Milk transcriptome biomarker identification to enhance feed efficiency and reduce nutritional costs in dairy ewes.

In recent years, rising prices for high-quality protein-based feeds have significantly increased nutrition costs. Consequently, investigating strategies to reduce these expenses and improve feed efficiency (FE) have become increasingly important for the dairy sheep industry. This research investigates the impact of nutritional protein restriction (NPR) during prepuberty and FE on the milk transcriptome of dairy Assaf ewes (sampled during the first lactation). To this end, we first compared transcriptomic differences between NPR and control ewes. Subsequently, we evaluated gene expression differences between ewes with divergent FE, using feed conversion ratio (FCR), residual feed intake (RFI), and consensus classifications of high- and low-FE animals for both indices. Lastly, we assess milk gene expression as a predictor of FE phenotype using random forest. No effect was found for the prepubertal NPR on milk performance or FE. Moreover, at the milk transcriptome level, only one gene, HBB, was differentially expressed between the NPR (n = 14) and the control group (n = 14). Further, the transcriptomic analysis between divergent FE sheep revealed 114 differentially expressed genes (DEGs) for RFI index (high-FERFI = 10 vs low-FERFI = 10), 244 for FCR (high-FEFCR = 10 vs low-FEFCR = 10), and 1 016 DEGs between divergent consensus ewes for both indices (high-FEconsensus = 8 vs low-FEconsensus = 8). These results underscore the critical role of selected FE indices for RNA-Seq analyses, revealing that consensus divergent animals for both indices maximise differences in transcriptomic responses. Genes overexpressed in high-FEconsensus ewes were associated with milk production and mammary gland development, while low-FEconsensus genes were linked to higher metabolic expenditure for tissue organisation and repair. The best prediction accuracy for FE phenotype using random forest was obtained for a set of 44 genes consistently differentially expressed across lactations, with Spearman correlations of 0.37 and 0.22 for FCR and RFI, respectively. These findings provide insights into potential sustainability strategies for dairy sheep, highlighting the utility of transcriptomic markers as FE proxies.

An electrochemical immunosensor for brain natriuretic peptide prepared with screen-printed carbon electrodes nanostructured with gold nanoparticles grafted through aryl diazonium salt chemistry.

A sensitive amperometric immunosensor has been prepared by immobilization of capture antibodies onto gold nanoparticles (AuNPs) grafted on a screen-printed carbon electrode (SPCE) through aryl diazonium salt chemistry using 4-aminothiophenol (AuNPs-S-Phe-SPCE). The immunosensor was designed for the accurate determination of clinically relevant levels of B-type natriuretic peptide (BNP) in human serum samples. The nanostructured electrochemical platform resulted in an ordered layer of AuNPs onto SPCEs which combined the advantages of high conductivity and improved stability of immobilized biomolecules. The resulting disposable immunosensor used a sandwich type immunoassay involving a peroxidase-labeled detector antibody. The amperometric transduction was carried out at -0.20V (vs the Ag pseudo-reference electrode) upon the addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate. The nanostructured immunosensors show a storage stability of at least 25 days, a linear range between 0.014 and 15ngmL-1, and a LOD of 4pgmL-1, which is 100 times lower than the established cut-off value for heart failure (HF) diagnosis. The performance of the immunosensor is advantageously compared with that provided with immunosensors prepared by grafting SPCE with p-phenylendiamine (H2N-Phe-SPCE) and attaching AuNPs by immersion into an AuNPs suspension or by electrochemical deposition, as well as with immunosensors constructed using commercial AuNPs-modified SPCEs. The developed immunosensor was applied to the successful analysis of human serum from heart failure (HF) patients upon just a 10-times dilution as sample treatment.

Data on clinical characteristics of a heart failure patients' cohort with reduced ejection fraction and analysis of the circulating values of five different heart failure biomarkers; high sensitivity troponin T, galectin-3, C-terminal propeptide of type I procollagen, soluble AXL and BNP.

