Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

Kuyas, C; Haeberli, André; Walder, Peristera; Straub, P W

doi:10.1055/s-0038-1645062

Cited by 35 publications

(30 citation statements)

References 4 publications

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“…Fresh blood was collected at Wing Lee Poultry (San Diego) into approximately 1͞10 volumes of cold 0.1 M trisodium citrate and centrifuged to remove cells. Crude fibrinogen was prepared by a modified cold ethanol method, after which the preparation was passed first over a Gly-Pro-ArgPro affinity column (13,14) and then lysine-Sepharose (15). The material was concentrated to 6 mg͞ml in 0.15 M NaCl͞0.05 M imidazole, pH 7.0 and stored in small aliquots at Ϫ70°C.…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of native chicken fibrinogen at 5.5-Å resolution

Yang

Mochalkin

Veerapandian

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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The crystal structure of native chicken fibrinogen has been determined at a resolution of 5.5 Å. The full-length molecule is 460 Å in length and sigmoidally shaped. The structure includes the full sweep of the coiled coils that connect the central and terminal domains; the chain paths of the central domain confirm a predicted scheme of planar disulfide rings in apposition with each other. Electron density maps have revealed the outlines of disordered ␣C domains nestled within the confines of the sinuous coiled coils. The amino-terminal segments of the ␣-and ␤-chains, including the fibrinopeptides A and B, are also disordered.central domain ͉ coiled coils ͉ x-ray structure F ibrinogen is a large (molecular mass ϭ 340 kDa) hexameric (␣ 2 ␤ 2 ␥ 2 ) glycoprotein found in the blood plasma of all vertebrate animals; it is converted into fibrin clots by the thrombin-catalyzed removal of peptides from the aminoterminal regions of the ␣-and ␤-chains. Tens of thousands of studies have been conducted on this system in the past, and its general properties are well known. Shadow-cast electron microscope images long ago revealed a triglobular structure approximately 470 Å in length (1), the subdomainal structure of which has been partially visualized by negative staining electron microscopy and low resolution x-ray crystallography (2, 3). The native protein has proved very difficult to crystallize, however, and no diffraction-grade crystals have been reported. There have been some recent successes in determining x-ray structures of certain core fragments. The first of these to be reported was a 30-kDa recombinant protein corresponding to the carboxylterminal domain of the ␥-chain of human fibrinogen (4); it was followed shortly thereafter by a structure for the 86-kDa fragment D, also from human fibrinogen, and its crosslinked equivalent from fibrin (5-7). The D fragments account for about half the mass of the fibrinogen molecule. Most recently, the partial structure of a 285-kDa moiety prepared from bovine fibrinogen was reported (8).One of the problems in obtaining good crystals of native fibrinogen has been attributed to the presumed mobility of the carboxyl domains of the ␣-chains. These domains (␣C) are the most variable parts of the molecule on a species-to-species basis (9). They are easily trimmed away by proteolysis and are often referred to as ''free-swimming appendages.'' Moreover, most contain a series of repeated sequences (10, 11) that are thought to add to the flexible nature of the region. As it happens, chicken fibrinogen ␣-chains lack these repeats (12), an attribute that led us to attempt crystallization of the native protein. Indeed, we were able to obtain large, easily managed crystals in short order. Like crystals of modified bovine fibrinogen (3, 8), these crystals have a high solvent content and diffract anisotropically.We now report a structure for native chicken fibrinogen at 5.5 Å, determined by the method of molecular replacement by using high-resolution structures of fragment D (5-7) as search mod...

