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Presence of a low molecular weight plasma fibrinogen stimulator and inhibitor in human urine
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PRESENCE OF A LOW MOLECULAR WEIGHT PLASMA FIBRINOGEN STIMULATORAND INHIBITOR IN HUMAN URINE Stephen H. Wentland, Timothy H. Carlson, BruceD. Leonard, Demerick C. Fradl and E. B. Reeve University of Colorado 14dical Center Denver, Colorado 80262 USA
(Received 5.l..l.977.
Accepted by Editor M.I. Barnhart)
ABSTRACT When male human urine was fractionatedby butanol extraction and cation-exchangechromatography,a substance lowering plasma fibrinogen levels (in rabbits) was separated from a substance elevating fibrinogen levels. When the urine was fractionatedby ethanol precipitationand gel permeation chromatography,these fibrinogen elevating and depressing substanceswere shown to have low molecular ’ weight ( < 1000 daltons). In still other fractionationprocedures, the two substanceswere found to be interconvertible. This pair of elevating and depressing substancesmay be unreported regulatorsof fibrinogen levels, and may be part of a new hormonal system of regulation.
INTRODUCTION It is well known that injury (e.g. inflannnation, tissue damage) results in increased fibrinogen synthesis (1). This laboratoryhas been concerned with the regulation of fibrinogenmetabolism and earlier studies showed that pharmacologicaldoses (ca. 50 IU) of ACTH (2) and milligram doses of various prostaglandins (3, 4, 576) stimulate fibrinogen synthesis. Using ACTH, the kinetics of fibrinogen stimulationhave been studied (7). In addition to investigatingknown, well-defined compounds,we have searched biological fluids for other components that influence fibrinogenmetabolism. We now wish to report evidence for the presence of substances in male human urine which raise and lower plasma fibrinogenlevels in rabbits. A preliminary account of this work was reported elsewhere (8).
MATERIALS AND METHODS All chemicals used were reagent grade. Ultrafilterswere obtained from Amicon Corp., Lexington,MA, and all chromatographicresins were obtained
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from Bio-Rad Laboratories,Richmond, CA. Cellulose sheets for thin-layer chromatographywere obtained from Eastman Kodak, Rochester,NY. The assay used was described previously (7) and consists of dissolving the substance to be tested in 20 ml physiologicalsaline, and infusing the solution at a constant rate into the marginal ear vein of the rabbit over 3 hr. Blood was collected immediatelybefore and 24 hours after the infusion, and fibrinogen concentrations,$, in the plasma samples were measured by a radioisotopedilution technique. The 24 hour plasma fibrinogenconcentration was corrected to constant plasma volume to give the corrected plasma fibrinogen concentration,$124,as described elsewhere (9). A$, the change in plasma fibrinogen concentrationwas given by $24-$. where $. is initial plasma fibrinogen concentration. The average increase in plasma fibrinogen,resulting from the infusion of 25 saline controls was .?47 mglml, SD .222. Stimulating fractions were defined as those which gave fibrinogenincreases greater than 0.791 mg/ml (the mean control increase plus two standard deviations),while fibrinogen inhibiting fractionswere defined as those which gave fibrinogen decreases falling below -0.097 mg/ml (the mean control increase minus two standard deviations). Since bacteria and bacterial endotoxins have been shown to elevate fibrinogen levels (1, lo), care was taken to avoid contaminationin all procedures used. All heat-resistantapparatus was autoclaved and all other apparatus was washed well with sterile solutions before use. Buffers were prepared from autoclaved distilledwater, glacial acetic acid and concentrated ammonium hydroxide, and were found not to cause significantchanges in fibrinogen levels when lyophilizedsamples were inf6sed into rabbits. Human male urine was collected in sterile containersand processed within two hours after collectionby either of two methods: In the first method, the urine was first acidified with concentratedHCl to pH 1.5, then saturated with salt, and finally extracted with an equal volume of E-butanol in small portions. The butanol extracts were neutralizedwith concentrated sodium hydroxide, and then concentratedunder reduced pressure at 37O, yielding a light-to-darkbrown solid we call "butanol extracted material". In the second method, the urine was diluted with three volumes of ethanol (95%) at 5O, and the resultingmixture was stored at 4O for 24-48 hours. The precipitate,obtained by decantationand centrifugation,was then extracted with cold physiologicalsaline using a Waring blender. The resulting extract was continuouslydialyzed against cold tap water for three hours, and then lyophilized,yielding a tan solid we call "ethanol precipitated material". We reported a preliminary account of this method previously (11). Ultrafiltrationwas typically carried out in a 60 ml Amicon Ultrafiltration cell, using Amicon UM type filters. In order to remove the protective film of glycerol, the filters were soaked in sterile water for 3 hours, with replacementof the water every hour. To prevent bacterial contamination, the filters were stored in 6 M urea containing 0.1% sodium azide. Before use, they were washed well with buffer, and then flushed with 20 ml buffer. The material to be ultrafilteredwas dissolved in 50 ml buffer and passed through the filter under 50 psig nitrogen. Filtrationproceeded until the volume in the cell was reduced to 3 ml. Filtrationwas then stopped, giving a 3 ml retentate and a 47 ml filtrate. If the filtrate was to be further fractionatedby a filter with a lower molecular weight cutoff, it was then passed through the filter as above, giving a 3 ml retentate and a 45 ml filtrate.
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Gel-permeation resins (Bio-Gel P-Z) were equilibrated with buffer, separated from'fines, and autoclaved before use. Ion-exchange resins (CMcellulose, DRAE cellulose, Bio-Rex 70) were first washed with dilute acid and base, separated from fines, equilibrated with starting buffer, and then autoclaved. All chromatographic columns were flushed overnight with sterile buffers before being washed. Thin-layer chromatography was carried out using plastic-backed sheets of cellulose, which were eluted several times with sterile buffer before use. The plates were dried in a dust-free environment and then spotted with a concentrated solution of the sample, using a gentle stream of nitrogen to facilitate evaporation. The eluting solvent was the upper layer obtained by mixing 10 parts sterile H20, 10 parts n-butanol, and 2 parts glacial acetic acid, shaking, and letting the layers separate. After elution, the plate was cut into seven sections, the adsorbant in each section was scraped off the backing, and was then extracted 3 times with 5 ml 2.0 M ammonium acetate buffer In all the above steps sterile technique was used. The buffer extracts were finally lyophilized and assayed as described above. All solutions to be analyzed in the rabbit assay were prepared with sterile physiological saline solutions and were cultured to check for bacterial contamination. Since endotoxins have been shown to be pyrogenic, their presence or absence in active fractions was assayed using the pyrogen test described in the U.S. Pharmacopeia (12). This consists of rapidly injecting the material to be tested into the marginal ear vein of the rabbit, and observing the rises in temperature (if any) after the lst, Znd, and 3rd hour. If the sum of the three temperature rises was less than 40, the fraction was taken to be free of pyrogen.
