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Biomedical subjects

D L Amrani

Publications and source records attributed to D L Amrani.

At least 19 recordsLinked to original sources
PubMed1995

Endocytosis of fibrinogen into hamster megakaryocyte alpha granules is dependent on a dimeric gamma A configuration.

Two species of fibrinogen that differ only in the structure of their gamma chains, gamma A and gamma', are present in normal plasma. Fibrinogen stored in platelet alpha granules does not contain gamma' chains. Because platelet fibrinogen was recently shown to be derived exclusively by receptor-mediated endocytosis from plasma and not by endogenous megakaryocyte synthesis, we postulated that the gamma' fibrinogen present in plasma is not endocytosed by megakaryocytes and platelets. We tested this hypothesis by studying endocytosis of peak 1 (containing two gamma A chains) and peak 2 (containing one gamma A and one gamma' chain) fractions of human fibrinogen obtained from diethyl aminoethyl (DEAE) cellulose chromatography in an in vivo hamster model. When 10 mg of biotinylated, unfractionated, or peak 1 fibrinogen was injected intravenously, each protein was endocytosed into megakaryocytes and platelets within 24 hours. In contrast, equivalent doses of biotinylated peak 2 fibrinogen and bovine serum albumin were barely detectable within megakaryocytes and platelets. We conclude that gamma' fibrinogen is not endocytosed and incorporated into megakaryocytes and platelet alpha granules. Furthermore, a dimeric gamma A-chain configuration is required for receptor-mediated endocytosis of fibrinogen into these organelles.

Amino Acid Sequence↗
PubMed1995

Development of a whole platelet ELISA to detect circulating activated platelets.

P-selectin is a granule membrane protein that is expressed on the surface of activated endothelial cells and platelets. Flow cytometry has been used as a means of detecting activated platelets in the circulation by using antibodies to P-selectin and other surface markers. In the study reported here, we developed a whole platelet ELISA for measuring P-selectin on platelets in platelet-rich plasma. Platelet-rich plasma samples for analysis were isolated from fresh blood by centrifugation, fixed with 1.0% paraformaldehyde, and used within 3 hours or after storage at -70 degrees C for up to 10 months. Paraformaldehyde-fixed, phorbol myristate acetate-activated or thrombin receptor peptide-activated platelets were used to construct a standard calibration curve. These platelets were stable after 10 months of storage at -70 degrees C. Interassay variability showed a high degree of correlation, with r = 0.98 +/- 0.03 (n = 12). The accuracy and specificity of the ELISA was verified by using fluorescence-activated flow cytometric analysis and is as sensitive (< or = 0.5%) as flow cytometry for detecting P-selectin expression on platelets. To assess the ability of the platelet ELISA to detect platelet activation in the systemic circulation, we examined 24 patients with unstable angina and 12 age-matched control subjects. Patients with unstable angina demonstrated significantly higher levels of circulating activated platelets than did age-matched control subjects. Although storage-dependent differences in absolute platelet activation levels were found, platelet ELISA results of samples evaluated within either 3 hours or after 10 months of storage were comparable to results obtained by fluorescence-activated flow cytometric analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Angina, Unstable↗
PubMed1994

Regulation of plasminogen activation by interleukin-6 in human lung fibroblasts.

We determined that exposure of cultured lung fibroblasts (HEL-299) to recombinant human interleukin-6 (0-400 ng/ml) resulted in a dose- and time-dependent increase in secreted and cell lysate PAI-1 and total tPA levels (maximal increase of 2.6-fold and 1.7-fold, respectively). Specificity of this response was indicated when increases in PAI-1 levels were inhibited by neutralizing polyclonal antibodies to IL-6, but not with non-specific antibodies. Inhibition of the response to IL-6 by cycloheximide and alpha-amanitin indicates that increases in PAI-1 are dependent on both protein and RNA synthesis. The addition of IL-6 to HEL-299 cells also stimulated a dose- and time-dependent increase in steady-state PAI-1 mRNA levels (3.8 to 15.1 pg/micrograms total RNA by 24 h). A rapid increase (5-6-fold) in PAI-1 mRNA levels was found between 3 and 12 h. Nuclear run-on assays using a maximum dose of IL-6 showed that IL-6 increases a 4-fold rate of transcription of the PAI-1 gene. We further showed that LPS induces a 70% increase in secreted IL-6 and a 50% increase in PAI-1 protein levels. Increasing doses of anti-IL-6 completely blocked the effect of LPS on PAI-1 while non-specific antibodies had no effect. These studies suggest an autocrine role for IL-6 in regulating localized proteolysis and modulating tissue remodeling during acute inflammatory conditions by fibroblasts.

