TR.ROYB0313 RESEARCH Printed in Great

Vol.

Britain

12,

pp. 6j'-665, Pergamon Press,

1978 Ltd.

'i'C=IZATIoN OF PIIODWTS OF PLASMIN CY..4R?.C.,i, COM?.AR4TiX iM~~tiOCSE"rICAL AND L~:'JKOCYT~ PROTEASE CLEP,VAC;E: OF H~JMANFIW.I~OGEN E.F. Plow and T.S. Edgington Department of kfolecular Immunology, Scripps Clinic and Research foundation, La Jolla, California 92037 USA

(Received

18.1.1978; in Accepted by Editor

revised form 6.2.1978. R.F. Doolittle)

ABSTRACT Plasmin-dependent as well as a plasmin-independent alternative pathways for fibrinolysis have been recognized, but means of discriminating between the products of these two pathways have not been established. To this end, a major product of the cleavage of human fibrinogen by neutral leukocyte proteases, a 270,000 M!! derivative designated Fragment I, has been immunochemically compared with the D:E complex produced by plasmic cleavage. Both classes of cleavage fragments exhibited absolute antigenic deficiency relative to native fibrinogen. Differences in the expression of residual native fibrinogen determinants were distinctive and localized to both the D and the E domains of the molecule; quantitative differences were predominantly localized to the D domain, whereas differences in competitive inhibition slopes were restricted to the E domain. The cleavageassociated neoantigens, fg-Dneo and fg-Eneo, were expressed differently by the two types of cleavage products. In contrast to the complete expression of fg-Eneo by the plasmic D:E complex, this neoantigen was minimally expressed by Fragment I but somewhat comparable to that of plasmic fragment X. Fragment I was clearly distinguishable from the plasmic cleavage products with respect to the expression of fg-Dneo. The competitive inhibition slopes differed, and Fragment I was antigenically incomplete for fg-Dneo. It is concluded that the cleavage products of fibrinogen generated by neutral leukocyte proteases and by plasmin are immunochemically distinguishable; and assays for fg-Dneo may provide a means for discrimination of the pathways of fibrinolysis mediated by these enzymes.

INTRODUCTION The fibrinolytic pathways represent compensatory proteolytic systems that serve a requisite biological role, i.e. the restoration of vascular integrity following local deposition of fibrin.

Within the vascular compartment the

653

CLEAVAGE BY LELXOCYTE

6-h 2

fluid (1,

phase activation 2);

with

however,

platelets

trast

alternative (3).

fibrinolytic

(6,

7).

products tivity

than plasmln,

include cells

that

are distinct (7).

and septicemia,

pathway may contribute

products

In con-

of plasmin,

fibrinolytic

in both

in the plasma of

patients

that

associated

10).

fibrin(ogen)

proteases

an al-

structure with

and bio-

fibrinolytic

with acute

associated

en-

degradation

the leukocyte-mediated

to the fibrino(geno)lysis

pathway

is plasminogen-indepen-

granulocytic

suggesting

(9,

and can secrete

pathway generates

from plasmic

Recently,

those

to activation

by leukocytes

contain

but this

have been demonstrated

eases

pathways lead

Vo1.12,No.4

fijrinolytic

and endothelial

pathways which

not only

activity

leukemia

ac-

myelocytic

alternative

with

these

dis-

(11). Discrimination

difficult, dent

(4-8)

pathway mediated

Leukocytes

zymes other logical

of

nay be the primary

fibrinolytic

leukocytes

to the majority

ternative dent

of plasminogen

PROTEXSE

but

of

it

dependent between

pathways by analysis

modifications

fibrinolytic of

of

of

information

of

pathway,

the products

products

for

fibrino(geno)lysis

to distinguish

1) provides

study:

conformational

responsible

may be possible

and independent

The present

pathways

between

fibrinogen

(fg)

these

pathways. and

by the leukocyte-

immunochemical

the leukocyte-dependent

the plasminogen-dependent

of

the structural

imparted

and 2) analyzes

is

plasminogen-depen-

the products concerning

in vivo

alternative

differences

pathway and the

pathways.

