Purification and some properties of autoprothrombin II-A: An anticoagulant perhaps also related to fibrinolysis

THROMBOSIS Printed

RESEARCH in the United

PURIFICATION

AND

AN ANTICOAGULANT Walter

SOME

I, pp. 443-460, 1372 Vol. Pergamon Press, Inc.

States

PROPERTIES

PERHAPS

ALSO

H. Seegers, Lowell Nobuo Sakuragawa,

OF AUTOPROTHROMBIN RELATED

TO

II-A:

FIBRINOLYSIS

E, McCoy, Hans D, Groben, and B. B. L. Agrawal

Department of Physiology, Thrombosis Specialized Center of Research, Wayne State University School of Medicine, Detroit, Michigan, U.S.A. [Received

8.8.1.972.

Accepted

by

Editor

L.

Vroman)

ABSTRACT Methods were developed for the assay and isolation of autoprothrombin II-A. The purified protein retarded blood coagulation in vitro as well as in vivo and induced fibrinolysis. It is tentatively concluded that the protein originated from prothrombin and possessed inhibitor-activator qualities respectively as anticoagulant and fibrinolytic agent. To obtain Auto II-A, concentrated prothrombin complex was digested with purified thrombin. Chromatography on a DEAE-cellulose column separated prethrombin, Auto II-A, and autoprothrombin III (Factor X1. The inhibitor-activator was then purified by using a Sephadex G-100 or G-100 superfine column. A single component was indicated by polyacrylamide gel electrophoresis and by ultracentrifugation. The equation for S;O w was 3.9 + 0.0114X. Isoelectric point = pH 3.9. It is a competitivd inhibitor of autoprothrombin C (Factor Xa). N-terminal amino acids were Ile and Gly. An amino acid analysis was compared with a new amino acid analysis of purified prothrombin. The latter had 518 amino acid residues per mole. The protein contained sialic acid, neutral sugars, and amino sugars. Serum from rabbits immunized with purified prothrombin crossreacted with purified Auto II-A. A working theory is proposed as follows: Thrombin functions in a negative feedback system which retards thrombin formation due to the production of prethrombin and Auto II-A. Platelet factor 3 also produced prethrombin and Auto II-A. This feedback system could be the molecular basis of latent coagulation and fibrinolysis. In accelerated coagula, tion, the latent coagulation sequences are by-passed with massive formation of fibrin. This investigation was supported by research grants HE-03424-15 and HE-14142-01 from the National Heart and Lung Institute, National Institutes of Health, U. S. Public Health Service. Hans D. Groben received a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, West Germany. Support was also given by the McGregor Fund, Detroit, Michigan,

444

AUTOPROTHROMBIN

In 1960, by Mammen,

Thomas,

and developed thrombin.

a previously

unrecognized

and Seegers

when purified

Under similar

(1).

conditions,

itor or autoprothrombin

II.

Later,

Seegers

It proved to be a competitive

conditions

In this paper, we describe

inhibition

either the inhib-

(2) fractionated

the activated

of autoprothrombin

In addition

by further work,

and fibrinolysis

of the inhib-

C (Factor X,1,

under which thrombin activity

generated

of autoprothrombin

to being an anticoagulant,

in blood samples from dog and man.

of coagulation

for obtaining

our work on the purification

of the product.

induced remains to be determined

generated,

column and obtained a concentrate (3,4)

II-A

with purified

II (Factor IX) activity

satisfactorily

inhibitor

to retard several

duced mild fibrinolysis

was found

was called autoprothrombin

and Marciniak

prothrombin complex on a DEAE-cellulose

and some of the properties

of blood coagulation

autoprothrombin

5

Vol.l,No.

prothrombin complex was digested at pH 7.9

to control conditions

thus functioned

inhibitor

The activity

but it was not possible

itor.

II-A

How the fibrinolysis

but tentatively

and (5).

II-A it inwas

it can be assumed that

was due to the same substance and that it was

derived from prothrombin.

METHODS Purified

by methods devised

(81, and sedimented

in this laboratory.

(8) were also according

gel electrophoresis cellulose

MATERIALS

bovine prothrombin complex (61, purified thrombin (71, purified prothrom-

bin (61, purified Ac-globulin

AC-globulin

AND

followed

was Whatman

Assays

amino acid analysis

microgranular,

preswollen

Fine Chemicals,

cellulose.

and 72 hours and for threonine,

serine,

instrumentation.

half-cystine,

Hydrolysis

methionine

to zero was made to correct for loss during hydrolysis,

hydrolysis

values were used.

method of Duggan and Udenfriend

made to contain

0.1

Sephadex

(13).

was determined N-terminal

A stock solution

mg protein per ml.