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Section: Methodsmentioning

confidence: 99%

Crystal structure of native chicken fibrinogen at 5.5-Å resolution

Yang

Mochalkin

Veerapandian

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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“…Fragment D was isolated from this lysate byaffinity chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel as described previously [25]. The purity of fragment D was >95% as verified by SDS-PAGE [24], and fragment D was identified as fragment D) with a molecular mass of90 kDa.…”

Section: Preparation Of Purified Fibrinogen and Fibronectin Fragmentsmentioning

confidence: 99%

“…After rinsing in PBS, the protein-coated cannula segments were exposed at 37°C to I J-Lg/mL plasminogen activated to plasmin by 800 units/ml, urokinase (Choay, Paris) in PBS supplemented with 1 mM Ca 2 + and 0.5 mM Mg2+. After either 10 or 60 min, the digestion was terminated by adding 10 J-LL of 10-2 M o-phenyl-prolyl-arginyl-chloromethyl ketone (PPACK; Bachem, Bubendorf, Switzerland) solution as previously described [25]. Zero time digestion controls were cannula segments incubated for 60 min with PPACK-inactivated plasminogen-urokinase mixture.…”

Section: Quantification Of Surface-bound Fibronectin or Fibrinogenmentioning

confidence: 99%

Fibronectin Is More Active than Fibrin or Fibrinogen in Promoting Staphylococcus aureus Adherence to Inserted Intravascular Catheters

Vaudaux¹,

Pittet²,

Haeberli³

et al. 1993

Journal of Infectious Diseases

136

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To further define the role offibrin(ogen) and fibronectin in Staphylococcus aureusadherence to central venous catheters, the amount, chemical integrity, and biologic activity of these proteins adsorbed on lines inserted in hospitalized patients were prospectively studied. Polyurethane cannulas promoted a significantly lower adherence of S. aureusthan polyvinyl chloride (P < .01) or Hickman (P < .001) cannulas and contained the lowest amount of immunologically assayed fibronectin but not offibrin(ogen). Fibrinogen showed an extensive loss of adherence-promoting activity on inserted cannulas, which was related to its proteolytic breakdown, as detected by SDS-PAGE and immunoblots with antifibrinogen antibodies and confirmed by in vitro studies with purified protein fragments. In contrast, either intact or fragmented fibronectin, although present in much lower amounts than fibrin(ogen), could actively promote S. aureus adherence onto intravenous catheters.Microbial colonization of intravascular devices is an important source of nosocomial infection and sepsis [1,2]. Such device-related infections are frequently due to staphylococci and represent a major risk to the patients receiving intravenous (iv) therapy [I, 2]. Several factors may explain the frequent colonization of iv devices by Staphylococcus aureus, Staphylococcus epidermidis, or other species ofcoagulase-negative staphylococci: First, the presence ofa transcutaneous cannula wound allowing skin microorganisms to migrate across the skin barrier [1,2]; second, the microbial production of mucoid exopolymeric substances enhancing the stickiness of colonizing organisms [3][4][5][6]; and third, the presence of plasma proteins adsorbed on iv devices, which might selectively promote staphylococcal attachment to their surfaces [7][8][9][10].We have reported that plasma proteins deposited on various categories of iv catheters removed from patients played a significant role in staphylococcal adherence [11]. Clinical isolates of S. aureus were more strongly promoted for adherence by cannula-adsorbed plasma proteins than clinical isolates of S. epidermidis [11]. This observation was explained in part by the more frequent occurrence ofclinical [8,12,13] 1993;167:633-41 of S. epidermidis on surface determinants recognizing some major plasma or matrix proteins, such as fibronectin [8,[10][11][12][14][15][16][17][18], fibrinogen [9,16,[19][20][21], collagen [13, 17], laminin [8, 17, 22], or vitronectin [23]. Each of these individual proteins is known to promote staphylococcal adherence when adsorbed in vitro on polymeric surfaces [8][9][10]. In our in vivo study, we also found that on cannulas inserted in patients, most of the adherence-promoting activity was mediated via surface-bound fibronectin and fibrinogen or fibrin [11]. However, the respective roles of each of these protein components in staphylococcal adherence could not be precisely evaluated [11].The objectives ofthis study were to quantify the respective content of surface-bound fibronectin versus fibrinoge...

