The role of the carbohydrate moiety in the biologic properties of fibrinogen

The carbohydrate moiety of some glycoproteins influences their secretion and functional properties. We have examined the importance of the oligosaccharide chains of fibrinogen in this regard. Fibrinogen was labeled de novo by the addition to rabbit hepatocyte monolayer cultures of either 3H-amino-acids or [2-3H] mannose, in the presence or absence of tunicamycin, a potent inhibitor of glycosylation. Inhibition of glycosylation, which ranged from 75 to 80%, was determined by incorporation of [2-3H]mannose as quantitated by gel filtration. Synthesis and secretion of fibrinogen were quantitated by 3H-amino-acid incorporation, using anti-fibrinogen immunoaffinity column chromatography of medium and cell homogenates. Tunicamycin did not appreciably inhibit fibrinogen synthesis, as compared to a 30-40% inhibition of overall protein synthesis, determined by incorporation of 3H-amino-acids into trichloroacetic acid-precipitable material. There was no evidence that secretion of fibrinogen was impaired. Fibrinogen from medium was copurified by adding cold plasma fibrinogen as carrier. Nonglycosylated fibrinogen was found to be functional as demonstrated by incorporation of radioactivity into clots of the copurified material at a rate identical to that of glycosylated fibrinogen. When clotted in the presence of Ca2+ and Factor XIII, cross-linking of glycosylated and nonglycosylated fibrin was demonstrable on fluorography of sodium dodecyl sulfate-polyacrylamide gels, showing disappearance of gamma-chain and appearance of gamma-gamma-dimers.     

Modification of fibrinogen by homocysteine thiolactone increases resistance to fibrinolysis: a potential mechanism of the thrombotic tendency in hyperhomocysteinemia

We have previously shown functional differences in fibrinogen from hyperhomocysteinemic rabbits compared to that in control rabbits. This acquired dysfibrinogenemia is characterized by fibrin clots that are composed of abnormally thin, tightly packed fibers with increased resistance to fibrinolysis. Homocysteine thiolactone is a metabolite of homocysteine (Hcys) that can react with primary amines. Recent evidence suggests that Hcys thiolactone-lysine adducts form in vivo. We now demonstrate that the reaction of Hcys thiolactone with purified fibrinogen in vitro produces fibrinogen (Hcys fibrinogen) with functional properties that are strikingly similar to those we have observed in homocysteinemic rabbits. Fibrinogen purified from homocysteinemic rabbits and Hcys fibrinogen are similar in that (1) they both form clots composed of thinner, more tightly packed fibers than their respective control rabbit and human fibrinogens; (2) the clot structure could be made to be more like the control fibrinogens by increased calcium; and (3) they both form clots that are more resistant to fibrinolysis than those formed by the control fibrinogens. Further characterization of human fibrinogens showed that Hcys fibrin had similar plasminogen binding to that of the control and an increased capacity for binding tPA. However, tPA activation of plasminogen on Hcys fibrin was slower than that of the control. Mass spectrometric analysis of Hcys fibrinogen revealed twelve lysines that were homocysteinylated. Several of these are close to tPA and plasminogen binding sites. Lysines are major binding sites for fibrinolytic enzymes and are also sites of plasmin cleavage. Thus, modification of lysines in fibrinogen could plausibly lead to impaired fibrinolysis. We hypothesize that the modification of lysine by Hcys thiolactone might occur in vivo, lead to abnormal resistance of clots to lysis, and thereby contribute to the prothrombotic state associated with homocysteinemia.     

