human fibrinogen and asialo-fibrinogen: a comparison of coagulation parameters

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 173, 50-57 (1976)

Human Fibrinogen and Asialo-Fibrinogen: A Comparison of Coagulation Parameters’

PATRICIA A. GENTRY2 AND BENJAMIN ALEXANDER

Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67th Street, New York, New York 10021

Received June 23, 1975

The effect of desialylation of fibrinogen on its conversion to fibrin has been investi- gated with particular reference to the kinetics of clot formation and structure. Also examined was the role of sialic acid in fibrinogen (factor I) poor in factor XIII (librin- stabilizing factor) and factor I containing F XIII. The removal of more than 90% of the sialic acid of fibrinogen does not alter the thrombin clotting time, the clot solubility in monochloroacetic acid, the extent of cross-linking in the fibrin polymer, or the firmness and elasticity of the evolved clot. The data indicate that the sialic acid residues of fibrinogen do not contribute significantly to its conversion to fibrin by thrombin.

In common with many of the blood clotting proteins, fibrinogen (FB3 is a glycopro- tein. The carbohydrate moiety, comprising about 3%, consists primarily of gala&se, mannose, glucosamine and sialic acid (1). There is still considerable controversy con- cerning the role of the carbohydrate in F I conversion to fibrin. Several workers have reported that carbohydrate-containing fragments are released during clotting (2, 3) and that factor XIII (F XIIL3 fibrin- stabilizing factor), in addition to its role in the conversion of a soluble fibrin monomer to a form insoluble in urea or acid, is probably responsible for the release of these fragments (4, 5). However, other in- vestigators could not detect any release of F I carbohydrate as a result of the conversion of F I to fibrin (6-11). Furthermore, the influence of F I sialic acid on fibrin

’ This research was supported by USPHS grants, No. HL 9011 and 11447, to the Community Blood Council of Greater New York, Inc.

2 Present address: Department of Biomedical Sci- ences, Ontario Veterinary College, University of Guelph, Guelph, Ontario NlG 2W2, Canada.

3 Nomenclature recommended by the Interna- tional Committee on Haemostasis and Thrombosis U’hromb. Diathes. Haemorrh. (Stattgart) Suppl. 13, 455 (1964). Abbreviations used: DEAE-, diethyl- aminoethyl; SDS, sodium dodecyl sulfate.

formation is uncertain. Chandrasekhar et al. (3) have reported that the removal of sialic acid from bovine F I increases its “clottability,” while it has also been demonstrated that the clot formed from asialo-F I exhibits an increased solubility in urea (4, 5).

The present studies are primarily con- cerned with reexamination of the discrepancy and clarification of the role of sialic acid in the conversion of human F I to soluble fibrin polymer and to cross-linked insoluble fibrin by investigating the kinetics of fibrin formation of normal and asialo-F I, and by examining the resulting clot structure.

MATERIALS AND METHODS

Fibrinogen. Two preparations were used: (a) A preparation of human F I that was shown to be essentially free of F XIII was prepared from F I (Grade L, A. B. Kabi, Stockholm) by DEAE-cellu- lose chromatography (12). The clottability of this F XIII-poor F I preparation was 92.60/o, as determined according to Blomback and Blomback (131, with the modification that, in order to assure maximum clottability, F XIII was added to the preparation before the addition of thrombin. (b) For F XIII-containing F I, Grade L (A. B. Kabi, Stockholm) was used. The F I sample was handled as described by Shamash and Alexander (14), and its clottability was 93.3%. Clot solubility tests (see below) indicated that this F I preparation still contained F XIII.

50 Copyright 0 1976 by Academic Press, Inc. All riohtr nf rmmmrh~rtion in anv form reserved.

CLOTTING OF HUMAN ASIALO-FIBRINOGEN 51

Thrombin (EC 3.4.4.13). Bovine thrombin (Parke- Davis, Kalamazoo, Mich.) was purified according to Lundblad (15). The specific activity of the final prod- uct was 250 NIH units/mg. Aliquots of a solution containing 0.4 mg/ml in 0.01 M NaCl were kept at -60°C until used.

