Dissemination of contact activation in plasma by plasma kallikrein.

The dissemination of contact activation of plasma was examined by measuring the cleavage of Hageman factor (HF) molecules on two separate sets of kaolin particles, one of which contained all of the components of the contact activation system, HF, prekallikrein (PK) and high molecular weight kininogen (HMWK) in whole normal plasma, and the second set of particles containing only HF and HMWK, being prepared with PK-deficient plasma. After mixing of the particles, cleavage of HF on the second set of particles occurred at a rate similar to that occurring on the first set of particles. This indicated that rapid dissemination and burst of activity of the contact reaction takes place in fluid phase. A supernatant factor, responsibel for the dissemination of the contact reaction, was identified as kallikrein. A rapid appearance of cleaved PK (kallikrein) and HMWK on both the kaolin surface and in the supernate was observed. Within 40 s, > 70-80% of the PK and HMWK in the supernate was cleaved. On the surface, approximately 70% of each radiolabeled protein was cleaved at the earliest measurement. Cleavage of PK by activated HF occurred at least 17 times faster on the surface than in the fluid phase, as virtually no cleavage of PK occurred in fluid phase. Each molecule of surface-bound, activated HF was calculated to cleave at a minimum, 20 molecules of PK per minute. It is concluded that the contact activaton of plasma may be divided into three phases: (a) the reciprocal activation of a few molecules of zymogen HF and PK on the surface, with HMWK acting as cofactor to bring these molecules into apposition; (b) the rapid release of kallikrein into the fluid phase and the continued conversion of PK to kallikrein by each surface-bound molecule of activated HF; and (c) the activation by fluid-phase kallikrein of multiple surface-bound HF molecules, and the cleavage of multiple molecules of MHWK both in fluid phase and on the surface by the soluble kallikrein. The evidence suggests that steps b and c account for a great majority of the generation of contact activation of plasma.

Activation of Hageman factor in solid and fluid phases. A critical role of kallikrein.

The activation of Hageman factor in solid and fluid phase has been analyzed. Activation of highly purified Hageman factor occurred after it interacted with and became bound to a negatively charged surface. Activation was observed in the absence of enzymes that are inhibitable with diisopropylfluorophosphate, phenyl methyl sulfonyl fluoride and epsilon-amino-n-caproic acid. The binding of [(125)I]Hageman factor to the negatively charged surface was markedly inhibited by plasma or purified plasma proteins. Activation of Hageman factor in solution (fluid phase) was obtained with kallikrein, plasmin, and Factor XI (plasma thromboplastin antecedent). Kallikrein was greater than 10 times more active in its ability to activate Hageman factor than plasmin and Factor XI. The data offer a plausible explanation for the finding that highly purified kallikrein promotes clotting of normal plasma. In addition, the combined results of this and previously reported data from this laboratory indicate that the reciprocal activation of Hageman factor by kallikrein in fluid phase is essential for normal rate of activation of the intrinsic-clotting, kinin-forming, and fibrinolytic systems. Activation of Hageman factor was associated with three different structural changes in the molecule: (a) Purified Hageman factor, activated on negatively charged surfaces retained its native mol wt of 80-90,000. Presumably a conformational change accompanied activation. (b) In fluid phase, activation with kallikrein and plasmin did not result in cleavage of large fragments of rabbit Hageman factor, although the activation required hydrolytic capacity of the enzymes. (c) Activation of human Hageman factor with kallikrein or plasmin was associated with cleavage of the molecule to 52,000, 40,000, and 28,000 mol wt fragments. Activation of rabbit Hageman factor with trypsin resulted in cleavage of the molecule into three fragments, each of 30,000 mol wt as noted previously. This major cleavage occurred simultaneously with activation.

Surface and fluid phase activities of two forms of activated Hageman factor produced during contact activation of plasma.