Cells, Cultured↗
PubMed1994

Characterization of adhesion of non-exogenously stimulated and resting platelets in normal plasma to fibrinogen and its fragments.

Platelet adhesion to various forms of fibrinogen was studied using platelets in plasma and washed platelets. The study was designed to determine if platelets prepared with minimal handling in plasma at physiological pH containing normal levels of Ca2+ have different requirements for adhesion to immobilized fibrinogen than do washed platelets tested in the absence of plasma. Exposure of platelets to citrate and low pH did not seem to affect the requirements of washed platelets for adhesion to fibrinogen. Nonetheless, behavioural differences between these two types of platelets were seen. Surprisingly, in the absence of exogenous activation normal platelets in plasma behaved qualitatively as stimulated washed platelets. That is, both types of platelets adhered to all forms of fibrinogen which possessed at least one gamma-chain carboxyl terminal platelet binding site. Platelets in plasma treated with prostaglandin E1 (resting platelets) adhered only to forms of fibrinogen which contained two gamma-chain platelet binding sites. These observations also demonstrate that the fibrinogen alpha-chain arginine-glycine-aspartic acid-phenylalanine and arginine-glycine-aspartic acid-serine sequences are not necessary or sufficient to mediate the adhesion of resting or stimulated platelets in plasma to fibrinogen. The presence of endogenous adenosine diphosphate appears to account, at least in part, for the ability of normal platelets in plasma to adhere to forms of fibrinogen which have only one gamma-chain platelet binding site.

Adenosine Diphosphate↗
PubMed1994

Further studies on the interaction of migrating keratinocytes with fibrinogen.

If glass implants placed under one edge of a skin wound in the adult newt are coated with fibrinogen (FGN), keratinocytes from the wound periphery migrate onto the implant. To learn more about the site(s) in FGN that permits this migration, we exposed keratinocytes to implants coated with forms of FGN containing modifications or deletions in the 3 most commonly studied cell binding sites; the RGDF sequence at A alpha 95-98, RGDS at A alpha 572-575 and the carboxy terminal 12 amino acids in the gamma A chain. Recombinant FGN with either RGD sequence altered to RGE supported migration as well as unmodified FGN did. Replacement of the carboxy terminal 4 amino acids in the gamma A chain by a 20 amino acid sequence that disrupts the ability of the gamma terminus to mediate platelet aggregation (the gamma' variant) likewise had no effect. Nor did simultaneous antibody blockade of the RGDS, RGDF, and gamma A sites have any effect. At its best, Dhem1, a fragment containing the RGDS and gamma A sites, produced only about half as much migration as the maximum obtained on intact FGN. Dhem2, a fragment differing from Dhem1 only by having a gamma' variant in place of gamma A, was even less active. Two other D fragments, both of which were missing a large part of the A alpha chain, and one of which contained none of the three major binding sites, supported considerable migration, suggesting that loss of the A alpha chain COOH terminus reveals a site that was not exposed in Dhem1 and 2. A alpha chain fragments containing the RGDF or RGDS sequence were active, but a much larger fragment without RGD was inactive. A soluble peptide consisting of the sequence, RGDS, was a potent inhibitor of migration on FGN but RGDF and the gamma A pentapeptide, KQAGD, were minimally effective. Longer versions of these peptides decreased the effectiveness in all cases. These results suggest that under certain circumstances, newt keratinocytes may interact with each of the 3 major binding sites in FGN as well as a site outside these sequences.

