Genetic and Pharmacological Inhibition of PAPP-A Protects Against Visceral Obesity in Mice.

Pathogenicity of visceral adipose tissue (VAT) has been linked to the metabolic stress of enlarging mature adipocytes and a limited ability to recruit new adipocytes. One of the major distinguishing features of VAT preadipocytes is the high expression of the zinc metalloprotease, pregnancy-associated plasma protein-A (PAPP-A), when compared to subcutaneous adipose tissue (SAT). In this study we used 2 different approaches to investigate the effect of PAPP-A inhibition on different fat depots in mice on a high-fat diet (HFD) for 15 weeks. Conditional knockdown of PAPP-A gene expression in female adult mice resulted in significant decreases of 30% to 40% in adipocyte size in VAT (mesenteric and pericardial depots) compared to control mice. There was no effect on SAT (inguinal) or intra-abdominal perigonadal fat. Liver lipid was also significantly decreased without any effect on heart and skeletal muscle lipid. We found similar effects when using a pharmacological approach. Weekly injections of a specific immunoneutralizing monoclonal antibody (mAb-PA 1/41) or isotype control were given to male and female wild-type mice on HFD for 15 weeks. Adipocyte size was significantly decreased (30%-50%) only in VAT with mAb-PA 1/41 treatment. In this model, cell number was significantly increased in mesenteric fat in mice treated with mAb-PA 1/41, suggesting hyperplasia along with reduced hypertrophy in this VAT depot. Gene expression data indicated a significant decrease in F4/80 (macrophage marker) and interleukin-6 (proinflammatory cytokine) and a significant increase in adiponectin (anti-inflammatory adipokine with beneficial metabolic effects) in mesenteric fat compared to inguinal fat in mice treated with mAb-PA 1/41. Furthermore, there was significantly decreased liver lipid content with mAb-PA 1/41 treatment. Thus, using 2 different models systems we provide proof of principle that PAPP-A inhibition is a potential therapeutic target to prevent visceral obesity and its metabolic sequelae, such as fatty liver.

Targeted Inhibition of Pregnancy-Associated Plasma Protein-A Activity Reduces Atherosclerotic Plaque Burden in Mice.

The metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), has been implicated in the development of cardiovascular disease in humans and mouse models. In the latter, genetic deletion or overexpression of PAPP-A confirmed a major role for PAPP-A in atherosclerosis. In this study, we tested the hypothesis that targeting PAPP-A proteolytic activity by an inhibitory monoclonal antibody (mAb-PA) reduces atherosclerotic plaque progression. Apolipoprotein E knock-out mice on high-fat diet were treated with mAb-PA or isotype control. Control mice had a 10-fold increase in aortic plaque after 10 weeks. Aortic plaque burden was reduced by ∼ 70% in mice treated with mAb-PA (P = 0.0002). Treatment was efficacious even in the face of elevated cholesterol and triglycerides. This study demonstrates proof-of-principle and provides feasibility for a novel therapeutic strategy to inhibit atherosclerotic plaque burden by selective targeting of PAPP-A.

Comparison of different blood collection, sample matrix, and immunoassay methods in a prenatal screening setting.

We compared how measurements of pregnancy-associated plasma protein A (PAPP-A) and the free beta subunit of human chorionic gonadotropin (fß-hCG) in maternal blood are influenced by different methods for blood collection, sample matrix, and immunoassay platform. Serum and dried blood spots (DBS) were obtained by venipuncture and by finger prick of 19 pregnant women. PAPP-A and fß-hCG from serum and from DBS were measured by conventional indirect immunoassay on an AutoDELFIA platform and by antibody microarray. We compared methods based on the recoveries for both markers as well as marker levels correlations across samples. All method comparisons showed high correlations for both marker concentrations. Recovery levels of PAPP-A from DBS were 30% lower, while those of fß-hCG from DBS were 50% higher compared to conventional venipuncture serum. The recoveries were not affected by blood collection or immunoassay method. The high correlation coefficients for both markers indicate that DBS from finger prick can be used reliably in a prenatal screening setting, as a less costly and minimally invasive alternative for venipuncture serum, with great logistical advantages. Additionally, the use of antibody arrays will allow for extending the number of first trimester screening markers on maternal and fetal health.

