British journal oi Haemalology, 1983, 53, 599-610

Stimulation of fibrinogen biosynthesis by fibrinogen fragments D and E

WILLIAM R. BELL, CRAIG M. KESSLER A N D R. R. TOWNSEND Department of Midicine, Division of Hematology, The johns Hopkins University School of Medicine, Baltimore, Maryland, U . S . A .

Received 26 May 1982; accepted for publication 22 October 1982

SUMMARY. Infusions of either fibrinogen fragment D or fibrinogen fragment E into rabbits were followed by increases in fibrinogen synthesis determined by the rate of incorporation of 7iSe-selenomethionine into circulating fibrinogen. The degree of stimulation was proportional to the amount of protein infused. When 4.5 mg of each fibrinogen fragment was administered separately to different groups of animals, fibrinogen fragment D was associated with a fourfold increase in fibrinogen synthesis above that in the control animals compared with l.5-fold increase induced by fragment E. Fragments D and E were assayed for bound sialic acid, the absence of which facilitates binding, transport and catabolism of many circulating glycopro- teins by the liver. Fibrinogen fragment D contained 1.3% sialic acid compared to 1.4% in fragment E. These data indicate conservation of sialic acid during plasmic digestion of fibrinogen. The capacity of these glycopolypeptide fragments to stimulate fibrinogen synthesis appears unrelated to the nearly identical quantities of N-acetyl neuraminic acid found in each fragment.

Previous experimental observations have indicated that the production of fibrinogen is responsive to many unrelated physiologic and non-physiologic stimuli. How these stimuli result in an increased rate of fibrinogen synthesis is at present unclear. In addition, the physiologic mechanism responsible for maintaining normal concentrations of fibrinogen in the circulating blood has not been identified. Recent studies have indicated neither hyper- nor hypofibrinogenaemic blood levels alter the rate of fibrinogen synthesis (Kropatkin & Izak, 1968; Atencio et al, 1969; Alving et al. 1977a, b). The duration of the hypo- or hyperfibrinogenaemia did not influence the basal rate of fibrinogen biosynthesis (Alving et al. 19 7 7a, b). Several investigators have demonstrated that crude preparations of degradation fragments of fibrinogen increase the rate of fibrinogen synthesis in dogs (Barnhart et al, 19 70; Young & Kolmen, 1970) and rabbits (Kropatkin & Izak, 1968; Bocci & Pacini, 1973). More Correspondence: Dr William R. Bell, Department of Medicine, Division of Hematology, The Johns Hopkins University School of Medicine, Baltimore. Maryland 2 1205, U.S.A. 0007-1048/83/0400-0599802.00 @ 1983 Blackwell Scientific Publications

599

600 W. R. Bell, C . M . Kessler and R. R. Townsend

recently, it has been observed that purified species specific 40 min and 16 h plasmin digests of fibrinogen causes stimulation of fibrinogen synthesis in rabbits (Kessler & Bell, 1979a, b). Particularly instructive are the findings that degradation fragments from cross-linked and non-cross-linked fibrin do not stimulate fibrinogen synthesis (Ittyerah et al, 1979; Kessler & Bell, 1980).

The current study evaluated the effects of infusions of purified homologous fibrinogen degradation products D and E on fibrinogen synthesis and correlated the differences in stimulatory capacity with the differences in the sialic acid content of the various fibrinogen degradation fragments. These two fragments were studied since previous studies have indicated that mixtures of fragments D and E stimulated in vivo fibrinogen synthesis (Kessler et al. 1978).

MATERIALS A N D METHODS

Animals. Healthy male New Zealand white rabbits, weighing between 2.0 and 2.5 kg, were caged individually in air-conditioned quarters for at least 3 d before use. Only rabbits with normal baseline fibrinogen concentrations, haematocrit values, white blood cell and platelet counts were used. The animals were allowed standard rabbit chow and water ad lib.

Purification offibrinogen. Rabbit fibrinogen was purified as described previously (Kessler & Bell, 19 79b) using the techniques of McFarlane (1 973) and Senior et aZ(1974). Clottability of the resulting fibrinogen was 89-98%. Purity was assessed also by gel electrophoresis (Weber & Osborn, 1969) and immunodiffusion (Ouchterlony, 19 58). Preparations were lyophilized and maintained at - 20°C until used.