Amino Acid Sequence↗
PubMed1993

Stimulation of chick hepatocyte fibronectin production by fibroblast-conditioned medium is due to interleukin 6.

Interleukin-6 (IL6) is produced by different cell types, including monocytes and fibroblasts. We show that recombinant human IL6 (rhIL6) and chick fibroblast conditioned medium stimulate plasma fibronectin (PFn) and PFn mRNA production by cultured chick hepatocytes in a dose-dependent manner. Lipopolysaccharide (LPS) treatment of fibroblast cultures induces higher levels of the PFn stimulating activity. These effects are blocked by preincubation of either rhIL6 or LPS-stimulated chick fibroblast conditioned medium with anti-rhIL6 antibody before treatment of hepatocytes, indicating that the conditioned medium contains chick fibroblast-derived IL-6 (cfIL6). Further, LPS induces fibroblast production of a proportional increase in cfIL6 detectable by a human IL6 ELISA. cfIL6 maximally stimulates chick hepatocyte PFn production by 24 h (4.5-fold). Dexamethasone acts more rapidly, but maximal stimulation is only 2.3-fold. Hepatocyte Fn mRNA levels are even more substantially stimulated by dexamethasone and cfIL6 (up to 8.9- and 18.5-fold by 12 h, declining to 2.3 and 4.2-fold by 24 h, respectively). The effect cfIL6 with or without dexamethasone on hepatocyte PFn levels are comparable. These observations are consistent with the role of IL6 as a major mediator of acute phase protein production.

Acute-Phase Proteins↗
PubMed1992

Characterization of the gamma chain platelet binding site on fibrinogen fragment D.

Glycoprotein (GP) IIb/IIIa on adenosine diphosphate (ADP)-activated human platelets interacts with specific sites on the fibrinogen molecule leading to aggregation. We characterized the platelet-binding site on the gamma chains of fibrinogen using plasmic fragments D gamma A and D gamma'. Fragment D gamma A, which contains the carboxy terminal gamma A400-411 platelet-binding sequence (HHLGGAKQAGDV), was 70-fold more active than the synthetic gamma A400-411 peptide in inhibiting ADP-induced platelet aggregation. Fragment D gamma A inhibited fibrinogen binding and also bound directly to ADP-activated platelets. The Kd values determined for fibrinogen and fragment D gamma A binding were 0.55 mumol/L and 1.2 mumol/L, respectively. In contrast, fragment D gamma', which differs from fragment D gamma A with respect to its gamma chain sequence from position 408 to the COOH-terminus at position 427, did not inhibit platelet aggregation or fibrinogen binding, and did not bind directly to the platelet surface. Denaturation of fragment D gamma A with guanidine-HCl caused a loss of inhibitory activity in platelet aggregation assays. These data indicate that the native conformation of the gamma chain platelet-binding site on fibrinogen is important for optimal binding to GPIIb/IIIa.

Adenosine Diphosphate↗
PubMed1990

Regulation of fibrinogen biosynthesis: glucocorticoid and interleukin-6 control.

Fibrinogen biosynthesis is regulated under normal and pathophysiological conditions by the constitutive, hormonal and cytokine-mediated mechanisms. As an acute-phase protein, fibrinogen biosynthesis is regulated by glucocorticoids and cytokines. Recent studies have defined glucocorticoid-consensus sequences on the beta-chain promoter. The cytokine mediating production of fibrinogen, originally termed leukocyte endogenous mediator and hepatocyte stimulatory factor, has now been demonstrated to be derived from a single gene family of cytokines called interleukin-6 (IL-6). IL-6 forms produced by mammalian fibroblasts, T-cells and endothelial cells, as well as monocytic cells, can stimulate basal levels of fibrinogen production in vitro and in vivo. Studies carried out in mammalian hepatocyte systems, with natural or recombinant IL-6, show a dependency on the presence of glucocorticoids to provide a maximal effect. Our results demonstrated the ability of purified human recombinant interleukin-6 (originally BSF-2) to stimulate fibrinogen production in primary chicken hepatocytes in a dose-dependent manner. Conditioned medium from primary chick fibroblasts unstimulated or stimulated with purified natural interleukin-1 (IL-1), induced a dose-dependent increase in fibrinogen levels in cultured chick hepatocytes. IL-1 alone had little or no direct effect on fibrinogen production.

