fibrin degradation products by chromatography on protamine-agarose

THROMBOSIS RESEARCH 48; 223-232,1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.

ISOLATION AND PURIFICATION OF FIBRINOGEN/FIBRIN DEGRADATION PRODUCTS BY CHROMATOGRAPHY ON PROTAMINE-AGAROSE C.E.Dempfle,

D.L.Heene,

Klinikum Mannheim, Fakultat fiir klinische Medizin Mannheim der Universitat Heidelberg, l.Medizinische Klinik, D-6800 Mannheim, Germany (Received 16.12.1986; Accepted in revised form 1.7.1987 by Editor A. Henschen) (Received by Executive Editorial Office 24.8.1987)

ABSTRACT Protamine-agarose chromatography is introduced as a rapid and efficient method for the isolation of fibrinogen/fibrin degradation products from biological fluids such as human plasma. All fibrinogen/fibrin fragabove a MW of 35000, including fragments D and E ments are quantitatively adsorbed to insolubilized protamine and can be eluted with a buffer coniaining 0.2 M sodium citrate/citric acid pH 5.3, following previou5 elution of non-fibrinogen proteins with a buffer containing 0.8 M NaCl at neutral pH. Fragments D and E are separated by stepwise elution. The efficiency of the method is evaluated by applying it to plasma samples obtained from healthy donors and from patients with clinical and laboratory evidence of disseminated intravscular coagulation. INTRODUCTION Protamine-agarose chromatography has been shown to be an efficient procedure for the isolation of fibrinogen from plasma and other biological fluids (1). Latallo et al. (2) presented results indicating that precipitation of fibrinogen by protamine sulphate was decreased in the presence of fibrinogen degradation products (FDP), although FDP were present in the precipitate only in negligible amounts. Similar observations were made by Okano et al. (3). The authors showed that protamine precipitation of fibrinogen is inhibited by fibrinogen fragment D, while fragment E had little influence. Hafter et al. (4) employed protamine precipitation for the purification of high molecular weight fiKey words: Protamine, Fibrinogen, Fibrinogen/Fibrin Degradation Products, Chromatography, Protein Purification. 223

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brin derivatives from ascitic fluid, achieving nearly complete recovery of fibrin fragments greater than MW 250000. Smaller fibrin fragments remained partially in solution. Fragment E was not precipitated at all by protamine sulphate. This investigation was carried out in order to evaluate if protamine-agarose chromatography, which has successfully been employed for purification of native fibrinogen, can also be used for isolation of fibrinogen/fibrin degradation products from biological fluids. Various in vitro preparations of fibrinogen/ fibrin degradation products, as well as plasma samples from patients with disseminated intravascular coagulation (DIC) were subjected to protamine-agarose chromatrography followed by gel filtration on Sephacryl S-300, and analysis of eluates by polyacrylamide gel electrophoresis in the presence of SDS and immunoblotting. MATERIALS CNBr-activated Sepharose 4B, Sephacryl S-300, (Pharmacia, Uppsala, Sweden); protamine sulphate, grade X, histone-free, from salmon, tris-hydroxymethyl aminomethan (Tris)(TRIZMA),(Sigma, St. Louis,MO, U.S.A.); human fibrinogen, grade L, human plasminoqen, plasmin,(KabiVitrum, Munich, Germany); streptokinase (Test-Streptokinase) factor XIII (Fibrogammin), thrombin (TestThrombin), polyclonal rabbit antisera Clotimmun-Fibrinogen, Clotimmun FSP-D and Clotimmun FSP-E (Behring, Marburg, Germany), epsilon-aminocaproic acid (eACA), ethylene diamine tetraacetic acid (EDTA), ethylene qlycol-bis-(2-aminoethyl ether)-N,N'tetraacetic acid (EGTA), acrylamide, methylenebisacrylamide (Bis), sodium dodecyl sulphate (SDS), Serva Blue G-250 and Amidoschwarz 10B extra, (Serva, Heidelberg, Germany); Minicon B15 microconcentrators (Amicon Corp., Danvers, MA, U.S.A.); goat anti-rabbit alkaline phosphatase conjugate, chromogenic substrates 5-bromo-4-chloro-3-indolylphosphate toluidine salt (BCIP) and p-nitroblue-tetrazolium chloride (NBT), and gelatin (BioRad, Richmond, CA, U.S.A.); 3-dimethylamino propionitrile (DMPN) (Fluka, Buchs, Switzerland); Nitrocellulose membranes, (Atlanta, Heidelberg, Germany); Aprotinin (Trasylol) (Bayer, Leverkusen, Germany). METHODS Protamine-aqarose was prepared from CNBr-activated Sepharose 4B and protamine sulphate as described previously (1). Maximal coupling ratio was 25 mq of protamine per 1 ml of wet beaded aqarose, ‘as determined by the decrease of protamine concentration in the supernatant following the coupling procedure. The following buffers were used for chromatography : Al: 0.05 M Tris, 0.005 M EDTA, 0.005 M eACA, pH 1.3 A2: 0.05 M Tris, 0.005 M EDTA, 0.005 M eACA, 0.8 M NaCl, pH 7.3 A3: 0.05 M Tris, 0.005 M EDTA, 0.005 M eACA, pH 5.0 A4: 0.05 M Tris, 0.005 M EDTA, 0.005 M eACA, pH 4.5 Bl: 0.20 M sodium citrate/citric acid, pH 5.3 El: 0.32 M arginine, 0.05 M Tris, 0.005 M EDTA, pH 5.3