The ability of the two forms of activated Hageman factor (HFa) produced during contact activation of plasma to activate prekallikrein and factor XI was studied. alpha-HFa, defined as an 80,000 mol wt two-chain enzyme which remains bound to the surface was capable of cleaving surface-bound prekallikrein and factor XI. beta-HFa, a 28,000 mol wt single chain molecule, released from the surface during contact activation was able to cleave prekallikrein but showed no activity on factor XI. Cleavage of prekallikrein by beta-HFa occurred irrespective of whether the substrate was surface-bound or in solution. Cleavage of factor XI occurred only when it was surface bound and only the alpha-form of HFa was capable of this proteolytic action. Factor XI was found to remain bound to the surface while prekallikrein and kallikrein rapidly dissociated from the surface into the supernate. These findings suggest that the initiation of intrinsic coagulation through the activation factor XI is a localized event occurring at the site of contact activation and is the result of the action of alpha-HFa. By contrast, kinin generation and fibrinolysis resulting from the formation of kallikrein can be initiated either at the site of contact activation, by alpha-HFa action, or throughout the plasma, by beta-HFa; further dissemination of these activities is assured by the rapid dissociation of kallikrein itself from the surface.

Pulmonary surfactant protein B (SP-B): structure-function relationships.

SP-B is a protein in pulmonary surfactant that is, in greatest part, responsible for resistance to surface tension and prevention of collapse of pulmonary alveoli. Peptides of 21 residues, synthesized following the sequence of SP-B or resembling the hydrophobic and hydrophilic domains of SP-B (such as RLLLLRLLLLRLLLLRLLLLR, R, Arg, and L, Leu), enhanced the abilities of phospholipids to reduce surface tension both in vitro and in vivo. Intermittent positively charged residues were essential for this activity. The SP-B-like peptides were found by tryptophan fluorescence to partition within the phospholipid layer in contact with both polar head groups and acyl side chains. These data, together with findings that the SP-B-related peptides increase inter- and intramolecular order of the phospholipid layer, suggest that SP-B resists surface tension by increasing lateral stability of the phospholipid layer.

The relationship of structure and function in human Hageman factor. The association of enzymatic and binding activities with separate regions of the molecule.

Three regions of the human Hageman factor molecule termed the c, d, and e regions have been defined. Division of the molecule into these three regions is based on the analysis of fragments obtained by enzymatic cleavage during fluid-phase activation. The three regions have the following properties: (a) the c region has a mol wt of 40,000, has the capacity to bind to negatively charged surfaces, and does not have detectable enzymatic activity; (b) the e region possess a mol wt of 28,000 has enzymatic activity, and does not bind to negatively charged surfaces; (c) the d region has a mol wt of 12,000, is located between the c and e fragments but has not been detected as a freely existing polypeptide, and can bind firmly to negatively charged surfaces. The preparation of antibodies specific for the c and e regions is described as well as their use in defining the electrophoretic characteristics of the cde, cd, de, c, and e polypeptide fragments of Hageman factor. Evidence is given showing that the e region, but not the c or d, is released from a negatively charged surface when bound Hageman factor is exposed to proteolytic enzymes or whole plasma and that when this occurs in the presence of normal plasma, the e fragment becomes bound to C1 esterase inhibitor.

Pathogenesis of the adult respiratory distress syndrome. Evidence of oxidant activity in bronchoalveolar lavage fluid.

Evidence is presented indicating that oxidants are generated in lungs of patients with the adult respiratory distress syndrome (ARDS). The evidence was derived from observations that alpha-1-PI, recovered in bronchoalveolar lavage (BAL) fluid, had been inactivated by oxidation, presumably oxidation of the methionyl residue in the reaction site of the molecule. This was indicated by findings that activity of the alpha-1-PI could be restored by exposure to the reducing agent, dithiothreitol in the presence of methionyl sulfoxide peptide reductase. The amount of activity restored was proportional to the amount of inactive alpha-1-PI present at 52,000 D. Oxidation of the 52,000-D alpha-1-PI was also revealed by the finding that the inactive molecule was subject to proteolytic cleavage to 47,000 D when exposed to porcine pancreatic elastase, a characteristic of alpha-1-PI with oxidized methionyl residues in the reactive site. Inactivation of the alpha-1-PI in vivo also resulted from complexing to an active enzyme, shown previously to be neutrophil elastase, and from proteolytic cleavage in vivo, that produced a fragment of 47,000 mol wt. In contrast to that in BAL fluids, the alpha-1-PI in plasma of patients with respiratory distress syndrome was found to be greater than 90% active in 14 of 22 cases and 50-90% active in 8 cases. This suggested that for the most part, alpha-1-PI was inactivated after leaving the vessels and entering the lung. The circulating alpha-1-PI in patients with the respiratory distress syndrome was found to be equally susceptible to oxidative inactivation as alpha-1-PI from normal individuals. It seems improbable therefore that patients develop ARDS because of labile alpha-1-PI inhibitor.

