Specific coagulation factor adsorption to insoluble heparin

Vol.50,No.2,

BIOCHEMICAL

1973

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

SPECIFIC CCAGDLATION FACTORADSORPTION TO INSOLUBLEHHPARIN. Patricia

W. Gentry and Benjamin Alexander

Coagulation Laboratory,

Received swnlarv:

The NewYork Blood Center, N.Y.,

N.Y.

10021

December 6,1972 An insoluble derivative

of heparin has been prepared by coupling to agarose beads, and its adsorption of certain blood clotting factors has been investigated. Factors IX*, XI, and heparin cofactor (He-Co)*are selectively adsorbed, and can be recovered by elution of the heparin-agarose (H-Ag) with an NaCl gradient. Fibrinogen (PI) adsorption is negligible. The affinity for F IX appears to be lower than for F XI and He-Co. Thrombin, F XI, and He-Co seemto be equally tightly bound to the insoluble H. Introduction:

Heparin (H)* acts predominantly as an antithrombin;

it also inhibits

F IX* (l-4).

antithrombogenicity

There is considerable recent interest

of H-coated surfaces (5-lo),

but it is difficult

evaluate the precise role of H in such preparations per se resemble H in being highly polar, factors

moreover, in the to

since many of the surfaces

and may thus affect

certain

clotting

Moreover, the H may be leached by blood from someH-surface

(11).

combinations (7,12). We report herein studies with an insoluble H derivative, an H-coated surface, which contribute coagulant activity

in contrast

to

to our understanding of the anti-

of H, and to assessing its affinity

for several coagulation

components. Materials

and Methods:

Heparin (Sigma, Grade 1, hog intestinal

mucosa, lot

numbers-57B 1450 and 79B 1610) was coupled to agarose beads (Biorad, 0.5M), according to Cuatrecasas (13). by toluidine

blue binding (14).

was consistently

of substitution

with H was determined

Amongst several preparations

the H incorporated

0.3 pgm/ml packed beads.

The II-Ag was equilibrated 0.01 M CaCl, and a

The degree

colunrn

with 0.05 M Tris buffer

(pH 7.5) containing

(0.7 cm diameter) of 4 ml bed volume was prepared.

*Romannumeral nomenclature as reconxnendedby the International Committee on Haemostasis and Thrombosis. Thromb. Diathes. haemorrh. (Stuttg.). Suppl. 13, 455 (1964). *Abbreviations used: H, He-Co, heparin cofactor; H-Ag, heparin-agarose; C.T., clotting time; NIH, National Institutes of Health; M.W., molecular weight; SDS, sodium dodecyl su1phat.e. 500

Copyright 0 I9 73 by Academic Press,Inc. All rights of reproduction in any form reserved.

BlOCHEMlCAL

Vol. 50, No. 2,1973

AND BIOPHYSICAL

RESEARCH COMMUNlCA-fiONS

Nine ml of venous blood from at least 8 random routine donors were mixed with 1 ml of 3.1% sodium citrate, fugation at 4" at 1200 g.

and platelet

The plasmas were pooled, and 22 ml were passed 4 ml to emerge, having

with the Ca* in the residual equilibrating

carded since the Ca+I-. interfered emerging plasma was collected non-adsorbed material, until

The first

0.2 mllmin.

through the column at 220 at been diluted

poor plasma was obtained by centri-

the effluent

with clotting

assays.

buffer,

were dis-

The rest of the To remove the

in 3 sequential 6 ml portions.

the column was then washed with 0.05 M Tris buffer

was essentially

protein

free.

The adsorbed components were eluted with a stepwiae gradient consisting of 25 ml of the buffer NaCl; and finally

containing 0.1 M NaCl; 25 ml buffer,

25 ml buffer,

containing 0.4 M NaCl.

containing

0.2 M

The eluates were

screened for protein by measuring adsorption at 282 nm in a Zeiss spectrophotometer.

