Cleavage of fibrinogen by the human neutrophil neutral peptide-generating protease.

The human neutrophil peptide-generating protease, which generates a low molecular weight vasoactive peptide from a plasma protein substrate, is directly fibrinolytic and cleaves human fibrinogen in a manner distinct from plasmin. Fibrinogen was reduced from 340,000 Mr to derivatives of 270,000-325,000 Mr during interaction with the protease at enzyme-to-substrate ratios of 0.3 or 1.0 microgram/1.0 mg. The 310,000-325,000 Mr cleavage fragments exhibited prolonged thrombin-induced clotting activity but were able to be coagulated, whereas the 270,000-290,000 Mr fragments were not able to be coagulated. Anticoagulants were not generated at either enzyme dose. As analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis in 4-30% gradient gels and 10% gels stained for protein and carbohydrate, the diminution to 310,000-325,000 Mr and the prolongation of thrombin-induced clotting time resulted from cleavage of the fibrinogen A alpha chain. The further decrease in size to 270,000-290,000 Mr was associated with B beta-chain and gamma-chain cleavage and an inability to form gamma-gamma dimers. The neutral peptide-generating protease, a distinct human neutrophil neutral protease with fibrinolytic and fibrinogenolytic activities comparable to those of plasmin on a weight basis, cleaves fibrinogen in a manner that is distinct from the action of plasmin, leukocyte elastase, and leukocyte granule extracts. It may be that the concerted action of this neutrophil protease to generate a vasoactive peptide and to digest fibrinogen and fibrin facilitates neutrophil movement through vascular and extravascular sites.     

Asbestos-induced release of a human alveolar macrophage-derived neutrophil chemotactic factor

Neutrophils accumulate in the alveoli of asbestos-exposed individuals. In determining whether asbestos fibers induce the release of neutrophil chemotactic factor (NCF) from human alveolar macrophages, alveolar macrophages (10(6) cell/mL) obtained by bronchoalveolar lavage from six non-asbestos-exposed control subjects were exposed to crocidolite (0.1 and 1 mg/mL), chrysotile (1 mg/mL), or medium alone for 4 h, and NCF activity was measured in the supernatants in a 48-well microchemotaxis chamber with polycarbonate membrane filters (pore size, 3 microns) and purified human neutrophils. Alveolar macrophages in medium alone released negligible amounts of NCF (4 +/- 1 neutrophils per high-power field [N/HPF]). When macrophages were exposed to crocidolite (0.1 and 1 mg/mL), significant NCF release occurred (43 +/- 9 and 105 +/- 32 N/HPF, respectively; p less than 0.01 for each amount compare to alveolar macrophages cultured in medium alone). Chrysotile (1 mg/mL) induced similar NCF release (96 +/- 14 N/HPF; p less than 0.01 compared to unstimulated alveolar macrophages). Partial characterization of the NCF by Sephadex G-25 fine gel filtration demonstrated a molecular size of less than 1,000 daltons. These results show that human alveolar macrophages release NCF after exposure to asbestos. Release of NCF by alveolar macrophages in asbestos-exposed individuals may play a central role in the pathogenesis of asbestosis.     

Cleavage of human von Willebrand factor by platelet calcium-activated protease

In human platelet lysates prepared by addition of nonionic detergent (Triton X-100) or by sonication, the multimer composition and electrophoretic mobility of platelet von Willebrand factor (vWF) were consistently modified under conditions that would favor activation of the endogenous calcium-activated, sulfhydryl-dependent neutral protease (CAP). By sodium dodecylsulfate-agarose gel electrophoresis, native platelet vWF contained some multimers that were larger than those characteristic of plasma vWF. Modified platelet vWF contained a multimer population equivalent to or smaller than that of plasma vWF plus an additional fast-migrating band. In crossed immunoelectrophoresis (CIE), modified platelet vWF was characterized by a more anodic distribution and the appearance of a distinct, cross- reactive, anodic component previously designated VIIIR:Ag fragment. In the presence of calcium, radiolabeled purified plasma vWF was also degraded by the protease in question, with a decrease in the apparent molecular weight of the reduced monomer from 230,000 to 205,000. The VIIR:Ag fragment isolated from the same degraded plasma vWF by preparative CIE was shown to be composed of an identical mol wt 205,000 subunit. Because cleavage of plasma or platelet vWF was inhibited by prior addition of leupeptin, EDTA, ethylene glycol bis (beta-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA), or N-ethylmaleimide (agents known to inhibit platelet CAP) but was unaffected by numerous other protease inhibitors, including soybean trypsin inhibitor, benzamidine, hirudin, phenylmethylsulfonyl fluoride, aprotonin, or epsilon-aminocaproic acid (none of which inhibits platelet CAP), we conclude that proteolysis of vWF observed in this study is a direct effect of CAP and is not mediated by way of secondary proteases.     