RESULTS Ultrafiltration Studies Our first experiments involved the fractionation of the ethanol precipitated material on the basis of molecular weight. The ethanol precipitated material from 500 ml urine was dissolved in 0.1 M ammonium acetate buffer, pH 8.0 and filtered through a series of Amicon ultrafiltration membranes having different molecular weight cutoffs. The resulting fractions were lyophilized, dissolved in physiological saline and infused as described above. Results are shown in Table 1. Three fractions were obtained, the first containing substances with molecular weights greater than 10,000; the second, substances with molecular weights between 10,000 and 1,000; and the third, substances with molecular weights less than 1,000. When assayed, the first and third fractions showed stimulatory activity, while the second showed no activity at all. Both active fractions were tested for pyrogens. While the first fraction gave a positive response, the third fraction gave a negative response. Thus, we have obtained two stimulatory substances having very different molecular weights. While the substance of higher molecular weight may be contaminated with bacterial pyrogens, the substance of lower molecular weight is free from this kind of contamination. In subsequent runs the low molecular weight product was obtained, but not consistently. Several modifications of the ultrafiltration procedure
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TABLE I Ultrafiltrationof Ethanol PrecipitatedMaterial
Molecular Weight Cutoff of Ultrafilter
A$ (mdml)
Retentate
MW > 10,000
1.05
Retentate
MW >
1,000
.07
Filtrate
MW <
1,000
1.60
were made, but the appearance of the desired substancewas still erratic. Gel Permeation ChromatographyStudies In an effort to overcome the erratic appearance of activity in the low molecular weight fraction, we further refined our fractionationtechnique by adding gel permeation chromatography. An inactive, low molecular weight ultrafiltration fraction (derived from 500 ml urine) was chromatographedon BioGel P-2 gel permeation resin (exclusionlimit, 1,800), with the results shown in Fig. 1. Two fractions of interest were obtained. Fraction number 4 showed an increase in fibrinogen of 1.36 mg/ml, but fraction number 5 showed a decrease_ in fibrinogen of -.28 mg/ml. Thus, from an inactive low molecular weight ultrafiltrationfraction both stimulatoryand inhibitory activity was obtained. Both fractions eluted around the total volume (35 ml) of the column thus demonstratingthat the active substanceshave molecular weights less than and inhibitory substancesquickly lost 1,000. But both the stimulator-y activity, and or&r of their appearance from the P-2 column and their presence in the eluate were both erratic. Similar results were obtained whqn the unfiltered crude ethanol precipitate was chromatographedon Bio Gel P-2 resin as above. Ion Exchange CnromatographyStudies A concurrent set of experiments involved fractionationof the butanol extracted material by CM-celluloseion-exchangechromatography. Butanol extracted material from 1 liter of urine was applied to the top of a 20 cm x 0.9 cm column of CM-cellulose,and elution was carried out as described in the legend to Fig. 2. The fractions thus obtained were assayed as described above, and the results are shown in Fig. 2. It is seen that inhibitorymaterial was eluted in fraction 6, (A$ = -.23) stimulatorymaterial in fractions 9 and 10, (A+ = .96, .92). Thus, a clean separation of the inhibitorymaterial from the stimulatorymaterial was obtained. But nevertheless,the appearance of both stimulatoryand inhibitory activity was again inconsistent.
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REGULATORS
0.8
9 2
0.6
3 %
0.4
I
I
10
20
I 30
I
I
40
SO
Fxution Volume bl)
FIG. 1 Bio-Rad P-2 gel permeation chromatographicresin, colunm dimensions 55 cm x 0.9 cm, flow rate 7.5 ml/hr., buffer 0.1 M ammonium acetate, pH = 8.0. The fractions shown above were lyophilized,dissolved in physiologicalsaline, and infused as described earlier. The dashed line at A$ = .347 represents the mean control values, the shaded area represents 2 standard deviations above and below the mean.
TABLE II StatisticalAnalysis of Fractions Obtained from CR-CelluloseChromatography
Number of Section of Inhibitory Fractions Column Analyzed Found
Nlnnberof Stimulatory Fractions Found
Number of Fractions in Control Range
Fractions 4-5
1
0
14
Fractions 6-7
2
1
13
Fractions 8-9
1
3
10
Fractions lo-11
0
3
12
Fractions 12-13,
0
4
5
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Bio Bad GM-cellulose ion-Exchangeresin, column dimensions 20 cm x 0.9 cm. Each fraction contained 5 ml buffer, flow rate 40 mljhr. Stepwise elution was carried out with aumnmium acetate buffers of molarity and pH as shown above. The above fractions were lyophilized,dissolved in physiological saline and infused as described earlier. The dashed line at A0 - .347 represents the mean control value, the shaded area represents 2 standard deviations above and below the mean.
StatisticalEvaluation In spite of the above inconsistencies,we have demonstratedstatistically that Fig. 2 gives a fair picture what happens during GM-cellulosechromatography. The GM-column described above was run eleven times, with fractions taken as above, and the results of these eleven runs were combined. There was some variation in intermediatesteps, though these did not significantly affect the chromatographicseparation. Thus, in some instances the butanol extracted material was precipitatedwith ether, back-extractedwith aqeuous base, or fractionatedon Bio-Gel P-2 gel permeation resin prior to GM-cellulose chromatography. Also to reduce the large number of rabbits required for the assay of activity in these staticticalstudies, adjacent fractionswere sometimes combined before being lyophylizedand infused. The results of examining fractions from each GM-cellulosecolumn for fibrinogenstimulating and inhibiting activity are shown in Table 2. It is seen that of a total of 69 fractions analyzed, 4 inhibitory and 11 stimulatory fractionswere found, the remaining fractionsbeing in the control range. Inspection of the data in Table 2 shows that the inhibitoryresponses clustered in the fractions eluting early from the column, the mean fraction number being 6.50, SD 1.64. In contrast, the stimulatoryresponses clustered in the fractions eluting late from the column, the mean fraction number being 10.32, SD 2.09. The differencebetween these two means was significantat the .Ol level using the two tailed t-test. So, in spite of the variations resulting from the instabilityof the active components,the above data demonstrate that a substance of inhibitory activity is regularlyeluted before a substance having stimulatory activity.