Indirect targeting of IGF receptor signaling in vivo by substrate-selective inhibition of PAPP-A proteolytic activity.

The insulin-like growth factor (IGF) signaling pathway is involved in certain human cancers, and the feasibility of directly targeting the IGF receptor has been actively investigated. However, recent evidence from clinical trials suggests that this approach can be problematic. We have developed an alternative strategy to indirectly inhibit the IGF signaling by targeting the metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). PAPP-A associated with the cell surface cleaves IGF binding protein-4 (IGFBP-4), when IGF is bound to IGFBP-4, and thereby increases IGF bioavailability for receptor activation in an autocrine/paracrine manner. We hypothesized that inhibition of PAPP-A would suppress excessive local IGF signaling in tissues where this is caused by increased PAPP-A proteolytic activity. To test this hypothesis, we developed an inhibitory monoclonal antibody, mAb 1/41, which targets a unique substrate-binding exosite of PAPP-A. This inhibitor selectively and specifically inhibits proteolytic cleavage of IGFBP-4 with an inhibitory constant (Ki) of 135 pM. In addition, it inhibited intracellular signaling of the IGF receptor (AKT phosphorylation) in monolayers of A549 cells, an IGF-responsive lung cancer-derived cell line found to express high levels of PAPP-A. We further showed that mAb 1/41 is effective towards PAPP-A bound to cell surfaces, and that it is capable of inhibiting PAPP-A activity in vivo. Using a murine xenograft model of A549 cells, we demonstrated that mAb 1/41 administered intraperitoneally significantly inhibited tumor growth. Analysis of xenograft tumor tissue recovered from treated mice showed penetration of mAb 1/41, reduced IGFBP-4 proteolysis, and reduced AKT phosphorylation. Our study provides proof of concept that IGF signaling can be selectively reduced by targeting a regulatory proteinase that functions extracellularly, upstream of the IGF receptor. PAPP-A targeting thus represents an alternative therapeutic strategy for inhibiting IGF receptor signaling.

Development of a recombinant antibody towards PAPP-A for immunohistochemical use in multiple animal species.

The metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), is increasingly recognized as a modulator of insulin-like growth factor (IGF) signaling; it cleaves IGF binding proteins causing the release of bioactive IGF. Accumulating evidence supports an important role of PAPP-A in both normal physiology and under different pathological conditions. However, antibodies for the detection of PAPP-A in non-human tissues have been lacking, although needed for use with several animal models which are currently being developed. To develop a monoclonal antibody suitable for the immunohistochemical detection of PAPP-A, we therefore selected a phage-derived scFv antibody, PAC1, specifically recognizing an epitope of PAPP-A, which is highly conserved between multiple animal species. We first converted this antibody into bivalent IgG, and verified its ability to recognize PAPP-A in sections of formalin-fixed and paraffin-embedded tissue. For increased sensitivity, affinity maturation to sub-nanomolar affinity was then carried out. The resulting recombinant antibody, PAC1-D8-mIgG2a, detects PAPP-A specifically and sensitively in human tissue. In addition, this antibody allows detection of PAPP-A in non-human species. We demonstrate its usefulness for the visualization of PAPP-A in murine and porcine tissues.

Pregnancy-associated plasma protein A values in patients with stable cardiovascular disease: use of a new monoclonal antibody-based assay.