The fractions

approximately

comprising the protein peaks were pooled, concentrated

3 fold in an Amicon ultrafiltration

PMlO, the NaCl concentration

cell with Diaflo

reduced to 0.1 M, and their

membrane

protein content was

estimated by the method of Lowry et al (15). The level of various clotting

factors,

before and after

the column, was measured by assessing the ability the defective

clotting

of plasma deficient

passage through

of test samples to correct

in the relevant

factors.

was estimated by interpolation

on a standard curve correlating

ity

prepared with the precolumn material.

VIII

with factor

concentration,

were measured against humanplasma hereditarily

deficient

F XI was also measured against plasma rendered F XI deficient with celite

(15 mg/ml) (Johns Manville)

using canine plasma hereditarily assayed with F X-deficient plasma artificially

clotting

activF XI and

in these factors. by adsorption

(16); F IX and VII were measured, in the respective

factors;

F X was

bovine plasma (Sigma) (17); F V was measured against

depleted in F V (18); F II,

modified by using purified plasma, and purified

deficient

Activity

by a two-stage procedure (19),

F V, VII and X (Sigma)' instead of bovine adsorbed

PI (AB Kabi, 93% clottable)

501

in the thrombin clotting

mix-

Vol. 50, No. 2,1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE I VARIOUSCLOTTINGFACTORACTIVITIES IN PLASMA PASSEDTHROUGH HEPARIN-AGAROSE COLUMN A c t i v i t y (Per Factor First

-VII

-IX

-x1*

a#

90

5

12

6

VIII

11

v

93

100

77

73

6 ml effluent

Cent)*

x

Second 6 ml

"

92

---

100

110

--

34

15

9

Third 6 ml

W

90

---

100

98

--

61

36

17

*

Expressed in % of the factor to contain 100%.

** Assayed by correction

in control

of clotting

parent plasma, arbitrarily

defect of F XI congenitally

# Assayed with plasma depleted in F XI by celite

ture.

He-Co activity

as follows:

of FI solution

adsorption (15 me/ml>.

time test,

in H20) and 10 ~1 thrombin

(4 mg/ml) was then added and the C.T. recorded. Various coagulation factor

sequential 6 ml fractions

of plasma collected

column are shown in Table I. depleted.

the celite-treated

three

passage through the H-Ag

with F XI since the F XI activity,

deficient

treated with 15 mg celite/ml,

as measured with

lower than that obtained

plasma. In this instance the normal plasma was an amount knownto remove F XI completely,

and

(16).

The fractions gradient

in the first

F IX and F XI appear to be the only factors

plasma, was always substantially

with the hereditarily

F XII partially

after

levels

Although F XII was not measured, we may assumethat it

was reduced concomitantly

tration

plasma.

in 0.15 M NaCl) were incubated at 370 for 30 seconds. 0.2 ml

Results and Discussion:

considerably

deficient

was measured in a one-stage thrombin clotting

5 ~1 sample, 5 ~1 H (31.3 units/ml

(25 NIH units/ml

considered

eluted from the H-Ag column with the stepwise NaCl concenshowedthree distinct

comprising in their

pooled material

Since the plasma effluents

and reproducible

peaks (Fig.

60, 24, and LOmg protein,

l),

each

respectively.

revealed predominant removal of F IX and XI, each

502

BIOCHEMICAL

Vol. 50, No. 2, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0.2M

M NaCI

NaCl

0.4M

No Cl

1.6

IO

20

30

40

50

Fig.

1

pool

were

calculated

was assayed

recovered

eluted

for

in the

in pool

activity

the

mixture

sample sample

each

individually;

ture

comprising

pool.

first

100

as He-Co.

curve in the

110

F IX and XI

prepared test

As evident

and second

1 and 2 could

interfered (Table

test of

SO

with

sample

and the

in Table

pools,

whereas

the

II,

levels

original total

F IX is

the major

portion

3.

The He-Co in pools

(a)

80

as well

of protein

in the particular

of F XI appears

He-Co

factors,

on a standard

of the amount

preponderantly

some FI which

these

by interpolation

on the basis

protein

70

Protein elution profile following application of plasma to H-Ag (-) or control agarose (l---1)column. Following washing of the columns with 0.05 M Tris buffer, pH 7.5, to remove nonadsorbed proteins, a step-wise increasing NaCl gradient at pH 7.5 was applied as indicated (see The flow rate was 0.5 mlfmin. Methods).

eluted

plasma

60

VOLfmll

ELUTION

with

the

not

assay

be estimated system.

