2M Complex Research Articles

The geometry of multi-qubit entanglement

This paper, a continuation of a previous one (Iwai 2007 J. Phys. A: Math. Theor. 40 1361), studies the geometry of multi-qubit entanglement with respect to bipartite partitions. An n-qubit system (C2)⊗n is isomorphic with , the linear space of 2ℓ × 2m complex matrices, where ℓ + m = n. According to the isomorphism, the local transformation group U(2ℓ) × U(2m) acts on the space of normalized states in . Let M and G denote the space of normalized states and the local transformation group acting on M, respectively. According to the orbit types, M is stratified into strata, among which a principal stratum and the sets of separable states and maximally entangled states will be identified. For , a function F(C) = det(I − CC*) proves to serve as a measure of entanglement, where I denotes the 2ℓ × 2ℓ identity matrix. The F(C) attains the minimal and the maximal values, respectively, on the sets of separable states and maximally entangled states, and further takes no extremal values on the principal stratum. The F(C) projects to a function on the factor space G\\M. A naturally defined metric on M also projects to that on G\\M, which serves to measure the distance between the separable states, F−1(0), and the states, F−1(k), of prescribed value k of measure. To be precise, one has to restrict M to the principal stratum, when projecting the metric. Three- and four-qubit systems will be studied intensively, and then multi-qubit systems discussed.

Classification of indefinite hyper-Kähler symmetric spaces

We classify indefinite simply connected hyper-Kahler symmetric spaces. Any such space without flat factor has commutative holonomy group and signature (4m, 4m). We establish a natural 1-1 correspondence between simply connected hyper-Kahler symmetric spaces of dimension 8m and orbits of the group GL(m,H) on the space (S4Cn)τ of homogeneous quartic polynomials S in n = 2m complex variables satisfying the reality condition S = τS, where τ is the real structure induced by the quaternionic structure of C2m = Hm. We define and classify also complex hyperKahler symmetric spaces. Such spaces without flat factor exist in any (complex) dimension divisible by 4.

Optical properties of substituted phthalocyanine rare-earth metal complexes

Comparative study of optical properties of alkylthio-group-substituted phthalocyanine rare-earth metal sandwich complexes ([(CnS)8Pc]2M,M=Eu,Lu,Tb) is presented. Photoluminescence and photoconductivity of [(CnS)8Pc]2M complex is very weak. Two photoluminescence bands were observed at around 400–650 and 720–800 nm in chloroform solution corresponding to the Soret and Q bands in the absorption spectra, respectively. However, the emission from Eu3+ ion (as well as Tb3+) was not found compared with other Eu complexes because the 5d levels of the Eu3+ ion lie higher than the triplet level of the ligand. The significant enhancement of the photoconductivity of [(C16S)8Pc]2M after C60 doping is reported. The photoconductivity is positive at the low electric field in the ohmic regime while it becomes negative at the high electric field upon photoexcitation with strongly absorbed light. The negative photoconductivity is attributed to space-charge effects. The mechanism of photoluminescence and photoconductivity are discussed by taking the electronic energy schemes of phthalocyanine ligands and lanthanide ion and C60 into consideration.