BACKGROUND: PAPP-A is promising in improving risk stratification and invasive treatment decisions in stable cardiovascular patients. We evaluated the prognostic value of pregnancy-associated plasma protein A (PAPP-A) measured by a novel assay in stable cardiovascular patients. METHODS: We investigated 228 stable cardiovascular outpatients. Blood was drawn for PAPP-A measurement after echocardiography and ergometry prior to heart catheterization. Angiographically we determined severity as well as qualitative characteristics suspect for vulnerability of coronary lesions. After 1108±297 days, follow-up information was obtained by questionnaire mailings and interviews by phone. RESULTS: 104 patients had coronary stenosis≥70%, 75 had B-type lesions≥50%, 46 showed complex lesions, and 68 were suspected to have vulnerable lesions. Median PAPP-A was 1.76 (interquartile range 1.21, 2.63) µIU/ml in the entire cohort. PAPP-A concentrations did not differ in dependence on coronary artery findings. A cutpoint of 2.7 µIU/ml was derived from receiver-operator characteristics for outcome measures. For this cutoff, Cox proportional hazard models with 19 further clinical variables showed that PAPP-A was predictive for all-cause death (HR 4.73, 95% CI 1.46-15.31, p=0.01), all-cause death or nonfatal infarction (HR 4.01, 95% CI 1.58-10.13, p=0.003) and all-cause death, nonfatal myocardial infarction or hospitalization (HR 1.96, 95% CI 1.03-3.70, p=0.04). The predictive value of PAPP-A did not change substantially after correction for values of cardiac troponin, using a highly sensitive cardiac troponin I research assay. CONCLUSIONS: PAPP-A, measured by a new, monoclonal antibody-based assay is a promising prognostic marker in patients with stable cardiovascular disease and an indication for heart catheterization.

Inflammatory biomarkers and coronary heart disease: from bench to bedside and back.

Inflammation plays a pivotal role in all stages of atherosclerosis from endothelial dysfunction and plaque formation to plaque destabilization and disruption. Inflammatory biomarkers, originally studied to better understand the pathophysiology of atherosclerosis, have generated increasing interest among clinicians, because of their utility in the challenging problems of diagnosis and risk assessment of patients with suspected or proved coronary heart disease. Moreover, in fascinating perspective, they could be used as therapeutic target, counteracting initiation, progression, and development of complications of atherosclerosis. In this review, we will provide an overview of the more promising inflammatory biomarkers, focusing on their utility and limitations in the clinical setting.

Resistance to age-dependent thymic atrophy in long-lived mice that are deficient in pregnancy-associated plasma protein A.

Pregnancy-associated plasma protein A (PAPPA) is a metalloproteinase that controls the tissue availability of insulin-like growth factor (IGF). Homozygous deletion of PAPPA in mice leads to lifespan extension. Since immune function is an important determinant of individual fitness, we examined the natural immune ecology of PAPPA(-/-) mice and their wild-type littermates reared under specific pathogen-free condition with aging. Whereas wild-type mice exhibit classic age-dependent thymic atrophy, 18-month-old PAPPA(-/-) mice maintain discrete thymic cortex and medulla densely populated by CD4(+)CD8(+) thymocytes that are capable of differentiating into single-positive CD4 and CD8 T cells. Old PAPPA(-/-) mice have high levels of T cell receptor excision circles, and have bone marrows enriched for subsets of thymus-seeding progenitors. PAPPA(-/-) mice have an overall larger pool of naive T cells, and also exhibit an age-dependent accumulation of CD44(+)CD43(+) memory T cells similar to wild-type mice. However, CD43(+) T cell subsets of old PAPPA(-/-) mice have significantly lower prevalence of 1B11 and S7, glycosylation isoforms known to inhibit T cell activation with normal aging. In bioassays of cell activation, splenic T cells of old PAPPA(-/-) mice have high levels of activation antigens and cytokine production, and also elicit Ig production by autologous B cells at levels equivalent to young wild-type mice. These data suggest an IGF-immune axis of healthy longevity. Controlling the availability of IGF in the thymus by targeted manipulation of PAPPA could be a way to maintain immune homeostasis during postnatal development and aging.

Discovery and characterization of human antibody inhibitors of pregnancy-associated plasma protein-A.

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease that cleaves insulin-like growth factor-binding proteins (IGFBPs) to release bioactive levels of free insulin-like growth factor. Specific and potent inhibitors of PAPP-A may further elucidate the biological functions of this protease and could prove to be of therapeutic value. Phage display was used to discover fully human antibody inhibitors of PAPP-A activity towards IGFBP4 cleavage. Estimates of the inhibition constants for these antibodies were subsequently determined using a novel continuous assay of PAPP-A protease activity that uses an internally quenched synthetic peptide substrate (DX-1655). DX-1655 was hydrolyzed by PAPP-A with a K(m) of 33 muM and a k(cat) of 0.3 s(-1) (k(cat)/K(m)=9.1x10(3) M(-1) s(-1)). PAPP-A activity towards DX-1655 displays a bell-shaped pH profile, with pK(a) values of 8.2 and 10.8 and a maximum rate at approximately pH 9.5. Using this continuous assay, we measured apparent K(i) values of 1.7+/-0.2 and 7.4+/-1.5 nM for the F2 and D9 antibodies, respectively.

