α-2-Macroglobulin: a physiological guardian.

Alpha macroglobulins are large glycoproteins which are present in the body fluids of both invertebrates and vertebrates. Alpha-2-macroglobulin (α2 M), a key member of alpha macroglobulin superfamily, is a high-molecular weight homotetrameric glycoprotein. α2 M has many diversified and complex functions, but it is primarily known by its ability to inhibit a broad spectrum of proteases without the direct blockage of the protease active site. α2 M is also known to be involved in the regulation, transport, and a host of other functions. For example, apart from inhibiting proteinases, it regulates binding of transferrin to its surface receptor, binds defensin and myelin basic protein, etc., binds several important cytokines, including basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), and modify their biological activity. α2 M also binds a number of hormones and regulates their activity. α2 M is said to protect the body against various infections, and hence, can be used as a biomarker for the diagnosis and prognosis of a number of diseases. However, this multipurpose antiproteinse is not "fail safe" and could be damaged by reactive species generated endogenously or exogenously, leading to various pathophysiological conditions.

Matrix metalloproteinases: do they play a role in mucosal pathology of the oral cavity?

Matrix metalloproteinases (MMPs) are critical factors in maintaining the integrity of mucosa and mediating normal biological processes. An imbalance between tissue levels of these mediators and their natural inhibitors is believed to underlie the pathophysiology of many diseases, including those affect the gastrointestinal and oral mucosae. The ongoing development of synthetic inhibitors of these mediators may provide opportunities to develop treatment modalities for patients suffering from these diseases. Understanding the role of MMPs in the pathophysiology of many diseases, however, is far from complete, and the improvement of pharmaceutical management strategies can only be achieved if the underlying process of these diseases is completely comprehended. This paper reviews the functions of matrix metalloproteinases and addresses their role in mediating mucosal pathologies with emphasis on oral mucosa.

The plasmin-antiplasmin system: structural and functional aspects.

The plasmin-antiplasmin system plays a key role in blood coagulation and fibrinolysis. Plasmin and α(2)-antiplasmin are primarily responsible for a controlled and regulated dissolution of the fibrin polymers into soluble fragments. However, besides plasmin(ogen) and α(2)-antiplasmin the system contains a series of specific activators and inhibitors. The main physiological activators of plasminogen are tissue-type plasminogen activator, which is mainly involved in the dissolution of the fibrin polymers by plasmin, and urokinase-type plasminogen activator, which is primarily responsible for the generation of plasmin activity in the intercellular space. Both activators are multidomain serine proteases. Besides the main physiological inhibitor α(2)-antiplasmin, the plasmin-antiplasmin system is also regulated by the general protease inhibitor α(2)-macroglobulin, a member of the protease inhibitor I39 family. The activity of the plasminogen activators is primarily regulated by the plasminogen activator inhibitors 1 and 2, members of the serine protease inhibitor superfamily.

Role of cardiac isoform of alpha-2 macroglobulin in diabetic myocardium.

Earlier studies from one of the investigator's laboratory have demonstrated the presence of a high molecular weight protein (182 kDa) in the blood serum of laboratory animals subjected to pressure-induced cardiac hypertrophy and suggested that this protein may be involved in the development of cardiac hypertrophy. Studies have shown that this protein is also involved in earlier stages of cardiac complications associated with diabetes, but the role of this protein in diabetic heart is less understood. So we aimed to check whether this protein is having any protective role in diabetic heart. The protein was purified from serum of rats induced with cardiac hypertrophy and the purified protein was injected through tail vein of diabetic rats for further studies. The results of various antioxidant enzymes and the TBARS levels have indicated the antioxidant activity of this protein. Real-time PCR analysis of gene expression revealed the upregulation of certain muscle-specific genes like ß-MHC, MLC-2, and skeletal α actin in diabetic group and also in presence of 182-kDa protein. The results further showed a down regulation of genes such as cardiac α-actin and α- MHC implicating the role of this protein in the development of cardiac hypertrophy in diabetes. Increased cardiac hypertrophy as revealed by the expression of various genes and improved antioxidant potential in presence of 182 kDa protein in diabetes at the earlier stages is beneficial for counteracting the myocardial damage associated with diabetes.

Activated α(2) macroglobulin induces matrix metalloproteinase 9 expression by low-density lipoprotein receptor-related protein 1 through MAPK-ERK1/2 and NF-κB activation in macrophage-derived cell lines.