Animals↗
PubMed1989

Identification of covalently linked trimeric and tetrameric D domains in crosslinked fibrin.

Following proteolytic conversion of fibrinogen to fibrin, clot assembly commences with formation of double-stranded fibrils that subsequently branch extensively in forming a three-dimensional network. Plasmin digests of fibrin clots that had first been covalently crosslinked by plasma transglutaminase (factor XIIIa) contained multimeric proteolytic fragments composed of crosslinked outer (D) domains of neighboring fibrin molecules. Two of these were larger than the well-known "D dimer" fragment and corresponded to D trimers and D tetramers, respectively. Whereas D dimers originate from crosslinked D domains at bimolecular junctions within two-stranded fibrils, D trimers and D tetramers evidently arise through crosslinking of contiguous D domains at trimolecular and tetramolecular junctions or at fibril branch points, respectively. Measurement of the widths of fibrils comprising trifunctional branches in thin fiber networks revealed tetramolecular branch points, which are formed by bifurcation of two double-stranded fibrils. In addition, another type of trifunctional structure, which we term the trimolecular branch point, was composed of three double-stranded fibrils. Crosslinking of D domains to form trimers may occur at this type of junction. These findings add to our understanding of the crosslinking arrangements that stabilize fibrin clot structure and the ways that fibrin molecules polymerize to form branches in the clot matrix.

Cross-Linking Reagents↗
PubMed1988

Evidence that binding to the carboxyl-terminal heparin-binding domain (Hep II) dominates the interaction between plasma fibronectin and heparin.

We assessed the participation of the three known heparin-binding domains of PFn (Hep I, Hep II, Hep III) in their interaction with heparin by making a quantitative comparison of the fluid-phase heparin affinities of PFn and PFn fragments under physiologic pH and ionic strength conditions. Using a fluorescence polarization binding assay that employed a PFn affinity-purified fluorescein-labeled heparin preparation, we found that greater than 98% of the total PFn heparin-binding sites exhibit a Kd in the 118-217 nM range. We also identified a minor (less than 2%) class of binding sites exhibiting very high affinity (Kd approximately 1 nM) in PFn and the carboxyl-terminal 190/170 and 150/136 kDa PFn fragments. This latter activity probably reflects multivalent inter- or intramolecular heparin-binding activity. Amino-terminal PFn fragments containing Hep I (72 and 29 kDa) exhibited low affinity for heparin under physiologic buffer conditions (Kd approximately 30,000 mM). PFn fragments (190/170 and 150/136 kDa) containing both the carboxyl-terminal Hep II and central Hep III domains retained most of the heparin-binding activity of native PFn (Kd = 278-492 nM). The isolated Hep II domain (33-kDa fragment) exhibited appreciable, but somewhat lower (2-5-fold), heparin affinity compared to the 190/170-kDa PFn fragment. Heparin binding to the 100-kDa PFn fragment containing Hep III was barely detectable (Kd greater than 30,000 nM). From these observations, we conclude that PFn contains only one major functional heparin-binding site per subunit, Hep II, that dominates the interaction between heparin and PFn.

Binding Sites↗
PubMed1988

The role of fibrinogen A alpha chains in ADP-induced platelet aggregation in the presence of fibrinogen molecules containing gamma' chains.