Quantification of proteolytically active pregnancy-associated plasma protein-A with an assay based on quenched fluorescence.

BACKGROUND: Maternal serum concentrations of pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1, EC 3.4.24.79) are used to predict the occurrence of Down syndrome. In pregnancy, PAPP-A primarily circulates as a covalent 2:2 complex with the proform of eosinophil major basic protein (proMBP), which inhibits the proteolytic activity of PAPP-A. At term, however, approximately 1% of PAPP-A exists as an active, uncomplexed dimer with proteolytic activity directed specifically toward insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5. No assays have been developed that allow quantification of PAPP-A proteolytic activity. METHODS: We developed a sensitive and specific immunocapture assay for PAPP-A activity based on intramolecular quenched fluorescence. We used a 26-residue synthetic peptide derived from IGFBP-4 in which specific positions on each side of the PAPP-A cleavage site were substituted with 3-nitrotyrosine and o-aminobenzoic acid. RESULTS: The assay detected the activity of recombinant PAPP-A as well as PAPP-A in serum samples from pregnant women. The limit of detection (mean signal of blank plus 3 SD) of the active PAPP-A subunit was 13 pmol/L, and the intra- and interassay CVs were <10% and <15%, respectively. Interestingly, the fraction of active PAPP-A decreased gradually from week 7 to week 19 of pregnancy. CONCLUSIONS: This method allows the measurement of PAPP-A enzymatic activity and because of its specificity it is relevant to the study of the biological function of PAPP-A. The method may also be useful in the diagnosis of pregnancy disorders.

Immunoassays developed for pregnancy-associated plasma protein-A (PAPP-A) in pregnancy may not recognize PAPP-A in acute coronary syndromes.

BACKGROUND: Pregnancy-associated plasma protein-A (PAPP-A) concentrations are increased in the circulation of patients with acute coronary syndromes (ACS) and are associated with future adverse cardiac events. PAPP-A in ACS differs from PAPP-A in pregnancy in that PAPP-A in ACS is not complexed with the proform of eosinophil major basic protein (proMBP). We investigated the effect of antibody selection on the utility of PAPP-A assays for measurement of PAPP-A in pregnancy and/or ACS, and whether immunoassays for PAPP-A in pregnancy are suitable for PAPP-A in ACS. METHODS: We constructed 2-site sandwich time-resolved immunofluorometric assays using 22 monoclonal antibodies raised against pregnancy serum PAPP-A. All antibodies were studied in pairs, with each antibody used as either capture or tracer. We compared the reactivity of each antibody combination with PAPP-A/proMBP complex derived from pregnancy sera or with uncomplexed PAPP-A extracted from atherosclerotic plaques. Recombinant human PAPP-A and proMBP were also used to determine the specificity of the antibodies. We confirmed all major findings with serum samples collected from patients with myocardial infarction. RESULTS: Six monoclonal antibodies reacted with the proMBP subunit of the PAPP-A/proMBP complex. Epitopes of 3 proMBP-reactive antibodies largely overlapped, but were well separated from those of another group of 3 proMBP-reactive antibodies. Assays using any of the 6 proMBP-reactive antibodies failed to detect PAPP-A in ACS. In addition, some 2-site assays capable of detecting PAPP-A in pregnancy were almost incapable of detecting PAPP-A in ACS, although the individual epitopes remained detectable in PAPP-A in ACS. CONCLUSIONS: Immunoassays developed for PAPP-A in pregnancy may not be suitable for PAPP-A in ACS. Assays for PAPP-A in ACS should be based on careful antibody selection and subjected to extensive testing with clinical ACS samples.

Pregnancy-associated plasma protein-A proteolytic activity in rat vertebral cell cultures: stimulation by dexamethasone--a potential mechanism for glucocorticoid regulation of osteoprogenitor proliferation and differentiation.