Macrophages under certain stimuli induce matrix metalloproteinase 9 (MMP-9) expression and protein secretion through the activation of MAPK-ERK and NF-κB signaling pathways. Previously, we demonstrated that activated α(2)-macroglulin (α(2)M*) through the interaction with its receptor low-density lipoprotein receptor-related protein 1 (LRP1) induces macrophage proliferation mediated by the activation of MAPK-ERK1/2. In the present work, we examined whether α(2)M*/LRP1interaction could induce the MMP-9 production in J774 and Raw264.7 macrophage-derived cell lines. It was shown that α(2)M* promoted MMP-9 expression and protein secretion by LRP1 in both macrophage-derived cell lines, which was mediated by the activation of MAPK-ERK1/2 and NF-κB. Both intracellular signaling pathways activated by α(2)M* were effectively blocked by calphostin-C, suggesting involvement of PKC. In addition, we demonstrate that α(2)M* produced extracellular calcium influx via LRP1. However, when the intracellular calcium mobilization was inhibited by BAPTA-AM, the α(2)M*-induced MAPK-ER1/2 activation was fully blocked in both macrophage cell lines. Finally, using specific pharmacological inhibitors for PKC, Mek1, and NF-κB, it was shown that the α(2)M*-induced MMP-9 protein secretion was inhibited, indicating that the MMP production promoted by the α(2)M*/LRP1 interaction required the activation of both signaling pathways. These findings may prove useful in the understanding of the macrophage LRP1 role in the vascular wall during atherogenic plaque progression.

Inhibition of ADAMTS-7 and ADAMTS-12 degradation of cartilage oligomeric matrix protein by alpha-2-macroglobulin.

OBJECTIVE: As we previously reported, ADAMTS-7 and ADAMTS-12, two members of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family, degrade cartilage oligomeric matrix protein (COMP) in vitro and are significantly induced in the cartilage and synovium of arthritic patients [Liu CJ, Kong W, Ilalov K, Yu S, Xu K, Prazak L, et al. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J 2006;20(7):988-90; Liu CJ, Kong W, Xu K, Luan Y, Ilalov K, Sehgal B, et al. ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein. J Biol Chem 2006;281(23):15800-8]. The purpose of this study was to determine (1) whether cleavage activity of ADAMTS-7 and ADAMTS-12 of COMP are associated with COMP degradation in osteoarthritis (OA); (2) whether alpha-2-macroglobulin (a(2)M) is a novel substrate for ADAMTS-7 and ADAMTS-12; and (3) whether a(2)M inhibits ADAMTS-7 or ADAMTS-12 cleavage of COMP. METHODS: An in vitro digestion assay was used to examine the degradation of COMP by ADAMTS-7 and ADAMTS-12 in the cartilage of OA patients; in cartilage explants incubated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1-beta (IL-1beta) with or without blocking antibodies; and in human chondrocytes treated with specific small interfering RNA (siRNA) to knockdown ADAMTS-7 or/and ADAMTS-12. Digestion of a(2)M by ADAMTS-7 and ADAMTS-12 in vitro and the inhibition of ADAMTS-7 or ADAMTS-12-mediated digestion of COMP by a(2)M were also analyzed. RESULTS: The molecular mass of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 were similar to those observed in OA patients. Specific blocking antibodies against ADAMTS-7 and ADAMTS-12 dramatically inhibited TNF-alpha- or IL-1beta-induced COMP degradation in the cultured cartilage explants. The suppression of ADAMTS-7 or ADAMTS-12 expression by siRNA silencing in the human chondrocytes also prevented TNF-alpha- or IL-1beta-induced COMP degradation. Both ADAMTS-7 and ADAMTS-12 were able to cleave a(2)M, giving rise to 180- and 105-kDa cleavage products, respectively. Furthermore, a(2)M inhibited both ADAMTS-7- and ADAMTS-12-mediated COMP degradation in a concentration (or dose)-dependent manner. CONCLUSION: Our observations demonstrate the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo. Furthermore, a(2)M is a novel substrate for ADAMTS-7 and ADAMTS-12. More significantly, a(2)M represents the first endogenous inhibitor of ADAMTS-7 and ADAMTS-12.

Potential roles of abundant extracellular chaperones in the control of amyloid formation and toxicity.