Human plasma fibrinogen (Fgn) is heterogenous with respect to the size of its gamma chains, which differ in that residues 408 to 411 of gammaA chains (93% of total) are replaced in gamma' chains by a unique 20 amino acid sequence (gamma408 to gamma427). In this study, we compared the contribution to adenosine diphosphate (ADP)-induced platelet aggregation of the A alpha chains in Fgn molecules containing predominantly (fraction 1-2) or exclusively (peak 1 Fgn) gammaA chains with that of molecules containing approximately 50% gamma' chains (peak 2 Fgn). Using washed human platelets, we confirmed that the number of peak 2 Fgn molecules binding to platelets in the presence of ADP was about half the number of peak 1 Fgn molecules (18,962 +/- 2,298 v 44,366 +/- 16,096 molecules per platelet), and that isolated S-carboxymethylated (SCM) gammaA chains supported ADP-induced platelet aggregation nearly as well as peak 1 Fgn. In contrast, SCM-gamma' chains alone supported aggregation poorly, whereas a mixture of SCM-gammaA and gamma' chains (1:1 ratio) gave intermediate results. Despite the findings with isolated SCM-gamma' chains, we found that peak 2 Fgn supported platelet aggregation nearly as well as peak 1 Fgn. However, peak 2 Fgn from which carboxy (COOH)-terminal A alpha chain segments had been removed by digestion with plasmin showed a markedly decreased platelet aggregation potential. Peak 1 Fgn core fraction from an 88% to 90% coagulable plasmin digest, or Fgn fraction 1-9, which has a high gammaA/gamma' chain ratio (93:7), but lacks COOH-terminal regions of A alpha chains, supported platelet aggregation to the same extent as did intact peak 2 Fgn. These findings indicate that Fgn molecules containing gamma' chains can approach the aggregation potential of Fgn molecules containing predominantly or exclusively gammaA chains only if intact A alpha chains are also present.

Adenosine Diphosphate↗
PubMed1987

Regulation of plasma fibronectin biosynthesis by glucocorticoids in chick hepatocyte cultures.

Plasma fibronectin is an acute-phase reactant synthesized by hepatocytes. Glucocorticoids are one of the major factors implicated in controlling the hepatic acute-phase response. To study the regulatory effects of glucocorticoids on plasma fibronectin biosynthesis, a model chick hepatocyte culture system under serum- and hormone-free conditions was used. In the presence of either dexamethasone or corticosterone, secreted plasma fibronectin increased maximally to 2.8-fold above basal levels. The stimulatory effect of the hormones was maintained only in their continuous presence, since plasma fibronectin production dropped to near basal levels within 16 h of glucocorticoid withdrawal. Pulse-chase studies indicated that pretreatment of cells with dexamethasone stimulated the level of secreted plasma fibronectin but had no effect on its rate of secretion. The increase in plasma fibronectin production by dexamethasone was abolished in a dose-dependent manner by the addition of progestin, an antagonist of dexamethasone known to compete specifically for the liver glucocorticoid receptor. Actinomycin D and alpha-amanitin, which are inhibitors of transcription, also blocked the early dexamethasone effect on plasma fibronectin synthesis. Slot blot hybridization of total RNA samples from dexamethasone-treated cultures revealed a 6-fold stimulatory rise in fibronectin mRNA during the first 6 h of treatment, which later declined and was no longer evident at 48 and 72 h. However, fibronectin mRNA levels were elevated to about the same extent in control and dexamethasone-treated cells at the later time points. During the same time period (0 to 72 h), plasma fibronectin protein levels rose and remained elevated. Evaluation of pulse-chase experiments following pretreatment with hormone for 48 h demonstrated that equal amounts of plasma fibronectin were translated by dexamethasone-treated and control cells, but only 42% of labeled plasma fibronectin was secreted by control cells compared with 93% for dexamethasone-treated cells. These findings suggest that the early phase of glucocorticoid regulation of plasma fibronectin biosynthesis occurs at the transcriptional level and is mediated through the specific action of the glucocorticoid receptor. A later phase of glucocorticoid-stimulated plasma fibronectin biosynthesis results from modulation of post-translational processing events leading to secretion of an increased amount of newly translated plasma fibronectin polypeptides.

Animals↗
PubMed1987

Studies on the ultrastructure of fibrin lacking fibrinopeptide B (beta-fibrin).