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DISCUSSION Our CM-cellulose studies have demonstratrd the presence in human urine of two distinct substances, one that elevates, and the other that depresses fibrinogen levels in rabbits. This procedure results in a high degree of purification. For example, from 1 liter of urlne, sub-milligram amounts of the active components are obtained. Our ultrafiltration and gel-permeation studies also demonstrated the presence of a stimulator and inhibitor, and established that these substances are of low molecular weight (< 1,000) and non-pyrogenic. It is quite likely that the pair of stimulating and inhibiting substances obtained through ultrafiltration and gel permeation chromatography is the very same pair obtained through CM-cellulose chromatography. As descrbied above, CM-cellulose chromatography of the butanol extracted material results in the elution of the inhibitor and stimulator in characteristic fractions. Active substances obtained from gel permeation chromatography were rechromatographed on CM-cellulose chromatography, and the inhibitor and stimulator were found to elute in these same characteristic fractions. This demonstrates that the stimulator-inhibitor pair derived from gel-permeation chromatography is identical to the pair derived from CM-cellulose chromatography with respect to their affinity for the cation-exchange resin. Furthermore, P-2 gelpermeation chromatography of the ethanol precipitated material results in the elution of inhibitor and stimulator in late-cluting fractions. When active samples obtained from CM-cellulose chromatography were rechromatographed on P-2 resin, active fractions were obtained in these same late-eluting fractions. This demonstrates that the stimulator-inhibitor pair obtained from CM-cellulose chromatography has the same molecular weight as the pair obtained from gelpermeation chromatography. Thus it is likely that the two pairs are identical. A problem this study has not resolved is the inconsistent appearance of activity in both the stimulating and inhibiting fractions. There may be several reasons for this. First, loss of activity is often encountered in the separation and purification of biological substances especially those initially present in very small amounts. This may be due to loss of cofactors or to denaturation due to large surface-to-mass ratios. Second, the stimulator and the inhibitor may not always be separated during ultrafiltration and gel permeation chromatography. Thus they may nullify each other's activity. However, this cannot account for the inconsistencies seen during CM-cellulose chromatography, in which the inhibitor is well separated from the stimulator. The following experiments further illustrate the erratic appearance of activity. Butanol extracted material was chromatographed on Bio-Rad 70 cation-exchange resin, a resin similar in composition to CM-cellulose, using the conditions described for CM-cellulose chromatography. While the resulting activity-elution profiles did not form a consistent pattern, active fractions were nevertheless obtained. When an inhibitory fraction (A$= -.34) was rechromatographed using the same conditions, stimulatory fractions (A$= 1.05, 0.98) were obtained. When a stimulatory fraction (A4= 1.33) obtained from another Bio-Rad chromatogram was rechromatographed, an inhibitory fraction (A$ = -.21) was obtained. This suggests that the stimulator can be converted into the inhibitor and vice-versa. This interconversion has also been observed when fractions obtained from Bio-Rad chromatography were rechromatographed on P-2 resin (A@= 1.11 + A$ = -.21), when fractions obtained from Bio-Rad chromatography were rechromatographed on DRAR-cellulose resin
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(A$= 1.11 + A@ = -.21), and when fractions obtained from U4-cellulosewere refractionatedby thin-layer chromatographyon cellulose plates (A$ = -.25 + A$ = 1.39). This interconversionmust contribute greatly to the inconsistentappearance of activity described earlier. The stimulator and inhibitor described above are active in sub-milligram amounts when infused into ca. 3 kg rabbits. It should be noted that our assay, based on the difference between final and initial fibrinogenlevels, is more sensitive to fibrinogenelevation than to fibrinogendepression. Thus, the inhibitormay have been present to a greater extent than the depression of plasma fibrinogen level suggests. In vitro studies have demonstratedthat several substances can stimulate -fibrinogen synthesis. Using a chick embryo liver culture, cortisol and other steroids was found to significantlyelevate fibrinogensynthesis (13). In perfused rat livers, a mixture of cortisol, growth hormone, and insulin stimulated fibrinogen synthesis,and in the absence of cortisol, this stimulation was greatly reduced (14). Incubation of suspensionsof rat hepatocytesshowed that mixtures of cortisol, triiodothyronine,glucagon, and growth hormone increased fibrinogen synthesis. Also, the inclusionor exclusion of insulin had no further effect (15). Other investigatorsusing this same technique reaffirmed that mixtures of cortisol and insulin increase fibrinogensynthesis, but in contrast to work cited above, insulin by itself significantlyelevated fibrinogen synthesis while cortisol by itself showed only a small effect (16). But in incubated rat liver slices, cortisol was actually found to decrease fibrinogen synthesis, as did histamine phosphate (17). In this latter system, sodium palmitate increased fibrinogen synthesis. Several substances, includingmost of those tested in vitro, have been shown to elevate fibrin0 en synthesis using various -in vivo assay'systems. Using incorporationof 1&C-carbonateas a measure of fibrinogensynthesis in rats, injection of cortisol (20 mg/kg, i.p.) caused significantincreases in fibrinogen synthesis (18), whereas injection of growth hormone (0.6 mg/kg, i.p.) led to moderate increases (19). Other studies in our laboratoriesusing this method in rabbits have shown that 50 IU ACTH (2) and milligram amounts of prostaglandinE-1 and E-2 (3, 4, 5, 6) also significantlystimulate fibrinogen synthesis. Using the incorporationof radioactiveglycine and other amino acids as measures of fibrinogen synthesis, injectionsof adrenalin (1 mg/kg, s.c.) (20), spironolactone (0.2 mnol/kg twice daily for 3 days, p.o.) (21), and phenobarbital (0.2 mmol/kg twice daily for 3 days, p.o.) (21) have significantly elevated fibrinogen synthesis in the rat. However, the last two substances do not occur naturally. Using the rate of incorporationof radioactivemethionine as well as the plot of fibrinogen concentrationvs. time as a measure of fibrinogensynthesis, cortisol (10 mg/kg, B.C.) and cortisone (10 mg/kg, i.m.) have shown significant elevations (22) in rabbits. Using these same assay systems, ACTH (ca, 20 U/kg, s.c.), growth hormone (l-2 U/kg, s.c.), and adrenalin (0.2 mg/kg, s.c.) also gave rise to significantelevations (23). However, glucagon (l-2 mg/kg, s.c.) failed to elicit a significantresponse. Using immunofluorescentmethods as well as the plot of fibrinogenconcentration vs. time as measures of fibrinogen synthesis, infusion of fibrin degradationproducts (11 mg/kg) into dogs led to significantelevations (24). Using only the plot of fibrinogen concentrationvs. time, injectionof 1 llg
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leukocytic endogenous mediator into rats i.p. gave rise to significant inin vitro by incubating rat liver creases (25). This compound was also tested -slices, with the same result. As measured by the 24-hour change in plasma fibrinogen concentration, adrenalin (2 x 3 prml/kg, i.p,) and noradrenalin (2 x 3 umol/kg i.e.) showed siginficant elevations in rats (26). In the same study, however, histamine (360 mg/kg, i.e.), corticosterone (25 mg/kg i.e.), dexamethasone (8 mg/kg, i.e.) and ACTH (80 units/kg, s.c.) led to no significant increases. It is unlikely that the stimulator we report here is glucagon, insulin, cortisol, histamine, adrenalin, or noradrenalin. For although some of these compounds have shown activity in some of the studies reported above, other studies in our laboratories demonstrated that the 24-hr plasma fibrinogen change resulting from the infusion of these compounds into rabbits at a dose level of 1 mg/kg were insignificant. As mentioned before, our stimulator displays activity at dose levels < 0.3 mg/kg. It is further unlikely that our stimulator is growth hormone, leukocytic endogenous mediator, or fibrin degradation products, since the molecular weight of our stimulator is less than 1000, while those of the latter three compounds exceed 10,000. Furthermore, our stimulator is active at lower dosage levels than required by either ACTH (2) or prostaglandin E-l or E-2 (3, 4, 5, 6). We conclude then that our.low molecular weight stimulator has not previously been described. Of particular interest, is our demonstration of an inhibitor and of the stimulator-inhibitor interconversion. This stimulatorinhibitor pair, of similar size and chromatographic behavior, may be part of a novel system of hormonal control, the actions of which depend on whether the control system is driven to produce primarily stimulator or primarily inhibitor. Further characterization of the stimulator and inhibitor has been hampered by their inconsistent appearance. Efforts are underway to find other ways of purifying and stabilizing these compounds, as well as to find other sources of them than urine.