The in vivo formation of fibrillar proteinaceous deposits called amyloid is associated with more than 40 serious human diseases, collectively referred to as protein deposition diseases. In many cases the amyloid deposits are extracellular and are found associated with newly identified abundant extracellular chaperones (ECs). Evidence is presented suggesting an important regulatory role for ECs in amyloid formation and disposal in the body. A model is presented which proposes that, under normal conditions, ECs stabilize extracellular misfolded proteins by binding to them, and then guide them to specific cell receptors for uptake and subsequent degradation. Thus ECs and their receptors may be critical parts of a quality control system to protect the body against dangerously hydrophobic proteins/peptides. However, it also appears possible that in the presence of a high molar excess of misfolded protein, such as might occur during disease, the limited amounts of ECs available may actually exacerbate pathology. Further advances in understanding of the mechanisms that control extracellular protein folding are likely to identify new strategies for effective disease therapies.

Hampered cumulus expansion of porcine cumulus-oocyte complexes by excessive presence of alpha2 -macroglobulin is likely mediated via inhibition of zinc-dependent metalloproteases.

In vitro maturation (IVM) in serum causes hampered expansion of porcine cumulus-oocyte complexes (COCs) due to excessive alpha2 -macroglobulin (A2M). This study investigated two hypotheses that could explain the effect of A2M: (i) binding of epidermal growth factor (EGF) to A2M, followed by its decreased availability; and (ii) inhibition of zinc-dependent metalloproteases. Cumulus expansion was evaluated based on the diameter of the COCs, the proportion of COCs participating in a floating cloud and the proportion of COCs with loss of cumulus cells. The first hypothesis of decreased EGF availability was tested by increasing the EGF concentration (20 and 50 ng/mL vs. 10 ng/mL), but was not confirmed because cumulus expansion did not improve. To verify the second hypothesis of inhibited zinc-dependent metalloproteases, the effect of tissue inhibitor of metalloproteases-3 (TIMP-3) on cumulus expansion during IVM with and without A2M was investigated. To immuno-neutralize A2M, serum was pre-incubated with A2M antibodies. Impaired cumulus expansion because of TIMP-3 could only be observed during IVM in 10% of serum with A2M antibodies. No effect of TIMP-3 was observed in medium without A2M antibodies. These results indicate that A2M and TIMP-3 share a common target, a zinc-dependent metalloprotease. Future research is directed toward the identification of the protease involved.

Morgagnian cataract resulting from a naturally occurring nonsense mutation elucidates a role of CPAMD8 in mammalian lens development.

To investigate the genetic basis of hereditary lens opacities we analyzed 31 cases of bilateral congenital cataract in Red Holstein Friesian cattle. A genome-wide association study revealed a significant association on bovine chromosome 7 at positions 6,166,179 and 12,429,691. Whole genome re-sequencing of one case and four relatives showed a nonsense mutation (g.5995966C>T) in the PZP-like, alpha-2-macroglobulin domain containing 8 (CPAMD8) gene leading to a premature stop codon (CPAMD8 p.Gln74*) associated with cataract development in cattle. With immunohistochemistry we confirmed a physiological expression of CPAMD8 in the ciliary body epithelium of the eye in unaffected cattle, while the protein was not detectable in the ciliary body of cattle with cataracts. RNA expression of CPAMD8 was detected in healthy adult, fetal and cataractous lenses.

Parasite cysteine proteinase interactions with alpha 2-macroglobulin or kininogens: differential pathways modulating inflammation and innate immunity in infection by pathogenic trypanosomatids.

Plasma extravasation is a common endothelium response to tissue injury provoked by pathogens. Herein I will review studies showing that host proteinase inhibitors (e.g., alpha2-macroglobulin (alpha2M) or kininogens) interact with protozoan cysteine proteinases (CPs) in extravascular infection sites, linking inflammation to innate immunity by different mechanisms. Using human monocytes as antigen presenting cells, we first demonstrated that alpha2M entrapment of cruzipain, a Trypanosoma cruzi CP, reduced the activation threshold of cruzipain-specific CD4 T cells due to facilitated uptake of alpha2M-cruzipain complexes by the multiscavenger receptor (CD91). More recently, studies of the mechanisms underlying inflammation elicited by T. cruzi revealed that kininogens, once bound to glycosaminoglycans, are not able to efficiently inactivate cruzipain via their inhibitory cystatin-like domains. Instead, we found that cruzipain readily processes surface-bound kininogens, liberating bioactive kinins. Acting as paracrine hormones, kinins vigorously activate host cells through bradykinin (BK) receptors, thus stimulating endocytic uptake of the pathogen. Rather than unilaterally enhancing parasite infectivity, the liberated kinins activate innate immunity by potently stimulating dendritic cell maturation via the BK B2 receptor. The discovery of chagasin, a novel family of endogenous inhibitors expressed by trypanosomatids, is likely another regulatory player involved in the dynamics of the inflammatory response.