In this article, the full description of a heart failure with reduced ejection fraction (HF_REF) cohort of 192 patients is provided. Tables with the baseline demographic, prior history, ECG parameters, echocardiographic parameters, laboratory values and pharmacological treatment of these patients are included. Also, the quartile values of the analyzed circulating biomarkers: high sensitivity Troponin T (hs-TnT), galectin-3 (Gal-3), C-terminal propeptide of type I procollagen (CICP), soluble AXL (sAXL) and Brain Natriuretic Peptide (BNP) are given. The main demographic and clinical features of the patients' subgroups that have hs-TnT, Gal-3, CICP or BNP above the third quartile are described. Tables with Pearson correlation analysis of the HF_REF patients' biomarker levels are included. And Pearson correlation analysis of the HF_REF patients' hs-TnT, Gal-3, CICP levels with patients' biochemical parameters, blood count and inflammation parameters are also described. These data are related to the research articles (AXL receptor tyrosine kinase is increased in patients with heart failure (M. Batlle, P. Recarte-Pelz, E. Roig, M.A. Castel, M. Cardona, M. Farrero, et al., 2014) [1] and Use of serum levels of high sensitivity troponin T, galectin-3 and C-terminal propeptide of type I procollagen at long term follow-up in Heart Failure patients with reduced ejection fraction: comparison with soluble AXL and BNP (M. Batlle, B. Campos, M. Farrero, M. Cardona, B. González, M.A. Castel, et al., 2016) [2].

Use of serum levels of high sensitivity troponin T, galectin-3 and C-terminal propeptide of type I procollagen at long term follow-up in heart failure patients with reduced ejection fraction: Comparison with soluble AXL and BNP.

BACKGROUND: Prognostic biomarkers are needed to improve the management of the heart failure (HF) epidemic, being the brain natriuretic peptides the most valuable. Here we evaluate 3 biomarkers, high sensitivity troponin T (hs-TnT), galectin-3 (Gal-3) and C-terminal propeptide of type I procollagen (CICP), compare them with a recently described new candidate (sAXL), and analyze their relationship with BNP. METHODS: HF patients with reduced ejection fraction (n=192) were included in this prospective observational study, with measurements of candidate biomarkers, functional, clinical and echocardiographic variables. A Cox regression model was used to determine predictors for clinical events, i.e. all-cause mortality and heart transplantation. RESULTS: Hs-TnT circulating values were correlated to clinical characteristics indicative of more advanced HF. When analyzing the event-free survival at a mean follow-up of 3.6years, patients in the higher quartile of either BNP, hs-TnT, CICP and sAXL had increased risk of suffering a clinical event, but not Gal-3. Combination of high sAXL and BNP values had greater predictive value (HR 6.8) than high BNP alone (HR 4.9). In a multivariate Cox regression analysis, BNP, sAXL and NYHA class were independent risk factors for clinical events. CONCLUSIONS: In this HF cohort, hs-TnT is a good HF marker and has a very significant prognostic value. The prognostic value of CICP and sAXL was of less significance. However, hs-TnT did not add predictive value to BNP, while sAXL did. This suggests that elevated troponin has a common origin with BNP, while sAXL could represent an independent pathological mechanism.

AXL receptor tyrosine kinase is increased in patients with heart failure.

BACKGROUND: AXL is a membrane receptor tyrosine kinase highly expressed in the heart and has a conspicuous role in cardiovascular physiology. The role of AXL in heart failure (HF) has not been previously addressed. METHODS AND RESULTS: AXL protein was enhanced 6-fold in myocardial biopsies of end-stage HF patients undergoing heart transplantation compared to controls from heart donors (P<0.0001). Next, we performed a transversal study of patients with chronic HF (n=192) and a group of controls with no HF (n=67). sAXL and BNP circulating levels were quantified and clinical and demographic data were collected. sAXL levels in serum were higher in HF (86.3 ± 2.0 ng/mL) than in controls (67.8 ± 2.0 ng/mL; P<0.0001). Also, sAXL correlated with several parameters associated with worse prognosis in HF. Linear regression analysis indicated that serum creatinine, systolic blood pressure and atrial fibrillation, but not BNP levels, were predictive of sAXL levels. Cox regression analysis indicated that high sAXL values at enrollment time were related to the major HF events (all-cause mortality, heart transplantation and HF hospitalizations) at one year follow-up (P<0.001), adding predictive value to high BNP levels. CONCLUSIONS: Myocardial expression and serum concentration of AXL is elevated in HF patients compared to controls. Furthermore, peripheral sAXL correlates with parameters associated with the progression of HF and with HF events at short term follow-up. All together these results suggest that sAXL could belong to a new molecular pathway involved in myocardial damage in HF, independent from BNP.