Glucocorticoids (GCs) at physiological concentrations stimulate osteoprogenitor proliferation and differentiation in rat bone cell populations, and this is mediated in part by an increased response to insulin-like growth factors (IGFs). Since IGF binding proteins (IGFBPs) modulate IGF actions, we evaluated whether the increased IGF responsiveness might be associated with decreased inhibitory IGFBP-4 peptide levels. Rat vertebral cells were cultured for up to 20 days with or without dexamethasone (Dex). Cell layer proteins were extracted at day 6, 8, 14, and 20, conditioned media (CM) collected at day 8, 14, and 20, and total RNA isolated at day 14 and 20 of culture. Western blotting showed that cell layer IGFBP-4 levels were lower, while IGFBP-4 protease activity in CM was higher, in Dex-treated cultures. Addition of pregnancy-associated plasma protein-A (PAPP-A) antibody to CM abrogated IGFBP-4 proteolysis. PAPP-A mRNA levels were the same in control and Dex-treated cultures as evaluated by RT-PCR. Our data demonstrate that activity of the IGFBP-4 protease, PAPP-A, in rat bone cell cultures is increased by Dex via post-transcriptional mechanisms. Since IGFBP-4 mRNA levels in Dex-treated cultures were the same as in controls at day 8, slightly lower than in controls at day 14, and higher than in controls at day 20 as shown previously, the decreased IGFBP-4 peptide levels in Dex-treated cultures likely result from increased IGFBP-4 proteolysis by the elevated PAPP-A enzymatic activity. Our findings underscore a novel mechanism whereby GCs increase IGF responses in rat bone cells via PAPP-A-induced IGFBP-4 proteolysis.

Molecular distinction of circulating pregnancy-associated plasma protein A in myocardial infarction and pregnancy.

BACKGROUND: In the blood of pregnant women, pregnancy-associated plasma protein A (PAPP-A) is present as a covalent complex with the proform of eosinophil major basic protein (proMBP). Recently, increased serum concentrations of PAPP-A have been found in acute coronary syndromes (ACS). The aim of this study was to investigate whether the circulating PAPP-A in ACS is the same as that in pregnancy. METHODS: We developed two time-resolved immunofluorometric assays based on a relative epitope map constructed by the use of 17 monoclonal antibodies. One assay, which measured total PAPP-A, used two PAPP-A subunit-specific antibodies. The other assay, which measured PAPP-A/proMBP complex, used one proMBP subunit-specific antibody and one PAPP-A subunit-specific antibody. Serum samples from four patients with myocardial infarction (MI), three pregnant women in their first trimester, and one in her third trimester were fractionated by gel filtration on a Superose 6 precision column. The two assays were used to analyze fractions obtained by gel filtration as well as serum samples serially collected from four other MI patients. RESULTS: Pregnancy-related PAPP-A was eluted as a single peak with a molecular mass of approximately 700 kDa, whereas ACS-related PAPP-A was also eluted as a single peak but with a molecular mass of approximately 530 kDa. Pregnancy-related PAPP-A was detected equally by the two assays, whereas increased ACS-related PAPP-A was detected only by the assay for total PAPP-A. CONCLUSIONS: Our results provide the first evidence that circulating ACS-related PAPP-A is different from circulating pregnancy-related PAPP-A in that it is not complexed with proMBP. These findings provide a solid foundation for the design of immunoassays to accurately measure atherosclerosis-associated plasma protein A in the circulation.

[Role of proteins of the macroglobulin family in the mechanisms of infection]. / Rol' belkov semeistva makroglobulinov v mekhanizmakh infitsirovaniia.

Information on the properties of proteins of the macroglobulin family taking part in the host protection from viral, fungal and bacterial pathogens is reviewed. High plasticity and polyfunctional character of these proteins makes it possible to realize different protective functions. They inhibit the lysis of the cell wall by binding the hydrolases of the pathogen thus blocking its penetration into the cell, directly participate in the presentation of antigens to immunocompetent cells, transport antibacterial substances (interferons, lysozyme) to the zone of infection. In addition, macroglobulins take part in the apoptosis regulation in infected cells, utilization of the lysosomal enzymes of annihilated pathogens. The complexes of macroglobulins with some proteins are powerful inductors of antibody production. Further studies of the properties of these proteins will result in a better understanding of the nature of infectious process. The possibility of artificial formation of macroglobulin complexes with pathogen components or with substances possessing protective or anti-inflammatory properties opens prospects for using these proteins in the fields of vaccinology, gene therapy and molecular biology.