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E2: E3: E4: E5: E6:

0.32 0.20 1.00 1.00 1.00

M M M M M

PROTAMINE-AGAROSE CHROMATOGRAPHY

glutamic acid, 0.05 M Tris, 0.005 M EDTA, calcium chloride, 0.05 M Tris, alanine, 0.05 M Tris, 0.005 M EDTA, glycine, 0.05 M Tris, 0.005 M EDTA, NaCl, 0.05 M Tris, 0.005 M EDTA, pH was adjusted by addition of 1 M HCl.

pH pH pH pH pH

5.3 5.3 5.3 5.3 5.3

The sample, on average 2 ml of plasma or 4 ml of FDP-containing solution, was applied to a column of protamine-agarose (PSagarose) via a peristaltic pump with a flow rate of approximately 20 ml/h. Column size was 1 x 4 cm (Pharmacia ClO/lO column). The effluent was monitored at 280 nm by means of an LKB UVicord S. Nonbound proteins were eluted with buffer Al, weakly adsorbed proteins were removed from the column with buffer A2. Elution of strongly adsorbed proteins was achieved with buffer Bl at a flow rate of 40 ml/h. When buffer Bl was applied to the column, the effluent was directed to a Sephacryl S-300 column (2.5 x 15 cm, Pharmacia C25) in order to achieve desalting and to obtain some preliminary information on the molecular weight of the proteins eluted from PS-agarose. The effluent of the Sephacryl S300-column was then passed to the UV-monitor and fraction collector. Eluates were concentrated with Minicon B15 microconcentrators. Before application of a new sample, both columns were extensively reeyuilibrated with buffer Al. Polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) was performed according to Laemmli (5), polymerization catalysts were ammonium persulphate and 3-dimethylaminopropionitrile (DMPN). Gels were stained with 0.08 % Serva Blue G250 and 0.1 % Amidoschwarz 10B in 50 % methanol, 10 % acetic acid. Immunoblotting was performed as described previously (1). Fibrinogen degradation products (FDP) were prepared from fibrinogen Kabi desalted by gel filtration. To 5 ml of fibrinogen in phosphate buffered saline pH 7.4 (5 mg/ml), and either 5 mM of calcium chloride or 5 mM EGTA, 1 unit of plasmin (0.1 ml) or 1 unit of plasminogen plus 50 units streptokinase (total of 0.1 ml) were added. Lysis was terminated after varying amounts of time by addition of 50000 KIU aprotinin (5 ml). Fibrin degradation products (fdp) were prepared by adding 5 ml of fibrinogen solution to 5 units of factor XIII (0.5 ml), and 5 units of thrombin and calcium chloride (0.5 ml) to a final concentration of 20 mM. After 60 minutes of incubation at 37OC, clots were harvested with a glass rod, pressed between filter pa per and immersed in 5 ml of phosphate buffered saline pH 7.4 containing 2.5 units of plasmin. Additional 2.5 units of plasmin were added after 60 minutes of incubation at 37OC. Incubation was continued until lysis was nearly complete. Lytic activity was quenched by adding 50000 KIU of aprotinin (5 ml). Citrated platelet-poor plasma was prepared from blood taken with 10 ml-syringes containing 1 ml of 0.11 M sodium citrate, 0.05 M eACA, pH 7.4, by 30 minutes of centrifugation at 4200 rpm. Fresh plasma was mixed with an equ;il amount of buffer Al and applied to the chromatography column. Sample size was 4 ml (2 ml of plasma + 2 ml of buffer). Frozen samples were not used in this study.