Release of fibrinopeptide B from fibrinogen by copperhead venom procoagulant enzyme results in a form of fibrin (beta-fibrin) with weaker self-aggregation characteristics than the normal product (alpha beta-fibrin) produced by release of fibrinopeptides A (FPA) and B (FPB) by thrombin. We investigated the ultrastructure of these two types of fibrin as well as that of beta-fibrin prepared from fibrinogen Metz (A alpha 16 Arg----Cys), a homozygous dysfibrinogenemic mutant that does not release FPA. At 14 degrees C and physiologic solvent conditions (0.15 mol/L of NaCl, 0.015 mol/L of Tris buffer pH 7.4), the turbidity (350 nm) of rapidly polymerizing alpha beta-fibrin (thrombin 1 to 2 U/mL) plateaued in less than 6 min and formed a "coarse" matrix consisting of anastomosing fiber bundles (mean diameter 92 nm). More slowly polymerizing alpha beta-fibrin (thrombin 0.01 and 0.001 U/mL) surpassed this turbidity after greater than or equal to 60 minutes and concomitantly developed a network of thicker fiber bundles (mean diameters 118 and 186 nm, respectively). Such matrices also contained networks of highly branched, twisting, "fine" fibrils (fiber diameters 7 to 30 nm) that are usually characteristic of matrices formed at high ionic strength and pH. Slowly polymerizing beta-fibrin, like slowly polymerizing alpha beta-fibrin, displayed considerable quantities of fine matrix in addition to an underlying thick cable network (mean fiber diameter 135 nm), whereas rapidly polymerizing beta-fibrin monomer was comprised almost exclusively of wide, poorly anastomosed, striated cables (mean diameter 212 nm). Metz beta-fibrin clots were more fragile than those of normal beta-fibrin and were comprised almost entirely of a fine network. Metz fibrin could be induced, however, to form thick fiber bundles (mean diameter 76 nm) in the presence of albumin at a concentration (500 mumol/L) in the physiologic range and resembled a Metz plasma fibrin clot in that regard. The diminished capacity of Metz beta-fibrin to form thick fiber bundles may be due to impaired use or occupancy of a polymerization site exposed by FPB release. Our results indicate that twisting fibrils are an inherent structural feature of all forms of assembling fibrin, and suggest that mature beta-fibrin or alpha beta-fibrin clots develop from networks of thin fibrils that have the ability to coalesce to form thicker fiber bundles.

Blood Coagulation↗
PubMed1986

Effect of hepatocyte-stimulating factor and glucocorticoids on plasma fibronectin levels.

We evaluated the effects of hepatocyte-stimulating factor (HSF) and a glucocorticoid (dexamethasone) on changes in the levels, in vivo and in vitro, of plasma fibronectin (Fn), a glycoprotein that is synthesized and secreted by hepatocytes. In turpentine-treated chickens, plasma levels of Fn, which peaked at 48 h (whereas fibrinogen levels were maximum at 72 h) rose 200-250% over basal levels, whereas albumin levels decreased by 20-40%. Corticosterone levels in serum samples taken between 5 and 48 h after injection revealed a 124% increase in hormone levels at 24 h in turpentine-treated chickens. We also showed that circulating HSF levels were maximal 8 to 12 h after injection and that HSF activity, as assessed by molecular-exclusion chromatography, was eluted in the 30-45 kDa range. Addition of either serum-derived HSF or dexamethasone (2 nM) to chick hepatocyte cultures resulted in a 130-150% increase in secreted Fn as well as in fibrinogen. When HSF and dexamethasone were added together, a 360-489% increase in the secreted levels of both proteins was found. Chicken mononuclear phagocytic cells treated with lipopolysaccharide secreted an HSF activity that was eluted in two peaks, a minor peak at approximately 70 kDa and a major peak in the 25-40 kDa range. Addition of mononuclear-cell-derived HSF resulted in a greater increase in Fn levels than did the addition of serum HSF. These findings indicate that Fn, like fibrinogen, is an acute-phase protein, the production of which, at least in chickens, is stimulated by HSF and glucocorticoids in an additive manner.