The contribution of leukocyte proteases to fibrinolysis

Polymorphonuclear leukocytes accumulate within blood clots and may contribute to fibrinolysis. The primary fibrinolytic enzymes of neutrophils are cathepsin G and elastase. Fibrin can be exposed to these granular enzymes as a result of cell lysis, phagocytosis of fibrin, or secretion of the enzymes from the cells. Neutrophil secretion occurs in association with blood coagulation and is dependent upon a plasma factor(s) and calcium. After secretion, the enzymes can degrade fibrin within a plasma environment. This is demonstrated by the inhibition of fibrinolysis by specific inhibitors of elastase and the augmentation of fibrinolysis by neutralization of the primary plasma inhibitor of elastase, alpha 1-proteinase inhibitor. A radioimmunoassay which discriminates elastase from plasmic degradation products of fibrinogen has been developed. In this assay, elastase elicited degradation products of fibrin(ogen) were detected in certain pathophysiologic plasma samples. Taken together, these findings indicate a role for leukocyte proteases in physiological fibrinolysis.     

Binding of hyaluronic acid to mammalian fibrinogens

We have postulated that the interaction of hyaluronic acid (HA), an extracellular matrix glycosaminoglycan, with fibrin is important during the early stages of wound healing and inflammation (J. Theor. Biol. 119:219; 1986), and have demonstrated the specific binding of 125I-labeled HA to human fibrinogen (J. Biol. Chem. 261:12 586; 1986). To determine whether HA binding is limited to human fibrinogen, we tested the ability of fibrinogens from various mammalian species to bind 125I-HA using a dot-blot assay. Increasing amounts of fibrinogen were adsorbed to nitrocellulose, and incubated with 125I-HA in the presence or absence of a 100-fold excess of nonradiolabeled HA to assess specific binding. In three independent experiments, the amount of 125I-HA bound/mg fibrinogen was determined from the slope derived by linear regression analysis of specifically bound 125I-HA versus protein concentration. A Student's t-test was performed to determine whether the slopes were statistically greater than zero. HA binding was considered statistically significant when P less than 0.05 was obtained by this analysis. Rabbit and dog fibrinogens significantly bound HA in all three trials. Baboon fibrinogen demonstrated significant HA binding in two of three trials. Pig, sheep and goat fibrinogens bound HA significantly in only one of three trials, whereas horse, rat and cow fibrinogens did not bind HA significantly at all. We conclude that fibrinogen from mammalian species other than human can specifically bind HA. The ability of fibrinogen to bind HA appears to correlate with an evolutionary divergence that separated human, baboon, dog, rabbit and rat from cow, pig, horse, goat and sheep.     

Conformational changes in human fibrinogen after in vitro phosphorylation and their relation to fibrinogen behaviour

The far-ultraviolet circular dichroism spectra of fibrinogens phosphorylated by protein kinase C or casein kinase II indicated a conformational change corresponding to an increase in ordered secondary structure. The spectra of protein kinase A- or casein kinase I-phosphorylated fibrinogens did not differ substantially from the control. Fluorescence studies indicated changes in the tertiary structure around tryptophan residues for protein kinase A- or C-phosphorylated fibrinogens, but failed to show any such change for fibrinogen phosphorylated by either of the casein kinases. This latter result was also confirmed by circular dichroism measurements in the near-ultraviolet region. The apparent increase in ordered structure was proposed as an explanation for the slower rate of plasmin degradation seen in fibrinogens after phosphorylation by protein kinase C [6], and casein kinase II, especially as both spectral changes and plasmin degradation rate were unaffected by alkaline phosphatase.     

Functional effects of asparagine-linked oligosaccharide on natural and variant human tissue-type plasminogen activator