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FIG.1 SDS-PAGE of eluates from PSagarose chromatography. Sample: FDP prepared in the presence of 5 mM EGTA. Separation of fragments D and E.

SDS-PAGE of eluates from PSagarose chromatography. Sample: fibrin degradation products DD (D-dimer) and E.

RESULTS Fibrinogen degradation products (FDP) above a molecular weight of 35000 are readily and completely adsorbed to protami ne-agarose. No immunologically detectable fibrinogen-related material above this molecular weight was detected in eluates Al and A2 eluted with with buffers containing up to 0.8 M NaCl at pH 7.3. Figure 1 shows the result of experiments with stepwise elution of FDP prepared from fibrinogen Kabi in the presence of 5 mM EGTA. The first gel presents eluate Al eluted with buffer Al, the second gel material eluted with buffer A3 (pH 5.0), and the third gel material eluted with buffer Bl (0.2 M citrate, pH 5.3). No fibrinogen-related material was eluted with buffer A2 (0.8 M NaCl pH 7.3). Fragment D and E of fibrinogen are effectively separated by adsorption to protamine-agarose and elution of fragment D with buffer A3. Fragment E, together with fragments X and Y present in the sample, is eluted with buffer Bl. Upon rechromatography of desalted eluates, the separated fibrinogen fragments D and E presented the same chromatographical behavior as when applied to the column together. Re-chromatography effectively eliminates cross-contamination of fragments D and E. As shown in figure 1, binding affinity of fragments Dl, D2 and D3 is similar. Figure 3 compares the protamine-agarose/ gel filtration patterns of fibrinogen and fibrinogen degradation products. Elution of FDP with buffer Bl was nearly complete, since very little protein could be removed from the protamine-agarose column with a

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!z r4 q

OS-

0.25

QOAl

I

A2

’

I

I

I,

0

5

10

B 1,

I

15

20

GELFILTRATION

I1

25

I

30

35

I

LO

ON SEPHACRY

I

CSImll L S - 300

FIG.3 Elution pattern of native fibrinogen (-----) and a mixture of fibrinogen degradation products X,Y,D and E (FDP) ) in protamine-agarose chromatography and gel fil( tration of Bl eluate on Sephacryl S-300. The material eluted with buffer Al in chromatography of FDP consisted of aprotinin and low MW peptides derived from fibrinogen. buffer containing 2.0 M urea plus 2.0 M NaBr, pH 5.5, after elution with buffer Bl (not shown). Fibrin degradation products prepared from fibrinogen Kabi were applied to protamine-agarose and eluted with buffer Al, A2, A3, A4 and Bl. The SDS-PAGE patterns of these eluates are presented in figure 2. Fragment DD (D-dimer) appears in the fraction eluted with buffer A4 (pH 4.5), although some DD is present in Bl-eluate as well. Fragment E of fibrin is present only in the fraction eluted with buffer Bl. Fragment DD not complexed with E is eluted with buffer A4 (not shown). Free fragment E of fibrin devoid of fibrinopeptides behaves identically to fragment E from fibrinogen in chromatography on protamine-agarose. Table 1 summarizes a number of experiments performed with mixtures of fibrinogen/fibrin degradation products and individual FDP/fdp purified by chromatography on protamine-agarose and gel filtration on Sephacryl S-300, which were applied to columns of protamine-agarose and eluted with various buffer systems. Buffer Bl is the only buffer capable of eluting all fibrinogen-related material above MW 35000 from protamine-agarose. Fragments D, DD (D-dimer), and native fibrinogen are eluted with 0.32 M arginine pH 5.3 (buffer El) and 0.32 M glutamic acid pH 5.3 (buffer E2) (molarities determined by gradient elution), whereas the neutral amino acids alanine and glycine at a concentration of 1.0 M and similar pH of 5.3 (buffers E4 and ES, respectively) fail to elute fibrinogen-related material. Fragments E, Y and X are not eluted with arginine, glutamic acid or 0.2 M calcium chloride (buffer

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E3). As mentioned above, a mere increase in ionic strength, e.g 1.0 M NaCl (buffer E6) has no effect concerning elution of fibrinogen-related matter above MW 35000 from protamine-agarose Rl-eluate of normal human plasma is comprised mainly of fibrinogen, and a small amount of zontaminatinq protein (1). Figure 4 shows Coomassie-stained SDS-PAGE patterns and immuno-