Role of tissue plasminogen activator receptor LRP in hippocampal long-term potentiation.

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional endocytic receptor that is expressed abundantly in neurons of the CNS. Both LRP and several of its ligands, including tissue plasminogen activator (tPA), apolipoprotein E/lipoproteins, alpha(2)-macroglobulin, and the beta-amyloid precursor protein, have been implicated in various neuronal functions and in the pathogenesis of Alzheimer's disease. It has been reported that induction of tPA expression may contribute to activity-dependent synaptic plasticity in the hippocampus and cerebellum. In addition, long-term potentiation (LTP) is significantly decreased in mice lacking tPA. Here we demonstrate that tPA receptor LRP is abundantly expressed in hippocampal neurons and participates in hippocampal LTP. Perfusion of hippocampal slices with receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, significantly reduced late-phase LTP (L-LTP). In addition, RAP also blocked the enhancing effect of synaptic potentiation by exogenous tPA in hippocampal slices prepared from tPA knock-out mice. Metabolic labeling and ligand binding analyses showed that both tPA and LRP are synthesized by hippocampal neurons and that LRP is the major cell surface receptor that binds tPA. Finally, we found that tPA binding to LRP in hippocampal neurons enhances the activity of cyclic AMP-dependent protein kinase, a key molecule that is known to be involved in L-LTP. Taken together, our results demonstrate that interactions between tPA and cell surface LRP are important for hippocampal L-LTP.

Characterization and serum inhibition of neutral collagenase from cultured dog gingival tissue.

1. Explants of dog gingiva, maintained in culture for 9 days in the absence of serum, released a collagenase (EC 3.4.24.3) into the medium. The yield of active enzyme reached a maximum after 5-8 days with concomitant release of collagen degradation products from the explants. 2. The enzyme attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of specific viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). Electron microscopy of segment-long-spacing crystallites of these reaction products showed the cleavage locus of the collagen molecule at interband 40. 3. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a molecular weight of approximately 35,000 was derived from gel filtration studies. EDTA, 1,10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. Proteoglycan derived from porcine and human cartilage did not inhibit the enzyme. 4. The enzyme was inhibited by the dog serum proteins alpha2-macroglobulin and a smaller component of molecular weight approximately 40,000. This small component was purified by column chromatography utilising Sephadex G-200, DEAE A-50, and G-100 (superfine grade). Agarose electrophoresis of the purified component showed it to represent a beta-serum protein. alpha1-Antitrypsin did not inhibit the enzyme. 5. The physiological importance of the natural serum inhibitors and gingival collagenase are discussed in relation to latent enzyme and periodontal disease.

The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors.

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.

Ineffectiveness of the inhibition of the main haemorrhagic metalloproteinase from Bothrops jararaca venom by its only plasma inhibitor, alpha 2-macroglobulin.

Observations that a haemorrhagic metalloproteinase (jararhagin) from Bothrops jararaca venom had less effect on platelets suspended in plasma than in washed platelet suspensions, suggested that plasma contains naturally occurring inhibitor(s) of this enzyme. By using radiolabelled jararhagin and crossed immunoelectrophoresis, we have demonstrated the binding of this enzyme to alpha 2-macroglobulin in plasma. SDS-PAGE analysis of this binding revealed the presence of radioactivity in four bands with relative molecular masses of 640, 570, 520 and 410 kDa; in addition a small amount of 47 kDa free enzyme was demonstrable. Reduced samples showed an additional non-complexed 90 kDa fragment of alpha 2-macroglobulin generated by jararhagin. These results are compatible with a model in which, upon multiple cleavages of alpha 2-macroglobulin, the enzyme becomes covalently bound to the inhibitor, and the two halves of the inhibitor become crosslinked. However, jararhagin activity was not completely inhibited even after long incubation (60 min) with a large (10-fold) molar excess of alpha 2-macroglobulin either in plasma or a purified alpha 2-macroglobulin preparation. Kinetic studies showed that inhibition was comparatively slow, although jararhagin readily cleaved alpha 2-macroglobulin in the bait region. Therefore, the ineffectiveness of the inhibition could have resulted from a low tendency of this proteinase to form covalent complexes with the inhibitor. We conclude that the pronounced haemorrhagic activity of jararhagin can be attributed to prolonged access of this enzyme to high molecular weight substrates, even in the presence of a large molar excess of alpha 2-macroglobulin.

