Biomedical subjects

W G Forssmann

Publications and source records attributed to W G Forssmann.

At least 37 records · Page 2Linked to original sources

PubMed2000

Dual function of the propeptide of prouroguanylin in the folding of the mature peptide: disulfide-coupled folding and dimerization.

Guanylyl cyclase activating peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, is produced via the processing of the precursor protein (prepro-GCAP-II). We have previously shown that the propeptide in pro-GCAP-II functions as an intramolecular chaperone in the proper folding of the mature peptide, GCAP-II (Hidaka, Y., Ohno, M., Hemmasi, B., Hill, O., Forssmann, W.-G., and Shimonishi, Y. (1998) Biochemistry 37, 8498-8507). Here, we report an essential region in pro-GCAP-II for the correct disulfide pairing of the mature peptide, GCAP-II. Five mutant proteins, in which amino acid residues were sequentially deleted from the N terminus, and three mutant proteins of pro-GCAP-II, in which N-terminal 6, 11, or 17 amino acid residues were deleted, were overproduced using Escherichia coli or human kidney 293T cells, respectively. Detailed analysis of in vivo or in vitro folding of these mutant proteins revealed that one or two amino acid residues at the N terminus of pro-GCAP-II are critical, not only for the chaperone function in the folding but also for the net stabilization of pro-GCAP-II. In addition, size exclusion chromatography revealed that pro-GCAP-II exists as a dimer in solution. These data indicate that the propeptide has two roles in proper folding: the disulfide-coupled folding of the mature region and the dimerization of pro-GCAP-II.

Amino Acid Sequence↗

PubMed2000

Electrospray ionization mass spectrometry of low-molecular-mass S-nitroso compounds and their thiols.

Low-molecular-mass S-nitroso compounds (R-S-N=O) are potent vasodilators and inhibitors of platelet aggregation. This work describes the electrospray ionization mass spectrometric (ESI-MS) analysis of physiological and synthetic low-molecular-mass S-nitroso compounds and their thiols including S-nitrosoglutathione, S-nitrosocysteine, glutathione and cysteine. Mass spectra of the unlabeled and S-15N-labeled low-molecular-mass S-nitroso compounds investigated are characterized by abundant cations due to [M+H]+, [M+Na]+, [(M+H)-NO]+, [2 M+H]+, and [(2 M+H)-2NO]+. Mass spectra of low-molecular-mass thiols are characterized by abundant cations due to [M+H]+, [M+Na]+ and [2M+H]+. Using off-line electrospray ionization tandem mass spectrometry we unequivocally identified S-[15N]nitrosoglutathione in human red blood cells formed after their incubation with S-[15N]nitrosocysteine. These results suggest that ESI-MS in combination with an appropriate liquid chromatographic system should be a useful analytical approach for the on-line quantitative determination of low-molecular-mass S-nitroso compounds in biological fluids in the presence of their thiols and nitrite. Considerations were made about on-line ESI-MS and quantitative measurements.

Artifacts↗

PubMed2000

Partial IGF affinity of circulating N- and C-terminal fragments of human insulin-like growth factor binding protein-4 (IGFBP-4) and the disulfide bonding pattern of the C-terminal IGFBP-4 domain.

Within the IGF axis, the insulin-like growth factor-binding proteins (IGFBPs) are known to play a pivotal role in cell proliferation and differentiation. Defined proteolysis of the IGFBPs is proposed to be an essential mechanism for regulating IGF bioavailability. The generated IGFBP fragments in part exhibit different IGF-dependent and -independent biological activities. Characterizing naturally occurring forms of IGFBPs in human plasma, we identified both a N- and a C-terminal fragment of IGFBP-4 by means of immunoreactivity screening. As a source for peptide isolation, we used large amounts of human hemofiltrate obtained from patients with chronic renal failure. Purification of the IGFBP-4 peptides from hemofiltrate was performed by consecutive cation-exchange and reverse-phase chromatographic steps. Mass spectrometric and sequence analysis revealed an M(r) of 13 233 for the purified N-terminal fragment spanning residues Asp(1)-Phe(122) of IGFBP-4 and an M(r) of 11 344 for the C-terminal fragment extending from Lys(136) to Glu(237). Proteolytic digestion and subsequent biochemical analysis showed that the six cysteines of the C-terminal IGFBP-4 fragment are linked between residues 153-183, 194-205, and 207-228 (disulfide bonding pattern, 1-2, 3-4, and 5-6). Plasmon resonance spectroscopy, ligand blot analysis, and saturation and displacement studies demonstrated a very low affinity of the C-terminal IGFBP-4 fragment for the IGFs (IGF-II, K(d) = 690 nM; IGF-I, K(d) > 60 nM), whereas the N-terminal fragment retained significant IGF binding properties (IGF-II, K(d) = 17 nM; IGF-I, K(d) = 5 nM). This study provides the first molecular characterization of circulating human IGFBP-4 fragments formed in vivo exhibiting an at least 5-fold decrease in the affinity of the N-terminal IGFBP-4 fragment for the IGFs and a very low IGF binding capacity of the C-terminal fragment.