II),

judged

on the basis

alone

retarded

F I-thrombin

and H markedly and (c) 15 pg protein

the

retarded degree from pool

503

Pool

3 eluate

they

contained

3 contained

of the

following

clotting

clotting,

to which

since

far

clotting and 0.62

only

appreciable criteria:

slightly;

beyond

the effect

was retarded H units

(b)

a of

by a mixwas found

to

BIOCHEMICAL

Vol. 50, No. 2,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II F IX, XI, ANDH-CO IN ELUATRSFROM H-AC COLUMNS EXPOSEDTO PLASMAORSERUM F XI Activity

F IX Activity Plasma Fraction Pool 1

Elut.

Units*/mg Protein

9;**

Units*/mg Protein

%**

Heparin Cofactor C.T. (sec.)

Agent

0.1 M.NaCl

58

4

10

--

II

2

0.2 M "

37

6

31

"

3

0.4 M "

4

53

60

144 (7.6)#

0.1 M NaCl

61

2

21

13 (32.4) 21 (21.4)

Serum Fraction Pool 1

Elut.

Agent

"

2

0.2 M "

30

5

26

II

3

0.4 M u

9

27

53

101 (7.4)

* One unit of F IX or XI is arbitrarily defined as that present in I'.0 ml of the original plasma or serum. F XI measured with celite-adsorbed plasma as substrate (16). * Also expressed in % of the total

eluted activity.

# Bracketed figures indicate pg protein in 10 ~1 of sample incubated for 30 set at 37 O with 5 ~1 H (0.16U) and 5 ~1 thrombin (0.25U), followed by the addition of 0.2 ml FI (0.8 mg). The control C.T. (10 ~1 buffer instead of sample) was 16 sec. increase during incubation, 19.8 set; after

as follo&:

30 set incubation,

after

15 set incubation the C.T. was

the C.T. was 143 set; and after

60 set, the

C.T. was 248 sec. The FI content of the three pools was measured by the procedure of Hawiger et al (20) as modified by Laevelle et al (21).

Pool 1 was found to contain

1.1 mg FI; pool 2, 0.3 mg; and pool 3, 0.2 mg - these amounting to 1.8, 1.3, and 2.0 % of the total

protein,

respectively.

To confirm that the selective

removal of F IX and XI from plasma was due to

504

BlOCHEMlCAL

Vol. 50, No. 2,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE III F IX ANDXI ACTIVITY IN THE FIRST FRACTIONSRECOVERED FROMAGAROSEORH-AG COLUMNS A c t i v i t Y (Per Cent)* F XI*

FIX Control-Agarose:

Plasma

p

82

--

76

H-Ag:

II

5

12

6

H-Ag:

Serum

13

42

37

ReusedH-Ag:

Plasma

24

--

11

the parent material arbitrarily

* Expressed in % of the factor, to be 100%. * Assayed against hereditarily

deficient

# Assayed against celite-adsorbed

specific

plasma.

plasma.

adsorption to the coupled H, and not attributable

to the agarose matrix

per se, plasma was passed through a column of non-H-treated conditions

otherwise identical

slightly

Tris buffer

since with Ca*,

a clot

forms

Comparedwith H-Ag, agarose alone reduces F IX and XI only

(Table III).

After

plasma passage, the agarose column was also eluted with a stepwise

gradient of increasing NaCl concentration protein collected was FI.

agarose beads under

to those for the H-Ag column except that Ca*

was omitted from the equilibrating on the column.

considered

(Fig.

in the activities

columns indicate

In contrast to

60 mg

in pool 1 from H-Ag, only 2 mg were recovered; of this 0.03 mg

No F IX, F XI or He-Co was detectable

differences

1).

in the eluate.

eluted from the control

that F IX, XI,

The striking

compared with the H-Ag

and He-Co are selectively

adsorbed by virtue

of

the coupled heparin. Except for FI, the factors One would expect, therefore,

removed by H-Ag are not consumedduring clotting.

that the sameresults

505

should be obtainable with

BJOCHEMICAL

Vol. 50, No. 2, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

serum. Non-anticoagulated blood from eight donors was collected tubes, these were kept at 37 ' for 1 hour, and thereafter the anticoagulated were similarly

whole blood.