Selection of the dominant follicle and insulin-like growth factor (IGF)-binding proteins: evidence that pregnancy-associated plasma protein A contributes to proteolysis of IGF-binding protein 5 in bovine follicular fluid.

Development of a dominant follicle is associated with decreased intrafollicular low molecular weight IGF-binding proteins (namely IGFBP-2, -4, and -5) and increased proteolysis of IGFBP-4 by pregnancy-associated plasma protein A (PAPP-A). In addition to IGFBP-4 proteolytic activity, bovine follicular fluid contains strong proteolytic activity for IGFBP-5, but not for IGFBP-2. Here we show that the IGFBP-5 protease present in bovine follicular fluid is a neutral/basic pH-favoring, Zn(2+) metalloprotease very similar to the previously described IGFBP-4 protease. We hypothesized that immunoneutralization and immunoprecipitation with anti-PAPP-A antibodies would result in abrogation of the IGFBP-4, but not the IGFBP-5, proteolytic activity in follicular fluid. As expected, anti-PAPP-A antibodies were able to neutralize and precipitate the IGFBP-4, but not the IGFBP-5, proteolytic activity of human pregnancy serum, which was used as a positive control for PAPP-A. Surprisingly, immunoneutralization and immunoprecipitation of follicular fluid from bovine preovulatory follicles with anti-PAPP-A antibodies abrogated both IGFBP-4 and IGFBP-5 proteolysis. Quantitative results derived from phosphorimaging revealed a complete inhibition of both IGFBP-4 and -5 proteolysis by follicular fluid incubated for 2 or 5 h in the presence of anti-PAPP-A antibodies. After 18 h of incubation, anti-PAPP-A antibodies still inhibited IGFBP-5 degradation, although with an efficiency lower than that for IGFBP-4 degradation. Both proteolytic activities have identical electrophoretic mobility, and a single band ( approximately 400 kDa) was detected by Western immunoblotting of bovine follicular fluid with anti-PAPP-A antibodies. Proteolysis of IGFBP-5 was readily detectable in follicular fluid from dominant follicles and was negligible in subordinate follicles from the same cohort. These results suggest that an active intrafollicular IGFBP-4/-5 proteolytic system, in which PAPP-A is the major protease involved, is an important determinant of follicular fate.

Pregnancy-associated plasma protein-A is involved in insulin-like growth factor binding protein-2 (IGFBP-2) proteolytic degradation in bovine and porcine preovulatory follicles: identification of cleavage site and characterization of IGFBP-2 degradation.

In mammalian ovaries, terminal follicular growth is accompanied by a decrease in levels of intrafollicular insulin-like growth factor binding protein-2 (IGFBP-2) and IGFBP-4. The decrease in IGFBP-4 is essentially due to an increase in proteolytic degradation by intrafollicular pregnancy-associated plasma protein-A (PAPP-A) in growing healthy follicles. In contrast, the decrease in IGFBP-2 is partly due to a decrease in mRNA expression by follicular cells and also to an increase in IGFBP-2 proteolytic degradation, as previously shown in ewes and sows. In the present work we show that bovine and porcine preovulatory follicular fluid contains a proteolytic activity that degrades IGFBP-2. Bovine and porcine preovulatory follicular fluids contain undetectable levels of native IGFBP-2 as assessed by Western ligand blotting in comparison with the corresponding serum. In contrast, much higher levels of 23- and 12-kDa proteolytic fragments were found by immunoblotting in bovine and porcine preovulatory follicular fluid than in the corresponding serum. Moreover, bovine and porcine preovulatory follicular fluids were able to induce proteolytic degradation of exogenous IGFBP-2, and this degradation was enhanced by insulin-like growth factors. Intrafollicular IGFBP-2 proteolytic activity was surprisingly immunoneutralized in both species by a polyclonal antibody raised against human PAPP-A. In addition, recombinant human PAPP-A (rhPAPP-A) was able to cleave IGFBP-2 between Gln165 and Met166 in vitro, generating 23- and 12-kDa proteolytic fragments. IGFBP-2 was shown to be less sensitive than IGFBP-4 to cleavage by rhPAPP-A in vitro. As in follicular fluid, cleavage of IGFBP-2 by rhPAPP-A was dose-dependently enhanced by IGFs and inhibited by a peptide derived from the heparin-binding domain of IGFBP-5 (P5). Finally, Biacore analysis showed that P5 peptide-induced inhibition of IGFBP-2 cleavage was due to a direct interaction of P5 with PAPP-A rather than with IGFBP-2. Overall, these data show that in bovine and porcine preovulatory follicles, PAPP-A is responsible for IGF-dependent IGFBP-2 degradation. During follicular growth, the increase in IGFBP-2 cleavage by PAPP-A, as well as the decrease in IGFBP-2 expression, are responsible for the decrease in intact IGFBP-2 levels and the increase in IGF bioavailability. In atretic follicles, the increase and decrease in IGFBP-2 and PAPP-A mRNA expression, respectively, as well as the inhibition of PAPP-A activity by heparin-binding domains present in IGFBP-5 or other proteins, might participate in higher IGFBP-2 levels and a decrease in IGF bioavailability.