Preparation of fibrinogen fragments D and E . Digestion of purified rabbit fibrinogen was accomplished at 37°C with 0.003 casein units of plasmin (Thrombolysin; Merck Sharp & Dohme Research Laboratories, Lot no. 0960W, West Point, PA) per mg of lyophilized fibrinogen dissolved in 30 ml aliquots of sterile water to a concentration of 1 mg/ml. The pH of the digestion mixture was 7.0 and was maintained during fibrinogenolysis by a pH stat with 0.5 N sodium hydroxide. The reaction was terminated by the addition of E-ACA 0.2 M final concentration (Marder & Shulman, 1969) at 16 h to provide optimal production of fragments D and E. To exclude the presence of residual fibrinolytic activity following addition of E-ACA, aliquots from all digestion mixtures were examined on heated fibrin plates (Enzo Diffusion fibrin plate test: Hyland Products, Costa Mesa, CA).

The effectiveness of E-ACA (0.2 M) as an inhibitor of fibrinogen proteolysis by activated plasmin was studied by comparing the termination of fibrinogenolysis in additional digestion mixtures with either di-isopropylflurophosphate (DFP) (Sigma Chemical Co., St Louis, MO) or phenylmethylsulfonyl fluoride (PMSF) (Sigma Chemical Co.) established potent serine protease inhibitors (Fahrney 81 Gold, 1963; Kaplan & Austen, 1972). These agents (E-ACA, DFP, PMSF) were incubated in a five- to ten-fold molar excess over the amount of plasmin present (Nussenzweig & Souza. 1962; Summaria et aZ, 1967, 1970: Bauer, 1969). The FDPs (70-1 50 pg of protein from each preparation) were identified in their unreduced form by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in 5% gels (Weber & Osborn, 1969) with appropriate standards.

Fibrinogen Biosynthesis 60 1 Separation of components in a 16 h digest: fragments D and E . The individual isolation of

fibrinogen fragments D and E was performed employing modifications of the technique described by Doolittle et aZ(l977). The results of a 16 h plasmic digest (200 mg protein) of rabbit fibrinogen were placed on a DE-52 DEAE cellulose column (2.5 x 38 cm) that was previously equilibrated with 0.01 M NaHC03/Na2C03 buffer, pH 8-9. Employing descending technique a sequential two-part gradient elution was started. The limit buffer solution was 200 mlO.01 M NaHC03/Na2C03 0.5 M NaCl, pH 8.9.

Fragment D2 (hereafter referred to as fragment D) and E were also identified by employing SDS-PAGE and the Ouchterlony double-immunodiffusion technique utilizing specific canine antihuman D and E antibodies (generously provided by Dr James P. Chen, University of Tennessee School of Medicine, Knoxville, TN) (Chen, 1977).

lnfusion studies to evaluate stirnulatory properties of fragment D and fragment E

Solutions containing varying concentrations of either fragment D or fragment E in a 30 ml volume [assayed for protein content (Lowry et al, 195 1) and fibrinogen-fibrin degradation products (FDP-fdp) (Merskey et aZ, 1969; Alving et al, 1977a, b)] were infused over 1 h into the right marginal ear vein by means of continuous infusion pump (B. Braun Perfusor: Ouigley-Rochester Inc., Rochester, N.Y.) of rabbits. Control animals received equal volumes of a 0.155 M sodium chloride 0.013 M sodium citrate solution, pH 7.5, over 1 h in the right marginal ear vein. At the end of the 1 h intravenous infusion a small quantity of the infusate was studied to assure that the contents of the infusate had not changed during the 1 h infusion.

Assessment of fibrinogen synthesis

In vivo biosynthesis of fibrinogen was measured by determining the incorporation of 7sSe selenomethionine (75SeM) (Sethotope, specific activity 350-450 mCi/mg: E. R. Squibb & Sons Inc.. New Brunswick, N.J.) into fibrinogen and other proteins was measured serially after the injection of 20 pCi of 75SeM as previously described (Kessler & Bell, 1979a, b, 1980) employing the techniques of Lerner et aZ(1968), Bocci & Viti (1 966), Atencio et al(1965) and Lowry et aZ (1951).

Additional evidence for the specific incorporation of 75SeM into fibrinogen synthesized de novo was obtained by precipitating the fibrinogen with 2 5% saturated ammonium sulphate from the plasma of rabbits administered 75SeM 6 h previously. This material was then examined on SDS polyacrylamide electrophoresis in reduced and nonreduced forms. Each gel band was cut and measured for radioactivity.

Multiple specimens of serum were collected from each rabbit before infusion of fragment D or fragment E and at 0, 3 and 5 h following 75SeM injection for determination of serum haptoglobin levels and incorporation of 75SeM into haptoglobin and FDP-fdp.

Determination of FDP-fdp titres. Titres of FDP-fdp were determined by the tanned red cell

602 W. R. Bell, C . M . Kessler and R . R. Townsend haemagglutination-inhibition immunoassay (Merskey et al, 1969) modified for use in rabbits (Alving et al, 1977a, b).