ACRNOWLEDGPlENTS This work was supported by NIH grant #HLO2262, The Colorado Heart Association, and NIH grant #RR00051 (GCRC).
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HAM, T.H. and CURTIS, F.C. Plasma fibrinogen response in man. Influence of the nutritional state, induced hyperpyrexia, infectious disease and liver damage. Medicine, II, 413, 1938.
2.
ATENCIO, A.C., CHAO, P.-Y., CHEN. A.Y., and REEVE, E.B. Fibrinogen response to corticotropin preparations in rabbits. Amer. J. Physiol. 216, 773, 1969.
3.
CARLSON, T.H., FRADL,, D.C., LEONARD, B.D., WENTLAND, S.H. and REEVE, E.B. Fibrinogen synthesis stimulation by prostaglandin E-l and some other vasodilators. Submitted for publication to Amer. J. Physiol.
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4.
FRADL, D.C. and REEVE, E.B. Plasma fibrinogenresponse to prostaglandin infusions. Fed. Proc, 32, 265, 1973. (abstract)
5.
CARLSON, T.H., FRADL, D.C., and REEVE, E.B. ProstaglandinE-l (PGE-1) stimulation of fibrinogen synthesis. Fed. Proc. 24, 404, 1975. (abstract)
6.
WENTLAND, s.H., CARLSON, T.H., LEONARD, B.D. and REEVE, E.B. Fibrinogen synthesis stimulationby several E prostaglandins(PCE). Fed. Proc. 35_,2431, 1976. (abstract)
7. CHEN, Y.,,BRIESE, F.W., and REEVE, E.B. Relation of fibrinogensecretion (synthesis)to ACTH dose. Amer. J. Physiol.227, 932, 1974. 8. WBNTLAND, S.H., CARLSON, T.H., and REEVE, E.B. Fibrinogensynthesis stimulatorsand inhibitors from urine. Fed. Proc. 33, 81, 1974. (abstract) 9.
CHEN, Y., BRIESE, F.W., and REEVE, E.B. Simplifiedmethod for serial estimation of fibrinogen secretion rate. Amer. J. Physiol.227, 927, 1974.
10. WYCOFF, H.D. Production of fibrinogen following an endotoxin injection. Proc. Sot. Fxptl. Biol. Med. 133, 940, 1970. 11.
FRADL, D.C. and REEVE, E.B. Fibrinogen synthesis stimulationby urine extract. Fee. Proc. -31, 261, 1972. (abstract)
12. United States Pharmacopeia,19th Revision, Rockville,MD, United States PharmacopeialConvention, Inc., 1975, p. 613. 13. PINDYCK, J., MOSESSON, M.W., ROOMI, M.W., and LEVERB, R.D. Steroid effects on fibrinogen synthesis by cultured embryonic chicken hepatocytes. Biochem. Med. l.2,22, 1975. 14. JOHN, D.W. and MILLER, L.L. Regulation of net biosynthesisof serum albumin and acute phase plasma proteins. J. Biol. Chem. 244, 6134, 1969. 15. JEEJEEBHOY, K.N., HO, J., GREENBERG, G.R., PHILLIPS,M.J., BRUCEROBERTSON, A., and SODTKE, U. Albumin, fibrinogen,and transferrin synthesis in isolated rat hepatocyte suspensions. Biochem. J. 146, 141, 1975. 16. CRANE, L.J. and MILLER, D.L. Synthesis and secretion of fibrinogenand albumin by isolated rat hepatocytes. Biochem. Biophys. Res. Comm. 60, 1269, 1974. 17. PILGERAM, L.O. and PICKART, L.R. Control of fibrinogenbiosynthesis: the role of free fatty acid. J. Atheroscler. Res. 8, 155, 1968. 18. JEEJEEBHOY, K.N., BRUCE-ROBERTSON,A., HO, J., and SODTKE, U. The effect of cortisol on the synthesis of rat plasma albumin, fibrinogenand transferrin. Biochem. J. 130, 533, 1972. 19. JEEJEEBHOY, K.N., BRUCE-ROBERTSON,A., SODTKE, U., and FOLEY, M. The effect of growth hormone on fibrinogen synthesis. Biochem. J. 2, 243, 1970.
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20.
HENRIQUES, S.B., HENRIQUES, O.B., and MANDELBAUM, F.R. Incorporation of glycine into glutathione and fibrinogen of rats under adrenalin treatment. Biochem. J. 66_, 222, 1957.
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T., and CSONGOR, J. Effect of spironolactone and T6TH, S., SZIIAGYI, phenobarbital on the plasma fibrinogen in rats. Fxperimentia 2, 1289, 1972.
22.
SELICSOIIN, U., RAPAPORT, S.I., SHEN, S.M.-C., and KUEFLER, P.R. IXCcct of corticosteroids upon fibrinogen metabolism in rabbits. Thrombos. Diathes. Haemorrh. 30, 531, 1973.
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SELIGSOHN, U., RAPAPORT, S.I., and ZIVELIN, A. Patterns of incorporation of 75Se-methionine into fibrinogen and other plasma proteins in rabbits stimulated by different test conditions. Thrombos. Diathes. Haemorrh. 29, 76, 1973.
24.
BARNHART, M.I., CRESS, D.C., NOONAN, S.M., and WALSH, R.T. Influence of fibrinolytic products on hepatic release and syhthesis of fibrinogen. Thrombos. Diathes. Haemorrh. 2, 143, 1970.
25.
