Differentiation of Leukocytic Fibrinolytic Enzymes from Plasmin by the Use of Plasmatic Proteolytic Inhibitors

A B S T R A C T We investigated the effect of elastaselike neutral protease isolated from human granulocytes on human fibrinogen. Dependent on enzyme concentration and time of incubation, the elastase-like protease induced a progressive degradation of fibrinogen. Analysis of the remaining polypeptide chains showed a high susceptibility of the Aa-and low susceptibility of the y-chain of fibrinogen towards the proteolytic action of the enzyme. The split products were characterized by polyacrylamide gel electrophoresis and two-dimensional immunoelectrophoresis. They showed antigenic determinants of fibrinogen and of plasmin-induced proteolysis products D and E. The cleavage fragments isolated by gel chromatography had distinct molecular weights. Coagulability of fibrinogen by thrombin was inhibited according to the concentration of the protease and the time of incubation. Split products of fibrinogen with higher molecular weight prolonged the coagulation time of native fibrinogen, whereas low molecular weight fragments were ineffective.

Section: Introductionmentioning

confidence: 99%

Degradation products of fibrinogen by elastase-like neutral protease from human granulocytes. Characterization and effects on blood coagulation in vitro.

Gramse¹,

Bingenheimer²,

Schmidt³

et al. 1978

“…Within the confines of the vascular compartment, leukocytes frequently accumulate in complex thrombi consisting of fibrin and platelets (1)(2)(3)(4)(5); whereas in extravascular sites, the migration of leukocytes to sites of injury where fibrin has accumulated is a central feature of the inflammatory response (5)(6)(7)(8)(9). In both circumstances, the interaction of these cells with fibrin may be active rather than passive, based on the morphologic identification of fibrin within leukocytes (5)(6)(7)(8)(9), the accumulation of leukocytes in thrombi relative to their concentration in blood (4), and the demonstration that fibrinolytic activity of leukocytes is not confined to low pH, at which many cathepsins are active, but also is observed at neutral pH (10)(11)(12)(13)(14)(15)(16).…”

Section: Introductionmentioning

confidence: 99%

“…This conclusion is based on the differential sensitivity of the systems to inhibitors, as well as comparisons of the molecular characteristics of the enzymes (11)(12)(13)(14). The purpose of the present study is to examine the alternative fibrinolytic system of leukocytes with respect to: (a) the relative contribution of the plasminogen and the plasminogen-independent system to the total fibrinolytic activity of the leukocytes; (b) the molecular characteristics of cleavage of fibrinogen by leukocyte proteases; (c) the biologic properties of the fibrinogen cleavage fragments generated by leukocyte protease; and (d) the release of protease from leukocytes when subjected to appropriate stimulus.…”

Section: Introductionmentioning

confidence: 99%

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

Plow¹,

Edgington²

1975

209

A B S T R A C T An alternative fibrinolytic system, active at physiological pH, is present in peripheral blood leukocytes. The fibrinolytic proteases localized predominantly in the leukocyte granules are capable of degrading both fibrinogen and fibrin, and plasmin activity does not contribute significantly to this proteolytic event.The specificity of the alternative fibrinolytic proteases for fibrinogen and the characteristics of the derivative cleavage fragments are clearly distinguishable from the classical plasmin system. The high molecular weight derivatives of fibrinogen, generated by the alternative system, under physiological conditions, are larger than the plasmin-generated X fragment, exhibit immunoelectrophoretic mobility comparable to native fibrinogen, and are not coagulable by thrombin. Analysis of the constituent polypeptide chains of the fragments reveals cleavage of the Aa, BP, and y chains of fibrinogen. The lower molecular weight derivatives of fibrinogen, generated by the alternative system, are structurally distinct from previously described fibrinogen degradation products and exhibit potent anticoagulant activity. This anticoagulant activity can be attributed to interference with normal fibrin polymerization. The proteases of the alternative fibrinolytic systems are actively secreted by leukocytes when stimulated to undergo a nonlytic release reaction. These results provide direct evidence for a fibrinolytic system resident in leukocyte granules that is associated with the leukocyte release reaction and is capable of generating unique fibrinogen cleavage fragments.

“…The fibrinolytic activity of leukocyte extracts is independent of the plasminogen system as plasmin (ogen) inhibitors and activators fail to inhibit or augment fibrinolytic activity (4,11) and fibrinogen degradation products generated by leukocyte proteases are structurally and immunochemically distinguishable from plasmin-derived degradation products (4,12,13). A variety of neutral proteases (5,(14)(15)(16)(17)(18) and acidic cathepsins (3) of neutrophils are capable of degrading fibrin (ogen).…”

mentioning

confidence: 99%

Leukocyte Elastase Release during Blood Coagulation

Plow¹

1982

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