Singlet oxygen (1O2) Inactivates Plasmatic Free and Complexed α2-Macroglobulin

α 2-macroglobulin (α 2M) is a broad-spectrum proteinase inhibitor and one of the major plasma proteins in humans. Activated phagocytes (especially granulocytes) generate large amounts of oxidants of the HOCl- and chloramine-type that release the mild nonradical, excited (light-emitting) oxidant singlet oxygen ( 1O 2). These oxidants have been shown to inactivate several specific serine protease inhibitors in human blood [e.g., α 1-antitrypsin or α 2-antiplasmin (plasmin inhibitor)]. The studies reported here demonstrate that nonradical oxidants also inactivate plasmatic α 2M. The effective dose for 50% inactivation (ED 50) of plasmatic α 2M is similar to that for plasmatic α 2-antiplasmin. Chloramines are about 1000-fold more effective than hydrogen peroxide (ED 50=0.75 μmol chloramine T/50 μl plasma). Serine protease–serine protease inhibitor complexes are resistant to oxidants. In contrast, here it is shown that α 2-macroglobulin, even after binding to serine proteases is sensitive to oxidation, the captured protease is released from the protease/α 2M complex. This is the first time that oxidative inactivation of a complexed (i.e., bound to a target protease) human protease inhibitor has be shown. The 1O 2 inhibitors methionine, cysteine, cystine, or ascorbate—in contrast to the oxy-radical scavengers mannitol, superoxide dismutase, or catalase—antagonize the chloramine/NaOCl-mediated inactivation of both uncomplexed and complexed α 2M. Thus, the oxidant involved here is of nonradicalic nature and has reaction characteristics of 1O 2. For the inhibitory function, critical oxidizable methionines or the internal thiol-ester might be targets for 1O 2. Consequently, α 2M can also be considered a carrier for proteases, since the α 2M-complexed proteases regain full activity in an oxidative environment. In local areas of inflammation or thrombolysis, activated phagocytes could create microenvironments of uncontrolled protease activity by generation of 1O 2.

Fluorogenic MMP activity assay for plasma including MMPs complexed to alpha 2-macroglobulin.

Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the proteinase inhibitor alpha 2-macroglobulin (alpha 2M). Reconstituted MMP-13/alpha 2M complex was unable to degrade collagen (MW 300,000) in contrast to the low-molecular-weight fluorogenic substrate (MW < 1500). Limited access of high-MW substrates to the active site of MMPs captured by alpha 2M presents the most likely explanation. Consistently, the high-MW inhibitor TIMP (MW approximately 28,000) was unable to inhibit MMP/alpha 2M enzyme activity, whereas the low-MW inhibitor BB94 (MW approximately 500) effectively suppressed enzyme activity. By using fluorogenic substrates with Dabcyl/Fluorescein as quencher/fluorophore combin-ation, sensitive MMP-activity assays in plasma were achieved. Spiking of active MMP-13 and MMP-13/alpha 2M complex, and inhibitor studies with TIMP-1 and BB94, indicated that active MMPs are efficiently captured by alpha 2M in plasma. MMP activity was even detected in control plasma, and was significantly increased in plasma from rheumatoid arthritis patients.

MHC class I phenotype and function of human beta 2-microglobulin transgenic murine lymphocytes.

Lymphoid cells from beta 2-microglobulin (beta 2m) knockout mice transgenic for human (h) beta 2m (C57BL/10 m beta 2m-/h beta 2m+) were compared with normal mice for their binding to exogenously added h beta 2m, binding to a H-2Db peptide and for functional activity in a one-way allogenic MLC. Based on data from cellular binding studies, Scatchard analyses and flow cytometry, it is concluded that exogenous h beta 2m does not bind to hybrid MHC class I (MHC-I) molecules composed of mouse heavy chain/h beta 2m molecules expressed on lymphocytes of transgenic mice. Immunoprecipitation and SDS-PAGE analysis of metabolically labelled normal C57BL/6 lymph node cells showed binding of exogenous h beta 2m to MHC-I, in particular, to the H-2Db molecule through an exchange with endogenous mouse beta 2m. In contrast to normal H-2Db molecules, hybrid H-2Db expressed on the surface of transgenic lymphocytes binds radiolabelled peptide in the absence of exogenous added h beta 2m suggesting that a stable fraction of hybrid H-2Db molecules is empty or contain peptides with very low affinity. In a one-way allogenic mixed lymphocyte culture, transgenic splenocytes were found to be far less stimulatory than normal splenocytes. In contrast, transgenic alloreactive cytotoxic T lymphocytes developed earlier in MLC than their non-transgenic counterparts. These data indicate that the hybrid mouse heavy chain/h beta 2m complex alters the alloantigenic repertoire and influences important aspects of T-cell activation.

Interleukin 1 beta (IL-1 beta) and the IL-1 beta-alpha 2-macroglobulin complex upregulate the platelet-derived growth factor alpha-receptor on rat pulmonary fibroblasts.