Review: immunomodulatory activity of pregnancy-associated plasma protein-A.

This mini-review highlights the growing number of indications for the immunological importance of pregnancy-associated plasma protein-A (PAPP-A), which is a remote member of the alpha-macroglobulin plasma protein family. PAPP-A can bind a variety of cytokines and specifically cleave a binding protein for insulin-like growth factors, thereby serving as a modulator of cytokine activity. Important immune functions, such as lymphocyte proliferation response to alloantigens and lectins and expression of HLA-DR molecules are predominantly suppressed in vitro by PAPP-A. It is likely that the immunoregulatory properties of PAPP-A are very similar to that of alpha 2-macroglobulin. The experimental data allows us to suppose that PAPP-A serves to prevent the recognition of the fetus by the maternal immune system and to suppress locally the host's immune response to the tumour.

Pregnancy associated plasma protein-A: ultrasensitive immunoassay and determination in coronary heart disease.

OBJECTIVES: Markers of myocardial injury have been vital in the assessment of patients with coronary heart disease. Pregnancy associated plasma protein A (PAPP)-A is an insulin-like growth factor (IGF) binding protein (IGFBP)-4 protease and a potential early indicator of unstable angina. We developed an ultrasensitive enzyme-linked immunosorbent assay (ELISA) for PAPP-A and measured serum PAPP-A in patients with biochemical evidence of acute coronary syndrome. DESIGN AND METHODS: Method development was based on pair-wise evaluation of a panel of antibodies and determination of PAPP-A specificity and sensitivity relative to those of a conventional method. Association of PAPP-A with myocardial damage was assessed in serum samples classified based on serum creatine kinase (CK)-MB or cardiac troponin-T levels. RESULTS: Serum PAPP-A was significantly higher in samples with elevated CK-MB or troponin-T than in samples with normal CK-MB (p < 0.001). Marker-association studies showed strong correlation between PAPP-A and troponin-T (r = 0.59, p < 0.001) in a subset of troponin-T positive samples. Indications for both parallel as well as divergence in the expression of PAPP-A and troponin-T was also evident when serial timed samples available from a number of patients were analyzed. CONCLUSIONS: The data are consistent with the conclusion that expression of PAPP-A is enhanced in patients with biochemical evidence of acute coronary syndrome and suggest strongly that demonstration of PAPP-A association with other cardiac markers might be influenced by their relative release dynamics (timing and duration). The availability of the ultrasensitive PAPP-A ELISA should facilitate systematic investigations of PAPP-A expression in this and other pathophysiological conditions that might involve altered expression of the IGF/PAPP-A system.

Pregnancy-associated plasma protein A proteolytic activity is associated with the human placental trophoblast cell membrane.