Amino Acid Sequence↗

PubMed2000

Solution structures of human parathyroid hormone fragments hPTH(1-34) and hPTH(1-39) and bovine parathyroid hormone fragment bPTH(1-37).

Parathyroid hormone (PTH) is involved in regulation of the calcium level in blood and has an influence on bone metabolism, thus playing a role in osteoporosis therapy. In this study, the structures of the human PTH fragments (1-34) and (1-39) as well as bovine PTH(1-37) in aqueous buffer solution under near physiological conditions were determined using two-dimensional nuclear magnetic resonance spectroscopy. The overall structure of the first 34 amino acids of these three peptides is virtually identical, exhibiting a short NH(2)-terminal and a longer COOH-terminal helix as well as a defined loop region from His14 to Ser17, stabilized by hydrophobic interactions. bPTH(1-37), which has a higher biological activity, shows a better-defined NH(2)-terminal part. In contrast to NH(2)-terminal truncations, which cause destabilization of helical structure, neither COOH-terminal truncation nor elongation significantly influences the secondary structure. Furthermore, we investigated the structure of hPTH(1-34) in 20% trifluoroethanol solution. In addition to its helix-stabilizing effect, trifluorethanol causes the loss of tertiary hydrophobic interactions.

Amino Acid Sequence↗

PubMed2000

Expression and cell-specific localization of the cholecystokinin B/gastrin receptor in the human stomach.

Gastrin stimulates gastric acid secretion by acting on the cholecystokinin B/gastrin receptor (CCK-BR). The localization of this receptor at the cellular level showed conflicting results in animal studies and has not been described in man by immunohistochemistry. The aim of the present study is to characterize the precise cellular location of the CCK-BR in the human stomach. Polyclonal antisera were raised against different epitopes of the CCK-BR molecule and used for immunohistochemical investigations. CCK-BR mRNA was detected in paraffin tissue sections by the highly sensitive method of in situ reverse transcriptase-polymerase chain reaction (RT-PCR). Using immunohistochemistry, CCK-BR could successfully be localized in gastric parietal cells. In the majority of parietal cells, CCK-BR immunoreactivity was present a he basolateral cell membrane domain. In some parietal cells, a granular pattern of immunoreactivity was exclusively confined to the cytoplasm of the cells. CCK-BR mRNA was found in parietal cells and in enterochromaffin-like (ECL) cells by means of in situ RT-PCR. No expression of CCK-BR was found in the gastric antral mucosa. Our data support the concept that gastrin stimulates gastric acid secretion directly via CCK-B receptors on parietal cells and indirectly by inducing histamine release from histamine-containing ECL cells, which contributes to acid secretion by parietal cells.

Amino Acid Sequence↗

PubMed2000

Effects of sildenafil on cAMP and cGMP levels in isolated human cavernous and cardiac tissue.