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“…Fragment D was prepared by digesting the fibrinogen with plasmin in the presence of calcium ions (10), thereby limiting the digest to the large molecular weight D usually referred to as fragment D1. Digestions were stopped by the addition of the plasmin inhibitor Trasylol (aprotinin from bovine lung), and fragments were purified either by ion-exchange chromatography on DEAE-cellulose (11, 12) or by affinity chromatography on a Gly-Pro-Arg-Pro-Lys agarose column similar to that described by Kuyas et al (13). In the latter case, elution of native fragment D was achieved with 1 M NaBr (pH 5.3).…”

mentioning

confidence: 99%

Photoaffinity labeling of the primary fibrin polymerization site: isolation and characterization of a labeled cyanogen bromide fragment corresponding to gamma-chain residues 337-379.

Shimizu

Nagel

Doolittle

1992

Proc. Natl. Acad. Sci. U.S.A.

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Human fibrinogen and the plasmin-generated fibrinogen fragment D were photoaffinity labeled specifically with the peptide [14C]Gly-Pro-Arg-N(4-azido-2-nitrophenyl)Lys amide. In the case of fibrinogen, >85% of the incorporated radioactivity was found in the y chain. Similarly, when fragment D (Mr, 90,000) was labeled with the same derivatized peptide, virtually all the radioactivity was found in the y-chain portion. The labeled fragment D was treated with CNBr and an initial purification was achieved by two gel.filtration steps. The labeled material was purified further by HPLC and was also compared with CNBr digests of unlabeled material. Amino acid analysis and gas-phase sequencing showed the labeled fragment to be y-chain residues 337-379.In vertebrates, the conversion of fibrinogen into fibrin is initiated by the thrombin-catalyzed removal of the fibrinopeptides A, the immediate consequence of which is the exposure of a pair of new amino termini beginning with the sequence Gly-Pro-Arg (for a review, see ref. 1). Synthetic peptides beginning with that sequence bind strongly to fibrinogen and are able to block fibrin formation (2). The binding has been localized to the terminal domains corresponding to the large plasmin-generated fragment designated D1 (3). In contrast, synthetic peptides beginning with the sequence Gly-His-Arg, which corresponds to the amino terminus of chains after release of the fibrinopeptides B, do not block fibrin formation, even though they do bind to fibrinogen. The Gly-His-Arg peptides bind to both fragments D1 and D3, the latter being a further degradation product of the former. Peptides with the sequence Gly-Pro-Arg do not bind to fragment D3, however, which lacks the carboxylterminal 109 residues of the y chain, leading to the supposition that the complementary binding site for the Gly-Pro-Arg "knob" is situated in the carboxyl-terminal third of the y-chain portion of fragment D (4). Previous efforts to localize further the complementary binding sites for the two sets of peptides have been a matter of dispute. Thus, Olexa and Budzynski (5) reported that the carboxylterminal 38 residues ofthe y chain (i.e., 'y374411) embodied the principal binding site; a similar claim about residues y374-396 was made by Horwitz et aL (6). Subsequently, Southan et al. (7) and Varadi and Scheraga (8) reported experiments indicating that these results were likely artifactual. We now report the results of a photoaffinity labeling study with the peptide Gly-Pro-Arg-N(4-azido-2-nitrophenyl)Lys amide that implicates residues 337-379 from the y chain. MATERIALS AND METHODSHuman fibrinogen was prepared from blood bank plasma as described (9). Fragment D was prepared by digesting the fibrinogen with plasmin in the presence of calcium ions (10), thereby limiting the digest to the large molecular weight D usually referred to as fragment D1. Digestions were stopped by the addition of the plasmin inhibitor Trasylol (aprotinin from bovine lung), and fragments were purified either by ion-exchange chromato...

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Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

Cited by 35 publications

References 4 publications

Crystal structure of native chicken fibrinogen at 5.5-Å resolution

Crystal structure of native chicken fibrinogen at 5.5-Å resolution

Fibronectin Is More Active than Fibrin or Fibrinogen in Promoting Staphylococcus aureus Adherence to Inserted Intravascular Catheters

Photoaffinity labeling of the primary fibrin polymerization site: isolation and characterization of a labeled cyanogen bromide fragment corresponding to gamma-chain residues 337-379.

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