Expression of the vitamin K-dependent proteins GAS6 and protein S and the TAM receptor tyrosine kinases in human atherosclerotic carotid plaques.

The GAS6/ProS-TAM system is composed of two vitamin K-dependent ligands (GAS6 and protein S) and their three protein tyrosine kinase receptors TYRO3, AXL and MERTK, known as the TAM receptors. The system plays a prominent role in conditions of injury, inflammation and repair. In murine models of atherosclerotic plaque formation, mutations in its components affect atherosclerosis severity. Here we used Taqman low-density arrays and immunoblotting to study mRNA and protein expression of GAS6, ProS and the TAM receptors in human carotid arteries with different degrees of atherosclerosis. The results show a clear down-regulation of the expression of AXL in atheroma plaques with respect to normal carotids that is matched by decreased abundance of AXL in protein extracts detected by immunoblotting. A similar decrease was observed in PROS1 mRNA expression in atherosclerotic carotids compared to the normal ones, but in this case protein S (ProS) was clearly increased in protein extracts of carotid arteries with increasing grade of atherosclerosis, suggesting that ProS is carried into the plaque. MERTK was also increased in atherosclerotic carotid arteries with respect to the normal ones, suggesting that the ProS-MERTK axis is functional in advanced human atherosclerotic plaques. MERTK was expressed in macrophages, frequently in association with ProS, while ProS was abundant also in the necrotic core. Our data suggest that the ProS-MERTK ligand-receptor pair was active in advanced stages of atherosclerosis, while AXL signalling is probably down-regulated.

Genetic loss of Gas6 induces plaque stability in experimental atherosclerosis.

The growth arrest-specific gene 6 (Gas6) plays a role in pro-atherogenic processes such as endothelial and leukocyte activation, smooth muscle cell migration and thrombosis, but its role in atherosclerosis remains uninvestigated. Here, we report that Gas6 is expressed in all stages of human and mouse atherosclerosis, in plaque endothelial cells, smooth muscle cells and macrophages. Gas6 expression is most abundant in lesions containing high amounts of macrophages, ie thin fibrous cap atheroma and ruptured plaque. Genetic loss of Gas6 does not affect the number and size of initial and advanced plaques in ApoE(-/-) mice, but alters its plaque composition. Compared to Gas6(+/+): ApoE(-/-) mice, initial and advanced plaques of Gas6(-/-): ApoE(-/-) mice contained more smooth muscle cells and more collagen and developed smaller lipid cores, while the expression of TGFbeta was increased. In addition, fewer macrophages were found in advanced plaques of Gas6(-/-): ApoE(-/-) mice. Hence, loss of Gas6 promotes the formation of more stable atherosclerotic lesions by increasing plaque fibrosis and by attenuating plaque inflammation. These findings identify a role for Gas6 in plaque composition and stability.

Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis.

The growth arrest-specific gene 6 product (Gas6) is a secreted protein related to the anticoagulant protein S but its role in hemostasis is unknown. Here we show that inactivation of the Gas6 gene prevented venous and arterial thrombosis in mice, and protected against fatal collagen/epinephrine-induced thrombo embolism. Gas6-/- mice did not, however, suffer spontaneous bleeding and had normal bleeding after tail clipping. In addition, we found that Gas6 antibodies inhibited platelet aggregation in vitro and protected mice against fatal thrombo embolism without causing bleeding in vivo. Gas6 amplified platelet aggregation and secretion in response to known agonists. Platelet dysfunction in Gas6-/- mice resembled that of patients with platelet signaling transduction defects. Thus, Gas6 is a platelet-response amplifier that plays a significant role in thrombosis. These findings warrant further evaluation of the possible therapeutic use of Gas6 inhibition for prevention of thrombosis.