TABLE 1 Elution buffer BI

(0

El E2 E3 E4 E5 E6 A3 A4

(0 (0 (0 (1 (1 (1 (0 (0

20 32 32 20 00 00 00 05 05

M M M M M M M M M

Fbg

t citrate pH 5.3) t arginine pH 5.3) glutamic acid pH 5.3) + t CaCl pH 5.3) alanine pH 5.3) gycine pH 5.3) NaCl pH 5.3) _ Tris pH 5.0) t Tris pH 4.5)

X

Y

D

DD

E

t

+ -

t t t +

t

-

+ t t t

_ _

_ _ _

t t

+

_

Elution oi native fibrinogen and fibrinogen/fibrin degradation products from protamine-agarose with various buffer systems. Exact composition of buffers is described in methods section. 'Fbg' denotes native fibrinogen, fragment E includes fragment E of fibrinogen as well as fragment E of fibrin devoid of fibrinopeptides. Fragment D includes all three species of this fibrinogen split product.

Fibrinogen

S

B A1

S

B

Bromphenolblue

S

B 81

A2

FIG.4 SDS-PAGE (S) and Immunoblot with polyclonal antifibrinogen antibody (B) of Al, A2 and Bl-eluates from human plasma.Fibrinogen presents as two major bands of MW 340000 and 305000, respectively, and a minor band of MW 290000.

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229

0.25

Al

I

A2

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I

5

1

10

0

15

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20

GELFILTRATION

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SEPHACRYL

S -300

FIG.5 Elution pattern of normal human plasma (----) and plasma from a patient with clinical and laboratory ). Presence of FDP/fdp broaevidence of DIC ( dens the Bl-gel filtration peak and shifts the apex towards the low MW side. blots of A1,AZ and Bl eluates of human plasma obtained with polyclonal rabbit anti-fibrinogen antiserum. SDS-PAGE of Bleluates from normal human plasma presented a double band for fibrinogen with a molecular weight of 340000 and 305000, respectively (6) some material of higher and lower MW reacting with anti-fibrinogen antiserum, and approximately 8-10 % non-clottable protei.n, consisting mainly of factor XIII. Fibrinogen is detected exclusively in fraction Bl. Figure 5 compares the elution pattern of normal human plasma with the elution pattern of plasma from a patient whose clinical condition and laboratory analysis (FDP-test, FM-test, antithrombin III, PTT, clottable fibrinogen, reptilase time, thrombocytopenia) revealed evidence of disseminated intravascular coagulation. Presence of fibrinogen/fibrin degradation products broadens the Bl-eluate peak in gel filtration and shifts its apex towards the low MW side. Electrophoretic patterns and immunoblots of Bl-eluates of plasma from patients with clinical and laboratory evidence of DIC are shown in figure 6. Individual FDP/fdp can be identified by molecular weight (7,8,9). All patients present a complex pattern of fibrinogen/fibrin degradation products in immunoblot, including high molecular weight fibrin complexes as well as fragments of lower MW than native fibrinogen. SDS-PAGE of native plasma with subsequent immunoblotting resulted in band patterns similar to those obtained with Bl-eluate, although in native plasma, concentration of fibrinogen and FDP/fdp is considerably lower than in Bl eluates. Therefore, protamine-agarose chromatography makes it possible to detect and analyse remarkably low titers of FDP/fdp is plasma samples. In addition, in SDS-PAGE of plasma samples, bands of FDP/fdp were considerably distorted in the vicinity of abundant plasma proteins such as albumin (not

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shown). Chromatography on protamine-agarose eliminates these problems by excluding these proteins from the electrophoresis sample.

234

56

FIG 6 L SDS-PAGE (left) and immunoblot with polyclonal antifibrinogen antibody (right) of fibrinogen Kabi (lane 1) and Bl-eluates from plasma of patients with various degrees of DIC (lanes 2 - 6).

DISCUSSION Protamine sulphatts has frequently been employed for precipitation of fibrinogen from biological fluids. Previous results have indicated that protamine covalently attached to beaded agarose can be used for isolation of fibrinogen from human plasma (1). Hafter et al. (4) have demonstrated the usefulness of protamine sulphate as precipitating agent for high MW fibrin derivatives. Fibrinogen fragments Y and D were precipitated only partially, and fragment E not at all in their experiments. Electron microscope studies by Stewart et al. (10) showed that fragments Y, D and E were unable to form extensive or highly ordered polymers upon addition of protamine. This evidence suggested that protamine interacts only with fibrinogen and FDP/fdp of high molecular weight. Detailed studies by Okano et al. (3) indicated that fragment D interacts with protamine, thereby intefering with the precipitation of fibrinogen with protamine, while fragment E did not cause a major decrease of fibrinogen-protamine precipitation. The results presented in this study show that all fibrinogen/fibrin degradation products above a MW of 35000 adsorb to protamine-agarose, including fragments D and E. Adsorption is nearly complete, since only negligible amounts of fibrinogen-related material above MW 35000 are eluted from protamine-agarose with buffers containing up to 1.0 M NaCl at neutral pH. Adsorp-