The role of Asn-linked oligosaccharide in the functional properties of both human tissue-type plasminogen activator (t-PA) and a genetic variant of t-PA was studied. Nonglycosylated and glycosylated wild-type t-PA were produced in mammalian cells which express recombinant t-PA. These proteins were compared in fibrin binding and 125I-labeled fibrin clot lysis assays, using purified components. The nonglycosylated form showed higher fibrin binding, as well as higher fibrinolytic potency than the glycosylated form. Subsequently, prevention of glycosylation of a t-PA variant which lacked the finger and epidermal growth factor domains (delta FE), was carried out in an attempt to enhance its fibrinolytic activity. Glycosylation was prevented by changing Asn to Gln; at Asn-117 to produce delta FE1X t-PA, and at Asn-117, -184, and -448 to produce delta FE3X t-PA. All variants were similar to wild-type t-PA in their catalytic dependence on fibrinogen fragments, fibrinolytic activity in fibrin autography analysis, and plasminogen activator activity. In a clot lysis assay, using citrated human plasma, the fibrinolytic potency of the variants were comparable to that of wild-type t-PA at activator concentrations of 17-51 nM (approximately 1-3 micrograms/ml). At 0.5-5.1 nM (approximately 0.03-0.3 micrograms/ml), however, the variant proteins had lower fibrinolytic potency than wild-type t-PA. Fifty percent lysis in 1.5 h for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, required 2.5, 10, 7.5, and 5.5 nM t-PA, respectively. The fibrinogenolytic activity in human plasma was measured for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, and showed significant fibrinogen depletion after 3 h of incubation at 51 nM, decreasing to 11, 11, 50, and 72% of basal levels, respectively. These data indicate that partial or total nonglycosylated t-PA variants have a higher fibrinolytic versus fibrinogenolytic ratio than their fully glycosylated counterparts.     

Recombinant human fibrinogen and sulfation of the gamma' chain

Human fibrinogen and the homodimeric gamma'-chain-containing variant have been expressed in BHK cells using cDNAs coding for the alpha, beta, and gamma (or gamma') chains. The fibrinogens were secreted at levels greater than 4 micrograms (mg of total cell protein)-1 day-1 and were biologically active in clotting assays. Recombinant fibrinogen containing the gamma' chain incorporated 35SO4 into its chains during biosynthesis, while no incorporation occurred in the protein containing the gamma chain. The identity of the sulfated gamma' chain was verified by its ability to form dimers during clotting. In addition, carboxypeptidase Y digestion of the recombinant fibrinogen containing the gamma' chain released 96% of the 35S label from the sulfated chain, and the radioactive material was identified as tyrosine O-sulfate. These results clarify previous findings of the sulfation of tyrosine in human fibrinogen.     

The carbohydrate prosthetic groups of rat fibrinogen plasmic fragment E.

Rat fibrinogen plasmic fragment E was found to contain one oligosaccharide chain per gamma-chain attached by a glycosylamine linkage. The oligosaccharide was composed of 1 sialic acid, 1 galactose, 2 mannose and 2 glucosamine residues. The probable sequence from the nonreducing end was sialic acid leads to galactose beta leads to mannose alpha leads to mannose alpha leads to glucosamine leads to glucosamine. No difference in the rate of clearance from the rat circulation could be detected between native and desialated fragment E. A non-denaturing method for the purification of fragment E is described.     

Carboxyl-terminal residues of mammalian fibrinogen and fibrin

The carboxyl-terminal residues of mammalian fibrinogens of six different species and the chain peptides, alpha(A), beta(B) and gamma, isolated from these fibrinogens were determined by hydrazinolysis, digestion with carboxypeptidases and selective tritium labelling. The C-terminal ends of bovine fibrinogen and fibrin were identified as proline and valine, in the molar ratio of approximately 1:2. Proline was identified as the C-terminus of the alpha(A)-chain, and C-terminal valine was found on both the beta(B)- and gamma-chains. On hydrazinolysis after selective tritium labelling of fibrinogen, radioactive C-terminal valine was also identified. The same C-terminal ends as those of bovine fibrinogen were found on the corresponding chain peptides isolated from sheep fibrinogen. The C-terminal residues of all the chain peptides of human and horse fibrinogens, however, were valine. In hog and dog fibrinogens, proline was identified at the C-termini of the alpha(A)-chains, and C-terminal valine and isoleucine were found on the beta(B)- and gamma-chains, respectively. Thus, the C-terminal amino acid residues of the fibrinogens of all mammalian species tested were very similar. It should be noted that hydrophobic amino acids, like isoleucine, valine and proline, are mainly located in the C-terminal ends of all three chain peptides in the fibrinogen molecule.     