Protein S Thr103Asn mutation associated with type II deficiency reproduced in vitro and functionally characterised.

Protein S functions as a cofactor to activated protein C (APC) in the degradation of FVa and FVIIIa. In protein S, the thrombin sensitive region (TSR) and the first EGF-like domain are important for expression of the APC cofactor activity. A naturally occurring Thr103Asn (T103N) mutation in the first EGF-like domain of protein S has been associated with functional (type II) protein S deficiency. To elucidate the functional consequences of the T103N mutation, recombinant protein S mutant was expressed in mammalian cells and functionally characterised. The expression level of protein S T103N from transiently transfected COS 1 cells was equal to that of wild type protein S. The mutant protein S and wild type protein S were also expressed in 293 cells after stable transfection, and the recombinant proteins purified. In APTT- and PT-based coagulation assays, the mutant protein demonstrated approximately 50% lower anticoagulant activity as compared to wild type protein S. The functional defect was further investigated in FVa- and FVIIIa-degradation assays. The functional defect of mutant protein S was attenuated at increasing concentrations of APC. The results demonstrate the region around residue 103 of protein S to be of functional importance, possibly through a direct interaction with APC.

The second laminin G-type domain of protein S is indispensable for expression of full cofactor activity in activated protein C-catalysed inactivation of factor Va and factor VIIIa.

Vitamin K-dependent protein S is a cofactor to the anticoagulant serine protease activated protein C (APC) in the proteolytic inactivation of the procoagulant, activated factor V (FVa) and factor VIII (FVIIIa). In the FVa degradation, protein S selectively accelerates the cleavage at Arg306, having no effect on the Arg506 cleavage. In the FVIIIa inactivation, the APC-cofactor activity of protein S is synergistically potentiated by FV, which thus has the capacity to function both as a pro- and an anticoagulant protein. The SHBG-like region of protein S, containing two laminin G-type domains, is required for the combined action of protein S and FV. To elucidate whether both G domains in protein S are needed for expression of APC-cofactor activities, chimeras of human protein S were created in which the individual G domains were replaced by the corresponding domain of the homologous Gas6, which in itself has no anticoagulant activity. In a plasma-based assay, chimera I (G1 from Gas6) was as efficient as wild-type recombinant protein S, whereas chimera II (G2 from Gas6) was less effective. The synergistic cofactor activity with FV in the inactivation of FVIIIa was lost by the replacement of the G2 domain in protein S (chimera II). However, chimera I did not exert full APC-cofactor activity in the FVIIa degradation, indicating involvement of both G domains or the entire SHBG-like region in this reaction. Chimera I was fully active in the degradation of FVa in contrast to chimera II, which exhibited reduced cofactor activity compared to protein S. In conclusion, by using protein S-Gas6 chimeric proteins, we have identified the G2 domain of protein S to be indispensable for an efficient inactivation of both FVIIa and FVa, whereas the G1 domain was found not to be of direct importance in the FVa-inactivation experiments.

Deficient APC-cofactor activity of protein S Heerlen in degradation of factor Va Leiden: a possible mechanism of synergism between thrombophilic risk factors.

