrane ◽  
Dose Response ◽  
Lysosomal Enzyme ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Microtubule Assembly ◽  
Enzyme Release ◽  
Thin Sections ◽  
Human Polymorphonuclear Leukocytes ◽  
In Cells

The dose-related inhibition by colchicine of both lysosomal enzyme release and microtubule assembly was studied in human polymorphonuclear leukocytes (PMN) exposed to the nonphagocytic stimulus, zymosan-treated serum (ZTS). Cells were pretreated with colchicine (60 min, 37 degrees C) with or without cytochalasin B (5 microng/ml, 10 min) and then stimulated with ZTS (10%). Microtubule numbers in both cytochalasin B-treated and untreated PMN were increased by stimulation and depressed below resting levels in a dose-response fashion by colchicine concentrations above 10(-7) M. These concentrations also inhibited enzyme release in a dose-response fashion although the inhibition of microtubule assembly was proportionately greater than the inhibition of enzyme release. Other aspects of PMN morphology were affected by colchicine. Cytochalasin B-treated PMN were rounded, and in thin sections the retracted plasma membrane appeared as invaginations oriented toward centrally located centrioles. Membrane invaginations were restricted to the cell periphery in cells treated with inhibitory concentrations of colchicine, and the centrioles and Golgi apparatus were displaced from their usual position. After stimulation and subsequent degranulation, the size and number of membrane invaginations greatly increased. They remained peripheral in cells pretreated with greater than 10(-7) M colchicine but were numerous in the pericentriolar region in cells treated with less than 10(-7) M. Similarly, untreated PMN that were permitted to phagocytose immune precipitates had many phagosomes adjacent to the centriole. After colchicine treatment, phagosomes were distributed randomly, without any preferential association with the centrioles. These data suggest that microtubules are involved in maintaining the internal organization of cells and the topologic relationships between organelles and the plasma membrane.

scholarly journals Functional activity of enucleated human polymorphonuclear leukocytes.

1983 ◽  
Vol 97 (2) ◽  
pp. 368-377 ◽  
Author(s):  
D Roos ◽  
A A Voetman ◽  
L J Meerhof
Keyword(s):  
Plasma Membrane ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Unit Area ◽  
Chemotactic Activity ◽  
Myristate Acetate ◽  
Intact Cells ◽  
Ficoll Gradient ◽  
Pmn Functions ◽  
Human Polymorphonuclear Leukocytes

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.

The Kinetics Of The Inhibition Of Human Plasma Kallikrein By Plasma Protease Inhibitors: Role Of High Molecular Weight Kininogen

1981 ◽  
Author(s):  
M Schapira ◽  
A James ◽  
C F Scott ◽  
F Kueppers ◽  
H L James ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Protease Inhibitors ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Reaction Rates ◽  
Plasma Kallikrein ◽  
High Molecular Weight Kininogen ◽  
Α2 Macroglobulin ◽  
Competitive Inhibitors

Plasma kallikrein (KAL) is inhibited by several plasma protease inhibitors, including C1-inhibitor (C1-INH), antithrombin III (ATIII), α1-antitrypsin (α1AT), and α2-macroglobulin (α2M). To assess the mechanism of action and the relative importance of these inhibitors, we have undertaken inhibition studies with purified proteins, using H-D-Pro- Phe-Arg-Nan as KAL substrate. Inhibition was competitive with C1INH, ATIII, and α1AT and noncompetitive with α2M. KAL retained 14% of its catalytic efficiency when complexed to α2M. The rate constants for inhibition by C1INH, ATIII, α1AT, and α2M were 28, 0.18, 0.003, and 6.9 M-ls-1(10-3) respectively. Michaelis-Menten kinetics was observed for the inhibition by ATIII, αlAT, and α2M. The constants for the rate-limiting formation of the irreversible complexes were 16, 0.27 and 2.0 s-1(xl02), while the KI’s for the reversible complex were 86, 63, and 0.29 γM, respectively for ATIII, α1AT and α2M. In_contrast, no Michaelis-Menten complex was observed when C1INH inhibited KAL. These results indicate that (a) C1INH is the most efficient inhibitor of KAL, (b) α2M is a significant inhibitor of KAL, (c) both ATIII and αlAT are probably not significant inhibitors of KAL. We have shown that high molecular weight kininogen (HMWK) decreases the inactivation rate of KAL by C1INH by forming a reversible complex with KAL. We now report that the reaction rates of KAL with ATIII and α1AT, which are competitive inhibitors, were decreased by 50%, when HMWK was 1 U/ml or 0.73 γM. When KAL was inhibited by α2M, a noncompetitive inhibitor, the inactivation rates were identical in the presence or absence of HMWK. Since HMWK protects KAL from being inhibited by competitive inhibitors but not by a noncompetitive one, these results confirm our previous observation indicating that the binding site for IMWK on KAL is closely linked to its catalytic site.

scholarly journals POTASSIUM AND AMINO ACID TRANSPORT IN HUMAN LEUKOCYTES EXPOSED TO PHAGOCYTIC STIMULI

1974 ◽  
Vol 63 (1) ◽  
pp. 215-226 ◽  
Author(s):  
Philip B. Dunham ◽  
Ira M. Goldstein ◽  
Gerald Weissmann
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Surface Binding ◽  
Ouabain Binding ◽  
Latex Particles ◽  
Contrast Exposure ◽  
Human Polymorphonuclear Leukocytes ◽  
Specific Membrane

Influxes of potassium and amino acids were measured in suspensions of human polymorphonuclear leukocytes (PMNs) under resting conditions and after various phagocytic stimuli. Both ouabain-sensitive (or pump) and ouabain-insensitive (or leak) influxes of K were determined. In 5 mM external K, mean total K influx was 0.69 nmol/106 cells x min, of which 52% was ouabain-sensitive. Ouabain binding was irreversible, and, as in erythrocytes, was inhibited by K. At external concentrations of 0.1 mM, influxes of lysine and leucine were entirely carrier-mediated, with means of 0.021 nmol/106 cells x min, and 0.019 nmol/106 cells x min, respectively. After incubation of PMNs with zymosan or latex particles, the K pump was reduced more than 60%, whereas amino acid influxes were inhibited only by 30%. PMNs were also exposed to cytochalasin B before challenge by particles: the drug prevented phagocytosis but not surface binding of zymosan, nor did it influence transport of K or amino acids. After pretreatment of PMNs with cytochalasin B, interaction of zymosan with their surface resulted in the same degree of inhibition of influxes of K and amino acids as when the cells were permitted to phagocytose the particles. In contrast, exposure of PMN to latex particles, which do not bind to cytochalasin B-treated cells, after pretreatment of cells with cytochalasin B did not result in inhibition of influxes. Treatment of cells with colchicine had no effect on either membrane transport or its inhibition after exposure to various phagocytic stimuli. These results indicate that the surface membranes of PMNs are functionally heterogeneous with respect to the association of transport sites for the different solutes. Moreover, loss of specific membrane functions from phagocytosing cells may result from the surface-at-tachment phase of particle-cell interactions, since the interactions of zymosan particles with PMNs in the absence of phagocytosis also inhibited transport of solutes.

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