MATERIALSAND METHODS Preparation

0E enzymes

Leukocytes

were isolated

dextran

(200,000

hypotonic prepared

From titrated

MN; Sigma Chemical

lysis

of

contaminating

by shearing

centrifugation

granule

obtained

fraction

was suspended freezing tion

following

brinolytic

in 1.0

a 2;s

(13),

0.01

Plasminogen chromatography

x 90 cm column of

purified

8% agarose

Leukocyte

a 19 gauge needle by Janoff at 40,000

lysate,

by molecular fine;

(12).

20 min at 4’C

frozen

as described exclusion

Biorad,

The

by repeated

as the supernatant

from fresh

on lysine-Sepharose

were

M sucrose

was used as the source

was isolated

(Al.5

in 0.34

and Sherer xg for

In

by brief

granules

and disrupted

recovered

as above,

sedimentation

followed

MO)

7).

M NaPOk, pH 7.3,

The granule

and further

Louis, (4,

as described

ultracentrifugation

proteases.

plasma by affinity and Mertz

through

by differential

St.

by centrifugation

M NaCl,

and thawing,

Co.,

red cells

leukocytes

and differential

blood

of

fracfi-

titrated by Deutsch

chromatography

Richmond,

CA)

on

equilibrated with 0.15 !-? SaCl, 0.131?i ?ia?O~, pE 7.3.

The plasninogen was ac-

tivated by incubation with 100 ?lough units urokinase/mg for 1 hr at 3i3C. Isolation of fibrinogen and degradation products Fibrinogen was isolated by differential ethanol fractionation followed by precipitation at 26% saturation with ammonium sulfate (14, 15).

Fibrinogen

was rendered plasminogen-free by passage of 100 - 300 mg through a 2.5 cm

column

of lysine-Sepharose 4B (16).

x

Reduced fg had intact Ail, BB and

40 Y

chains when analyzed by polyacrylamide gel electrophoresis in sodium dodecylsulfate, and exhibited no loss in coagulability by thrombin or change in constituent chain size after incubation with 100 IU streptokinase (Calbiochem, La Jolla, CA) per mg fg for 6 hr at 37°C. Fibrinogen was digested with l/600 part (w/w) plasmin at 37°C for 1, 3 or 18 hr for the preparation of fg-X, fg-Y and fg-D:E complex, respectively. The fragments were isolated by molecular exclusion chromatography on a 2.5 x 80 cm column of A1.5, fine, agarose and recycled for relative homogeneity. The constituent fg-D and fg-E fragments of the fg-D:E complex were separated by ion exchange chromatography on DEAE-cellulose (Type 40; Schleicher and Schuell, Keene, NH) followed by molecular exclusion chromatography of each separately on a 2.5 x 80 cm column of Sephadex G-100 in 10% acetic acid. Fragments were dialyzed against saline or lyophilized and stored frozen at -20°C. Leukocyte protease digests of fg were prepared at 37°C using the granular lysate from 1 x lo6 leukocytes per mg fg.

The relative rate of digestion

of fg in this incubation mixture was comparable to the above plasmic digest with respect to the loss of coagulability.

Both digests were coagulable by

thrombin at 10 U/ml at 30 min but not at 60 min of incubation (thrombin time >180 set).

The major high molecular weight derivative present in the 24 hr

leukocyte protease digest (see Fig. 1) was designated Fragment I and was exhaustively dialyzed against phosphate-buffered saline at 4°C to remove small peptides. Antisera Fibrinogen, fg-D, fg-E and Fragment I, were injected at 50 - 200 ug doses in complete Freund's adjuvant initially into the rear footpads of rabbits, then bimonthly in incomplete Freund.'sadjuvant in multiple subcutaneous sites. Antisera represented pools from two or more animals and had the following characteristics by immunoelectrophoresis and Ouchterlony gel diffusion analysis.

Antiserum to fg (anti-fg) produced a single precipitin with plasma but

not serum, and yielded reactions of non-identity between fg-D and fg-E with one another but gave reactions of partial identity of each fragment with fg.

6j6

CLEAVAGE

BY

LEL-KOCYTE

PROTEASE

Vo1.12,so.4

FIGURE 1. The electrophoretic characteristics of Fragment I produced by leukocyte proteases and the D:E complex produced by plasmic cleavage of Eg. A. Electrophoresis on 5% polyacrylamide gel in the presence of sodium dodecylsulfate under non-reducing conditions. B. Electrophoresis on 7.5% polyacrylamide gel in the presence of sodium dodecylsulfate following reduction with 1% 2-mercaptoethanol. C. Immunoelectrophoresis in 1% agar at pH 8.8.