Isolation of functional human coagulation factor V by using a hybridoma antibody

Spleen cells obtained from mice immunized with partially purified human coagulation Factor V were fused with NS-1 mouse myeloma cells, and hybrids were selected. Culture media were screened for anti-Factor V activity, and an antibody-positive clone was obtained and passaged as an ascites tumor in mice. The ascitic fluid from the hybridoma-bearing mouse could be diluted 1:10(6) before losing reactivity in an anti-Factor V radioimmunoassay. When immobilized on agarose, the monoclonal antibody quantitatively removed Factor V activity from human plasma. Factor V activity could be eluted with 1.2 M NaCl at pH 6.5. Homogeneous Factor V was isolated by chromatography of barium citrate-adsorbed, polyethylene glycol 6000 precipitated plasma on the antibody column followed by chromatography on phenyl-Sepharose. The isolated Factor V exhibited a single band upon gel electrophoresis in sodium dodecyl sulfate with an apparent Mr comparable to that of bovine Factor V (330,000). Upon exposure to thrombin, the activity of Factor V increased 53-fold when measured in Factor V-deficient plasma. This increased activity was associated with discrete proteolytic cleavages of the parent molecule.     

Activation/inactivation of human factor V by plasmin

The effect of human plasmin on human coagulation factor V was studied using isolated proteins. Incubation of factor V with plasmin resulted in a rapid increase in procoagulant activity, followed by a subsequent decline in the ability of factor V to serve as a cofactor in the prothrombinase complex. Identical results were obtained when these reactions were conducted in the presence of dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA), indicating that the changes observed could not have occurred as a consequence of cleavage by alpha-thrombin. Analysis of the products of the reaction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a temporal correlation between the rise and fall in factor V activity and the presence of several transient intermediates. These fragments are distinct from the subunits of alpha-thrombin-activated factor V (factor Va). The activation phase of the reaction was not significantly affected by the presence of phospholipid. In contrast, the rate of degradation of active fragments of factor V and the accompanying loss of activity were markedly enhanced in the presence of phospholipid vesicles. These data suggest that the action of plasmin upon factor V results in the transient formation of proteolytic fragments which express significant procoagulant activity.     

The regulation of neutrophil phospholipase A2 by granulocyte-macrophage colony-stimulating factor and its role in priming superoxide production

Experiments were performed to investigate the relative role of phospholipase A₂(PLA₂) in the activation and cytokine-mediated priming of neutrophil superoxide production. PLA₂ activity was measured with a radiometric assay which discriminates between PLA₂ and the downstream enzyme, 5-lipoxygenase. In cells that had not been primed by prior incubation with granulocyte-macrophage collony stimulating factor (GM-CSF), PLA₂ and NADPH oxidase were differentially stimulated by the chemotactic peptide N-formyl-met-leu-phe (FMLP), calcium ionophore, or phorbol ester. In addition, inhibition of PLA₂ by mepacrine (0–100 μmol/l) did not concomitantly inhibit FMLP-stimulated superoxide production. These findings suggest that the activity of PLA₂ and NADPH oxidase may be uncoupled in the unprimed cell. In cells preincubated with GM-CSF, time- and dose-dependent priming of FMLP-stimulated PLA₂ responses were observed and inhibition of PLA₂ by mepacrine was accompanied by the inhibition of FMLP-stimulated superoxide production down to the level of unprimed cells. The effect of mepacrine was not due to inhibition of FMLP receptor expression. These data suggest that a mepacrine-sensitive PLA₂ may have a role in the GM-CSF mediated priming of superoxide production. Using ionophore-stimulated PLA₂ activity as a model, we showed that Bordatella pertussis toxin did not inhibit GM-CSF mediated priming, demonstrating that a pertussis-sensitive GTP-binding protein does not mediate signal transduction from the GM-CSF receptor to PLA₂. The tyrosine kinase inhibitor, genestein, selectively inhibited GM-CSF primed but not unprimed PLA₂ activity, demonstrating that GM-CSF-mediated priming requires tyrosine kinase activity.     