In protein S Heerlen, an S-to-P (single-letter amino acid codes) mutation at position 460 results in the loss of glycosylation of N458. This polymorphism has been found to be slightly more prevalent in thrombophilic populations than in normal controls, particularly in cohorts of patients having free protein S deficiency. This suggests that carriers of the Heerlen allele may have an increased risk of thrombosis. We have now characterized the expression in cell cultures of recombinant protein S Heerlen and investigated the anticoagulant functions of the purified recombinant protein in vitro. Protein S Heerlen was synthesized and secreted equally well as wild-type protein S by transiently transfected COS-1 cells. The recombinant protein S Heerlen interacted with conformation-dependent monoclonal antibodies and bound C4b-binding protein to the same extent as wild-type protein S. Protein S Heerlen displayed reduced anticoagulant activity as cofactor to activated protein C (APC) in plasma-based assays, as well as in a factor VIIIa-degradation system. In contrast, protein S Heerlen functioned equally well as an APC cofactor in the degradation of factor Va as wild-type protein S did. However, when recombinant activated factor V Leiden (FVa:Q506) was used as APC substrate, protein S Heerlen was found to be a poor APC cofactor as compared with wild-type protein S. These in vitro results suggest a possible mechanism of synergy between protein S Heerlen and factor V Leiden that might be involved in the pathogenesis of thrombosis in individuals carrying both genetic traits. (Blood. 2000;96:523-531)

The first laminin G-type domain in the SHBG-like region of protein S contains residues essential for activation of the receptor tyrosine kinase sky.

Vitamin K-dependent protein S and the product of growth-arrest-specific gene 6 (Gas6) both possess the ability to phosphorylate members of the Axl/Sky subfamily of receptor tyrosine kinases. However, Gas6 appears to be the bona fide ligand for these receptors in man, as human protein S has been demonstrated to activate murine Sky but not the human orthologue. In contrast, bovine protein S is able to stimulate human Sky despite its high degree of sequence identity with human protein S. The domain organisations of protein S and Gas6 are virtually identical and the C-terminal SHBG-like region, containing two globular (G) domains, has been shown to play a crucial role in the receptor stimulation. In order to further localise the area responsible for the interaction, a number of protein chimeras were used to stimulate human Sky. Each chimera had one part of the human protein S SHBG-like region replaced by the corresponding part of bovine protein S or human Gas6. We found that human protein S may indeed activate human Sky but only above physiological plasma concentrations. The human-bovine protein S chimeras provided new information implying that the first G domain contains critical residues for the interaction with the Sky receptor. Moreover, these residues do not seem to be clustered but rather to be distributed at various positions in the first G domain.

Protein S secretion differences of missense mutants account for phenotypic heterogeneity.

To elucidate the molecular background for the heterogeneity in protein S plasma concentrations observed in protein S deficient individuals, the in vitro synthesis of recombinant protein S missense mutants was investigated. Six different naturally occurring mutations identified in the protein S gene (PROS1) of thrombosis patients were reproduced in protein S cDNA by site directed mutagenesis. Two mutants, G441C and Y444C (group A), were associated with low total plasma concentration of protein S. Modestly low protein S was found in families with R520G and P626L (group B) mutants. T57S and I518M (group C), which was associated with marginally low protein S, did not segregate with protein S deficiency in the respective families, raising doubts as to whether they were causative mutations or rare neutral variants. The 6 protein S mutants were transiently expressed in COS 1 cells. The Y444C mutant showed the lowest level of secretion (2.5%) followed by the G441C mutant (40%). Group B demonstrated around 50% reduction in secretion, whereas group C mutants showed normal secretion. Pulse-chase experiments demonstrated impaired protein S processing with intracellular degradation and decreased secretion into the culture media of group A and B mutants. Interestingly, there was a good correlation between in vitro secretion and the concentration of free protein S in the plasma of heterozygous carriers. These results demonstrate impaired protein S secretion to be an important mechanism underlying hereditary protein S deficiency and that variations in protein secretion is a major determinant of the phenotypic heterogeneity observed in protein S deficiency. (Blood. 2000;95:173-179)

Both G-type domains of protein S are required for the high-affinity interaction with C4b-binding protein.