Anti-fg-D precipitated fg and fg-D, but not fg-E; and anti-fg-E precipitated fg and Eg-E but not fg-D.

Antiserum to Fragment I produced a single precipi-

tin line with Eg, plasma and Fragment I but not with serum or leukocyte granular extracts. Immunochemical analysis Radioimmunoassays of the double antibody type 2rere performed as previously described (15).

The 1251-labelled ligands were introduced at a concentration

of 5 x lO-'O M and each was >90% precipitable in 10% trichloroacetic acid, and was >90% bound by a 1:lOO dilution of each antiserum.

Competing antigen

concentrations were determined from micro-Kjeldahl nitrogen determinations, and molar concentrations were calculated from estimated molecular weights of 340,000 for fg, 270,000 for fg-X, 165,000 for fg-Y, 130,000 for fg-D:E complex, 80,000 for fg-D, 50,000 for fg-E and 270,000 for Fragment I.

Results

of radioimmunochamicai analysis vere interpreted with respect to three inde1) concentration of competing antigen required for 50%

pendent parameters:

inhibition (CIss), 2) inhibition slope at serial concentrations of competing antigen representing the linear regression where [A% bound/-A log10 competing antigen] x 100 (CI,), and 3) maximum inhibition produced by a given competing antigen expressed as % inhibition at the maximum tested concentration (cxmax) (17, 18).

The coefficients of variation of CI, and CIga were 3.8 and

5.2%, respectively.

Comparisons of CI, were analyzed by variance with two-

tailed t tests, and analyzed for significance in both directions according to the formula: b - b' t= SY.X"'1 1 ssx + SSX' where b is the slope, sy.x is the error variance and SSx is the sum of the squares (19). Electrophoretic techniques Polyacrylamide gel electrophoresis was performed in 5 x 75 mm cylindrical gels in the presence of sodium dodecylsulfate as described by Weber and Osborn (20).

The molecular weight of Fragment I was estimated under non-re-

ducing conditions utilizing fg and its plasmic derivatives for calibration. Molecular weights of constituent chains were estimated following reduction with 1% 2-mercaptoethanol from the relative mobilities of serum albumin (68,000), the heavy (50,000) and light (23,500) chains of IgC, ovalbumin (43,000), pepsin (35,000) and myoglobulin (18,000).

Immunoelectrophoresis

was performed in 1% purified agar (Oxoid Ltd., London, England) in 25 mN barbital buffer, pH 8.8, on 7.5 x 2.5 cm slides.

Precipitation arcs were de-

veloped at 20°C with anti-fg following electrophoresis at 6 V/cm for 75 min.

RESULTS The characteristics of fg-D:E complex present in advanced plasmic and Fragment I in neutral leukocyte protease digests of Eg are compared in Figure 1. When the plasmic derivatives are analyzed on 5% polyacrylamide gels in the presence of 0.1% sodium dodecylsulfate, only bands characteristic of the D and E fragments are observed indicating complete degradation of the intermediate fragments X and Y.

Analysis of the leukocyte protease digest demon-

strates a broad band, indicative of considerable size heterogeneity, of approximately 270,000 W.

Previous studies (7) and unpublished observations

have demonstrated that this high molecular weight derivative, which we have termed Fragment I, is reasonably resistant to further degradation at this concentration of leukocyte proteases.

Only at a much higher concentration of

enzymes is further degradation observed.

5iien reduced in the presence of 1Z

2-mercaptoethanol, the plasmic digest contains chains at 44,000 and 38,000 consistent with DB remnants, a chain at 25,000 consistent with Dy, and smaller constituent chains of fg-D and fg-E in the 10,000 - 14,000 range. Reduction of Fragment I demonstrates a pattern consistent with extensive Ae chain cleavage, slight degradation of BE chains, and intact y chains.

In the

immunoelectrophoretic patterns (Fig. 1C) of the plasmic digest, fg-D andfg-E migrate in opposite directions.

Fragment I yields a broad precipitin arc

which may represent fusion of two or more precipitin arcs in reactions of identity.