The binding and cleavage characteristics of human Hageman factor during contact activation. A comparison of normal plasma with plasmas deficient in factor XI, prekallikrein, or high molecular weight kininogen.

The ability of human Hageman factor (coagulation factor XII) to bind to a glass surface and its susceptibility to limited proteolytic cleavage during the contact activation of plasma have been studied using normal human plasma and plasmas genetically deficient in factor XI, prekallikrein, or high molecular weight kininogen (HMWK). When diluted normal plasma containing (125)I-Hageman factor was exposed to a glass surface for varying times, the Hageman factor was found to bind to the surface, and within 5 min became maximally cleaved from its native 80,000 mol wt to yield fragments of 52,000 and 28,000 mol wt. Hageman factor in factor XI-deficient plasma behaved similarly. In prekallikrein-deficient plasma, the binding of Hageman factor to the glass surface occurred at the same rate as in normal plasma but the cleavage was significantly slower, and did not reach maximum until 60 min of incubation. Cleavage of Hageman factor in HMWK-deficient plasma occurred at an even slower rate, with greater than 110 min of incubation required for maximal cleavage, although the rate of binding to the glass was again the same as in normal plasma. Normal rates of cleavage of Hageman factor were observed for the deficient plasmas after reconstitution with purified human prekallikrein or HMWK, respectively. These observations suggest that normal contact activation in plasma is associated with proteolytic activation of surfacebound Hageman factor. The cleavage of the surface-bound Hageman factor molecule responsible for the formation of the 52,000-and 28,000-mol wt fragments occurred at two closely situated sites, one of which was within a disulfide loop. Cleavage at the site external to the disulfide bond resulted in the release from the surface of the 28,000-mol wt fragment. Cleavage at the site within the disulfide loop resulted in the formation of a 28,000-mol wt fragment which remained surface bound, presumably by virtue of the disulfide linkage to the larger fragment.

Proteases and oxidants in experimental pulmonary inflammatory injury.

We have examined various biochemical parameters of pulmonary inflammation in experimental animals. Intrabronchial instillation of glucose oxidase-glucose (GO/G) to produce oxidants or formylated norleu-leu-phe (FNLP) or phorbol myristate acetate (PMA) as leukocytic stimuli induced severe acute pulmonary injury in New Zealand white rabbits. PMA also induced inflammation when administered intravenously. Each stimulus induced transudation of protein from the vascular space into the pulmonary tissues, and an influx of leukocytes during the 4-6 h period of the experiment. Pathophysiologic changes were measured by edema formation (transudation of 125I-bovine serum albumin), and histologic examination. Biochemical analysis was performed by measuring concentrations of potentially injurious agents in bronchoalveolar lavage (BAL) fluid. Increased acid protease and myeloperoxidase levels were found in the BAL fluid after administration of either of the stimuli. Evidence of oxidant generation in vivo was obtained in two different ways. In the first, specific activities for catalase were measured in the BAL fluid in the presence or absence of 3-amino, 1,2,4 triazole (AT), injected at intervals before obtaining BAL fluid. In the presence of AT, specific activities for catalase dropped to 0.22 after a double instillation of FNLP and to 0.15 in the presence of GO/G. In neutrophil-depleted FNLP animals, catalase was not greatly inhibited by AT (sp act 0.90). In the second, intracellular levels of total glutathione (GSH + GSSG) in whole lung tissue and alveolar macrophages decreased when stimuli of neutrophils were administered. Intrabronchially instilled PMA, e.g., caused a drop of glutathione in whole lung tissue from the control value of 2.3 mumol GSH equivalent/100 mg dry wt to 0.54 mumol GSH equivalent/100 mg dry wt at 4 h. Neutrophil depletion and superoxide dismutase protected from this effect. From these results, we conclude that O-2 or its metabolites can initiate severe pulmonary injury as shown by the effect of GO/G and that, during development of pulmonary injury, stimulated neutrophils generate oxidants and release proteolytic enzymes into the surrounding tissues.

Structural changes accompanying enzymatic activation of human Hageman factor.