Allosteric regulation of the cofactor-dependent serine protease coagulation factor VIIa

The integration of structure and function analysis of the tissue factor–factor VIIa complex has provided a detailed view of the functional surface of the extrinsic activation complex. An incomplete zymogen to enzyme transition is responsible for the strict cofactor dependence of catalytic function of factor VIIa. The mutational analysis demonstrates that factor VIIa is allosterically regulated by specific conformational linkages that involve the cofactor binding site, the catalytic cleft, and the macromolecular substrate exosite. Regions of the flexible activation domain appear to play an important role in the allosteric regulation of this cofactor-dependent coagulation serine protease.     

Biology of human megakaryocyte factor V

To learn more about human megakaryocyte coagulation cofactor V (FV), we studied the expression of this protein in normal bone marrow megakaryocytes and in megakaryocytes cloned from their colony-forming unit in FV-depleted plasma clot cultures. Mouse monoclonal antibodies directed against either the light chain or an activation peptide of human FV and a rabbit polyclonal, monospecific FV antiserum were used as probes for these experiments in conjunction with a variety of immunochemical detection techniques. All morphologically recognizable megakaryocytes were shown to contain FV. The origin of this protein appeared to be both from FV bound to the cell as well as from endogenous FV in the majority of cells examined. The existence of a population of small bone marrow mononuclear cells that simultaneously expressed platelet glycoproteins and FV was also noted. Such cells represented approximately 70% of all small cells positive for platelet glycoproteins. In contrast, only about 40% of megakaryocyte colonies cloned in FV-deficient medium contained cells with immunochemically detectable FV. FV expression was most clearly demonstrated in large cells in the colonies, whereas smaller, presumably less mature cells labeled weakly or not at all. Synthesis of FV by human megakaryocytes was documented using elutriation-enriched cells incubated in 35S-methionine-containing medium. Megakaryocyte lysates and medium conditioned by these cells were subjected to immunoaffinity column purification. Column eluates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed radioactive bands comigrating with the heavy and light chains of thrombin-activated FV. These studies suggest that human megakaryocytes both bind and synthesize FV. Expression of these traits appears to be related to cell maturation, with binding ability appearing earlier than the ability to synthesize this protein. Finally, although the ability to bind FV appears to be universal among megakaryocytes, our culture data suggest that synthesis may be a restricted, or constitutively expressed property of these cells.     

Cell-surface heparan sulfate proteoglycan-mediated regulation of human neutrophil migration by the serpin antithrombin III

The serpin antithrombin III (AT III) is reported to have hemostasis-regulating and anti-inflammatory properties. To determine its ability to influence thrombin-independent leukocyte responses, the direct effects of the AT III concentrate Kybernin P and a monoclonal antibody-purified AT III on neutrophil migration were studied. Chemotactic activity of human neutrophils isolated from the blood of healthy donors was determined in modified Boyden microchemotaxis chambers, and binding studies were performed according to standard experimental protocols. Preincubation in vitro of neutrophils with Kybernin P or immune-adsorbed AT III significantly deactivated migration toward fMet-Leu-Phe, or interleukin-8 (IL-8), in a concentration-dependent manner. In the absence of additional attractants, neutrophils exhibited a migratory response toward gradients of AT III preparations. True chemotaxis was confirmed in checkerboard assays. Analyses revealed that the AT III heparin-binding site interacts with neutrophil membrane-associated heparan sulfate proteoglycan receptors. Mechanisms of intracellular signaling differed; the deactivation of IL-8-induced chemotaxis resulted from tyrphostin-sensitive interactions of AT III-signaling with the IL-8 signal transduction pathway, whereas AT III-induced chemotaxis involved protein kinase C and phosphodiesterases. Signaling similarities between AT III and the proteoglycan syndecan-4 may suggest the binding of AT III to this novel type of membrane receptor. Under physiological conditions, AT III may prevent neutrophils from premature activation. Moreover, the systemic administration of AT III concentrate could have beneficial effects in combating systemic inflammation.     