Factor XIII. This was prepared from either the commercial F I preparation (Grade L, A. B. Kabi, Stockholm) or the residual F XIII-containing material following chromatography of the commercial F I preparation described under (a) above. The purifica- tion procedure involved heat treatment and precipi- tation with polyethylene glycol, as described by oth- ers (16-18). The resultant precipitate containing F XIII activity was dissolved in 0.1 M Tris buffer (pH 7.51, dialyzed overnight against this buffer, and stored in aliquots at -60°C.

Factor XIII activity. Activity was assayed according to McDonagh et al. (19) using the F XIII-poor F I as substrate. The control clot, with titrated saline substituted for F XIII, always dissolved in less than 4 min. The dilution of F XIII which yielded a clot that dissolved in 10 min was arbitrarily defined as having 0.1 unit of activity. The method was modified for the assay of the clot solubility of F XIII-poor F I and its asialo derivative as follows: The F I concentration was varied by dilution with 0.15 M NaCl and F XIII was added in excess at a constant amount of 2.7 units.

Fibrin cross-linking. To examine fibrin cross-linking utilizing SDS-gel electrophoresis, the reaction mixtures were similar to those described by Schwartz et al. (20): 0.5 M Tris-maleate (pH 6.8) containing 0.05 M CaCl, was used, and the clots were incubated at 37°C for 2 h before the addition of denaturing reagent (20).

SDS-gel electrophoresis. Electrophoresis was per- formed by techniques previously described (20-221, slightly modified: 7.5% polyacrylamide gels were prepared containing 50%’ 10 M urea, 4% 0.05 M EDTA and 0.1% SDS. The reducing agent was 1% mercapto- ethanol, and the protein load applied to each gel was 100-140 /.Lg.

Thrombelastograms. These were obtained with a Hartert Model 2601D instrument and with a similar instrument modified so that the clot firmness and elasticity could be observed directly as a printout on a recorder (Bausch and Lomb, VOM 5) via a dual- output-regulated power module (Acopian).* A mixture of 0.2 ml of F XIII-containing F I (12.6 mg/ml) and 0.1 ml of 0.5 M Tris-maleate buffer (pH 6.8) containing 0.05 M CaCI, was brought to 37°C in the cup before adding 0.05 ml (5 units) of thrombin. The

4 This instrument, including a constant voltage transformer and power supply, was constructed by Mr. M. N. Leeming, Department of Anesthesia, Sloan-Kettering Institute, New York, N.Y.

standard notation is used to describe the thrombelastograms: t = time (minutes); a = amplitude (millimeters); ma = maximum amplitude (millimeters); k, the coagulation time, is the interval required for a clot of definite solidity to develop, which is arbitrarily defined as the interval between an amplitude of 1 mm and an amplitude of 20 mm); and E which reflects the elastic properties of the fibrin clot and is numerically calculated from the formula (100 X mall (100 - ma).

Thrombin clotting times. These were determined in duplicate at 20°C on native and asialo-F I samples as follows: To a mixture of 0.1 ml of 0.2 M Tris buffer (pH 7.5) and 0.1 ml of an F I solution (6.0 mg/mll were added 0.01 ml of thrombin (25 NIH units/ml in 0.1 M phosphate buffer, pH 7.1),5 and the clotting time was recorded.

Protein. Protein was measured according to Gor- nail et al. (23) or, when the protein concentration was relatively low, according to Lowry et al. (24).

Sialic acid. This was determined by the thiobarbi- turic acid method (25). To determine the total F I sialic acid, 0.1 ml of the F I solution was hydrolyzed with 0.1 ml of 0.1 N H,SO, at 75°C for 1 h. N-acetyl neuraminic acid (General Biochemicals, Chagrin Falls, Ohio) was used as a standard.