Fibroblasts are the primary proliferating cell type in pulmonary fibrosis. We previously showed that inorganic, fibrogenic particles alter the platelet-derived growth factor (PDGF) receptor system on rat lung fibroblasts (Bonner, J. C., et al. 1993, J. Clin. Invest 92:425-430). In lung fibroblasts, PDGF is the most potent proliferative cytokine, and the responses to PDGF isoforms depend on the relative amounts of two PDGF receptors (PDGF-R alpha and PDGF-R beta). Interleukin 1 beta (IL-1 beta) production by lung macrophages is increased following exposure to fibrogenic particles. We have examined the role of IL-1 beta in regulating the lung fibroblast PDGF receptor system. IL-1 beta induced a 10-fold increase in the number of binding sites for [125I]PDGF-AA, caused a 2-fold increase in affinity of [125I]PDGF-AB, but it had no effect on [125I]PDGF-BB binding. PDGF-R alpha gene expression was increased 5-fold after 4 h of IL-1 beta treatment. IL-1 beta increased the proliferative and chemotactic response to PDGF isoforms in the following order of potency: AA > AB > BB. IL-1 beta was tested for its ability to cause increased [125I]PDGF-AA binding when complexed to its binding protein, alpha 2-macroglobulin (alpha 2M). IL-1 beta bound covalently to fast methyl-amine-activated alpha 2M (alpha 2M-MA). IL-1 beta-alpha 2M-MA or alpha 2M-MA alone possessed minimal activity for inducing an increase in [125I]PDGF-AA binding. However, treatment of the IL-1 beta-alpha 2M complex with thioredoxin, which released bioactive IL-1 beta that was covalently bound to alpha 2M, maximally increased [125I]PDGF-AA binding to the same extent as free IL-1 beta. These results indicate that the fibroblast response to PDGF isoforms is modulated by a complex interaction involving IL-1 beta, alpha 2M, and thioredoxin, all of which are produced in vivo by activated macrophages.

Species-specific differences in chaperone interaction of human and mouse major histocompatibility complex class I molecules.

Previous studies have shown that immature mouse class I molecules transiently associate with a resident endoplasmic reticulum protein of 88 kD that has been proposed to act as a chaperone for class I assembly. Subsequently, this protein was demonstrated to be identical to calnexin and to associate with immature forms of the T cell receptor complex, immunoglobulin, and human class I HLA heavy chains. In this paper we define further the interaction of human class I HLA heavy chains with chaperone proteins and find key differences with the complexes observed in the mouse system. First, calnexin and immunoglobulin binding protein (BiP) both associate with immature HLA class I heavy chains. The two chaperones are not found within the same molecular complex, suggesting that calnexin and BiP do not interact simultaneously with the same HLA class I heavy chain. Second, only free HLA class I heavy chains, and not beta 2-microglobulin (beta 2m)-associated heavy chains are found associated with the chaperones. Indeed, addition of free beta 2m in vitro induces dissociation of chaperone-class I HLA heavy chain complexes. The kinetics for dissociation of the class I HLA heavy chain-chaperone complexes and for formation of the class I HLA heavy chain-beta 2m complex display a reciprocity that suggests the interactions with chaperone and beta 2m are mutually exclusive. Mouse class I heavy chains expressed in human cells exhibit the mouse pattern of interaction with human chaperones and human beta 2m and not the human pattern, showing the difference in behavior is purely a function of the class I heavy chain sequence.