KAMPSCHMIDT, R.F. and UPCHURCH, H.F. Effect of leukocytic endogenous mediator on plasma fibrinogen and haptoglobin. Proc. Sot. Exptl. Biol. Med. I&, 904, 1974.
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MCKENZIE, J.M. and FOWLER, P.R. Supporting role of the adreanl cortex in the induction of hyperfibrinogenemia. Amer. J. Physiol. 214, 786, 1968.
R IISlihRCll
Votumc
10,
Pngcs
337-317. Pcrgnmon
Press,
1977.Printccl inCt.ISritnin.
PRESENCE OF A LOW MOLECULAR WEIGHT PLASMA FIBRINOGEN STIMULATORAND INHIBITOR IN HUMAN URINE Stephen H. Wentland, Timothy H. Carlson, BruceD. Leonard, Demerick C. Fradl and E. B. Reeve University of Colorado 14dical Center Denver, Colorado 80262 USA
(Received 5.l..l.977.
Accepted by Editor M.I. Barnhart)
ABSTRACT When male human urine was fractionatedby butanol extraction and cation-exchangechromatography,a substance lowering plasma fibrinogen levels (in rabbits) was separated from a substance elevating fibrinogen levels. When the urine was fractionatedby ethanol precipitationand gel permeation chromatography,these fibrinogen elevating and depressing substanceswere shown to have low molecular ’ weight ( < 1000 daltons). In still other fractionationprocedures, the two substanceswere found to be interconvertible. This pair of elevating and depressing substancesmay be unreported regulatorsof fibrinogen levels, and may be part of a new hormonal system of regulation.
INTRODUCTION It is well known that injury (e.g. inflannnation, tissue damage) results in increased fibrinogen synthesis (1). This laboratoryhas been concerned with the regulation of fibrinogenmetabolism and earlier studies showed that pharmacologicaldoses (ca. 50 IU) of ACTH (2) and milligram doses of various prostaglandins (3, 4, 576) stimulate fibrinogen synthesis. Using ACTH, the kinetics of fibrinogen stimulationhave been studied (7). In addition to investigatingknown, well-defined compounds,we have searched biological fluids for other components that influence fibrinogenmetabolism. We now wish to report evidence for the presence of substances in male human urine which raise and lower plasma fibrinogenlevels in rabbits. A preliminary account of this work was reported elsewhere (8).
MATERIALS AND METHODS All chemicals used were reagent grade. Ultrafilterswere obtained from Amicon Corp., Lexington,MA, and all chromatographicresins were obtained
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from Bio-Rad Laboratories,Richmond, CA. Cellulose sheets for thin-layer chromatographywere obtained from Eastman Kodak, Rochester,NY. The assay used was described previously (7) and consists of dissolving the substance to be tested in 20 ml physiologicalsaline, and infusing the solution at a constant rate into the marginal ear vein of the rabbit over 3 hr. Blood was collected immediatelybefore and 24 hours after the infusion, and fibrinogen concentrations,$, in the plasma samples were measured by a radioisotopedilution technique. The 24 hour plasma fibrinogenconcentration was corrected to constant plasma volume to give the corrected plasma fibrinogen concentration,$124,as described elsewhere (9). A$, the change in plasma fibrinogen concentrationwas given by $24-$. where $. is initial plasma fibrinogen concentration. The average increase in plasma fibrinogen,resulting from the infusion of 25 saline controls was .?47 mglml, SD .222. Stimulating fractions were defined as those which gave fibrinogenincreases greater than 0.791 mg/ml (the mean control increase plus two standard deviations),while fibrinogen inhibiting fractionswere defined as those which gave fibrinogen decreases falling below -0.097 mg/ml (the mean control increase minus two standard deviations). Since bacteria and bacterial endotoxins have been shown to elevate fibrinogen levels (1, lo), care was taken to avoid contaminationin all procedures used. All heat-resistantapparatus was autoclaved and all other apparatus was washed well with sterile solutions before use. Buffers were prepared from autoclaved distilledwater, glacial acetic acid and concentrated ammonium hydroxide, and were found not to cause significantchanges in fibrinogen levels when lyophilizedsamples were inf6sed into rabbits. Human male urine was collected in sterile containersand processed within two hours after collectionby either of two methods: In the first method, the urine was first acidified with concentratedHCl to pH 1.5, then saturated with salt, and finally extracted with an equal volume of E-butanol in small portions. The butanol extracts were neutralizedwith concentrated sodium hydroxide, and then concentratedunder reduced pressure at 37O, yielding a light-to-darkbrown solid we call "butanol extracted material". In the second method, the urine was diluted with three volumes of ethanol (95%) at 5O, and the resultingmixture was stored at 4O for 24-48 hours. The precipitate,obtained by decantationand centrifugation,was then extracted with cold physiologicalsaline using a Waring blender. The resulting extract was continuouslydialyzed against cold tap water for three hours, and then lyophilized,yielding a tan solid we call "ethanol precipitated material". We reported a preliminary account of this method previously (11). Ultrafiltrationwas typically carried out in a 60 ml Amicon Ultrafiltration cell, using Amicon UM type filters. In order to remove the protective film of glycerol, the filters were soaked in sterile water for 3 hours, with replacementof the water every hour. To prevent bacterial contamination, the filters were stored in 6 M urea containing 0.1% sodium azide. Before use, they were washed well with buffer, and then flushed with 20 ml buffer. The material to be ultrafilteredwas dissolved in 50 ml buffer and passed through the filter under 50 psig nitrogen. Filtrationproceeded until the volume in the cell was reduced to 3 ml. Filtrationwas then stopped, giving a 3 ml retentate and a 47 ml filtrate. If the filtrate was to be further fractionatedby a filter with a lower molecular weight cutoff, it was then passed through the filter as above, giving a 3 ml retentate and a 45 ml filtrate.
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Gel-permeation resins (Bio-Gel P-Z) were equilibrated with buffer, separated from'fines, and autoclaved before use. Ion-exchange resins (CMcellulose, DRAE cellulose, Bio-Rex 70) were first washed with dilute acid and base, separated from fines, equilibrated with starting buffer, and then autoclaved. All chromatographic columns were flushed overnight with sterile buffers before being washed. Thin-layer chromatography was carried out using plastic-backed sheets of cellulose, which were eluted several times with sterile buffer before use. The plates were dried in a dust-free environment and then spotted with a concentrated solution of the sample, using a gentle stream of nitrogen to facilitate evaporation. The eluting solvent was the upper layer obtained by mixing 10 parts sterile H20, 10 parts n-butanol, and 2 parts glacial acetic acid, shaking, and letting the layers separate. After elution, the plate was cut into seven sections, the adsorbant in each section was scraped off the backing, and was then extracted 3 times with 5 ml 2.0 M ammonium acetate buffer In all the above steps sterile technique was used. The buffer extracts were finally lyophilized and assayed as described above. All solutions to be analyzed in the rabbit assay were prepared with sterile physiological saline solutions and were cultured to check for bacterial contamination. Since endotoxins have been shown to be pyrogenic, their presence or absence in active fractions was assayed using the pyrogen test described in the U.S. Pharmacopeia (12). This consists of rapidly injecting the material to be tested into the marginal ear vein of the rabbit, and observing the rises in temperature (if any) after the lst, Znd, and 3rd hour. If the sum of the three temperature rises was less than 40, the fraction was taken to be free of pyrogen.