Preparation of asialo derivatives ofF I. Two preparations of neuraminidase (EC 3.2.1.181 were used: (i) neuraminidase from Clostridium perfringens (Wor- thington Biochemical Corp., Freehold, N. J., NEU- PILA, 0.8 unit/mg); and (ii) neuraminidase from Vibrio cholerae (24, Schwarz/Mann, Orangeburg, N. Y., W 4318, 500 units/ml).

Preparation conditions were as follows: (a) The F I preparations, both F XIII-containing and F XIII- poor, were brought to pH 6.2 by adding 0.1 M acetate buffer pH 5.0, incubated at 37°C for 3 h with 0.02 unit of neuraminidase (Cl. perfringens) per mg of F I and the pH adjusted to 7.0 before the samples were frozen. As control, neuraminidase was omitted. (b) To obtain asialo-F I at a higher, more physiological pH, F XIII-containing F I was incubated for 3 h at 37°C at pH 6.8 with 0.008 unit of neuraminidase (CI. perfringens) per mg of F I or with 0.034 unit of neuraminidase W. cholerae) per mg. To one sample was added an additional 0.11 unit of neuraminidase (V. cholerae) per mg of F I and incubation was continued for 3 more h at 37°C. For controls, the F XIII-containing F I solutions were incubated for the same time periods without additions.

All neuraminidase preparations were devoid of

5 This thrombin concentration was purposely se- lected on the basis of observations reported earlier by Bray and Alexander (26) in which the clotting kinetics of F I and its asialo derivative were examined under conditions of varying thrombin concentrations. Our results confirm the earlier findings.

52 GENTRY AND ALEXANDER

protease (caseinolytic) or e&erase (tosylarginine methyl ester, pH stat) activity.

RESULTS

Total sialic acid of F XIII-poor F I was initially determined after hydrolysis in 0.1 N H,SO, at 80°C for 1 h. The mean value obtained for three different preparations was 5.69 + 0.30 cLg/mg of protein (range, 5.2-6.0). When determined on the same samples after hydrolysis at 75”C, a mean value of 8.24 ? 0.89 @/mg was found (range, 7.4-9.1). A mean value of 9.09 + 0.87 pg/mg was found for four preparations of F XIII-containing F I after hydrolysis at 75°C. This value was used to calculate the percentage of sialic acid released after F I incubation with neuraminidase.

Except for the F I sample incubated with the V. cholerue neuraminidase for 3 h, all the other asialo-F I preparations were

found to have had almost the entire por- tion of their sialic acid content removed (Table I). Only these samples were used for further study.

The thrombin clotting times of the three asialo-F I samples from which more than 90% of the sialic acid residues had been removed were compared with the corresponding control and preincubation samples. No significant differences were observed (Table II).

The solubilities of the clots obtained from the control and asialo-F XIII-poor F I were compared over a broad range of concentrations (0.2-1.0 mg of F I/ml in 0.15 M NaCl) since under our conditions we observed a linear relationship in this range between the initial F I concentration and the clot dissolution time (Fig. 1). No significant difference was evident.

The extent of cross-linking in the fibrin

TABLE I SIALIC ACID RELEASED FROM FIBRINOGEN BY NEURAMINIDASE

Sample Incubation conditions Sialic Acid cleaved (pg/mg of

protein)

Sialic acid re- leaseda (%I

F XIII-poor F I

F XIII-containing F I

3 hr, 37”C, pH 6.2, Cl. perfrin- gem enzyme

3 hr, 37”C, pH 6.2, Cl. perfrin- gen.s enzyme

3 hr, 37”C, pH 6.8, Cl. perfringens enzyme

3 hr, 37”C, pH 6.8, V. cholerae enzyme

6 hr, 37”C, pH 6.8, V. cholerae enzyme

8.08 98.1

a.72 95.9

8.80 96.8

2.21 24.3

6.43 70.7

(2 Based on the total (acid hydrolyzable) sialic acid content of 8.24 pg/mg of protein for F XIII-poor F I and 9.09 pg/mg for F XIII-containing F I.