OBJECTIVES: To further investigate the mechanism of action of sildenafil, a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase 5 (PDE5), that has been proved to be effective in the treatment of male erectile dysfunction. We assessed the effects of sildenafil on the in vitro formation of cGMP and cyclic adenosine monophosphate (cAMP) in isolated human corpus cavernosum and cardiac muscle. METHODS: Isolated segments of human corpus cavernosum and cardiac muscle were exposed to increasing concentrations of sildenafil. The dose-dependent accumulation of cGMP and cAMP was determined in the tissue samples by means of radioimmunoassays. Responses of the isolated tissue preparations to sildenafil were compared with those obtained with the reference compounds sodium nitroprusside, forskolin, and milrinone. RESULTS: In the concentration range 0.01 to 1 microM, there was only a minor effect of sildenafil on cGMP levels in isolated human cavernous and cardiac tissues. In contrast, sildenafil was found to increase cAMP significantly in both cavernous and cardiac tissue in physiologic and supraphysiologic concentrations. The stimulation of cAMP by sildenafil was more pronounced in cavernous than in cardiac tissue. Concentrations of cGMP in the cardiac strips were unaltered by milrinone; cAMP was stimulated starting at a concentration of 0.05 microM. In the range of 0.1 to 1.0 microM, the in vitro effect of sildenafil on cAMP levels in the cardiac samples was almost equivalent to that of milrinone. CONCLUSIONS: Our findings provide a potential mechanism for the cardiovascular side effects that have been reported with sildenafil use, highlighting the fact that a "cross-talk" between cGMP and cAMP-dependent signal transduction pathways might exist in human cavernous and cardiac muscle that may be of pharmacologic significance.

Colforsin↗

PubMed2000

Gene expression of the phosphodiesterases 3A and 5A in human corpus cavernosum penis.

OBJECTIVE: The following study was performed to evaluate the importance of phosphodiesterases 3A (PDE3A) and 5A (PDE5A) for the regulation of penile smooth muscle tone. Furthermore, indications of side effects of specific inhibitors in certain tissues as well as of a possible relation between the level of PDE3A and PDE5A gene expression and erectile dysfunction should have been deduced from the results obtained. METHODS: Total ribonucleic acid was isolated from different human tissues (urogenital tract, gastrointestinal tract, cardiovascular system and central nervous system) and subjected to RTPCR analysis and Northern blotting using primers and probes specific for PDE3A and PDE5A. RESULTS: As shown by RT-PCR and Northern blotting, PDE3A and PDE5A mRNAs exhibit a distinct distribution throughout the tissues examined but were 2-fold higher in cavernous tissue than in all other tissues investigated. However, there were no significant differences in the levels of gene expression between the two subgroups of patients. CONCLUSIONS: Very high expression levels of PDE3A and PDE5A in human cavernous tissue underscore the physiological importance of these enzymes for the regulation of penile erection, emphasizing their therapeutical and pharmacological relevance. The distribution pattern of the mRNA for the isoenzymes PDE3A and PDE5A may explain the pharmacological effects as well as the side effects of milrinone and sidenafil.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1999

Fibroblast growth factor protects the kidney against ischemia-reperfusion injury.

Ischemia-reperfusion injury, a common source of renal dysfunction in adults, is associated with tubular epithelial cell damage. Since fibroblast growth factors (FGF) attenuated tissue injury after transient myocardial ischemia, we hypothesized that acidic fibroblast growth factor (aFGF; FGF-1) would attenuate renal ischemia-reperfusion injury. We studied the effects of FGF-1 in a rat model of acute renal failure induced by bilateral renal ischemia (60 min) and 1, 2 or 7 days reperfusion. After FGF-1 administration at the onset of renal reperfusion, there was less functional impairment of the kidneys. The histological changes were not as severe as in controls. Increases in serum creatinine and blood urea nitrogen 24 h after reperfusion were attenuated by 35% (p< 0.01) and by 53% (p< 0.001), respectively, in FGF-1-treated animals compared to vehicle-treated rats. The ischemia/reperfusion-induced increase in tissue myeloperoxidase, a marker of neutrophil infiltration, was mitigated (67% reduction, p< 0.05) with FGF-1 treatment. As shown by histology, neutrophil infiltration and tubular cell necrosis in medulla were less pronounced (p< 0.0001 and p< 0.05, respectively) in animals receiving FGF-1. Furthermore, ischemia-induced apoptosis, prevalent in tubular cells of the cortex, was also attenuated by FGF-1-treatment (83% reduction, p< 0.0001). Pretreatment of animals with Nw-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase, abolished the attenuating effects of FGF-1 on neutrophil infiltration, suggesting that nitric oxide might participate in the anti-inflammatory effects of FGF-1 in this experimental design. Our data support a role for FGF-1 in attenuation of renal damage or failure after ischemia-reperfusion injury of the kidney, in part at least by inhibition of neutrophil infiltration.

Animals↗

PubMed1999

Bronchodilation using combined urodilatin - albuterol administration in asthma: a randomized, double-blind, placebo-controlled trial.