RESULTS Ultrafiltration Studies Our first experiments involved the fractionation of the ethanol precipitated material on the basis of molecular weight. The ethanol precipitated material from 500 ml urine was dissolved in 0.1 M ammonium acetate buffer, pH 8.0 and filtered through a series of Amicon ultrafiltration membranes having different molecular weight cutoffs. The resulting fractions were lyophilized, dissolved in physiological saline and infused as described above. Results are shown in Table 1. Three fractions were obtained, the first containing substances with molecular weights greater than 10,000; the second, substances with molecular weights between 10,000 and 1,000; and the third, substances with molecular weights less than 1,000. When assayed, the first and third fractions showed stimulatory activity, while the second showed no activity at all. Both active fractions were tested for pyrogens. While the first fraction gave a positive response, the third fraction gave a negative response. Thus, we have obtained two stimulatory substances having very different molecular weights. While the substance of higher molecular weight may be contaminated with bacterial pyrogens, the substance of lower molecular weight is free from this kind of contamination. In subsequent runs the low molecular weight product was obtained, but not consistently. Several modifications of the ultrafiltration procedure
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TABLE I Ultrafiltrationof Ethanol PrecipitatedMaterial
Molecular Weight Cutoff of Ultrafilter
A$ (mdml)
Retentate
MW > 10,000
1.05
Retentate
MW >
1,000
.07
Filtrate
MW <
1,000
1.60
were made, but the appearance of the desired substancewas still erratic. Gel Permeation ChromatographyStudies In an effort to overcome the erratic appearance of activity in the low molecular weight fraction, we further refined our fractionationtechnique by adding gel permeation chromatography. An inactive, low molecular weight ultrafiltration fraction (derived from 500 ml urine) was chromatographedon BioGel P-2 gel permeation resin (exclusionlimit, 1,800), with the results shown in Fig. 1. Two fractions of interest were obtained. Fraction number 4 showed an increase in fibrinogen of 1.36 mg/ml, but fraction number 5 showed a decrease_ in fibrinogen of -.28 mg/ml. Thus, from an inactive low molecular weight ultrafiltrationfraction both stimulatoryand inhibitory activity was obtained. Both fractions eluted around the total volume (35 ml) of the column thus demonstratingthat the active substanceshave molecular weights less than and inhibitory substancesquickly lost 1,000. But both the stimulator-y activity, and or&r of their appearance from the P-2 column and their presence in the eluate were both erratic. Similar results were obtained whqn the unfiltered crude ethanol precipitate was chromatographedon Bio Gel P-2 resin as above. Ion Exchange CnromatographyStudies A concurrent set of experiments involved fractionationof the butanol extracted material by CM-celluloseion-exchangechromatography. Butanol extracted material from 1 liter of urine was applied to the top of a 20 cm x 0.9 cm column of CM-cellulose,and elution was carried out as described in the legend to Fig. 2. The fractions thus obtained were assayed as described above, and the results are shown in Fig. 2. It is seen that inhibitorymaterial was eluted in fraction 6, (A$ = -.23) stimulatorymaterial in fractions 9 and 10, (A+ = .96, .92). Thus, a clean separation of the inhibitorymaterial from the stimulatorymaterial was obtained. But nevertheless,the appearance of both stimulatoryand inhibitory activity was again inconsistent.
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REGULATORS
0.8
9 2
0.6
3 %
0.4
I
I
10
20
I 30
I
I
40
SO
Fxution Volume bl)
FIG. 1 Bio-Rad P-2 gel permeation chromatographicresin, colunm dimensions 55 cm x 0.9 cm, flow rate 7.5 ml/hr., buffer 0.1 M ammonium acetate, pH = 8.0. The fractions shown above were lyophilized,dissolved in physiologicalsaline, and infused as described earlier. The dashed line at A$ = .347 represents the mean control values, the shaded area represents 2 standard deviations above and below the mean.
TABLE II StatisticalAnalysis of Fractions Obtained from CR-CelluloseChromatography
Number of Section of Inhibitory Fractions Column Analyzed Found
Nlnnberof Stimulatory Fractions Found
Number of Fractions in Control Range
Fractions 4-5
1
0
14
Fractions 6-7
2
1
13
Fractions 8-9
1
3
10
Fractions lo-11
0
3
12
Fractions 12-13,
0
4
5
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Bio Bad GM-cellulose ion-Exchangeresin, column dimensions 20 cm x 0.9 cm. Each fraction contained 5 ml buffer, flow rate 40 mljhr. Stepwise elution was carried out with aumnmium acetate buffers of molarity and pH as shown above. The above fractions were lyophilized,dissolved in physiological saline and infused as described earlier. The dashed line at A0 - .347 represents the mean control value, the shaded area represents 2 standard deviations above and below the mean.
StatisticalEvaluation In spite of the above inconsistencies,we have demonstratedstatistically that Fig. 2 gives a fair picture what happens during GM-cellulosechromatography. The GM-column described above was run eleven times, with fractions taken as above, and the results of these eleven runs were combined. There was some variation in intermediatesteps, though these did not significantly affect the chromatographicseparation. Thus, in some instances the butanol extracted material was precipitatedwith ether, back-extractedwith aqeuous base, or fractionatedon Bio-Gel P-2 gel permeation resin prior to GM-cellulose chromatography. Also to reduce the large number of rabbits required for the assay of activity in these staticticalstudies, adjacent fractionswere sometimes combined before being lyophylizedand infused. The results of examining fractions from each GM-cellulosecolumn for fibrinogenstimulating and inhibiting activity are shown in Table 2. It is seen that of a total of 69 fractions analyzed, 4 inhibitory and 11 stimulatory fractionswere found, the remaining fractionsbeing in the control range. Inspection of the data in Table 2 shows that the inhibitoryresponses clustered in the fractions eluting early from the column, the mean fraction number being 6.50, SD 1.64. In contrast, the stimulatoryresponses clustered in the fractions eluting late from the column, the mean fraction number being 10.32, SD 2.09. The differencebetween these two means was significantat the .Ol level using the two tailed t-test. So, in spite of the variations resulting from the instabilityof the active components,the above data demonstrate that a substance of inhibitory activity is regularlyeluted before a substance having stimulatory activity.