Quantijcation of haptoglobin levels. Nonhaemolysed serum was obtained from each rabbit immediately before infusion of the dialysed fragments and at 0, 3 and 5 h following the injection of "%eM, for determination of haptoglobin levels by the immunodiffusion technique of Mancini et al (1965) as modified by Fahey & McKelvey (1965). Several samples were evaluated simultaneously with the techniques outlined by Mouray et al (1964) for determination of haptoglobin concentration and incorporation of labelled amino acids into haptoglobin.

After 18 h diffusion, areas of agar corresponding to the circumference of immunoprecipi- tin rings which had developed around each sample well were measured for quantification (Lowry et al, 1951; Mancini et al, 1965), carefully cut out, placed in a gamma-well scintillation counter (Picker Nuclear Autowell 11; Intertech Inc., North Haven, Conn.) set for an energy range of 350-450 keV, and counted over 15 min to estimate haptoglobin radioactivity.

Lirnulus lysate assay. The Lirnulus lysate assay for endotoxin contamination was performed on each preparation infused in this study, according to the method of Levin & Bang (1 968). Aerobic and anaerobic cultures of all infusates were negative for bacterial growth.

Determination ofsialic acid. The sialic acid content of fibrinogen, fragment D and fragment E was quantified by the method of Warren (1959).

0.51

Fraction Number (6rnl-Aliquots)

Fig 1. Descending D52-DEAE column chromatography of 16 h plasmic digest of rabbit fibrinogen. On the abscissa are the fraction members and la), tb), tc) and fd) are the starting points for the first (400 rn10.01 M N ~ H C O ~ pH 8 ~ 9 / 4 0 0 m l 0 ~ 0 1 M N ~ H C O ~ - N ~ ~ C O ~ O . O ~ M N ~ C ~ ~ H 8~9) , second( l50ml0~01 M NaHCO3-NarCO3 0.09 M NaClI150 mlO.01 M NaHCO3-NazCO3 0.1 7 M NaCl pH 8.9). third (1 50 rnl 0 . 0 1 ~ NaHCO3-Na2CO3 0.1 7 M NaCl pH 8.9/150 ml0-01 M NaHC03-Na2C03 0.34 M NaCl pH 8.9) and fourth (150 mlO.01 M NaHCO3-Na2CO3 0.34 M NaCl pH 8.9/150 mlO.01 M NaHC03-Na~C03 0.5M NaCl pH 8.9) portions of the buffer gradient. On the left ordinate is absorbance at 280 mm and right ordinate is conductance K (mho) and indicated by the interrupted line.

Fibrinogen Biosynthesis 603

A B C Fig 2. Non-reduced SDS-tube gels of rabbit fibrinogen fragment E (lane A) Mr 55 000, rabbit fibrinogen fragment D (lane B) Mr 80 000, and standards in lane C. Number 1 is B-galactosidase Mr 90 000. number 2 is bovine serum albumin Mr 65 000: number 3 is ovalbumin Mr 4 5 000: number 4 is chymotrypsin Mr 24 000; and number 5 is myoglobin Mr 16 000.

Statistical evaluation

Mean, standard deviation, standard error, and comparative group analysis were computed by conventional methods (Snedecor & Cochran, 1965).

RESULTS

The elution profile of the ion exchange chromatogram of the plasmic digest of fibrinogen is shown in Fig 1 . Fractions under peaks D1 (designated D in this paper) and E were pooled separately, dialysed, lyophilized and characterized by SDS-PAGE under non-reducing conditions (Fig 2 ) . It is apparent from these studies where 10 pg of protein were applied to each gel that the fractions selected from the digest contained a single species of fragment D with estimated Mr of 80 000 and fragment E with an estimated Mr of 5 5 000. On reduced SDS gels the u, j and y chain segments of both fragment D and E were intact. A single arc was

604 W. R. Bell, C . M . Kessler and R. R. Townsend observed on immunoelectrophoresis employing the monospecific anti D and E peaks, respectively.

