An alternative pathway for fibrinolysis. I. The cleavage of fibrinogen by leukocyte proteases at physiologic pH.

184

A B S T R A C T Immunological detection of elastase, an enzyme present within leukocyte granules, has been used as a marker for polymorphonuclear leukocyte activation. Polymorphonuclear leukocytes contained 4.6 gg/107 cells, whereas erythrocytes, mononuclear cells, and platelets contained < 1% of this level. In plasma that was separated from blood cells after 1 h at 22°C, the mean level of elastase-related antigen in seven normal donors was 25±6 ng/ml. This level was unaltered by immediate separation of the plasma from the cells, by inclusion of protease inhibitors, or by anticoagulation of the plasma with either EDTA or acidcitrate-dextrose (the level in heparinized plasma was approximately threefold higher). In serum, the level of elastase-related antigen was 288±125 ng/ml, representing an 11.5-fold increase above plasma levels. The antigen detected in serum was immunochemically indistinguishable from the leukocyte enzyme. Release of elastase was observed when isolated polymorphonuclear leukocytes were added to nonanticoagulated platelet-rich or platelet-poor plasma, recalcified plasma, or to serum. Addition of a chelating agent to serum prevented elastase release, but calcium or magnesium did not induce release in the absence of plasma. Coagulation induced by addition of thrombin to plasma also failed to induce release. In whole blood or in anticoagulated plasma reconstituted with polymorphonuclear leukocytes and then recalcified, initial release of elastase occurred concomitantly with or slightly after clotting and reached maximal levels within 20-40 min after clot formation. The data indicates that early events in coagulation or other pathways that occur in parallel with coagulation induce leukocyte release. The release of elastase, a major fibrinolytic protease of leukocytes, from the cells provides a mechanism Portions of this study were presented at the

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Leukocyte Elastase Release during Blood Coagulation

Plow¹

1982

184

“…Coincident with fibrin formation and platelet activa- tion, a variety ofchemotactic and growth promotion substances are released, including complement fiagments (27,28), proteases (29), and platelet proteins such as platelet factor 4 and PDGF (19,25,30). Once formed, the fibrin clot undergoes remodeling by proteolysis, notably through the actions of plasmin (31) and PMN elastase (32). Thus, substances released during clot formation, platelet activation, and clot resolution have the capacity to recruit inflammatory cells and other cell types including fibroblasts, and also to exert mitogenic activity.…”

Section: Discussionmentioning

confidence: 99%

Effects of fibrinogen derivatives upon the inflammatory response. Studies with human fibrinopeptide B.

Senior¹,

Skogen²,

Griffin³

et al. 1986

218

104

Fibrin formation and turnover are intimately associated with inflammation and wound healing. To explore whether fibrin(ogen)-derived peptides exert direct effects upon cells involved in inflammation and tissue repair we examined the capacity of human fibrinopeptide B (hFpB), a thrombin-derived proteolytic cleavage product of the fibrinogen B ,8-chain, to stimulate neutrophils (PMN), monocytes, and fibroblasts. hFpB caused directed cell migration of PMN and fibroblasts that was optimal at -810 M. This chemotactic activity was blocked by preincubating hFpB with antiserum to hFpB. hFpB was not chemotactic for monocytes. The chemotactic potency of hFpB for PMN was equivalent to that of anaphylatoxin from the fifth component of human complement (C5a), leukotriene B4 (LTB4), and formyl-methionylleucyl-phenylalanine (fMLP), and for fibroblasts its chemotactic activity was comparable to that of platelet-derived growth factor. hFpB did not interact with PMN receptors for C5a, LTB4, or fMLP as (a) desensitization with 10-7 M hFpB abolished chemotaxis to hFpB but had no effect upon chemotaxis to C5a, LTB4, or fMLP and (b) induction of chemotactic responses to fMLP and LTB4 in neutrophilic leukemic cells (HL-60 cells) by incubation with dimethylsulfoxide did not extend to hFpB. Like fMLP, hFpB caused a rapid, dose-dependent increase in PMN cytoskeletal associated actin, but unlike fMLP, hFpB did not cause PMN aggregation, release of lysosomal enzymes (lysozyme and ,l-glucuronidase), or the production of superoxide anion.These results suggest that hFpB may have a role in recruiting PMN and fibroblasts at sites of fibrin deposition and turnover.

“…Purification and antigenic identification of enzymes from different tissue sources have shown a broad substrate specificity for at least two of the lysosomal neutral proteases. Elastase hydrolyzes elastin, collagen, proteoglycan, azo-casein (2), hemoglobin, fibrinogen (3,4), and histone (5). Cathepsin G, which is separable from elastase, hydrolyzes hemoglobin and fibrinogen (3), casein (6), azo-casein, collagen, and proteoglycan (7).…”

Section: Introductionmentioning

confidence: 99%

Purification and Characterization of a Human Neutrophil Neutral Protease

Coblyn¹,

Austen²,

Wintroub³

1979

A B S T R A C T A human neutrophil neutral protease which generates a low molecular weight peptide from a plasma protein substrate and cleaves the basic amino acid ester substrates a-N-p-tosyl-L-arginine methyl ester HC1, a-N-benzoyl-L-arginine-methyl ester HC1, and a-N -carbobenzoxy-L-lysine-p -nitrophenyl ester has been purified to homogeneity and distinguished from the known lysosomal neutrophil proteases. The starting activity was obtained from purified human neutrophils by homogenization, sedimentation by lowspeed centrifugation, and high salt elution of the insoluble material. Purification was achieved by aprotinin-affinity chromatography, precipitation at low ionic strength, and gel filtration. The overall recovery, relative to the activity in the starting eluate of the neutrophil fraction, was m50% with a 200-to 400-fold increase in specific activity. After treatment with diisopropylfluorophosphate to eliminate autodegradation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced and unreduced protein gave a single protein band of 29,000-30,000 mol wt. The isoelectric point determined in sucrose gradients ranged from pH 7.8 to 8.3 with a peak at pH 8.0. This neutrophil protease, like cathepsin G and elastase, is composed ofa single polypeptide chain of _30,000 mol wt, but differs from cathepsin G and elastase in its less cationic isoelectric point and its failure to cleave synthetic substrates presenting an aromatic amino acid ester linkage and alanyl peptide bonds, respectively.Dr.