Lymphocyte suppressive peptides from fibrinogen are derived predominantly from the A alpha chain

Cellular immune responses can elicit local deposition of fibrin at the site of immunologic reactions, as well as the formation of intravascular fibrin in disseminated reactions. The subsequent physiologic proteolysis of fibrinogen and fibrin by plasmin results in small peptides that suppress lymphocyte functions in vitro and in the immune response in vivo. The intramolecular origin of lymphocyte suppressive activity and the proteolytic events responsible for the release of active peptides have been analyzed. Plasmic peptides from the isolated B beta and gamma constituent chains of fibrinogen did not inhibit mitogen-driven responses of human peripheral blood mononuclear cells. In contrast, plasmic digests of the A alpha chain, but not the intact A alpha chain were suppressive. Advanced plasmic digests of fibrinogen and the A alpha chain were suppressive at similar concentrations, suggesting that biological activity is derived predominantly from the A alpha chain. Limited plasmic digests of fibrinogen were fractionated to yield a heat-precipitable 250,000 dalton fragment X and heat-soluble proteolytic products containing fragments derived from the carboxyl-terminal region of the A alpha chain including a 42,000 dalton major A alpha chain derivative. Neither fragment X nor derivatives produced by its additional plasmic proteolysis were suppressive. In contrast, the heat-soluble fraction from limited plasmic cleavage was suppressive, and this activity was enhanced 10-fold by additional plasmic cleavage of this fraction. The isolated 42,000 dalton A alpha chain fragment was devoid of activity, but plasmic digestion of this derivative generated peptides of less than 8000 daltons that inhibited mitogen-stimulated thymidine uptake by lymphocytes. Two synthetic peptides corresponding to A alpha 220-230 and B beta 43-47, peptides with known vasoactive activities, suppressed lymphocyte thymidine uptake at very high concentrations. Based on their maximal yield from plasmic digests of fibrinogen, these two peptides would account for only 1% of the immunosuppressive activity of fibrinogen derivatives. In summary, the results indicate that the suppressive activity of fibrinogen is predominantly derived from the 42,000 dalton carboxyl terminal region of the A alpha chain of the molecule and is not attributable to the known vasoactive peptides. Initial proteolytic release of this region from the core of fibrinogen does not result in suppressive activity, but additional cleavage releases small peptides with the lymphocyte inhibitory function.     

Characterization of the terminal degradation products of canine fibrinogen by plasmin.

Fibrinogen, isolated from canine plasma by the successive procedures of (1) freezing and thawing, (2) fractional precipitation with 25% saturated (HN4)2SO4 and (3) Sepharose 6B gel-filtration, had a molecular weight of 282 000 by the rapid sedimentation equilibrium method. However, a molecular weight for canine fibrinogen of 332 000, which is closer to that reported for human and bovine fibrinogens (340 000 plus or minus 20 000), was obtained from the sum of the molecular weights of the Aalpha, Bbeta and gamma chains, determined from dodecylsulfate gel electrophoretic patterns of reduced fibrinogen. Canine fibrinogen, subjected to proteolysis by urokinase-activated plasminogen for 24 h, contained degradation fragments D and E which were isolated by starch block electrophoresis and Sephadex G-200 gel-filtration. The purified D and E fragments with sedimentation coefficients of 5.0 S and 2.5 S had weight average molecular weights of 89 000 and 42 000, respectively by the rapid sedimentation equilibrium method. The ratio of D to E was 2:1 per parent fibrinogen molecule. Antigenic analysis according to anti-fibrinogen antiserum showed that both D and E fragments were antigenically deficient to native fibrinogen and revealed a reaction of non-identity with each other. Upon immunoelectrophoresis at pH 8.2, D and E had different electrophoretic mobilities. Preliminary studies indicate that based on thrombin time alone, D has anticoagulant activity while E appears to be a coagulation potentiator. Canine fibrinogen apparently consist of two core fragments with dissimilar chemical characteristics in common with the fundamental structures of human and bovine fibrinogens.     