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DISCUSSION Our CM-cellulose studies have demonstratrd the presence in human urine of two distinct substances, one that elevates, and the other that depresses fibrinogen levels in rabbits. This procedure results in a high degree of purification. For example, from 1 liter of urlne, sub-milligram amounts of the active components are obtained. Our ultrafiltration and gel-permeation studies also demonstrated the presence of a stimulator and inhibitor, and established that these substances are of low molecular weight (< 1,000) and non-pyrogenic. It is quite likely that the pair of stimulating and inhibiting substances obtained through ultrafiltration and gel permeation chromatography is the very same pair obtained through CM-cellulose chromatography. As descrbied above, CM-cellulose chromatography of the butanol extracted material results in the elution of the inhibitor and stimulator in characteristic fractions. Active substances obtained from gel permeation chromatography were rechromatographed on CM-cellulose chromatography, and the inhibitor and stimulator were found to elute in these same characteristic fractions. This demonstrates that the stimulator-inhibitor pair derived from gel-permeation chromatography is identical to the pair derived from CM-cellulose chromatography with respect to their affinity for the cation-exchange resin. Furthermore, P-2 gelpermeation chromatography of the ethanol precipitated material results in the elution of inhibitor and stimulator in late-cluting fractions. When active samples obtained from CM-cellulose chromatography were rechromatographed on P-2 resin, active fractions were obtained in these same late-eluting fractions. This demonstrates that the stimulator-inhibitor pair obtained from CM-cellulose chromatography has the same molecular weight as the pair obtained from gelpermeation chromatography. Thus it is likely that the two pairs are identical. A problem this study has not resolved is the inconsistent appearance of activity in both the stimulating and inhibiting fractions. There may be several reasons for this. First, loss of activity is often encountered in the separation and purification of biological substances especially those initially present in very small amounts. This may be due to loss of cofactors or to denaturation due to large surface-to-mass ratios. Second, the stimulator and the inhibitor may not always be separated during ultrafiltration and gel permeation chromatography. Thus they may nullify each other's activity. However, this cannot account for the inconsistencies seen during CM-cellulose chromatography, in which the inhibitor is well separated from the stimulator. The following experiments further illustrate the erratic appearance of activity. Butanol extracted material was chromatographed on Bio-Rad 70 cation-exchange resin, a resin similar in composition to CM-cellulose, using the conditions described for CM-cellulose chromatography. While the resulting activity-elution profiles did not form a consistent pattern, active fractions were nevertheless obtained. When an inhibitory fraction (A$= -.34) was rechromatographed using the same conditions, stimulatory fractions (A$= 1.05, 0.98) were obtained. When a stimulatory fraction (A4= 1.33) obtained from another Bio-Rad chromatogram was rechromatographed, an inhibitory fraction (A$ = -.21) was obtained. This suggests that the stimulator can be converted into the inhibitor and vice-versa. This interconversion has also been observed when fractions obtained from Bio-Rad chromatography were rechromatographed on P-2 resin (A@= 1.11 + A$ = -.21), when fractions obtained from Bio-Rad chromatography were rechromatographed on DRAR-cellulose resin
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(A$= 1.11 + A@ = -.21), and when fractions obtained from U4-cellulosewere refractionatedby thin-layer chromatographyon cellulose plates (A$ = -.25 + A$ = 1.39). This interconversionmust contribute greatly to the inconsistentappearance of activity described earlier. The stimulator and inhibitor described above are active in sub-milligram amounts when infused into ca. 3 kg rabbits. It should be noted that our assay, based on the difference between final and initial fibrinogenlevels, is more sensitive to fibrinogenelevation than to fibrinogendepression. Thus, the inhibitormay have been present to a greater extent than the depression of plasma fibrinogen level suggests. In vitro studies have demonstratedthat several substances can stimulate -fibrinogen synthesis. Using a chick embryo liver culture, cortisol and other steroids was found to significantlyelevate fibrinogensynthesis (13). In perfused rat livers, a mixture of cortisol, growth hormone, and insulin stimulated fibrinogen synthesis,and in the absence of cortisol, this stimulation was greatly reduced (14). Incubation of suspensionsof rat hepatocytesshowed that mixtures of cortisol, triiodothyronine,glucagon, and growth hormone increased fibrinogen synthesis. Also, the inclusionor exclusion of insulin had no further effect (15). Other investigatorsusing this same technique reaffirmed that mixtures of cortisol and insulin increase fibrinogensynthesis, but in contrast to work cited above, insulin by itself significantlyelevated fibrinogen synthesis while cortisol by itself showed only a small effect (16). But in incubated rat liver slices, cortisol was actually found to decrease fibrinogen synthesis, as did histamine phosphate (17). In this latter system, sodium palmitate increased fibrinogen synthesis. Several substances, includingmost of those tested in vitro, have been shown to elevate fibrin0 en synthesis using various -in vivo assay'systems. Using incorporationof 1&C-carbonateas a measure of fibrinogensynthesis in rats, injection of cortisol (20 mg/kg, i.p.) caused significantincreases in fibrinogen synthesis (18), whereas injection of growth hormone (0.6 mg/kg, i.p.) led to moderate increases (19). Other studies in our laboratoriesusing this method in rabbits have shown that 50 IU ACTH (2) and milligram amounts of prostaglandinE-1 and E-2 (3, 4, 5, 6) also significantlystimulate fibrinogen synthesis. Using the incorporationof radioactiveglycine and other amino acids as measures of fibrinogen synthesis, injectionsof adrenalin (1 mg/kg, s.c.) (20), spironolactone (0.2 mnol/kg twice daily for 3 days, p.o.) (21), and phenobarbital (0.2 mmol/kg twice daily for 3 days, p.o.) (21) have significantly elevated fibrinogen synthesis in the rat. However, the last two substances do not occur naturally. Using the rate of incorporationof radioactivemethionine as well as the plot of fibrinogen concentrationvs. time as a measure of fibrinogensynthesis, cortisol (10 mg/kg, B.C.) and cortisone (10 mg/kg, i.m.) have shown significant elevations (22) in rabbits. Using these same assay systems, ACTH (ca, 20 U/kg, s.c.), growth hormone (l-2 U/kg, s.c.), and adrenalin (0.2 mg/kg, s.c.) also gave rise to significantelevations (23). However, glucagon (l-2 mg/kg, s.c.) failed to elicit a significantresponse. Using immunofluorescentmethods as well as the plot of fibrinogenconcentration vs. time as measures of fibrinogen synthesis, infusion of fibrin degradationproducts (11 mg/kg) into dogs led to significantelevations (24). Using only the plot of fibrinogen concentrationvs. time, injectionof 1 llg
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leukocytic endogenous mediator into rats i.p. gave rise to significant inin vitro by incubating rat liver creases (25). This compound was also tested -slices, with the same result. As measured by the 24-hour change in plasma fibrinogen concentration, adrenalin (2 x 3 prml/kg, i.p,) and noradrenalin (2 x 3 umol/kg i.e.) showed siginficant elevations in rats (26). In the same study, however, histamine (360 mg/kg, i.e.), corticosterone (25 mg/kg i.e.), dexamethasone (8 mg/kg, i.e.) and ACTH (80 units/kg, s.c.) led to no significant increases. It is unlikely that the stimulator we report here is glucagon, insulin, cortisol, histamine, adrenalin, or noradrenalin. For although some of these compounds have shown activity in some of the studies reported above, other studies in our laboratories demonstrated that the 24-hr plasma fibrinogen change resulting from the infusion of these compounds into rabbits at a dose level of 1 mg/kg were insignificant. As mentioned before, our stimulator displays activity at dose levels < 0.3 mg/kg. It is further unlikely that our stimulator is growth hormone, leukocytic endogenous mediator, or fibrin degradation products, since the molecular weight of our stimulator is less than 1000, while those of the latter three compounds exceed 10,000. Furthermore, our stimulator is active at lower dosage levels than required by either ACTH (2) or prostaglandin E-l or E-2 (3, 4, 5, 6). We conclude then that our.low molecular weight stimulator has not previously been described. Of particular interest, is our demonstration of an inhibitor and of the stimulator-inhibitor interconversion. This stimulatorinhibitor pair, of similar size and chromatographic behavior, may be part of a novel system of hormonal control, the actions of which depend on whether the control system is driven to produce primarily stimulator or primarily inhibitor. Further characterization of the stimulator and inhibitor has been hampered by their inconsistent appearance. Efforts are underway to find other ways of purifying and stabilizing these compounds, as well as to find other sources of them than urine.