Pregnancy-associated plasma protein-A (PAPP-A) is a product of the placenta and decidua and is secreted into the maternal circulation during human pregnancy. It recently has been identified as an IGF binding protein (IGFBP)-4 protease. Presumed functions at the maternal-fetal interface are to proteolyze IGFBP-4 and thus increase IGF bioavailability locally in the placenta, to promote IGF-II-mediated trophoblast invasion into the maternal decidua, and to modulate IGF regulation of steroidogenesis and glucose and amino acid transport in the villous. Herein, we have investigated the possibility that IGFBP-4 proteolysis may occur on the trophoblast cell membrane, presumably to increase local bioavailable IGF for interactions with cognate IGF membrane receptors. Human trophoblasts were cultured, trophoblast plasma membranes were isolated and solubilized, and IGFBP-4 protease activity and PAPP-A immunoreactivity in the solubilized plasma membrane fraction were investigated. IGFBP-4 protease activity was detected in solubilized human trophoblast membranes, resulting in cleavage of recombinant human IGFBP-4 into 18- and 14-kDa fragments, detected by Western immunoblot analysis. This protease activity was dependent on the presence of IGF-II, and its metal ion dependence was demonstrated by inhibition of the protease by the metal chelators, EDTA and EGTA. The presence of PAPP-A in solubilized human trophoblast membranes was demonstrated by Western immunoblotting. Trophoblast membrane PAPP-A had a relative molecular weight of approximately 200 kDa and comigrated on (reducing) SDS-PAGE with recombinant human PAPP-A and PAPP-A secreted into media conditioned by cultured human trophoblasts. IGFBP-4 protease activity was not detected after immunodepletion of PAPP-A from the trophoblast membrane fraction with PAPP-A polyclonal antibodies, suggesting the identity of the membrane-derived IGFBP-4 protease as PAPP-A. Immunocytochemistry revealed PAPP-A on the cell membrane and in the cytoplasm of human trophoblasts in culture. Together, these data demonstrate the presence of an IGF-II- and metal-dependent IGFBP-4 protease activity in human trophoblast plasma membranes, identified as PAPP-A, which is well situated to proteolyze IGFBP-4 at the maternal-placental interface to facilitate IGF action at the villous surface and/or the invading extravillous cytotrophoblast.

Point-of-care time-resolved immunofluorometric assay for human pregnancy-associated plasma protein A: use in first-trimester screening for Down syndrome.

BACKGROUND: Screening for Down syndrome in the first trimester by a combination of fetal nuchal translucency thickness and maternal serum pregnancy-associated plasma protein A (PAPP-A) and free beta-human chorionic gonadotropin has been shown to be effective and efficient. We aimed to develop a fast point-of-care assay that could be placed in one-stop clinics for the measurement of PAPP-A. METHODS: We developed a two-site, one-step assay that uses two monoclonal antibodies (mAbs) to PAPP-A, based on a dry-reagent, all-in-one immunoassay concept with a stable fluorescent lanthanide chelate and time-resolved fluorometry. One antibody (mAb 10E1) was biotinylated, and the other (mAb 234-5) was europium-labeled, both via the epsilon-amino groups of surface lysine residues. The assay was performed on an AIO immunoanalyzer at 36 degrees C in single, streptavidin-coated microtitration wells that contained the dry reagents. PAPP-A, either in free or complexed form, was detected by the antibodies used. RESULTS: The assay procedure required 20 min and used 10 microL of sample. The calibration curve was linear from 5 to 10 000 mIU/L. The detection limit was 0.5 mIU/L. Intra- and interassay imprecision (CV) was < or = 4.3% and 8.3%, respectively, for whole blood, plasma, or serum samples. Recovery was 93-96% for serum, 95-108% for heparin-derived whole blood, and 98-103% for heparin-derived plasma. Parallelism was observed in all three matrices. Results correlated [slope = 0.85 (confidence interval, 0.82-0.87); intercept = -33 (confidence interval, -58 to -9); S(y:x) = 85 mIU/L; r = 0.991; n = 100] with those obtained by a Delfia assay. Heparin did not affect the assay, but EDTA markedly reduced PAPP-A values. PAPP-A was stable at 4 degrees C for at least 18 days in serum and for 8 days in heparin-derived whole blood or plasma. CONCLUSIONS: The present assay appears suited for use in one-stop clinics for screening for Down syndrome in the first trimester, with results available within 1 h.