Anticoagulant protein S interacts with the complement regulatory protein C4b-binding protein (C4BP) via its sex-hormone-binding globulin (SHB6)-like region, which contains two globular (G) domains. Similar G domains are found in Gas6, a protein homologous to protein S, which is not known to bind C4BP or to have any anticoagulant activity. To determine the relative importance of the two G domains in protein S for C4BP protein binding, three recombinant protein S chimeras were produced having either of the two globular domains, or the whole SHB6-like globulin region, replaced by corresponding parts from Gas6. The chimeras were tested for binding to immobilized C4BP using surface-plasmon-resonance technology and microtiter plate-based assays. In both systems, chimeras containing either only globular domains G1 or G2 from protein S were found to bind C4BP. Binding was stimulated by Ca2+ in a manner similar to that found for wild-type protein S. The affinities for C4BP of both chimeras containing individual G domains from protein S, were lower than that of wild-type protein S. Chimera II, containing the G1 domain from protein S, consistently bound C4BP more efficiently than chimera I, which had the protein S-derived G2 domain. The chimera containing the whole SHB6-like globulin region from Gas6 interacted considerably more weakly with C4BP. Our results demonstrate that both G domains of protein S are involved in the interaction between protein S and C4BP and that full affinity binding is dependent on contributions from both domains.

In vitro characterisation of two naturally occurring mutations in the thrombin-sensitive region of anticoagulant protein S.

The molecular consequences of two naturally occurring mutations in the thrombin-sensitive region of protein S were investigated using a combination of recombinant protein expression, functional analysis and molecular modelling. Both mutations (R49H and R70S) have been found in thrombosis patients diagnosed as having type I protein S deficiency. Molecular modelling analysis suggested the R49H substitution not to disrupt the structure of thrombin-sensitive region, whereas the R70S substitution could affect the 3D structure mildly. To elucidate the molecular consequences of these substitutions experimentally, site directed mutagenesis of protein S cDNA and expression in mammalian cells created the two mutants. The secretion profiles and functional anticoagulant activities of the protein S mutants were characterised. Secretion of the R49H mutant was similar to that of wild type protein S, whereas the R70S mutant showed moderately decreased expression. Neither of the mutants showed any major functional defects as cofactors to activated protein C (APC) in an APTT-based assay or in degradation of factor Va. However, both mutants demonstrated decreased activity in a factor VIIIa degradation assay, which in addition to APC and protein S also included factor V as synergistic APC cofactor. In conclusion, the R49H substitution did not produce a quantitative abnormality in vitro, raising doubts as to whether it caused the type I deficiency. In contrast, the experimental data obtained for the R70S mutant agrees well with the observed type I deficiency. Our study illustrates that in vitro experimental characterisation together with computer-based structural analysis are useful tools in the analysis of the relationship between naturally occurring mutations and clinical phenotypes.

Thrombophilia as a multigenic disease.

BACKGROUND AND OBJECTIVE: Venous thrombosis is a common disease annually affecting 1 in 1000 individuals. The multifactorial nature of the disease is illustrated by the frequent identification of one or more predisposing genetic and/or environmental risk factors in thrombosis patients. Most of the genetic defects known today affect the function of the natural anticoagulant pathways and in particular the protein C system. This presentation focuses on the importance of the genetic factors in the pathogenesis of inherited thrombophilia with particular emphasis on those defects which affect the protein C system. INFORMATION SOURCES: Published results in articles covered by the Medline database have been integrated with our original studies in the field of thrombophilia. STATE OF THE ART AND PERSPECTIVES: The risk of venous thrombosis is increased when the hemostatic balance between pro- and anti-coagulant forces is shifted in favor of coagulation. When this is caused by an inherited defect, the resulting hypercoagulable state is a lifelong risk factor for thrombosis. Resistance to activated protein C (APC resistance) is the most common inherited hypercoagulable state found to be associated with venous thrombosis. It is caused by a single point mutation in the factor V (FV) gene, which predicts the substitution of Arg506 with a Gln. Arg506 is one of three APC-cleavage sites and the mutation results in the loss of this APC-cleavage site. The mutation is only found in Caucasians but the prevalence of the mutant FV allele (FV:Q506) varies between countries. It is found to be highly prevalent (up to 15%) in Scandinavian populations, in areas with high incidence of thrombosis. FV:Q506 is associated with a 5-10-fold increased risk of thrombosis and is found in 20-60% of Caucasian patients with thrombosis. The second most common inherited risk factor for thrombosis is a point mutation (G20210A) in the 3' untranslated region of the prothrombin gene. This mutation is present in approximately 2% of healthy individuals and in 6-7% of thrombosis patients, suggesting it to be a mild risk factor of thrombosis. Other less common genetic risk factors for thrombosis are the deficiencies of natural anticoagulant proteins such as antithrombin, protein C or protein S. Such defects are present in less than 1% of healthy individuals and together they account for 5-10% of genetic defects found in patients with venous thrombosis. Owing to the high prevalence of inherited APC resistance (FV:Q506) and of the G20210A mutation in the prothrombin gene, combinations of genetic defects are relatively common in the general population. As each genetic defect is an independent risk factor for thrombosis, individuals with multiple defects have a highly increased risk of thrombosis. As a consequence, multiple defects are often found in patients with thrombosis.