ACRNOWLEDGPlENTS This work was supported by NIH grant #HLO2262, The Colorado Heart Association, and NIH grant #RR00051 (GCRC).
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2.
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3.
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FRADL, D.C. and REEVE, E.B. Plasma fibrinogenresponse to prostaglandin infusions. Fed. Proc, 32, 265, 1973. (abstract)
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CARLSON, T.H., FRADL, D.C., and REEVE, E.B. ProstaglandinE-l (PGE-1) stimulation of fibrinogen synthesis. Fed. Proc. 24, 404, 1975. (abstract)
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WENTLAND, s.H., CARLSON, T.H., LEONARD, B.D. and REEVE, E.B. Fibrinogen synthesis stimulationby several E prostaglandins(PCE). Fed. Proc. 35_,2431, 1976. (abstract)
7. CHEN, Y.,,BRIESE, F.W., and REEVE, E.B. Relation of fibrinogensecretion (synthesis)to ACTH dose. Amer. J. Physiol.227, 932, 1974. 8. WBNTLAND, S.H., CARLSON, T.H., and REEVE, E.B. Fibrinogensynthesis stimulatorsand inhibitors from urine. Fed. Proc. 33, 81, 1974. (abstract) 9.
CHEN, Y., BRIESE, F.W., and REEVE, E.B. Simplifiedmethod for serial estimation of fibrinogen secretion rate. Amer. J. Physiol.227, 927, 1974.
10. WYCOFF, H.D. Production of fibrinogen following an endotoxin injection. Proc. Sot. Fxptl. Biol. Med. 133, 940, 1970. 11.
FRADL, D.C. and REEVE, E.B. Fibrinogen synthesis stimulationby urine extract. Fee. Proc. -31, 261, 1972. (abstract)
12. United States Pharmacopeia,19th Revision, Rockville,MD, United States PharmacopeialConvention, Inc., 1975, p. 613. 13. PINDYCK, J., MOSESSON, M.W., ROOMI, M.W., and LEVERB, R.D. Steroid effects on fibrinogen synthesis by cultured embryonic chicken hepatocytes. Biochem. Med. l.2,22, 1975. 14. JOHN, D.W. and MILLER, L.L. Regulation of net biosynthesisof serum albumin and acute phase plasma proteins. J. Biol. Chem. 244, 6134, 1969. 15. JEEJEEBHOY, K.N., HO, J., GREENBERG, G.R., PHILLIPS,M.J., BRUCEROBERTSON, A., and SODTKE, U. Albumin, fibrinogen,and transferrin synthesis in isolated rat hepatocyte suspensions. Biochem. J. 146, 141, 1975. 16. CRANE, L.J. and MILLER, D.L. Synthesis and secretion of fibrinogenand albumin by isolated rat hepatocytes. Biochem. Biophys. Res. Comm. 60, 1269, 1974. 17. PILGERAM, L.O. and PICKART, L.R. Control of fibrinogenbiosynthesis: the role of free fatty acid. J. Atheroscler. Res. 8, 155, 1968. 18. JEEJEEBHOY, K.N., BRUCE-ROBERTSON,A., HO, J., and SODTKE, U. The effect of cortisol on the synthesis of rat plasma albumin, fibrinogenand transferrin. Biochem. J. 130, 533, 1972. 19. JEEJEEBHOY, K.N., BRUCE-ROBERTSON,A., SODTKE, U., and FOLEY, M. The effect of growth hormone on fibrinogen synthesis. Biochem. J. 2, 243, 1970.
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HENRIQUES, S.B., HENRIQUES, O.B., and MANDELBAUM, F.R. Incorporation of glycine into glutathione and fibrinogen of rats under adrenalin treatment. Biochem. J. 66_, 222, 1957.
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T., and CSONGOR, J. Effect of spironolactone and T6TH, S., SZIIAGYI, phenobarbital on the plasma fibrinogen in rats. Fxperimentia 2, 1289, 1972.
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SELICSOIIN, U., RAPAPORT, S.I., SHEN, S.M.-C., and KUEFLER, P.R. IXCcct of corticosteroids upon fibrinogen metabolism in rabbits. Thrombos. Diathes. Haemorrh. 30, 531, 1973.
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SELIGSOHN, U., RAPAPORT, S.I., and ZIVELIN, A. Patterns of incorporation of 75Se-methionine into fibrinogen and other plasma proteins in rabbits stimulated by different test conditions. Thrombos. Diathes. Haemorrh. 29, 76, 1973.
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BARNHART, M.I., CRESS, D.C., NOONAN, S.M., and WALSH, R.T. Influence of fibrinolytic products on hepatic release and syhthesis of fibrinogen. Thrombos. Diathes. Haemorrh. 2, 143, 1970.
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KAMPSCHMIDT, R.F. and UPCHURCH, H.F. Effect of leukocytic endogenous mediator on plasma fibrinogen and haptoglobin. Proc. Sot. Exptl. Biol. Med. I&, 904, 1974.
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MCKENZIE, J.M. and FOWLER, P.R. Supporting role of the adreanl cortex in the induction of hyperfibrinogenemia. Amer. J. Physiol. 214, 786, 1968.