Correlation between the stimulation of human neutrophil function by monoclonal antibody and by colony-stimulating factor

Purified human neutrophils from 48 individuals were tested for their capacity to kill antibody-coated target cells in vitro in the absence or presence of stimulating agents. The agents used to stimulate cytotoxic capacity were the monoclonal antibody (MAb) WEM-G1, colony- stimulating factor (CSF-alpha), or mononuclear cell supernatant (MNC- SN). There existed an heterogeneity among the neutrophils of different individuals in the capacity to kill target cells both in the unstimulated ("resting") or the stimulated state. A positive correlation was found between the ability of neutrophils to kill in the "resting" state and their capacity to be stimulated by MAb WEM-G1, CSF- alpha, or MNC-SN. Furthermore, a strong positive correlation in the ability of neutrophils to be stimulated by the MAb WEM-G1 and either CSF-alpha (r = .76) or MNC-SN (r = .68), as well as between CSF-alpha and MNC-SN (r = .79) was demonstrated. No correlation was seen, however, between stimulation of neutrophil function in vitro and total blood leukocyte counts, neutrophil counts, monocyte counts, or intensity of binding of MAb WEM-G1. The observation that neutrophils respond to a similar extent to different types of stimulators, -such as cytokines (CSF-alpha and MNC-SN) and MAb, suggests that these two factors may be operating through a common mechanism and the degree of stimulation may reflect an intrinsic responsiveness of neutrophils that differs among individuals. Our results also suggest a potential clinical use of WEM-G1 in measuring neutrophil functional capacity in vitro and predicting the capacity to respond to CSF-like cytokines.     

Identification of human megakaryocyte coagulation factor V

Specific monoclonal and polyclonal antibody reagents and a double antigen indirect immunofluorescence microscopy technique were used to visualize coagulation factor V in human bone marrow. Marrow aspirates were smeared directly on glass slides, or washed and cytospun onto glass slides, or processed and plated into a plasma/methylcellulose cell culture system. Morphologically identifiable colonies of megakaryocytes, erythrocytes, granulocytes, or monocytes/macrophages were removed from 14- to 18-day marrow culture dishes by micropipette and streaked onto glass slides. Smears of marrow cell preparations were air-dried, fixed, washed, and incubated sequentially with primary IgG antibody reagents and with secondary anti-IgG antibody reagents conjugated with either fluorescein or rhodamine. Preparations were examined and photographed through a microscope suitably equipped for two-color fluorescence and phase contrast analysis. Cells of megakaryocytic lineage were identified by their immunofluorescent reactivity with murine monoclonal antibody HP1-1D, specific for human platelet plasma membrane glycoprotein IIb/IIIa (GP IIb/IIIa), or by their immunofluorescent reactivity with monoclonal or polyclonal antibodies specific for von Willebrand factor (vWF) or for platelet factor 4 (PF4). Coagulation factor V in bone marrow was detected by simultaneous immunofluorescent staining with polyclonal burro anti-human factor V antibody or with a panel of murine monoclonal anti-human factor V antibodies. The double antigen immunofluorescence staining technique, incorporating appropriate controls, revealed that coagulation factor V was principally located in marrow cells simultaneously identified as megakaryocytes by antibodies to GP IIb/IIIa, vWF, or PF4. The specific immunofluorescence of factor V in megakaryocytes and platelets was eliminated when excess purified factor V antigen was preincubated with anti-factor V antibody. Our observations establish the presence of human megakaryocyte coagulation factor V, confirm the presence of human platelet factor V, and indicate that human megakaryocyte/platelet coagulation factor V is a lineage-associated protein.     

Covalent crosslinking of human coagulation factor V by activated factor XIII from guinea pig megakaryocytes and human plasma

Coagulation factor V (FV) has been shown to be synthesized in both the liver and megakaryocytes. We now present evidence that FV can be covalently crosslinked by an enzyme originating from megakaryocytes to form polymeric multimers of factor V. The guinea pig megakaryocyte enzyme appears to be factor XIIIa since the FV-crosslinking activity (1) had an absolute requirement for Ca++, (2) was completely inhibited by iodoacetamide, 5,5'-dithiobis- (2-nitrobenzoic acid), p- chloromercuribenzene sulfonic acid, and N-ethylmaleimide, all known alkylators of the thiol group at the active site of the factor XIIIa, (3) was blocked by known pseudoamine donor substrates of factor XIIIa including dansylcadaverine and putrescine, and (4) could be directly demonstrated in the guinea pig megakaryocyte lysate by a specific activity staining procedure. No tranglutaminase was detected in guinea pig megakaryocytes in contrast to red cells and liver. A similar pattern of covalent crosslinking of human FV by purified activated human plasma factor XIII was also demonstrated. Analysis of the crosslinked products of FV formed by the guinea pig enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicates the formation of intermediate as well as higher molecular weight polymers, suggesting that the crosslinking is a stepwise polymerization process.     