TABLE II THROMBIN CLOTTING TIMES OF FIBRINOGEN AND ITS ASIALO DERIVATIVES

Sample

Preincubation sample”

Clotting time (seconds)

Control sampleb Asialo samplec

F XIII-poor F I 17.1, 18.3 20.2, 21.6 20.3, 21.5 F XIII-containing F I 18.4, 18.4 17.6, 18.4 17.3, 18.3

15.1, 15.7 14.7, 15.7 15.2, 16.0

a No incubation; clotting time determined at pH 6.8. b Sample incubated 3 h at pH 6.2; clotting time determined at pH 6.8. c F I incubated 3 h at pH 6.2 with Cl. perfringens enzyme; clotting time determined at pH 6.8.


FIG. 1. The relationship between the clot solubility in 2% monochloroacetic acid and the F XIII-poor F I content of the samples used to prepare control fibrin and asialo-fibrin clots. The ordinate indicates the time required for clot dissolution; control samples (0); asialo samples (0). An excess of F XIII (2.7 units) was provided in each experiment (see Mate- rials and Methods).

formed from both control and asialo-F I was examined by SDS-gel electrophoresis (Figs. 2-4). No difference in the migration of the Aa, B/3 and y chains of the F I molecules6 was observed between the control and asialo samples (Figs. 2a and 3 and 3a and 3). When thrombin alone was added to the F XIII-poor F I sample there was apparently no cross-linking between the chains (Fig. 2b). When F XIII was added, extensive cross-linking of the evolved fibrin was observed (Fig. 2~1. The extent of cross-linking in the fibrin formed from asialo-F XIII-poor F I was the same as that of the control sample (Figs. 2c and d), although in both cases the y chains were only partially converted to y chain dirners. The degree of cross-linking was not im- proved either by prolonging the incubation time of the F XIII-poor F I, F XIII and thrombin mixture or by preactivating the F XIII with calcium before its addition to the F XIII-poor F I. Incomplete polymerization of F XIII-containing F I which had also been exposed to pH 6.2 for 3 h during the preparation of the asialo derivative was also observed (Figs. 3a and b), although here the addition of F XIII resulted

6 Nomenclature of chains adopted by the Interna- tional Committee on Haemostasis and Thrombosis at its meeting in Washington, D.C. in 1972.

in almost complete cross-linking (Fig. 3~). As with the F XIII-poor samples, there was no significant difference between the extent of cross-linking observed for the fi-

FIG. 2. Electrophoretic patterns of F XIII-poor F I and fibrin on 7.5% polyacrylamide gels in SDS. Sam- ples applied: (a), F XIII-poor F I; (b), F XIII-poor fibrin formed in the absence of added F XIII; (c), F XIII-poor fibrin formed in the presence of added F XIII; (d), asialo-F XIII-poor fibrin formed in the presence of added F XIII; and (e), asialo-F XIII-poor F I. The anode was at the bottom.

FIG. 3. Electrophoretic profiles of control F XIII- containing F I and fibrin and their asialo counter- parts on 7.5% polyacrylamide gel in SDS. The asialo-F I was prepared by incubating native FI with Cl. perfringens neuraminidase at pH 6.2 (see text). (a), Control F I; (b), fibrin formed in the absence of added F XIII; (cl, fibrin formed in the presence of added F XIII; cd), asialo-fibrin formed in the presence of added F XIII; and (e), asialo-F I. The anode was at the bottom.


brin formed from the control and asialo-F XIII-containing F I samples (Figs. 3c and d).