Urodilatin, the renal form of natriuretic peptide type A, induces bronchodilation, increasing intracellular cyclic guanosine monophosphate (cGMP), whereas the bronchorelaxant effect by b2-agonists is triggered by cyclic adenosine monophosphate (cAMP). The objective of this investigation is to demonstrate the efficacy of urodilatin in inducing bronchodilation, and to show this activity alone or in combination with albuterol. Therefore, a randomized, double-blind, placebo-controlled, dose-finding study with cross-over design was carried out including 12 stable, mild to severe (step 2 to 4, definition by NIH/NHLBI guideline 1997) asthmatics. 96 treatments were thus performed. The intervention was comprised of an intravenous infusion of urodilatin (0, 10, 30, or 60 ng/kg/min) combined with inhaled albuterol (0 or 200 microg). As primary objective, the increase in forced expiratory volume in one second (FEV subset1) was measured. - The trial shows that urodilatin at all applied doses or 200 microg albuterol significantly increases FEV subset1 (p < 0.05). Combination of urodilatin and albuterol treament significantly improves FEV subset1 (p < 0.05) compared to either monotherapy and results in maximum bronchodilation. - From the results, the following conclusions can be drawn. In stable asthmatics, the combined activation of cGMP- and cAMP-mediated pathways results in a significantly improved, maximal bronchodilation in comparison to either type of monotherapy. This shows that urodilatin combined with albuterol improves lung function and ameliorates the therapy in asthmatics.

Adult↗

PubMed1999

Novel glycosylated forms of human plasma endostatin and circulating endostatin-related fragments of collagen XV.

Circulating elongated forms of the angiogenesis inhibitor and potential anti-cancer drug endostatin were isolated from human blood filtrate. Immunoreactive endostatin was identified by a polyclonal rabbit antiserum raised against an N-terminal epitope of the polypeptide and purified by consecutive chromatographic steps and immunoblotting. N- and C-terminal sequence analyses of the isolated molecules revealed different forms of endostatin starting with V(117)HLRPAR. lacking the last and final three residues of the noncollagenous domain 1 (NC-1) of collagen XVIII, respectively. These polypetides are found to be O-glycosylated at T(125) (residue 9) with a glycan structure of the mucin type consisting of galactose N-acetylgalactosamine and N-acetylneuraminic acid residues. Carbohydrate analyses were performed via the semiquantitative HPLC-electrospray ionization mass spectrometry (ESMS) technique after exoglycosidase hydrolysis. Circulating endostatins are present as sialoglycoprotein (22 000 and 21 841 Da +/- 0.02%) and asialoglycoprotein structures (21 710 and 21 549 Da +/- 0.02%), while the two completely deglycosylated forms are obtained only after enzymatic incubation. The described glycosylated endostatins may represent intermediates in the proteolytic pathway of the NC-1 domain of collagen XVIII resulting in bioactive endostatins. Furthermore, immunoreactive endostatin-related C-terminal fragments of human collagen XV are found in the hemofiltrate. These polypeptides exhibit the N-terminal sequences P(66)HLLPPP. and Y(81)EKPALH. of the collagen XV NC-1 domain. ESMS and immunoblotting analyses reveal three glycosylated polypeptides with a molecular mass ranging from 16 to 21 kDa. Due to the high degree of homology between collagen XV and collagen XVIII as well as their analoqous proteolytic processing, functional similarities of collagen XVIII- and XV-related fragments should be revealed in future experiments.

Amino Acid Sequence↗

PubMed1999

Strategy for identifying circulating fragments of insulin-like growth factor binding proteins in a hemofiltrate peptide bank.

A differentiated strategy was established to isolate circulating forms of the six human insulin-like growth factor binding proteins (IGFBPs). As starting material we used our peptide bank, a comprehensive blood plasma peptidoma generated from human blood filtrate. The peptides were initially identified in the fractions of the hemofiltrate peptide bank by their immunoreactivity, their capacity to bind the insulin-like growth factors (IGFs), and their molecular masses determined by polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Fractions revealing both immunoreactivity and IGF-binding capacity were analyzed by direct sequencing of immunoreactive bands from a Coomassie-stained gel. Further purification of the IGFBP peptides was performed by consecutive chromatographic steps guided by sensitive MALDI-MS. Using this strategy, different fragments of IGFBP-3, -4, and -5 were identified and a fragment of IGFBP-4 was purified to homogeneity.