Impaired activity of protease inhibitors towards neutrophil elastase bound to human articular cartilage.

OBJECTIVE: To investigate the effects of protease inhibitors on the ability of free and cartilage bound neutrophil elastase to degrade cartilage proteoglycan in vitro. METHODS: Cryostat sections of human articular cartilage were used as substrate, and proteoglycan loss induced by free or cartilage bound elastase was quantified by alcian blue staining, followed by scanning and integrating microdensitometry. RESULTS: High molecular mass protease inhibitors (alpha 1 protease inhibitor, alpha 2 macroglobulin, and soya bean trypsin inhibitor) and synovial fluid from patients with rheumatoid arthritis were effective in blocking proteoglycan loss from sections treated with free elastase, but their activity towards cartilage bound elastase was much reduced. In contrast, low molecular mass elastase inhibitors (N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone and ONO-5046 (N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulphonylamino]benzoyl] amino-acetic acid) were effective against free and cartilage bound elastase. CONCLUSION: The binding of elastase to cartilage appears to be a mechanism whereby the enzyme can remain active in the presence of high molecular mass protease inhibitors.     

Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V

Purified human factor VIII procoagulant protein (VIII:C) was treated with purified human activated protein C (APC) and the loss of VIII:C activity correlated with proteolysis of the VIII:C polypeptides. APC proteolyzed all VIII:C polypeptides with mol wt = 92,000 or greater, but not the doublet at mol wt = 79-80,000. These results and our previous thrombin activation studies of purified VIII:C, are analogous with similar studies of factor V and form the basis for the following hypothesis: activated VIII:C consists of heavy and light chain polypeptides [mol wt = 92,000 and mol wt = 79-80,000 (or 71-72,000), respectively] which are similar in Mr to the heavy and light chains of activated factor V. Thrombin activates VIII:C and V by generating these polypeptide chains from larger precursors and APC inactivates both molecules by cleavage at a site located in the heavy chain region of activated VIII:C and V.     

Monoclonal antibodies to human coagulation factor V and factor Va

BALB/c mice were immunized with human factor V. The immunogen was a mixture of procofactor (factor V) and thrombin-activated cofactor (factor Va). Spleen cells were obtained from an immunized animal and fused with NS-1 murine myeloma cells. Hybrid cell cultures were assayed for the production of antibodies to human factor V and factor Va by a solid-phase radioimmunoassay. Factor V and/or factor-Va-specific antibodies were detected in 38 of the 96 cultures assayed. The cells from 10 of these positive cultures were subcloned by limiting dilution and grown as ascites tumors in BALB/c mice. Ascitic fluids were obtained and characterized with respect to their binding interaction with human factor V and factor Va. Three hybridoma cell lines produce monoclonal antibodies that react equally well with factor V and factor Va. Another antibody reacts with both antigens, but the reactivity with factor V is better than with factor Va. An additional two antibodies react with factor Va better than factor V in the radioimmunoassay (RIA). The remaining four antibodies react exclusively with factor V. A previously described murine monoclonal antibody to human factor V (alpha HFV-1) has been used to study the peptides produced during the thrombin-catalyzed activation of human factor V. This antibody binds both factor V and factor Va, releases them at high ionic strength, and has an apparent dissociation constant for factor Va of 3 x 10(-9)M. When human factor V (mol wt 330,000) is activated by thrombin and passed over an alpha HFV-1-Sepharose affinity resin, factor Va binds and subsequently can be eluted. The eluate in 1.2 M NaCl contains two fragments of apparent mol wt 93,000 and 70,000. EDTA, which inactivates factor Va, promotes release of the mol wt 93,000 fragment from factor Va bound to the antibody. Subsequent elution with 1.2 M NaCl releases the mol wt 70,000 fragment. These observations indicate that human factor Va is a two subunit protein and that the epitope for alpha HFV-1 is on the mol wt 70,000 fragment.