The structure of Hageman factor, isolated from human plasma, was analyzed before and after enzymatic activation. The purified molecule is a single polypeptide chain of 80,000 molecular weight (mol wt) sedimenting at 4.5S. An amino acid analysis has been performed. The concentration of Hageman factor in normal human plasma was found to be 29 mug/ml with variation between individuals ranging from 15 to 47 mug/ml. Treatment of the molecule with kallikrein, plasmin, or trypsin resulted in cleavage at two primary sites, yielding fragments of 52,000, 40,000, and 28,000 mol wt. No further changes occurred in the fragments with subsequent reduction. Prekallikrein-activating ability was associated exclusively with the 28,000 moiety.

Use of human surfactant low molecular weight apoproteins in the reconstitution of surfactant biologic activity.

Two low molecular weight (LMW) apoproteins were isolated from human pulmonary surfactant. SDS polyacrylamide gel analysis showed one protein (SP 18) to have an apparent molecular weight of 18,000 when unreduced and 9,000 D after reduction. The second protein (SP 9) migrated at approximately 9,000 D in the presence or absence of reducing agents. Both proteins contain a high number of hydrophobic amino acids. The NH2-terminal sequence of SP 18 was determined to be: NH2-phe-pro-ile-pro-leu-pro-tyr-. A cDNA clone isolated from a human adult lung cDNA library contained a long open reading frame encoding at an internal position the human SP 18 amino-terminal sequence. Mixtures of phospholipids (PL) and SP 9 and SP 18 were assessed for their capacity to reduce surface tensions on a pulsating bubble surfactometer. The addition of 1% apoprotein resulted in a reduction of surface tension after 15 s from 42.9 dyn/cm for PL alone to 16.7 and 6.3 dyn/cm for preparations containing SP 9 and SP 18, respectively. In vivo assessment of reconstituted surfactant activity was performed in fetal rabbits. Reconstituted surfactant consisting of PL + 0.5% SP 18 instilled intratracheally at delivery resulted in a marked increase in lung compliance, while the incorporation of 0.5% SP 9 yielded a moderate increase. These data show the ability to produce biologically active surfactant by the addition of isolated LMW apoproteins to defined PL.

The interaction of Hageman factor and immune complexes.

The possible interaction of Hageman factor from human or rabbit plasma with a variety of immunologic reactants was studied. Evidence of an interaction was not obtained and neither binding of radiolabeled Hageman factor to immune aggregates nor depletion of the Hageman factor from the supernate was observed. Cleavage of the labeled Hageman factor molecule into its 30,000 molecular weight-active fragments was not detectable after incubation with immune complexes. Isolated Hageman factor was far more sensitive to activation than Hageman factor in plasma or serum. There was no consistent activation of isolated Hageman factor by immunologic reactants as determined by conversion of prekallikrein to its enzymatic form or by shortening of the clotting time of factor XII-deficient plasma. A variety of immunologic stimuli were tested: (a) antigen-antibody complexes in soluble or precipitated form; (b) particulate antigen-antibody complexes, i.e., zymosan-anti-zymosan in which a surface was presented for activation; (c) human IgM-IgG and IgG-IgG (rheumatoid factor) complexes; (d) immune aggregates consisting of heat or bis-diazotized benzidine-aggregated myeloma proteins of all human immunoglobulin classes and subclasses: IgG(1,2,3,4), IgA, IgD, IgM, and IgE. Absorption with immune aggregates did not reduce the quantity of Hageman factor in solution, nor was the Hageman factor bound to the precipitates. The presence of plasma or serum with immune aggregates did not generate activity of the Hageman factor. The only preparations of immunoglobulins capable of activating Hageman factor were found to be contaminated with bacteria. These bacteria, upon isolation, activated Hageman factor.

Experimental pulmonary inflammatory injury in the monkey.