Blood-testis barrier dynamics are regulated by {alpha}2-macroglobulin via the c-Jun N-terminal protein kinase pathway.

The blood-testis barrier (BTB), in contrast to the blood-brain and blood-retina barriers, is composed of coexisting tight junctions, gap junctions, and basal ectoplasmic specializations, a testis-specific type of adherens junction. Recent studies showed that BTB restructuring that facilitates germ cell migration during spermatogenesis involves proteolysis, an event that is usually restricted to the cell-matrix interface in other epithelia. For instance, a surge in alpha(2)-macroglobulin (alpha(2)-MG), a protease inhibitor produced by Sertoli cells, was detected at the Sertoli-Sertoli and Sertoli-germ cell interface in the epithelium during cadmium chloride-induced BTB disruption in adult rats. It is thus proposed that the increase in alpha(2)-MG is crucial for protecting the epithelium from unwanted proteolysis as well as regulating the availability of cytokines that affect junction turnover. Although both tight junction and adherens junction dynamics at the BTB are regulated via the p38 MAPK signaling pathway, the mechanism(s) that regulates alpha(2)-MG is entirely unknown. In this study, we have shown that by administering dimethylaminopurine, a c-Jun N-terminal protein kinase (JNK) inhibitor, to the testis, JNK activity was blocked specifically and alpha(2)-MG production was inhibited, worsening the cadmium chloride-induced damage to the epithelium. Studies coupled with inhibitors, immunoblottings, and immunofluorescent and electron microscopy have unequivocally demonstrated that the JNK signaling pathway is a putative regulatory pathway for alpha(2)-MG production in the testis. This finding illustrates for the first time that a cell-matrix restructuring event occurs in normal cell physiology at the cell-cell interface in the testis, highlighting the significance of alpha(2)-MG in the regulation of BTB function.

In vitro preparation of nonenzymatically glucosylated human transferrin, alpha 2-macroglobulin, and fibrinogen with preservation of function.

Human transferrin, alpha 2-macroglobulin, and fibrinogen were incubated with [3H]-glucose. After a 7-day, 37 degrees C incubation at 20 mM glucose, transferrin incorporated 1.1 mol of glucose/mol protein; alpha 2-macroglobulin, 10 mol of glucose/mol; and fibrinogen, 3.8 of glucose/mol, or approximately 14 mumol of glucose/g for each protein. These results were the same for glucose labeled in the 1 or 6 position. No radiolabel was incorporated into the proteins during incubations with glucose labeled in the 2 position. The rate and extent of iron binding were identical for both glucosylated and nonglucosylated transferrin. Glucosylated transferrin bound to Wil-2 human lymphoblast cells with a Kd = 33 nM and receptor number of 3.4 X 10(5) receptors/cell; nonglucosylated transferrin with a Kd = 31 nM and receptor number of 3.9 X 10(5) receptors/cell. Glucosylated and nonglucosylated alpha 2-macroglobulin showed the same conformational change as determined on native PAGE after reaction with trypsin, plasmin, or methylamine and had the same activity in the Ganrot assay after reaction with trypsin or plasmin. The clearance of 125I-labeled, methylamine-treated alpha 2-macroglobulin from the mouse circulation was identical for both glucosylated and nonglucosylated alpha 2-macroglobulin, t1/2 = 3 min. alpha 2-Macroglobulin that was first glucosylated then reacted with methylamine bound to mouse peritoneal macrophages with a Kd of 2.5 nM and receptor activity of 370 fmol/mg cell protein. alpha 2-Macroglobulin that was first reacted with methylamine and then glucosylated bound with a Kd of 3 nM and receptor activity of 320 fmol/mg cell protein. Glucosylated fibrinogen had the same clotting time as control fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmin inhibitors in the prevention of systemic effects during thrombolytic therapy: specific role of the plasminogen-binding form of alpha 2-antiplasmin.