Classification of alpha 2-macroglobulin-cytokine interactions based on affinity of noncovalent association in solution under apparent equilibrium conditions.

alpha 2-Macroglobulin (alpha 2M) binds numerous cytokines; however, since binding affinities have not been determined, it is difficult to compare various alpha 2M-cytokine interactions or predict whether alpha 2M-cytokine complexes will form in the presence of other cytokine-binding macromolecules. In this investigation, we used a novel method to demonstrate that transforming growth factor-beta 1 (TGF-beta 1), TGF-beta 2, nerve growth factor-beta (NGF-beta), platelet derived growth factor-BB (PDGF-BB), tumor necrosis factor-alpha (TNF-alpha), and basic fibroblast growth factor (bFGF) reversibly associate with alpha 2M-methylamine to form noncovalent complexes. Apparent equilibrium was achieved in less than 15 min. Noncovalent alpha 2M-cytokine complexes were converted into covalent complexes; however, this occurred slowly. Therefore, a rapid equilibrium assumption was applied and equilibrium dissociation constants were determined using a single binding site model. KD values for the binding of cytokines to alpha 2M-methylamine varied by 2 orders of magnitude. The rank order of affinity was TGF-beta 2 (13 +/- 2 nM) > TGF-beta 1, NGF-beta > PDGF-BB > or = bFGF > TNF-alpha. Native alpha 2M bound TGF-beta 1, TGF-beta 2, NGF-beta, PDGF-BB, and TNF-alpha. Interferon-gamma did not bind to native alpha 2M or alpha 2M-methylamine. Each cytokine bound native alpha 2M with lower affinity than alpha 2M-methylamine except for TGF-beta 2 which bound both forms with equal affinity. In non-equilibrium systems, alpha 2M-methylamine appeared to bind more TGF-beta 2 due to the more rapid dissociation of TGF-beta 2-native alpha 2M complex. The classification of alpha 2M-cytokine complexes according to binding affinity should predict which complexes are most likely to form in cell culture and under various conditions in vivo.

Developmental changes in nerve growth factor level in rat serum.

In serum, nerve growth factor (NGF) forms a complex with alpha 2-macroglobulin (alpha 2M), which formation inhibits the immunoreactivity between NGF and its antibodies. For measuring the serum level of NGF, it is thus necessary to liberate NGF from the NGF-alpha 2M complex and prevent reformation of such complex. The pretreatment of rat serum with 1 M guanidine hydrochloride for a few hours and operation of the enzyme immunoassay (EIA) in the presence of guanidine hydrochloride provided a reliable means for determination of the NGF level in serum. By this procedure we followed the serum NGF level in rats developmentally. It increased from prenatal day 2 to postnatal day 5 and decreased slightly at postnatal week 3, thereafter remaining constant throughout adulthood. In pregnant rats, the NGF level in serum increased threefold to fivefold before birth and then decreased rapidly. These data suggest that serum NGF level may reflect the demand for this molecule during establishment of the peripheral nervous system.

Reversible binding of platelet-derived growth factor-AA, -AB, and -BB isoforms to a similar site on the “slow” and “fast” conformations of alpha 2-macroglobulin.

The mechanism by which the platelet-derived growth factor (PDGF)-binding protein, alpha 2-macroglobulin (alpha 2M), modulates PDGF bioactivity is unknown, but could involve reversible PDGF-alpha 2M binding. Herein we report that greater than 70% of 125I-PDGF-BB or -AB complexed to alpha 2M was dissociated by SDS-denaturation followed by SDS-polyacrylamide gel electrophoresis, i.e. most of the binding was noncovalent. Reduction of the PDGF.alpha 2M complex following denaturation dissociated the cytokine from alpha 2M by greater than 90%, suggesting covalent disulfide bond formation. Approximately 50% of the growth factor was dissociated by lowering the pH from 7.5 to 4.0. 125I-PDGF-BB bound alpha 2M in a time-dependent manner (t1/2 = approximately 1 h), reaching equilibrium after 4 h. The 125I-PDGF.BB/alpha 2M complex dissociated more slowly (t1/2 = approximately 2.5 h). "Slow" and "fast" alpha 2M bound nearly equal amounts of PDGF-AB or -BB. Trypsin treatment converted PDGF-BB/alpha 2M complex to the fast conformation but did not release bound 125I-PDGF-BB. All PDGF-isoforms (AA, -AB, and -BB) competed for binding with 125I-PDGF-BB binding to slow alpha 2M and fast alpha 2M-methylamine by 65-80%. Other cytokines that bind alpha 2M (transforming growth factor-beta 1 and -beta 2, tumor necrosis factor-alpha, basic fibroblast growth factor, interleukin -1 beta, and -6) did not compete for 125I-PDGF-BB binding slow alpha 2M, but transforming growth factor-beta 1 and basic fibroblast growth factor inhibited 125I-PDGF-BB binding alpha 2M-methylamine by 30-50%. The reversible nature of the PDGF.alpha 2M complex could allow for targeted PDGF release near mesenchymal cells which possess PDGF receptors.