The resulting

applied to an H-Ag column.

found markedly depleted in the first profile

and total

protein

into glass

handled the sameas

sera were pooled, and aliquots

As with plasma, F IX and F XI were

6 ml effluent

(Table III).

Moreover, the

eluted by the stepwise NaCl gradient was identical

with that for plasma. The distribution

of F IX and XI activity

were also the

same(Table II). Since the eluates obtained from the serum adsorbed column were devoid of FI, He-Co could be measured in all

three pools.

No activity

was detectable

in

pool 1, someappeared in pool 2, and the major portion was found in pool 3 (Table II). To see whether protein had been eluted completely by the NaCl gradient, the columns were washed with 0.15 M NaCl, and samples were examined with the sensitive

ninhydrin

procedure (22, 23).

No reactive

material was found.

more, when a column that had already been eluted was re-equilibrated plasma sample applied, F IX and XI were again effectively and subsequent gradient elution depicted in Fig. 1. specifically

gave a profile

The data thus indicate

Further-

and a second

removed (Table III),

and distribution

similar

to that

that F IX, XI, and He-Co are

adsorbed by H-Ag, can be quantitatively

and readily

desorbed, and

that the desorbed column is reusable. Since H acts as an antithrombin, respect by He-Co, the affinity

of H-Ag for thrombin was also examined. Two ml

of a bovine thrombin (Parke-Davis) purified

although markedly augmented in this

solution

in 0.01 M NaCl (200 NIH U/ml),

according to Lundblad (24), were passed through an H-Ag column (1 ml

bed volume) equilibrated was adsorbed.

with the Tris buffer.

Virtually

all of the thrombin

Following extensive washing with the buffer,

eluted with the samegradient system described above. devoid of thrombin. eluates contained

The fraction

the column was

Pool 1 was essentially

corresponding to the pool 2 plasma and serum

0.5 thrombin units,

and

150 units were found in.pool 3.

Thrombin adsorption by H-Ag is apparently also specific

506

since with agarose beads

BIOCHEMICAL

Vol. 50, No. 2, 1973

2

Fig.

alone,

H-Ag affinity

cent for

The profile

of the

thrombin

thrombin

appears

of the various

electrophoresis

(25),

and is

of known M.W. were

mobility

probably

represent

but

they

absent

also

Except exhibiting conforms

RESEARCH COMMUNICATIONS

Electrophoretic patterns of the three pools eluted from the H-Ag plasma and serum columns on 5% polyacrylamide gels in SDS. a and b represent pool 1 from plasma and serum, respectively; c (plasma) and d (serum), pool 2; and e (plasma) and f (serum), pooi 3. The numbers on the left indicate M.W. x 104. The protein load was 50 pg incubated for,2 hours in 0.01 M sodium phosphate buffer (pH 7.0) containing 1% SDS. The gels were stained with Coomassie blue.

93 per

Mann)

AND BIOPHYSICAL

are for

these,

the highest approximately

run

appeared

in the

for

was examined

in Fig.

2.

not

only

Furthermore,

F XI and He-Co. by SDS polyacrylamide

Eight

protein

The two bands do they

markers

(Schwartz-

exhibiting

conform

gel

lowest

in M.W. with

FI,

serum profile.

serum and plasma

mobility with

to that

as standards. FI since

the

similar

eluates shown

in the wash effluent.

is

likely

patterns He-Co

6.5 x 104 reported

507

are

since for

its

purified

identical. M.W.(app)

The band of 8 x lo4

antithrombin

III

(26).

Vol. 50, No. 2, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Assuming that the results obtained with H-Ag truly of native H with clotting

factors,

many factors tested H specifically under our conditions, thrombin)

F IX>) FI.

binds and complexes with only a few, and, order of binding tightness:

It would thus appear that the anticoagulant

II).

The altered

in H affinity

affinity

acts at

induced by H-Ag in plasma F XI com-

pared with serum, where presumably F XI exists

evident in the difference

F XI = He-Co =

sequence.