Amino Acid Sequence↗

PubMed1999

Proteolytic cleavage of glucagon-like peptide-1 by pancreatic beta cells and by fetal calf serum analyzed by mass spectrometry.

Fetal calf serum and a beta-cell line exhibit a proteolytic activity essential for the biological function of glucagon-like peptide-1 (GLP-1). This process of cleavage was investigated using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). To generate processing products, GLP-1 was subjected to rat insulinoma m5F (RINm5F) cell cultures or to fetal calf serum (FCS). For detection of processing products, a standardized extraction method including ion-exchange batch extraction, ultrafiltration, gel filtration, and reversed-phase chromatography was used. The RP fractions were analyzed by MALDI-TOF-MS. Processed proteolytic products were detected by comparing the resulting mass spectra of cell media or FCS after 2 h incubation with GLP-1 (7-36) amide with these of 2 h controls. To perform the comparison of the resulting mass spectra, software (MASSSPECANALYST) based on Microcal Software, Origins C-like language LABTALK was developed. GLP-1 fragments were purified by RP-HPLC, and characterized by sequence analysis. As insulin is the major secretory product of beta cells depending on GLP-1 stimulation, the insulin and insulin fragments of the cell culture supernatants were also analyzed by this method.

Amino Acid Sequence↗

PubMed1999

Application of a peptide bank from porcine brain in isolation of regulatory peptides.

Over the past years, the introduction of biological assay systems, random peptide sequencing and orphan receptor screening has led to the isolation and identification of new regulatory peptides with potential clinical impact. We have developed a method for separating peptides into about 300 fractions from large amounts of porcine brain tissue. The preparation of this peptide bank consists of three steps including ultrafiltration followed by cation-exchange separation and reversed-phase chromatography. These fractions represent the peptide bank with desalted and lyophilized peptides from brain tissue. Molecular masses of the peptides in the fractions are determined by matrix-assisted laser desorption ionization MS and a mass data bank is subsequently generated. For systematic analysis of the peptides, a subsequent two-step purification procedure is followed by Edman sequencing resulting in the identification of different peptides. A survival assay with a neuronal cell line revealing the stimulatory and inhibitory activities is applied as a model to test the 300 fractions. This primary screen indicates that the biological activities of the extracted peptides are easily characterized and, moreover, can be related to the biochemical entities. We conclude that the established peptide bank is an efficient and useful tool for the isolation of regulatory brain peptides applying different purification strategies.

Animals↗

PubMed1999

LEKTI, a novel 15-domain type of human serine proteinase inhibitor.

Proteinase inhibitors are important negative regulators of proteinase action in vivo. We have succeeded in isolating two previously unknown polypeptides (HF6478 and HF7665) from human blood filtrate that are parts of a larger precursor protein containing two typical Kazal-type serine proteinase inhibitor motifs. The entire precursor protein, as deduced from the nucleotide sequence of the cloned cDNA, exhibits 15 potential inhibitory domains, including the Kazal-type domains, HF6478, HF7665, and 11 additional similar domains. An inhibitory effect of HF7665 on trypsin activity is demonstrated. Because all of the 13 HF6478- and HF7665-related domains share partial homology to the typical Kazal-type domain but lack one of the three conserved disulfide bonds, they may represent a novel type of serine proteinase inhibitor. The gene encoding the multidomain proteinase inhibitor, which we have termed LEKTI, was localized on human chromosome 5q31-32. As shown by reverse transcriptase-polymerase chain reaction and Northern blot analysis, it is expressed in the thymus, vaginal epithelium, Bartholin's glands, oral mucosa, tonsils, and the parathyroid glands. From these results, we assume that LEKTI may play a role in anti-inflammatory and/or antimicrobial protection of mucous epithelia.

Amino Acid Sequence↗

PubMed1999

Porcine guanylin and uroguanylin: cDNA sequences, deduced amino acid sequences, and biological activity of the chemically synthesized peptides.