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231

tion to protamine is not dependent upon presence of fibrinopeptides since fragment E of fibrin presents a chromatographical behavior similar to fragment E derived from fibrinogen, and fragments Dl - D3 are also adsorbed to the chromatography gel. Fragments D and E can be effectively separated by stepwise elution from protamine-agarose, fragment D being eluted prior to fragment E. The fact that Okano et al. (3) found only minor influence of fragment E on fibrinogen-protamine precipitation implies that the interaction between native fibrinogen and protamine is mediated primarily via a D-domain binding site, and that binding of fragment E occurs via a different mechanism. This evidence is supported by our observation that fibrinogen, as well as fragments D and DD (D-dimer), can be eluted from protamine-agarose with buffers containing 0.32 M arginine pH 5.3, or 0.32 M glutamic acid pH 5.3, or 0.2 M calcium chloride pH 5.3, whereas fragments E, X and Y do not respond to these buffers in chromatography. Precipitation of FDP/fdp with protamine appears to require the presence of at least two D-like binding sites on the individual molecule (2,3,4,10), as only fragments X, DD (D-dimer), and fragments of high MW share both features with native fibrinogen. The presence of exposed E-domain on the molecule, as in fragments X, Y and E itself, as well as DD/E complex, provides an additional point of attachment to protamine. Since all FDP/fdp above MW 35000, including fragment D and E are quantitatively adsorbed to protamine-agarose and can be separated from nearly all other plasma proteins, as well as easily eluted from the chromatography gel, protamine-agarose chromatography presents as an easy and useful method for isolation and purification of FDP/fdp. Subsequent analysis of eluates with SDSPAGE and immuinoblotting provides reliable identification and quantitation of individual fibrinogen derivatives. If higher degrees of purification are desired, protamine-agarose chromatography may be combined with other procedures such as gel filtration or isoelectric focusing. REFERENCES 1.

DEMPFLE,C.E., HEENE,D.L., Purification of human plasma fibrinogen by chromatography on protamine-agarose, Thromb. Res.46, 19-27, 1987

2.

LATALLO,Z.S., WEGRZYNOWICZ,Z., BUDZYNSKI,A.Z., KOPEC,M., Effect of protamine on the solubility of fibrinogen, its derivatives and other plasma proteins, Scand.J.Haematol. supp1.13, 151-162, 1971

3.

OKANO,K., SAITO,Y., MATSUSHIMA,A., INADA,Y., Protamine interacts with the D-domains of fibrinogen. Biochim.Biophys. Acta 671, 164-167, 1981

4.

HAFTER,R., KLAUBERT,W., GOLLWITZER,R., VonHUGO,R., GRAEFF, H ., Crosslinked fibrin derivatives and fibronectin in ascitic fluid from patients with ovarian cancer compared to ascitic fluid in liver cirrhosis, Thromb.Res.35, 53-64, 1984

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5.

Cleavage of structural LAEMMLI,U.K., assembly of t.he head of bacteriophage 685, 1970.

6.

HOLM ,R . , NILSEN,D.W.T., KIERULY,P., GODAl,,H.i:., P\lr-ific‘.jt,i.oIland characl.erization of 3 fibrinoqens with different molecular weiqhts obtained from normal human plasma. Thromb.Res.37, 165-176, 2.985

8.

HAVERKATE,F., TIMAN,G., Protective effect 171tc:alc.i urn i.r~t TIC, plasmic degradation of fibrinogen and fihl--in tt'aqmc~nts I? Thromb.Res.10, 803-812, 1977

9.

OLEXA,S.A., BUDZYNSKI,A.Z., Bi ndirrq phr~~~~-lrr~+~na t-ifi :,I tlated unique plasmic deqradatio~~ product:; of human CL clsslinked fibrin.
10.

STEWART,G.J., NIEWIAROWSKI,S., and its degradation products Diat.h. Haem.25, 566-579, 1971

pL c-~tc‘ ins duL irllj!ilc'I'4 Nature .,';!7 , c;no

Aggregation of fibrinogen by basic proteins. Thrnmh