The presence of heat-labile factors interfering with binding analysis of fibrinogen with ferritin in horse plasma

Background

Horse fibrinogen has been identified as a plasma specific ferritin-binding protein. There are two ways in the binding of ferritin-binding protein with ferritin: one is direct binding and the other is indirect binding which is heme-mediated. The aim of this study was to analyze the binding between horse fibrinogen and ferritin.

Findings

Although fibrinogen in horse plasma did not show the binding to ferritin coated on the plate wells, after following heat-treatment (60°C, 30 min) of horse plasma, plasma fibrinogen as well as purified horse fibrinogen bound to plates coated with horse spleen ferritin, but not with its apoferritin which lost heme as well as iron after the treatment of reducing reagent. Binding of purified or plasma fibrinogen to ferritin was inhibited by hemin and Sn-protoporphyrin IX (Sn-PPIX), but not by PPIX or Zn-PPIX.

Conclusions

Heat-treatment of horse plasma enabled plasma fibrinogen to bind to plate well coated with holo-ferritin. From the binding analysis of fibrinogen and ferritin, it is suggested that horse fibrinogen recognized iron or tin in complexed with the heme- or the hemin-ring, and also suggest that some fibrinogens circulate in the form of a complex with ferritin and/or heat-labile factors which inhibit the binding of fibrinogen with ferritin.

     

Fibrinogens Kawaguchi and Osaka: an amino acid substitution of A alpha arginine-16 to cysteine which forms an extra interchain disulfide bridge between the two A alpha chains

Structural analyses of fibrinogens from patients with congenital dysfibrinogenemia, designated as fibrinogens Kawaguchi and Osaka, have been performed to identify the difference responsible for the lack of fibrinopeptide A release. For the structural studies, a new strategy was employed. Amino acid sequence analysis of one of the lysyl endopeptidase-peptides isolated from the abnormal fibrinogens indicated that in both fibrinogens, arginine-16 of the A alpha chain had been replaced by cysteine. To characterize the chemical nature of the sulfhydryl group of cysteine-16, a tryptic peptide containing cysteine-16 of the A alpha chain was prepared from intact fibrinogen Kawaguchi. The amino acid composition and the molecular weight determination of this aberrant peptide revealed that it was a dimeric NH2-terminal peptide corresponding to residues 1-19 derived from the abnormal A alpha chain. These results indicate that the half-cystine at position 16 in the abnormal A alpha chain forms an intramolecular disulfide bridge with the same residue in the other abnormal A alpha chain and that fibrinogen Kawaguchi is a homo dimer composed of two identical abnormal halves.     

Enhanced degradation of cathepsin D synthesized in the presence of the threonine analog beta-hydroxynorvaline

The threonine analog beta-hydroxynorvaline is an inhibitor of asparagine-linked glycosylation. In the presence of the analog human fibroblasts synthesized cathepsin D molecules containing two, one, or no oligosaccharides. The nonglycosylated cathepsin D precursor was but a minor species and was degraded within 45 min of its synthesis, presumably in the lumen of the endoplasmic reticulum. The polypeptides with one or two oligosaccharides were normally segregated into lysosomes and their proteolytic maturation was not affected. The stability of mature glycosylated and nonglycosylated cathepsin D polypeptides within the lysosomes, however, was markedly decreased. The recovery of cathepsin D polypeptides was increased in the presence of inhibitors of cysteine and aspartyl-proteinases. These data suggest that the absence of carbohydrate side chains in cathepsin D results in an enhancement of the degradation rate of the precursor in the endoplasmic reticulum, and the replacement of threonine by beta-hydroxynorvaline in an enhanced degradation of the mature cathepsin D in lysosomes.     