Inflammatory pulmonary injury was induced in Macaca mulatta rhesus monkeys by the intrabronchial instillation of the formylated peptide norleu-leu-phe (FNLP) or phorbol myristate acetate (PMA). Indicators of pulmonary injury included an increase in mean protein content of bronchoalveolar lavage (BAL) fluid from 0.51 mg/ml in untreated animals to 3.74 mg/ml and 6.64 mg/ml in FNLP- and PMA-treated animals, respectively, the appearance of a diffuse pulmonary infiltrate in chest roentgenograms, and histologic evidence of a predominantly neutrophilic leukocytic infiltration. Concomitant with the appearance of pulmonary injury was the generation of proteases and oxidants in the BAL fluids. Neutrophil elastase, bound to alpha 1-protease inhibitor (alpha 1-PI), was found to increase from 0.47 micrograms/ml in untreated monkeys to 0.99 micrograms/ml in FNLP-treated animals and 1.23 micrograms/ml in monkeys receiving PMA. Radioiodinated human prekallikrein, instilled for 2 min into the inflammatory site and retrieved by lavaging, was found to have undergone proteolytic cleavage; this cleavage was not consistently inhibitable with the inclusion of antibody to elastase. BAL fluids were shown to contain an amidolytic activity when tested on the synthetic substrate H-D-pro-phe-arg-pNA. This activity was partially inhibitable with known inhibitors of active Hageman factor and kallikrein. beta-Glucuronidase levels in the BAL fluids increased from 0.85 U/ml to 4.36 U/ml and 8.25 U/ml in FNLP- and PMA-treated animals, respectively. Myeloperoxidase (MPO) levels also increased from 1.37 OD U/ml X min to 16.59 and 30.47 OD U/ml X min in the same groups of animals. Oxidant generation was also assessed in several different ways. The specific activity of the oxidant-sensitive inhibitor alpha 1-PI recovered in the BAL fluid decreased from 0.80 in control samples to 0.57 and 0.65 in FNLP- and PMA-treated animals. That this inactivation was due to oxidant injury of the molecule was confirmed by the return to full activity of four out of five BAL samples after their incubation with the reducing agent dithiothreitol in the presence of methionine sulfoxide peptide reductase. The specific activity of catalase in the BAL fluids of animals given 3-amino, 1,2,4 triazole (AT) 1 h before lavaging showed drops from 0.97 in untreated monkeys to 0.04 in FNLP-treated and 0.49 in PMA-treated monkeys. MPO levels also fell in the AT-treated injured animals from 16.59 to 0.85 delta OD/min X ml in FNLP animals in the absence and presence of AT, and 30.47 to 0.60 delta OD/min X ml in PMA-treated animals. Inhibition of MPO by AT was shown in vitro to be H2O2 dependent. Total glutathione levels in the BAL fluids did not change appreciably after FNLP or PMA treatment. These studies present substantial evidence of the generation of both proteases and oxidants during the establishment of acute pulmonary inflammatory injury in an experimental primate model.

Protein-phospholipid interactions in pulmonary surfactant. The Parker B. Francis Lectureship.

SP-B is the protein in pulmonary surfactant with the greatest capacity to augment the phospholipids, ability to resist surface tension, and capability to prevent collapse of pulmonary alveoli. Synthetic peptides derived from the structure of SP-B and simplified analogues of these SP-B-derived peptides were found by tryptophan fluorescence to partition within the phospholipid layer in contact with both polar head groups and acyl side chains of the phospholipids. The intermittent hydrophilic basic residues were found to be essential for full activity, probably because of electrostatic interactions formed with phosphates of the polar head groups. The hydrophobic stretches of residues in SP-B and the related peptides supplement the activity through interaction with the phospholipid acyl side chains. By increasing intermolecular and intramolecular order of the phospholipid layer and thereby stability of the layer, the SP-B analogues provide strong surfactant activity. Simplified peptide analogues of SP-B, dispersed in DPPC and POPG, provide strong surfactant activity in vitro and in the lungs of premature infant rabbits, rhesus monkeys, and humans.

Activation of human Hageman factor by a leukocytic protease.

We earlier reported the IgE-mediated release of a basophil kallikrein of anaphylaxis (BK-A) which, like plasma kallikrein, is an arginine esterase and cleaves human plasma kininogen generating immunoreactive kinin. We herein report that, like plasma kallikrein, preparations rich in this basophil protease also activate human Hageman Factor by proteolytic cleavage of the zymogen molecule into light and heavy chains. These fragments of 28,000 and 52,000 daltons are similar in size to those produced during activation of Hageman Factor by plasma kallikrein. Exposure of Hageman Factor (bound to a negatively charged surface) to BK-A led to the proteolytic cleavage of Hageman Factor producing a 28,000 molecular weight fragment (HFa) which is functionally active and capable of activating prekallikrein to kallikrein. We conclude that, during anaphylaxis, basophils may release a protease that is capable of cleaving and activating Hageman Factor, thus providing a mechanism for initiating the in vivo activation of the Hageman Factor dependent systems.