A portion of the Fragment I arc migrates equivalent to fg-D but

none of the rather heterogeneous Fragment I has an electrophoretic mobility as anodic as fg-E.

Native fibrinogen determinants Expression of native fg determinants by Fragment I and plasmic derivatives is analyzed by equilibrium competitive inhibition in a radioimmunoassay using 12'I-fg and anti-fg (Fig. 2).

Native

-to-10

Fitirinwen

19-9 Competing

10-7 10-E Antigen Concentration EM

m-6

1

FIGURE 2. Analysis of expression of native fibrinogen determinants by equllibrium competitive inhibition with 1251-fg at 5 x lo-lo M as ligand and antiserum to native fg.

Doti fg-D:Z complex and Fragment I exhibit absolute antigenic deficient;?relative to fg whiei! is indicated by failure to produce complete competitive inhibition.

This is in contrast to the complete inhibition produced by fg-X

even though Fragments I and fg-X are generally somewhat similar in chain constituency.

Quantitative and qualitative differences in expression of residu-

al native determinants b:: Fragment I and fg-D:E complex are evident from differences in CIsc and CI,.

A Z-fold molar excess of Fragment I is required to

give a C150 equivalent to fg-D:E complex; and differences in CI, of fg-D:E complex and Fragment I are significant (p ~0.05) suggesting relative differences in antibody affinity for these derivatives. Domains of fibrinogen The relative degree of modification of the D and E domains of the molecule by plasmic as compared to leukocyte protease cleavage was analyzed by radioimmunoassay using lz51-fg as the ligand and antisera to either fg-D or fg-E.

In

t'leD domain (Fig. 3) the CI, for fg, Fragment I and fg-D:E complex are comparable (p 1.1).

The CI50 are significantly different and a 3-fold molar

excess of Fragment I is required for inhibition equivalent to fg-~:~

60

1

complex.

D Domain

50 i 40 FrI 30

D:E

20 10

;y,’ rs

0 ~~,~ 10-l’

g

C: c::,; Cr.%, I.15 ;:i;; z.15 3.r0 z.*a3

($1

z

0

;:: 132

lD-lo Competing

1D-g Antigen

10-8 Concentration

10-T

10-6

[MI

FIGURE 3. Analysis of expression of nativei: determinants in the D domain 8 I-fg at 5 x 10-l' X as ligand by equilibrium competitive inhibition with and antiserum specific for fg-D.

vo1.12,xo.4

This indicates differences in structure and/or conformation of the D region present in these two types of fragments.

In analysis of the E domain (Fig.

4), fg-D:E complex and Fragment I are comparable on a quantitative basis (CI53); but differences in CI, are significant (p ~0.001) providing additional evidence for differences in modification of the structure of the E domain and its antigenic determinants. E Domain s0I

Competing

Antigen

Concentration

[M

1

FIGURE 4. Analysis of expression of native fibrinogen determinants in the E domain by equilibrium competitive inhibition with 1-51-fg at 5 x lo-l3 X as ligand and antiserum specific for fg-E. Cleavage-associated neoantigens The relative expression of the cleavage-associated neoantigenic expressions, fg-Dneo and fg-En,, (15, 21), by the plasmic derivatives as compared to Fragment I is illustrated in Figures 5 and 6. he0

Competing

Antigen

Concentration

[Ml

FIGURE 5. Expression of the cleavage-associated neoantigen, fg-Dneo. The system consists of 12'I-fg-D at 5 x lo-lo M and antiserum specific for fgDneo*

Competing

Antigen

Concentration

[MI

FIGLmE 6. Expression of the cleavage-associated, fg-En,.. The system consists of l*'I-fg-E at 5 x lo-10 M and antiserum specific for fg-En,o. Intact fibrinogen does not express fg-Dn,, (CI,,, = O), but fg-D:E complex and all plasmic derivatives containing a D region produce a CI,,, = 100 and virtually identical (2150 and CI, indicating complete expression of these determinants in a quantitatively comparable fashion.

In contrast, Fragment I

produces a CImax of only about 80X, indicating that there is significant but incomplete exposure of the fg-Dn,, determinants.

A CI50 of 20 nM for Frag-

ment I is approximately a 2.5 molar excess relative to the plasmic derivatives.

The differences in CI, for Fragment I and the plasmic derivatives are

significant at the p