Guanylin and uroguanylin are structurally related intestinal peptide hormones which were purified from a limited number of mammals and are capable of activating the particulate guanylate cyclase-C. Although the biological functions of guanylin and uroguanylin are not yet clarified in detail, they are involved in the regulation of the intestinal water and electrolyte balance. In order to verify the general importance of this hormone system in mammals, we cloned the corresponding cDNAs from pig. Here, we present the nucleotide sequences and the deduced amino acid sequences representing porcine guanylin and uroguanylin. The expression patterns of the corresponding genes, as shown by Northern hybridization and RT-PCR analysis, resemble those of the human homologues. Further, we demonstrate the bioactivity of both porcine peptide hormones by inducing the intracellular cGMP production in human T84 cells and by ion transport experiments using porcine intestinal mucosa in the Ussing chamber.

Amino Acid Sequence↗

PubMed1999

Molecular biological characterization of phosphodiesterase 3A from human corpus cavernosum.

The hydrolysis of the second messenger cyclic AMP (cAMP) by phosphodiesterase 3 (PDE3) is known to play an important regulatory role in the context of relaxation of cavernous smooth muscle of the penis. Thus, we investigated the PDE3A isoform from penile cavernous tissues of male patients with and without symptoms of erectile dysfunction at the molecular biological level. As revealed by reverse transcriptase polymerase chain reaction, of all tissues of the urogenital tract analyzed the expression of the PDE3A gene was highest in the corpus cavernosum. However, significant differences in the levels of gene expression were not found between the two subgroups of patients. Also, the determined nucleotide sequences of the cloned penile PDE3A cDNAs of all patients were absolutely identical. Surprisingly, some deviations could be detected in the cDNA sequences of PDE3A from human myocard and platelets. The data obtained indicate that neither the expression levels nor the sequence deviations of PDE3A are the main reasons for erectile dysfunction in men.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1999

Solution structure of the human CC chemokine 2: A monomeric representative of the CC chemokine subtype.

HCC-2, a 66-amino acid residue human CC chemokine, was reported to induce chemotaxis on monocytes, T-lymphocytes, and eosinophils. The three-dimensional structure of HCC-2 has been determined by 1H nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics calculations on the basis of 871 experimental restraints. The structure is well-defined, exhibiting average root-mean-square deviations of 0.58 and 0.96 A for the backbone heavy atoms and all heavy atoms of residues 5-63, respectively. In contrast to most other chemokines, subtle structural differences impede dimer formation of HCC-2 in a concentration range of 0.1 microM to 2 mM. HCC-2, however, exhibits the same structural elements as the other chemokines, i.e., a triple-stranded antiparallel beta-sheet covered by an alpha-helix, showing that the chemokine fold is not influenced by quaternary interactions. Structural investigations with a HCC-2 mutant prove that a third additional disulfide bond present in wild-type HCC-2 is not necessary for maintaining the relative orientation of the helix and the beta-sheet.

Amino Acid Sequence↗

PubMed1999

Composition of the peptide fraction in human blood plasma: database of circulating human peptides.

A database was established from human hemofiltrate (HF) that consisted of a mass database and a sequence database, with the aim of analyzing the composition of the peptide fraction in human blood. To establish a mass database, all 480 fractions of a peptide bank generated from HF were analyzed by MALDI-TOF mass spectrometry. Using this method, over 20000 molecular masses representing native, circulating peptides were detected. Estimation of repeatedly detected masses suggests that approximately 5000 different peptides were recorded. More than 95% of the detected masses are smaller than 15000, indicating that HF predominantly contains peptides. The sequence database contains over 340 entries from 75 different protein and peptide precursors. 55% of the entries are fragments from plasma proteins (fibrinogen A 13%, albumin 10%, beta2-microglobulin 8.5%, cystatin C 7%, and fibrinogen B 6%). Seven percent of the entries represent peptide hormones, growth factors and cytokines. Thirty-three percent belong to protein families such as complement factors, enzymes, enzyme inhibitors and transport proteins. Five percent represent novel peptides of which some show homology to known peptide and protein families. The coexistence of processed peptide fragments, biologically active peptides and peptide precursors suggests that HF reflects the peptide composition of plasma. Interestingly, protein modules such as EGF domains (meprin Aalpha-fragments), somatomedin-B domains (vitronectin fragments), thyroglobulin domains (insulin like growth factor-binding proteins), and Kazal-type inhibitor domains were identified. Alignment of sequenced fragments to their precursor proteins and the analysis of their cleavage sites revealed that there are different processing pathways of plasma proteins in vivo.

Amino Acid Sequence↗