F XIII-containing F I which had not been exposed to acid pH required no additional F XIII in order to exhibit extensive cross-linking of the fibrin chains (Figs. 4a and b). The corresponding asialo derivative prepared with both Cl. perfringens and V. cholerae neuraminidase showed the same degree of cross-linking as the parent molecule, as judged by the electrophoretic profiles (Figs. 4b-d).

Fibrin clots from both the control F XIII- containing F I and its asialo derivatives, obtained by incubation with each enzyme at pH 6.8, were examined in a thrombelas- tograph in order to compare the rate of formation and elasticity. Typical profiles are illustrated in Fig. 5 and data are shown in Table III. No gross differences were evident. Although the kinetics of clot formation vary somewhat (k value, Table III) as do firmness and elasticity of the fibrin clot formed from the control F I (E value, Table III), the values for the clots formed from the asialo samples are similar to those of the controls.

s

n lcm

FIG. 5. Thrombelastograms obtained with control and asialo-F XIII-containing F I. (11, Asialo-F I prepared with Cl. perfringens neuraminidase by incubation at pH 6.8 and 37°C for 3 h; (21, F I similarly incubated with cholera enzyme for 3 h; (31, asialo-F I obtained after 6 h incubation with V. cholerae enzyme. These profiles are typical of and included in the detailed data in Table III.

DISCUSSION

The 0.91% value for total sialic acid of F XIII-containing F I, found by acid hydrolysis at 75”C, agrees closely with the 0.89% found by extensive F I digestion with neuraminidase (26). It is considerably higher, however, than the values obtained by acid hydrolysis at 80°C (about 0.67%) reported earlier (26-30). A similarly low value (0.57%) was also found by us (for F XIII- poor F I) under the same conditions. The reason for the discrepancy remains ob- scure. That it can be entirely attributed to destruction of the liberated sialic acid at the higher temperature was excluded. ’ Al- though this may explain it in part, the sensitivity of the neuraminosidic linkages in F I to acid and/or to neuraminidase may also be involved, as alluded to in a meticu- lous study by Gibbons (311.’

FIG. 4. Electrophoretic patterns of control and asialo-F XIII-containing F I and fibrin, on 7.5% polyacrylamide gels in SDS. (a) and (b), Control F I and fibrin respectively; (cl, polymerization pattern of fibrin formed from asialo-F I which was prepared by incubation of native F I with Cl. perfringens neuraminidase at pH 6.8; (d), polymerization pattern of fibrin formed from asialo-F I prepared with the V. cholerae enzyme. The anode was at the bottom.

’ Alexander, Bray, and Pereira (unpublished observations) found that only about 4-14% of pure sialic acid was destroyed in 0.1 N H,SO, at 80°C in 1 h at concentrations of 17-125 pmoliml. On one occa- sion 31% was destroyed at a concentration of 17 ymollml. Except for this inexplicably high value, these findings agree fairly well with the figure of about 7% reported by Gibbons (31) who found the degradation process kinetically complex, irrespec-


TABLE III

PARAMETERS” OF THROMBELASTOGRAMS OF CONTROL AND ASIALO FIBRINOGEN

Sample Number of as- says

mu (mm) k (min)

Control F I 9 35.0 t 2.8 3.1 + 0.7 53.9 _f 7.6 Asialo-F I* 6 39.2 + 2.9 3.0 2 0.7 64.5 t 8.2

Asialo-F I” 4 34.8 + 3.6 3.0 + 0.6 53.4 ? 8.7

Asialo-F I” 4 31.5 + 1.2 3.7 + 0.2 46.0 + 2.4

a For definitions of parameters, see text. Observations were made with the Hartert Model 2601 D instrument attached to a moving recorder (Bausch and Lomb, VOM 5) via a dual-output-regulated power module (Acopian); see text footnote 4.

* F I digested with Cl. perfringens enzyme, pH 6.8, 3 h, 37°C. c Same as a, with V. cholerue enzyme, pH 6.8, 3 h, 37°C. d Same as b, 6 h.