Plasma fibrinogen in female patients with genital cancer and its early stage

Changes of plasma fibrinogen derivates in 54 female patients suffering from carcinomas of the genital system (36 with cervix carcinoma, 9 with ovarian carcinomas, 7 with carcinomas of the corpus uteri, 1 woman with a vulva and 1 patient with vaginal carcinoma) and 6 females with cervical intraepithelial neoplasia were investigated. It was shown, that the fibrinogen content and the concentration of soluble fibrin monomer complexes in blood plasma depend on the extension of the clinically diagnosed tumor. Furthermore, differently degraded fibrinogens were demonstrated to exist in the blood circulation. The extent of fibrinogenolysis did not correlate with the clinically diagnosed tumor stage.     

Isolation and characterization of a specific antibody population against human fibrinogen

A specific antibody population against human fibrinogen was isolated from a rabbit antiserum by affinity chromatography on fibrinogen-bound Sepharose gel. Using a sensitive competitive radioimmunoassay, the antibody population was found to recognize epitopes on native fibrinogen but crossreacted minimally with fibrinogen fragment D and an early plasmin-degraded fibrinogen A alpha-chain product, but not at all with fragment E or fibrinopeptides A and B. Fibrin monomers shared part of these epitopes. The antibody population crossreacted to a small extent with bovine, horse and baboon fibrinogens and not at all with fibrinogens from sheep, rat, pig, goat, guinea pig, dog and rabbit.     

Glycosylation reduces the bioactivity of calcitonin

The biological significance of peptide hormone glycosylation is uncertain. To examine the effect of Asn-linked glycosylation on calcitonin's bioactivity we purified glycosylated calcitonin from a transplantable rat medullary thyroid carcinoma. Glycosylated calcitonin constituted 2.3% of the total extracted immunoreactive calcitonin. The structure of this peptide differed from nonglycosylated calcitonin only by the oligosaccharide modification of asparagine 3. Affinity of glycosylated calcitonin for lentil lectin indicated that the oligosaccharide was a complex processed form. In a standard in vivo bioassay glycosylated calcitonin had a markedly reduced hypocalcemic activity compared to nonglycosylated calcitonin, an effect most likely due to the presence of the oligosaccharide.     

Lysosomal cysteine protease, cathepsin B, is targeted to lysosomes by the mannose 6-phosphate-independent pathway in rat hepatocytes: site-specific phosphorylation in oligosaccharides of the proregion

Cathepsin B, a lysosomal cysteine protease, is synthesized as a glycoprotein with two N-linked oligosaccharide chains, one of which is in the propeptide region while the other is in the mature region. When cultured rat hepatocytes were labeled with [(32)P]phosphate, (32)P-labeled cathepsin B was immunoprecipitated only in the proform from cell lysates and medium. Either Endo H or alkaline phosphatase treatment of (32)P-labeled procathepsin B demonstrated the acquisition of a mannose 6-phosphate (Man 6-P) residue on high mannose type oligosaccharides. To identify the site of phosphorylation, immunoisolated (35)S- or (32)P-labeled procathepsin B was incubated with purified lysosomal cathepsin D, since cathepsin D cleaves 48 amino acid residues from the N-terminus of procathepsin B, in which one N-linked oligosaccharide chain was also included [Kawabata, T. et al. (1993) J. Biochem. 113, 389-394]. Treatment of intracellular (35)S-labeled procathepsin B with a molecular mass of 39-kDa with cathepsin D resulted in the production of the 31-kDa intermediate form, but the (32)P-label incorporated into procathepsin B disappeared after treatment with cathepsin D. These results indicate that the phosphorylation of procathepsin B is restricted to an oligosaccharide chain present in the propeptide region. Interestingly, cathepsin B sorting to lysosomes was not inhibited by NH(4)Cl treatment and about 90% of the intracellular procathepsin B initially phosphorylated was secreted into the medium without being dephosphorylated intracellularly, and did not bind significantly to cation-independent-Man 6-P receptor, suggesting the failure of Man 6-P-dependent transport of procathepsin B to lysosomes. Additionally, about 50% of the newly synthesized (35)S-labeled cathepsin B was retained in the cells in mature forms consisting of a 29-kDa single chain form and a 24-kDa two chain form, while part of the procathepsin B was associated with membranes in a Man 6-P-independent manner. Taken together, these results show that in rat hepatocytes, cathepsin B is targeted to lysosomes by an alternative mechanism(s) other than the Man 6-P-dependent pathway.     