The discrepancy between the depletion

difference

the interaction

we can for the time being conclude that of

in the following

several steps in the clotting

a striking

reflect

in activated

form (XIa),

for F XI compared with XIa.

suggests

This is also

in recovery of F XI from the two He-Ag columns (Table suggests a structural

change in F XI during its acti-

vation. The data also indicate in the eluates, presently

substantial

protein

under investigation.

H-Ag is thus a valuable tool, entities,

amounts of as yet uncharacterixed

highly useful for studying certain clotting

as well as somenon-clotting

functions of H.

Acknowledgments: This research was supported by USPHSGrants HL 9011 and 11447. Blood samples were voluntarily provided by donors in the Blood Procurement Program of the NewYork Blood Center. We wish to thank Dr. H. Al-Mondhiry of Memorial Hospital, N.Y. for the samples of humanF XI deficient plasma, and Dr. Jean W. Dodds, N.Y. State Department of Health, for F VII and IX deficient canine plasmas. REFERENCES 1. 2. 2: 5. 6. 7. 8. 9. 10. 11. 12.

Shanberge, J. N., Sarelis, A., and Regan, E. E., 2. -Lab. -Clin. Med. 54, 501 (1959). O'Brien, J. R.,. Nature 181, 1801 (1958). Ratnoff, 0. D., and Davie, E. W., Biochemistry 1, 967 (1962). Pitlick, F. A., Lundblad, R. L., and Davie, E. W., J. -Biomed. Mater. b. 2, 95 (1969). Gott,-v. L., Whiffen, J. D., and Dutton, R. C., Science 142, 1297 (1963). Leininger, R. I., Cooper, C. W., Falb, R. D., and Grode, G. A., Science 152, 1625 (1966). Usdin, V. R., and Fourt, L., 2. Biomed. Mater. Res. 3, 107 (1969). Merrill, E. W., Salsman, E. W., Wong, P.E.,xhford, T. P., Brown, A. H., and Austen, W. G., J. u. Phvsiol. 29, 723 (1970). 30, 1679 (1971). Get, v. L., and Fur-use, A., Fed. w. Nossel, H. L., Wilner, G. A., and LeRoy, E. C., Nature 221, 75 (1969). Oja, P. D., Holmes, G. W., Perkins, H. A., and Love, J., J. -Biomed. Mater. k. 3, 165 (1969). Falb, R. D., Grode, G. A., -Fed. Proc. 30, 1688 (1971).

508

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13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cuatracasas, P., J. Biol. Chem. 245, 3059 (1970). Macintosh, F. C., -Bis.J.35 --3 776 (1941). Lowry, 0. II., Rosebrough, N. J., Farr, A. L., and Randall, R. J., 2. w. Chem. 193, 265 (1951). SoulicJ. P., and Prou-Wartelle, O., Brit. 2. Haemat. a, 88 (1960). Bachman, F., Duckert, P., and Keller, Fxhromb. Diath. Haemorrhag. 2, 24 -(1958). Gwen, C. A., Bowie, E. J. W., Didisheim, P., and Thompson, J. H., in The Diagnosis of Bleeding Disorders 1st Ed., pp. 105-106, Boston: Little, Brown and Company, 1969. Ware, A. G., and Seegers, W. H., &. 2. -Clin. Path. l9, 471 (1949). Hawiger, J., Niewiarowski, S., Gurewich, V. and Thomas, D. P., J. Lab. Clin. Med. 75, 93 (1970). LaeveK Dx., Mertens, B. F., Bowie, E. J. W., and Owen, C. A., Am. 2. Clin. Path. 55, 452 (1971). -Matheson, A. T., Tigane, E., and Hanes, C. S., Can. 2. Biochem. Physiol. 39, 417 (1961). Awad, W. M., and Wilcox, P. E., Biochim. Biophys. &. 73, 285 (1963). Lundblad, R. L., Biochemistry l.0, 2501 (1971). Weber, K., and Osborn, M., J. Biol. Chem.244, 4406 (1969). Andersson,L. -O., and Miller-Andersson, M., III Congress, Washington, D.C.: The International Society on Thrombosis and Haemostasis, 1972.

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