Anti‐HLA antigen class I heavy chain monoclonal antibodies inhibit human immunodeficiency virus production by peripheral blood mononuclear cells

We have examined the capacity of monoclonal antibodies (mAb) specific for HLA class I heavy chain to interfere with the human immunodeficiency virus (HIV) replicative cycle in human T cells. Among six anti-HLA class I heavy chain-specific mAb assayed, two mAb, RL4-24-6 and W6/32, were able to delay HIV1 and HIV2 cytopathic effect on MT4 cells, a human T cell leukemia virus type I (HTLVI) immortalized T cell line, mAb RL4-24-6, chosen for further studies, also inhibited HIV1 production by peripheral blood mononuclear cells (PBMC), and this inhibition was dose dependent. However, no effect was observed when mAb treatment was performed with either the CEM or Jurkat T cell lines. Our investigation of how RL4-24-6 interferes with the HIV replicative cycle revealed that: (a) incubation of PBMC with RL4-24-6 prior to HIV exposure did not change the susceptibility of these cells to HIV infection, (b) syncytia formation between CD4+ MT4 cells and HIV chronically infected PBMC was not affected by RL4-24-6 and (c) treatment of freshly infected PBMC with RL4-24-6, however, inhibited viral production. These data, together with those we previously reported using anti-beta 2-microglobulin (beta 2m) mAb, suggest that anti-HLA class I/beta 2m complex mAb can modify an early step of the HIV replicative cycle without affecting the viral entry.

Molecular distribution of elastase between its two main inhibitors: direct quantitation of elastase-α2-macroglobulin complex with a novel ELISA technique

A novel enzyme-linked immunosorbent assay for the quantitation of elastase linked to alpha 2-macroglobulin (alpha 2M) (elastase-alpha 2M complex, EMC) in body fluids is presented. The assay has a lower detection limit of 1.5 ng bound elastase per ml. The critical factor allowing immunological detection of alpha 2M-bound elastase is the addition of phenyl methyl sulphonyl fluoride (PMSF) to the assay buffer. The assay has been used to quantitate EMC in plasma and serum of healthy volunteers, and assess the distribution of elastase between its two main inhibitors, alpha 2M and alpha 1-proteinase inhibitor (API). EMC levels in serum samples from volunteers were significantly higher than in plasma (26.9 vs 21.9 ng/ml, p = 0.05, Student's t-test for paired samples). The API-bound elastase levels were 70.2 and 170.1 ng/ml in plasma and serum respectively (statistically significantly higher in serum, p less than 0.0001). The ratio of macroglobulin to alpha 1-proteinase-bound elastase levels was 0.333 in plasma and 0.168 in serum (p less than 0.0001). As alpha 2M bound elastase retains some proteolytic activity, direct measurement of circulating levels in inflammatory conditions should be of interest.

Pathogenic potentials of bacterial proteases

Six separate molecular mechanisms for pathogenesis attributed to bacterial proteases are described. (I). Enhancements of vascular permeability and edema formation which result from the activation of kinin generating cascade such as Hageman factor by the proteases. (II). Degradation of defense oriented proteins including IgG and IgA as well as destruction of structural matrices such as fibronectin, proteoglycan and collagen. (III). Inactivation of complement system and generated chemotactic factor from C3 and C5. (IV). Degradation of regulatory plasma protease inhibitors (serpins) including α 1protease inhibitor, α 2-macroglobulin (α 2M), C1-esterase inhibitor, α 2-antiplasmin and antithrombin-III. (V). The protease forms a transitory stable enzyme/inhibitor(α 2M) complex. It binds to and internalizes into the cells which possess α 2M-receptor such as fibroblasts via the α 2M-receptor, and the protease activity is regenerated in cells, and subsequently intracellular integrity is destroyed resulting in cell killing. (VI). The serratial 56 kDa (56K) protease is found to potential viral yield 100 fold more when influenza virus infected mice were subjected to administrations of this protease intranasally. This results in rapid and much elevated lethality.