Topological studies of the amino terminal modules of vitamin K-dependent protein S using monoclonal antibody epitope mapping and molecular modeling.

Protein S is an important anticoagulant protein acting as cofactor to activated protein C (APC) in the degradation of membrane-bound factors Va and VIIIa. Binding of protein S to the membrane depends on the Gla-domain, whereas sites for APC-interaction are located in the thrombin-sensitive region (TSR) and the first EGF domain. The aims of the present investigation were to localize the sites on protein S which are involved in APC-cofactor function and to elucidate possible orientations of the TSR in relation to the membrane. For these purposes, we determined the epitope for a calcium-dependent monoclonal antibody (HPS67) against the TSR, which inhibits APC cofactor activity even though it does not impede protein S binding to the membrane. HPS67 did not recognize wild-type mouse protein S but gained reactivity against a recombinant mouse protein in which G49 and R52 were mutated to R and Q (found in human protein S), respectively, suggesting these two residues to be part of a surface exposed epitope for HPS67. This information helped in the validation and refinement of the structural model for the Gla-TSR-EGF1-modules of protein S. The X-ray structure of a Fab-fragment mimicking HPS67 was docked onto the protein S model. The observation that HPS67 did not inhibit phospholipid binding of protein S has implications for the possible orientation of protein S on the membrane surface. In the proposed model for membrane-bound protein S, there is no contact between the TSR and the membrane. Rather, the TSR is free to interact with membrane-bound APC.

The SHBG-like region of protein S is crucial for factor V-dependent APC-cofactor function.

Activated protein C (APC) regulates blood coagulation by degrading factor Va (FVa) and factor VIIIa (FVIIIa). Protein S is a cofactor to APC in the FVa degradation, whereas FVIIIa degradation is potentiated by the synergistic APC-cofactor activity of protein S and factor V (FV). To elucidate the importance of the sex-hormone-binding globulin (SHBG)-like region in protein S for expression of anticoagulant activity, a recombinant protein S/Gas6 chimera was constructed. It comprised the amino-terminal half of protein S and the SHBG-like region of Gas6, a structurally similar protein having no known anticoagulant properties. The protein S/Gas6 chimera expressed 40-50%, APC-cofactor activity in plasma as compared to wild-type protein S. In the degradation of FVa by APC, the protein S/Gas6 chimera was only slightly less efficient than wild-type protein S. In contrast, the protein S/Gas6 chimera expressed no FV-dependent APC-cofactor activity in a FVIIIa-degradation system. This demonstrates the SHBG-like region to be important for expression of APC-cofactor activity of protein S and suggests that the SHBG-like region of protein S interacts with FV during the APC-mediated inactivation of FVIIIa.

Structural investigation of C4b-binding protein by molecular modeling: localization of putative binding sites.

C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one beta-chain and seven alpha-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its alpha-chains and with protein S through its beta-chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP alpha-chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the alpha-chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary-structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein-protein interactions.