To delineate the role of plasmin inhibitors, especially the two molecular forms of alpha 2-antiplasmin (that is, the plasminogen-binding and the nonplasminogen-binding forms), in the control of systemic effects during thrombolytic therapy, the consumption of plasmin inhibitors and the degree of fibrinogen breakdown were studied in 35 patients with acute myocardial infarction treated with recombinant tissue-type plasminogen activator (rt-PA) or streptokinase. At a low degree of plasminogen activation (in six patients treated with rt-PA), plasminogen-binding alpha 2-antiplasmin was consumed first. At a higher degree of plasminogen activation (in 20 patients), plasminogen-binding alpha 2-antiplasmin became exhausted (less than 20%) and other plasmin inhibitors (that is, nonplasminogen-binding alpha 2-antiplasmin and alpha 2-macroglobulin) were consumed. After extensive plasminogen activation (in nine patients treated with streptokinase), plasminogen-binding alpha 2-antiplasmin consumption was complete and nonplasminogen-binding alpha 2-antiplasmin and alpha 2-macroglobulin were consumed to about 30% to 50% of the pretreatment level. No significant C1-inactivator consumption occurred, even at extreme degrees of plasminogen activation. Fibrinogen breakdown as a marker for systemic effects correlated strongly with consumption of plasminogen-binding alpha 2-antiplasmin. Fibrinogen breakdown did occur, but only when the amount of plasminogen-binding alpha 2-antiplasmin was decreased to less than 20% of the pretreatment level. The other plasmin inhibitors could not prevent fibrinogen breakdown. These results were confirmed by in vitro studies. It is concluded that plasminogen-binding alpha 2-antiplasmin is the most important inhibitor of plasmin in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Functions of alpha 2 macroglobulins in pregnancy.

The alpha 2 macroglobulins (A2M) are a family of abundant plasma proteins produced predominantly by the mammalian liver. Pregnancy zone proteins (PZP) of humans and rats are A2M family members that bind a wide variety of macromolecules including the important pregnancy-associated molecules such as vascular endothelial growth factor, placenta growth factor and glycodelin (also called PP14). Recently, a mouse gene analogous to PZP (A2M of pregnancy or A2Mp) was cloned. A2Mp has a unique pattern of expression in reproductive and cardiovascular tissues and, unexpectedly, is not expressed by liver. Since changes in heart function and remodeling of renal and uterine vasculature are amongst the earliest maternal responses to pregnancy, the product of the A2Mp gene has been postulated to systemically regulate these changes. A2Ms with and without non-covalently bound ligands also down regulate immune cell activation but promote immune cell migration, additional features associated with gestational success. Here, we review the A2M gene families of mice and humans, the predicted structural relationships between A2M and its pregnancy induced forms and the postulated roles for this gene family in normal pregnancy.

Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies.

Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein.

A Pro-Nerve Growth Factor (proNGF) and NGF Binding Protein, α2-Macroglobulin, Differentially Regulates p75 and TrkA Receptors and Is Relevant to Neurodegeneration Ex Vivo and In Vivo.

Nerve growth factor (NGF) is generated from a precursor, proNGF, that is proteolytically processed. NGF preferentially binds a trophic tyrosine kinase receptor, TrkA, while proNGF binds a neurotrophin receptor (NTR), p75(NTR), that can have neurotoxic activity. Previously, we along with others showed that the soluble protein α2-macroglobulin (α2M) is neurotoxic. Toxicity is due in part to α2M binding to NGF and inhibiting trophic activity, presumably by preventing NGF binding to TrkA. However, the mechanisms remained unclear. Here, we show ex vivo and in vivo three mechanisms for α2M neurotoxicity. First, unexpectedly the α2M-NGF complexes do bind TrkA receptors but do not induce TrkA dimerization or activation, resulting in deficient trophic support. Second, α2M makes stable complexes with proNGF, conveying resistance to proteolysis that results in more proNGF and less NGF. Third, α2M-proNGF complexes bind p75(NTR) and are more potent agonists than free proNGF, inducing tumor necrosis factor alpha (TNF-α) production. Hence, α2M regulates proNGF/p75(NTR) positively and mature NGF/TrkA negatively, causing neuronal death ex vivo. These three mechanisms are operative in vivo, and α2M causes neurodegeneration in a p75(NTR)- and proNGF-dependent manner. α2M could be exploited as a therapeutic target, or as a modifier of neurotrophin signals.