When these purified fragments were infused directly intravenously into rabbits in quantities ranging from 1 to 12 mg per infusion: the degree of stimulation of fibrinogen synthesis, as indicated by the increase of the per cent of 75SeM into newly synthesized fibrinogen, was directly related to the quantity of the fragment infused (Figs 3 and 4). The degree of stimulation of fibrinogen synthesis was dose related for both fragment D and fragment E but was observed to a lesser degree with fragment E. When 4.5 mg of each fibrinogen digestion fragment was administered separately to different groups of rabbits, fragment D was associated with a fourfold increase in fibrinogen synthesis, while fragment E produced a 1.5-fold increase as compared to controls (Fig 5). Increase in plasma fibrinogen concentration was first observed 4 h after the 75SeM but was progressive up to 24 h going from a baseline of 2*40*0.20 (SE) g/1 to 3.65fi0.18 (SE) g/1 at 24 h in those animals

3.60

3.20

5 8 2.80

P

g 2.00

d 2 = 1.60 8

g 1.20

5 2 0.80

z m

z

K

U 2.40

2

z 2

Ln

I-

w a

0.40

- 4.00mgl

- 2.00mgl

0 1 2 3 4 5 24

HOURS AFTER ADMINISTRATION OF 75SEM

Fig 3. Rabbit fibrinogen fragment D dose dependent response in the stimulation of fibrinogen synthesis in treated animals with respective doses in brackets (dotted lines) and control animals (solid lines). Each point plotted is the mean of 10 animals with standard error (I). The numbers in mg in brackets are the range in mg of fragment D that induced the response plotted in the adjacent curve.

2.80

5 8 2.40

e L a m

z

LL 2.00

605 -

FDP-E -- --- Control

(11.0 - 12.00mgl -

-

H % 0.80

In

- 9.00mgl

- 3.00mgl

0 1 2 3 4 5 24

HOURS AFTER ADMINISTRATION OF 75SEM

Fig 4. Rabbit fibrinogen fragment E dose dependent response in the stimulation of fibrinogen synthesis in treated animals with respective doses in brackets (dotted lines) and control animals (solid lines). Each point plotted is the mean of 10 animals with standard error (I). The numbers in mg in brackets are the range in mg of fragment E that induced the response plotted in the adjacent curve.

receiving fragment D and 2 .20 f0 .14 (SE) g/1 to 2.61 f0.14 (SE) g/1 in animals receiving fragment E (Fig 5). Control animals received solutions containing saline, E-ACA and plasmin in quantities employed in the digestion of fibrinogen and did not demonstrate an increase in fibrinogen synthesis above that seen in the basal state in animals receiving only 20 pCi of the cohort label 75SeM.

Examination of the terminal aliquot of the solution containing either fragment D or fragment E at the end of the 1 h infusion revealed no change in the character of these fragments as seen on SDS polyacrylamide gel electrophoresis and immunoelectrophoresis. Haematocrit values remained stable throughout the in vivo studies. Rabbits receiving the various infusates did not evidence any adverse reaction such as fever, rigors, hypotension or anaphylaxis.

In the animals receiving fragments D and E which demonstrated an increase in fibrinogen biosynthesis, a concomitant increase above control in "SeM incorporation into haptoglobin, or into TCA precipitable proteins (after removal of fibrinogen from plasma) or an increase in the serum haptoglobin level was not observed.

All infusions contained less than 10 pg/ml of endotoxin. This level is below the minimal concentration of endotoxin that will stimulate fibrinogen synthesis (Alving et al, 1979).

Intact rabbit fibrinogen contained 0.6% sialic acid by weight or 6.13 residues/mole. The

606 W. R . Bell, C . M . Kessler and R. R. Townsend 2.80 -

Control - FDP-0

z 3.20- FDP-E

0

..... ....... ---- %

- U

0 1 2 3 4 5 24

HOURS AFTER ADMINISTRATION OF '%EM

Fig Effect ( FDP-D and FDP-E on fibrinogen synthesis. Incorporation of 75S~. . - into fibrinogen was measured 4 h after infusions of 4.5 mg of fibrinogen degradation fragment D (dotted line) 5.6 x lo-' mol or 4.5 mg of fragment E (interrupted line) 8.2 x lo-* mol. Fibrinogen synthesis and rate of 75SeM incorporation into fibrinogen were stimulated to a much greater degree in rabbits administered FDP-D than in those rabbits given FDP-E. The fibrinogen production and 75SeM incorporation induced by FDP-E resembled control values (solid line). Each point represents the mean f 1 SE. The per cent increase change from baseline in plasma fibrinogen concentrations 24 h following administration of 75SeM is indicated within parentheses.

Table I. Sialic acid content of rabbit fibrinogen and derived fragments D and E

Molar ratiof Compound Sialic acid* mole sugar/mole specimen

Fibrinogen 0.56 fO.O2% 6.13 Fragment D 1.22f0.02% 3.09 Fragment E 1.31&0-07% 2-12

*Based on protein concentration by the method of Lowry et a1 (1951) using bovine serum albumin as a standard and sialic acid assayed by the method of Warren (1959).

t Calculated using Mr = 340 000. 80 000 and 5 5 000 for fibrinogen, fragment D and fragment E respectively.