Almost complete desialylation of either F XIII-poor F I or F XIII-containing F I has no significant effect on the thrombin clotting time (Table II), a result in agreement with that previously noted for F XIII-containing F I (26). Laki and Mester (32) reported that the oxidation of the sugar moiety of bovine F I compromised coagulabil- ity but whether this was the result of oxidation of the carbohydrate or amino acid residues is not clear (33,34). Chandrasekhar et al. (4) have reported that enzymatic desialylation of bovine F I accelerates its clotting by thrombin. It is to be noted that species difference may be significant in comparing results obtained by various in- vestigators since bovine F I was used in earlier studies whereas we used human F I. However, Bray and Alexander found no difference in the thrombin clotting times of either bovine or human asialo-F I, compared with the controls (26).

No difference was evident in the extent of the solubility in monochloroacetic acid of the clots formed from control and asialo-

tive of the analytical method used to, follow the reaction. Also to be considered in attempting to resolve the anomalous F I sialic acid values is the sensitivity of the FI neuraminosidic linkage(s) either to neuraminidase or acid, as Gibbons pointed out. This may vary in individual F I molecules, F I lots or disease states. Although a wide range of F I sialic acid has been reported from individual to individual (46) and from pool to pool of F I (47), our high values cannot be attributed to material from a particular unusually high group since the material was bulk from large random donor plasma pools,

F XIII-poor F I in the presence of excess F XIII (Fig. 1). This is in discord with the findings of Laki and Chandrasekhar who reported that clots obtained from both human and bovine asialo F I dissolve more readily in urea than those from control F I and that the addition of F XIII to human asialo-F I does not alter its increased solubility (4, 5). This discrepancy may be due to different experimental conditions.

Partially or completely soluble fibrin evolves when only partial polymerization of the fibrin chains has occurred (20). That such was the reason for the solubility observed for our asialo-fibrin preparation formed from F XIII-poor F I was amply demonstrated by the electrophoretic patterns (Figs. 2c and d). The incomplete polymerization of the fibrin chains would ap- pear to be a function of the prolonged incubation of the F I molecule at an acid pH (Fig. 3b) since complete polymerization was found with a similar preparation which had not been exposed to an acid pH (Fig. 4b). Furthermore, a marked increase in the heterogeneity of the Aa band was also observed in those samples exposed to an acid pH (Fig. 2a, 3a, and 4a). However, the results clearly indicate that desialylation does not significantly affect the electrophoretic mobility of the F I molecule or the extent of cross-linking in the fibrin formed since identical results were obtained with each asialo derivative and its parent preparation.

Our studies do not exclude an important biological function for F I sialic acid. Like


several circulating sialo-glycoproteins (35) including prothrombin (361, F I sialic acid appears to be important in regulating in uiuo T/2 of rabbit asialo-F I is markedly shortened compared with native material.* There is one reported case of congenital dysfibrinogenemia (fibrinogen Detroit) associated with impaired fibrin polymerization and subnormal F I sialic acid (37). However, Blomback et al. (38) have sug- gested that the impaired polymerization might be directly or indirectly associated with a demonstrated amino acid substitu- tion that could affect the primary or ter- tiary structure of the molecule as well as the observed low sialic acid. Conversely, there have been several reported cases of congenital dysfibrinogenemia in which the sialic acid is normal (39-41) or even elevated (42). Indeed, Mester et al. have reported that in many cases of both acquired (43) and congenital dysfibrinogenemia (44, 45) the F I sialic acid to hexosamine ratio is elevated. It would be of considerable inter- est to compare fibrin polymerization of normal and sialic acid-enriched F I.

ACKNOWLEDGMENTS

We thank Ms. Claude Chaptal for her excellent technical assistance and Dr. Birger Blombiick for his helpful suggestions and critical review of the manuscript.

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