Expression and function of HLA‐A2.1 in transgenic mice

We have derived a number of transgenic mouse lines which express the human major histocompatibility complex class I gene HLA-A2.1. Two lines carry the complete human HLA-A2.1, the others bear a recombinant gene in which the HLA-A2.1 coding regions are fused to the H-2Kb promoter. Analysis of transgenic spleen cells by immunofluorescence demonstrates that these mouse cells express HLA-A2.1 on their surface in association with mouse beta 2-microglobulin (beta 2m), confirming that HLA-A2 does not require human beta 2m to be expressed at the cell surface. The cells contain more HLA mRNA than endogenous H-2 class I mRNA. There is also a large pool of non-beta 2m-associated HLA heavy chain inside the cell. In contrast the amount of HLA:beta 2m complex is low. Thus, in transgenic mice HLA-A2 seems to compete poorly with H-2 heavy chains for mouse beta 2m. The HLA-A2.1 transgenic mice do not produce influenza-virus-specific cytotoxic T cells (CTL) restricted to the HLA transgene, at least in sufficient numbers to be measured in a direct bulk CTL assay. The dominance of H-2-restricted clones may be the result of quantitative rather than qualitative factors. However, HLA-A2.1 transgenic spleen cells are effective in stimulating an allogeneic CTL response in normal mice. This response is not H-2 restricted. Cold target inhibition studies show that there are at least two populations of CTL, one of which is specific for HLA-A2.1 on mouse cells. This result suggests that at least some allo-CTL are directed against major histocompatibility complex plus "self-peptide".

Human transforming growth factor beta.alpha 2-macroglobulin complex is a latent form of transforming growth factor beta.

125I-Labeled human platelet-derived transforming growth factor beta (125I-TGF-beta) and human alpha 2-macroglobulin (alpha 2M) formed a complex as demonstrated by 5% native polyacrylamide gel electrophoresis. The 125I-TGF-beta.alpha 2M complex migrated at a position identical to that of the fast migrating form of alpha 2M. Most of the 125I-TGF-beta.alpha 2M complex could be dissociated by acid or urea treatment. When 125I-TGF-beta was incubated with serum, the high molecular weight form of 125I-TGF-beta could be immunoprecipitated by anti-human alpha 2M anti-sera as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 2M purified from platelet-rich plasma also showed the latent transforming growth factor activity and immunoreactivity of TGF-beta. These results suggest that TGF-beta.alpha 2M complex is a latent form of TGF-beta.

Covalent and non-covalent interaction of chymotrypsin with α 2-macroglobulin

The pattern of covalent crosslinking between human α 2-macroglobulin (α 2M) and chymotrypsin has been investigated by chromatography and polyacrylamide gel electrophoresis in denaturing medium. Reaction with a single mol of chymotrypsin per mol α 2M results in the formation of a 95% covalent 1:1 chymotrypsin-α 2M complex and in the proteolytic cleavage of both 180 kDa monomers in one α 2M subunit. Proteolytic cleavage in the other α 2M subunit requires the presence of a second mol of chymotrypsin; part (20%) of the protease in the 2:1 chymotrypsin-α 2M complex thus formed appears to be non-covalently bound to the α 2M chains. Covalent binding is abolished when the reaction of α 2M with the protease is carried out in the presence of hydroxylamine. A single mol of the protease is then able to cleave all four 180 kDa monomers in α 2M.