Fibrinogen Biosynthesis 60 7 content of sialic acid in fragments D and E are shown in Table I. The total sialic acid content is retained on these fragments following in vitro plasminolysis of rabbit fibrinogen.

DISCUSSION

Earlier studies from our laboratory (Alving et al, 1977a, b; Kessler & Bell, 1979a, b, 1980) employing the cohort label 75SeM to assess the rate of de novo synthesis of fibrinogen have indicated that mixtures of fibrinogen-fibrin degradation products were capable of stimulating fibrinogen synthesis. These studies identified that 16 h plasmic digests of fibrinogen containing mixtures of fragments D and E stimulated fibrinogen biosynthesis. Short-term plasmic digests containing fragments X and Y and not fragments D and E did not stimulate fibrinogen biosynthesis. Separation of these two terminal digestion fragments has made possible the observation that both fragment D and fragment E when studied individually can stimulate fibrinogen synthesis. The in vivo infusion of fragment D results in a synthesis rate increase in fibrinogen that is 4-5-fold greater than observed with identical quantities of fragment E. For both a dose dependent response in fibrinogen synthesis was observed over a wide range of protein concentrations infused. In addition to an increase in the incorporation of "SeM into fibrinogen reflecting an increase in synthesis, this was substantiated by an increase in plasma fibrinogen concentration. However, the increase in plasma fibrinogen lagged by several hours behind the evident increase in the rate of synthesis. The stimulatory effect observed following the infusion of fragment D and fragment E is not the result of a non-specific generalized stimulus since a concomitant increase in serum haptoglobin, or increase in 75SeM into haptoglobin. or increase in 75SeM into TCA precipitable serum proteins was not observed. Similar findings by cthers (Franks et al, 1981) employing different techniques in rats have confirmed our earlier report (Kessler et al, 1978) on the stimulatory capacity of fragment D and fragment E.

The reason for the striking difference between the stimulatory property of fragment D in contrast to the minimal stimulatory capacity of fragment E is not known. We considered the possibility that this may be due to the sialic content of each fragment. It is known that survival time of sialated proteins in the plasma is considerably different from their asialo counterparts (Amir et al, 1966; Morel1 et al, 1971; Hudgin et al, 1974). Data from other laboratories (Prose et al, 1965; Barnhart & Noonan, 1973) have demonstrated that fibrinogen degradation fragments are removed from the circulating blood by the liver. Conceivably, this step may precede stimulation of fibrinogen synthesis by the hepatocytes. Receptors on hepatocytes that specifically bind asialoglycoproteins have been isolated (Morrell et al, 1971) and may potentially play a role in the regulation of plasma glycoprotein homeostasis. Our studies revealed that the total sialic content of fragment D and fragment E were very similar. Although obvious differences in total sialic acid are not present between fragment D and fragment E. this does not eliminate the possibility that differences in stimulatory capacity are not related to the carbohydrate moiety on these fragments. Differences may exist in the other components of the carbohydrate moiety on these fragments. Possibly the carbohydrate moiety on these fragments must be cleaved before the fragments undergo endocytosis. Fragment D may be more susceptible to this in the

608 W. R . Bell, C . M . Kessler and R. R. Townsend

circulation than the terminal fragment E. Further studies are needed to clarify these speculations.

Intact rabbit fibrinogen (the parent molecule) contains 0.6% sialic acid by weight or 6- 13 residues/mole. Our studies indicate that the total sialic acid content is preserved during in vitro plasminolysis of fibrinogen. It is apparent that if cleavage of the carbohydrate moiety is necessary for endocytosis that this takes place via some proteolytic enzyme other than plasmin. If stimulation of fibrinogen synthesis is dependent upon the clearance of desialylated fibrinogen degradation fragments by the hepatocytes, alteration of the intact digestion fragments must occur in v iva with an increased specificity for fragment D.

In the present studies we elected to infuse the homologous purified fibrinogen degradation fragments D and E directly into the intravascular compartment. This is the compartment where they exist in the physiologically normal metabolic steady state. These fragments are normally not present outside the vascular compartment. Placement of these fragments in extravascular compartments may result in non-physiologic alteration of these peptides. Such an alteration may not be possible in the intravascular compartment where these fragments are normally present.

ACKNOWLEDGMENTS

This work was supported in part by research grants HL-01601-01, HL24898-01 and Training Grants HL-0714303 and HL-06188 from NHLBI of the National Institutes of Health and The Whitehall Foundation. William R. Bell is a Hubert E. and Ann E. Rogers Scholar in Academic Medicine.

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