Biochemical events associated with pulmonary failure in shock and trauma.

Evidence obtained by biochemical analysis of BAL fluids from patients with ARDS indicates that at least 2 important pathogenic events take place in the pulmonary tissues. These are the release of neutrophil elastase and the generation of oxidants. Both events can lead to severe pulmonary injury as has been demonstrated in experimental animals. To better understand the mechanisms of oxidant damaged cells, H2O2 was added to cultured cells. H2O2 compromises a multitude of cellular functions, the combination of which leads to cell death. DNA is an important target for oxidant-induced injury. The formation of DNA strand breaks leads to activation of pADP-RP which in turn causes depletion of NAD and ATP, followed by Ca++ influx and eventually cell lysis. Inhibition of pADP-RP prevented cell lysis, but not DNA damage. A similar sequence of events has been described for cell injury following DNA damage induced by gamma-irradiation and alkylating agents and was proposed to be a suicide mechanism for cells with irreversibly damaged DNA. Sublethal doses of H2O2 will delay cell replication, but not necessarily prevent it.

Biochemical factors in pulmonary inflammatory disease.

Various biochemical events taking place during pulmonary inflammation were examined in the bronchoalveolar lavage (BAL) fluids from patients with acute respiratory distress syndrome (ARDS) and in experimental animal models. In patients with ARDS, active neutrophil elastase was found in the BAL fluids. In these fluids, inactivation of the major elastase inhibitor alpha 1-protease inhibitor (alpha 1-PI) occurred. This was caused by oxidation of a methionine residue at the active site of the alpha 1-PI, and offered indirect evidence of oxidation occurring in the inflamed pulmonary tissues. Studies with experimental animals have been initiated to gain understanding of the relative roles of proteases, oxidants, arachidonate metabolites, complement and contact system components, and other mediators in the pathogenesis of pulmonary inflammation. Intrabronchial instillation of glucose oxidase/glucose to produce oxidants or formylated norleucylleucylphenylalanine or phorbol myristate acetate as leukocytic stimuli induced severe acute pulmonary injury in New Zealand white rabbits and rhesus monkeys. The injury was accompanied by leukocytic protease (acid cathepsins) release in rabbit lungs and oxidant formation, and could be inhibited by neutrophil depletion. Oxidant formation was demonstrated by the inactivation of catalase by 3-amino-1,2,4-triazole in the presence of H2O2, a drop in intracellular glutathione levels, and in the rhesus monkey by inactivation of alpha 1-PI.

Raman spectroscopic studies of model human pulmonary surfactant systems: phospholipid interactions with peptide paradigms for the surfactant protein SP-B.

The temperature dependence of dipalmitoylphosphatidylcholine (DPPC)/phosphatidylglycerol (PG) multilayers, reconstituted with various synthetic peptides for modeling human lung surfactant, was monitored by vibrational Raman spectroscopy. The synthetic peptides consisted, respectively, of residues 59-81 of the human surfactant protein SP-B and 21 amino acid residue peptides containing repeating units of arginine separated by either four or eight leucines (RL4 or RL8). Each peptide demonstrated the ability to reduce significantly the surface tension of analogues of the phospholipid mixture used in the Raman studies. Raman spectroscopic integrated band intensities and relative peak height intensity ratios, two spectral parameters used to determine bilayer disorder, provided sensitive probes for characterizing multilayer perturbations in the reconstituted liposomes. Temperature profiles derived from the various Raman intensity parameters for the 3100-2800-cm-1 carbon-hydrogen (C-H) stretching mode region, a spectral interval representative of acyl chain vibrations, reflected lipid reorganizations due to the bilayer interactions of these peptides. For the three reconstituted multilamellar surfactant systems, the gel-to-liquid-crystalline phase-transition temperatures Tm, defined by acyl chain C-H stretching mode order/disorder parameters, increased from 35 degrees C in the peptide free system to 37-38 degrees C, indicating increased lipid headgroup constraints for the model liposomes. Although the values of Tm were similar for the three recombinant lipid/peptide assemblies, individual phase-transition cooperativities varied significantly between systems and between spectroscopically derived order/disorder parameters.