Binding of alpha-2-macroglobulin trypsin complex to human monocytes in culture

125I-labelled alpha 2-macroglobulin-trypsin (alpha 2MT) complex bound specifically to freshly isolated human blood monocytes at 4 degrees C but not to B or T lymphocytes, polymorphonuclear leucocytes, erythrocytes or thrombocytes. Binding of 12 pmol/l labelled alpha 2MT to freshly isolated monocytes was low. However, when monocytes were cultured in vitro, binding increased, reaching 10- to 20-fold higher specific binding by 2-4 weeks. Non-specific binding was absent. Considerable variations were observed in binding to monocytes from different cultures (individuals). The half-time of 125I-labelled alpha 2MT complex association to 14-days-old monocyte cultures was about 6 h at 4 degrees C. Dissociation of labelled complex after the addition of a saturing concentration of unlabelled complex was biphasic. About half of the labelled complex dissociated with a half-time of about 1 h, whereas the other half dissociated extremely slowly. At near steady state, half of the receptors were occupied at a complex concentration of about 200 pmol/l and Scatchard analysis showed that the data were adequately described by assuming one class of receptors. It is concluded that human monocytes express a marked increase in binding of alpha 2M complex when developing to macrophage-like cells in tissue cultures.

The kinetics of inhibition of alpha-thrombin in human plasma.

Methods have been developed for kinetic studies of the inhibition of exogenous unmodified thrombin in human plasma containing normal levels of fibrinogen and calcium ion. To prevent interference by other proteases, factor VIII-deficient plasma was used and contained 50 nM Phe-Phe-Arg-chloromethyl ketone and 1 kallikrein-inactivating unit/ml aprotinin; neither inhibited thrombin at these levels. Two independent assays were used. The first was the discontinuous amidolytic assay of thrombin activity, which measures both free thrombin and thrombin-alpha 2-macroglobulin complex, and was used to estimate the rates of inhibition both by "inactivating" inhibitors, such as anti-thrombin and alpha 1-protease inhibitor, and by alpha 2-macroglobulin (alpha 2M). The contribution of alpha 2M was confirmed by a second method, which measured with time the generation of amidolytic activity due to the thrombin-alpha 2M complex. The total rate of thrombin inhibition in plasma containing 4 mM free Ca2+ was of the order of 1.9 min-1, of which 0.4 min-1 was due to alpha 2M and 0.9 min-1 was due to inhibitors that were removed when plasma was passed through heparin-agarose. Thrombin inhibition was also measured in varying dilutions of plasma and confirmed that total inhibition rate is approximately linearly related to plasma (and thus inhibitor) concentration. Negatively charged phospholipid micelles had very little effect on thrombin inhibition rate, but platelets accelerated inhibition to more than 2.5 min-1.

Detection of alpha 2-macroglobulin-associated proteases in the plasma of patients with rheumatoid arthritis.

In previous work a polyclonal B cell activator has been detected in the serum of patients with rheumatoid arthritis (RA). This activator is associated with alpha 2-macroglobulin (alpha 2M) and its activity is blocked by low-Mr trypsin inhibitors, which suggests that it may be a protease-alpha 2M complex. Here we determined the possibility of developing a routine clinical chemistry test for detection of this complex in patients' blood. We measured with chromogenic substrates the total proteolytic activity of citrated plasma and of the alpha 2M immunoabsorbed from plasma. Low-Mr substrates containing Arg were degraded much better by plasma from RA patients than by plasma from patients with other arthritides. Low-Mr substrates containing Leu, Lys, or Gly or the large-Mr substrate Azocoll were not degraded by RA patients' plasma. alpha 2M from RA patients' plasma attached to a solid-phase immunoabsorbent degraded an Arg-containing tripeptide much better than did the alpha 2M from normal donors, from patients with systemic lupus erythematosus, or from those with joint inflammation of other, "non-autoimmune" origin. Although the enzyme associated with alpha 2M in the plasma from RA patients appeared to be similar to trypsin, the differences in optimal pH, cation concentration, degradation of Lys-containing substrates, and biological activity suggest otherwise. We speculate that the alpha 2M-protease complexes are generated in the immune system and contribute to the inflammatory and autoimmune phenomena in RA.