The identification of N-linked oligosaccharides on the human CR2/Epstein-Barr virus receptor and their function in receptor metabolism, plasma membrane expression, and ligand binding

Human complement receptor type 2 (CR2) was biosynthetically labeled by pulsing SB B lymphoblastoid cells for 25 min with [35S]methionine followed by chase in the presence of excess unlabeled methionine. An Mr 134,000 polypeptide represented the major form of the receptor at the end of the pulse period, and within 1 h of chase this disappeared coincident with the appearance of the Mr 145,000 mature form of CR2. Precursor, but not mature, CR2 was sensitive to endoglycosidase H, indicating that maturation of CR2 represented processing of N-linked high mannose oligosaccharides to the complex type. The processing of precursor CR2 was impaired by monensin. In the presence of tunicamycin an Mr 111,000 form of CR2 was synthesized by SB cells, and this did not chase into either precursor or mature CR2. This Mr 111,000 form of CR2 did not incorporate [3H]glucosamine, indicating that it lacked both N- and O-linked oligosaccharide. The half-lives of mature CR2 and nonglycosylated CR2 pulse-labeled in the presence of tunicamycin were 13.8 and 2.8 h, respectively; the turnover rate of B1, a membrane protein normally lacking carbohydrate, was unaffected by the presence of the antibiotic. The percentage of pulse-labeled, nonglycosylated CR2 that was expressed at the cell surface after 1 h of chase in the presence of tunicamycin was 30%, identical to that of mature CR2 in cells chased in the absence of the antibiotic. However, after 6 h of chase there was no additional net accumulation of nonglycosylated CR2 at the plasma membrane, while the proportion of pulse-labeled mature CR2 at this site had risen to 81%. Therefore, N-linked oligosaccharides are essential for the stability of CR2 and have some role in its plasma membrane expression. In contrast, the observation that all three forms of CR2 bound to Sepharose C3 indicates that oligosaccharides are not necessary for the interaction between CR2 and its complement ligand.     

Thrombin-induced fibrinopeptide B release from normal and variant fibrinogens: influence of inhibitors of fibrin polymerization

Thrombin preferentially cleaves fibrinopeptides A (FPA) from fibrinogen resulting in the formation of desAA-fibrin from which most of the fibrinopeptides B (FPB) are then released with an enhanced rate. Kinetics of fibrinopeptide release from normal and dysfunctional fibrinogens were investigated in order to further characterize the mechanism of accelerated FPB release during desAA-fibrin polymerization. Dysfunctional fibrinogens London I and Ashford, exhibiting primary polymerization abnormalities (i.e., an abnormality present when all fibrinopeptides have been cleaved), which in the case of fibrinogen London I is believed to be caused by a defect in the D-domain, were shown to exhibit a decreased rate of FPB release compared with normal fibrinogen. While Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, was shown to decrease the rate of FPB release from normal fibrinogen by a factor of 5, normal fragment D1, although inhibiting clot formation of normal fibrinogen, did not influence the acceleration of FPB release. On the other hand, the presence of fragment D1 did not enhance FPB release from fibrinogen London I, suggesting that interaction of D-domains in functional isolation with desAA-fibrin E-domains is not sufficient to enhance FPB release. Although clot formation was inhibited by the concentrations of fragment D1 used, the formation of small desAA-fibrin oligomers was hardly affected. Thus, small fibrin polymers, but not desAA-fibrin monomers, act as optimal substrates for the release of FPB by thrombin.