Acute-Phase Reaction↗
PubMed1985

Evidence that chicken antithrombin III is a developmentally regulated glycoprotein synthesized by hepatocytes.

Antithrombin III is the principal circulating active-site inhibitor of thrombin and other serine proteinases. We studied a protein synthesized and secreted by cultured chick embryo hepatocytes that has very similar immunological, structural and functional properties to adult antithrombin III. Its presence was demonstrated by; immunodiffusion analysis of a 100-fold concentrate of culture medium, which produced a single precipitin line of identity with adult and 1-day-old hatchling plasma antithrombin III; immunoprecipitation of a metabolically labelled protein from culture medium, having the same molecular size as adult chicken antithrombin III; conversion of antithrombin activity in culture medium to a faster acting thrombin inhibitory activity in the presence of heparin. Antithrombin III antigen levels were increased 3- to 4-fold in the presence of dexamethasone (2 nM) during a 3-day culture period. Plasma antithrombin III antigen levels from unhatched chicks increased from 26 +/- 6 micrograms/ml at 16 days of development to 104 /+- 6 micrograms/ml at 20 days, whereas 1-day-old hatchlings (21 days) had levels similar to that in adults (135 +/- 7 micrograms/ml vs. 143 +/- 24 micrograms/ml). In contrast to immunodiffusion and immunoelectrophoretic analysis of hepatocyte or hatchling plasma antithrombin III, which showed lines of identity with adult antithrombin III, 16- and 20-day-old embryonic plasma antithrombin III yielded lines of partial identity and migrated less anodally than adult antithrombin III. Consistent with this finding, embryonic plasma antithrombin III had no sialic acid (less than 0.01 residue/mol) in contrast with the adult form (3.5 residues/mol). These studies show that the increase in adult antigen levels and sialation of antithrombin III occurs rapidly after hatchling, suggesting developmental changes in expression at the transcriptional and translational levels in addition to post-translational carbohydrate processing.

Animals↗
PubMed1985

Preparation of functionally intact monomers by limited disulfide reduction of human plasma fibronectin dimers.

Most (90 to 95%) human plasma fibronectin (PFn) molecules exist as 450-kDa disulfide-rich dimers comprised of two major types of subunits (A, 220 kDa; B, 215 kDa) that are joined near the COOH terminus by two disulfide bonds. Smaller PFn species (Zone II; 190-235 kDa) consist mainly of monomers and/or a monomeric subunit joined covalently to a smaller peptide remnant presumably derived by proteolysis of a parent 450-kDa molecule. A relatively simple and selective method for preparing functionally active, partially reduced monomeric fibronectin subunits (PR-PFn) by limited and selective reduction of dimeric plasma fibronectin (PFn) has been developed. PR-PFn was prepared by incubating PFn in phosphate-buffered saline, pH 7.4, for 2 h at room temperature in the presence of 17 mM dithiothreitol (DTT). Following S-carboxymethylation or S-carboxyamidomethylation, the material was passed through a gelatin-Sepharose column and nonbinding material was discarded; gelatin-bound material was eluted using a 0 to 2 M KSCN gradient. Residual dimeric species (10-20%) could be separated from monomers in high yield by gel-sieving chromatography on a Sepharose 6B-Cl in the presence of a chaotropic salt, 0.3 M KSCN. Most new SH groups (74-81%) in that fraction of PR-PFn binding to gelatin were localized in proteolytic fragments containing the COOH terminus, thus suggesting that selective cleavage of the interchain disulfide bridges had taken place. The binding affinity of PR-PFn to gelatin- and fibrin-Sepharose was lower than that of dimeric PFn, but the same as that of Zone II PFn and other monomeric gelatin-binding proteolytic derivatives. PR-PFn also bound to heparin-Sepharose and promoted cell attachment and spreading. We conclude that PR-PFn monomers possess the same functional activities as those of the parent chains.

Chromatography, Affinity↗