DEAEproducts

and the column effluents

trapolation

Tryptophan

and

Poiyacrylamide

with an LKB Uvicord II, recording at 280

was by chromatography

by the methods of Edman (14).

prothrombin (111,

procedures as adapted to our needs (12).

and columns were obtained from Pharmacia were monitored automatically

for thrombin (101,

(9) were obtained

to the methods of this laboratory.

described

DE-52

brain thromboplastin

nm. Quantitative was for 24,48,

and tyrosine,

an ex-

Otherwise,

22 hour

by the spectrophotofluorometric

amino acid analyses

were performed

of purified soybean trypsin inhibitor

The product was from Worthington

Biochemical

was _

AUTOPROTHROMBIN

Vol.l,No.5

For ihe ultracentrifuge was dissolved

work,

in 0.1

Phosphate

the Spinco

M potassium

buffers

we list the composition

were

analytical

chloride

reagents

445

instrument

was used,

The

protein

at pH 7.2.

made from stock

of stock

II-A

reagent

solutions

and then the buffers

Reagent

A:

One M dipotassium

Reagent

B:

One M potassium

Reagent

C:

One M sodium

hydrogen

to be at pH 7.0.

made from these

First

solutions:

phosphate

dihydrogen

phosphate

chloride

Buffer

1:

50

ml A +

50

ml B; 1.0

M phosphate

Buffer

2:

50

ml A +

50

ml B + water

Buffer

3:

50

ml A +

50

ml B +

140

ml C + water

to 2 liters;

0.07

M NaCl

Buffer

4:

50

ml A +

50

ml B +

260

ml C + water

to 2 liters;

0.13

M NaCl

Buffer

5:

50

ml A +

50

ml B +

360

ml C + water

to 2 liters;

0.18

M NaCl

Buffer

6:

50

ml A +

50

ml B +

800

ml C + water

to 2 liters;

0.40

M NaCl

to 2 liters;

CHROMATOGRAPHY PROTHROMBIN The column A stock

preparation

A sufficient layer

used was

was removed

COMPLEX 2.5

was

poured

and replaced About

Then

applied

outflow.

#2 was again

This

the buffer

complex

ml were

until

and in water thrombin

as described a million

solution at 37’

to the DEAE-cellulose

DEAE-CELLULOSE was done in a cold room (3 to 8’ C), with After

required

overnight

M phosphate

settling #l

until

buffer

to 20-25 at pH 7.0.

the effluent

of the inflow

operation.

and the column

#2.

cm,

the top

This

buffer

was

pH 7.0.

equaled

that of the

The flow was stopped

was now ready

when

for application

below.

units

of prothrombin,

(110-120

ml),

were

C and at pH 7.0. column.

0.05

buffer

the conductivity

the cellulose,

ml) of purified applied

300

had just entered

Approximately

Work

M phosphate

ACTIVATED

1 M phosphate

a convenient

prepared

cm.

into the column.

was usually

of the material

ON

was mixed

with

was at room temperature. buffer

x 20

of DEAE-cellulose

quantity

OF

0.05

in the form of purified mixed After

with 90

50,000

minutes,

prothrombin units

(in 2-3

tile mixture

was

446

AU'~OPl~O'I'IllIOMl3IN II-A

At 4’ C in the cold room, the activated lulose,

As soon as it went in, 0.05

nitrogen pressure. less,

absorbance

prothrombin was poured on top of the cel-

M phosphate buffer #2 was applied

This force was used throughout,

the buffer was changed to #3.

under 10 psi

When the absorbance

This eluted the prethrombin (Fig.

was again less than 0.1,

1).

was 0.1

or

When the

the buffer was changed to 44 and, in like manner,

to buffers 5 and 6.

The latter eluted inhibitor

column work usually

required 4-5

tions were used for special

Vol.l,No.5

hours.

and autoprothrombin

III respectively.

The prethrombin and autoprothrombin

The

III frac-

purposes.

+.07M+."M

FIG.

TUBE NUMBER

The inhibitor do this,

Prothrombin complex activated with thrombin and chromatographed on a DEAE-cellulose column. At pH 7.0 the prethrombin, inhibitor, and autoprothrombin III were eluted respectively with 0.07, 0.18 and 0.4 M sodium chloride in phosphate buffer, Cold room.

(I9 ML PER TUBE)

fraction was prepared for fractionation

we worked under cold conditions

solution.

The precipitate

dissolved

in a small volume of water.

was collected

night before precipitation,

and added 0.4

Sometimes

OF INHIBITOR

BY SEPHADEX G-100

ward flow at an approximate bonate at pH 7.8 applied.

to 8.0.

lnert material

in a refrigerated

the inhibitor

the dissolved

column.

To

per ml

centrifuge

and

fraction was frozen over-

ammonium sulfate

precipitate

was

days before further fractionation.

PURIFICATION

A Sephadex

on a Sephadex

gm ammonium sulfate

by centrifugation

and sometimes

stored frozen for several

1

column 2.5

FILTRATION

x 183

rate of 25 ml/l-u. The inhibitor

(Fraction

cm was used at 20 to 25’ C with upThe buffer was 0.1

from the DEAE-cellulose

1) appeared first,

later more inert protein was obtained

(Fig.

FRACTION

2).

followed

M ammonium bicarfractionation

by the desired

work was

inhibitor,

and

AUTOPROTHROMBIN

Vol.l,No.5

II-A

447

2

n

E 0.30 1

-=f

300

200

n

INHIBITOR

400

EFFLUENT

VOLUME

a

EFFLUENT

VOLUME

(ml)

(ml) FIG. Fig. 2.5

2

FIG.

2. Purification of autoprothrombin II-A on Sephadex G-100 x 183 cm. Center peak corresponds to active material.

Fig. 3. Purification of autoprothrombin II-A on Sephadex column 2.5 x 95 cm. Center peak represents inhibitor.

We also ward ties

flow

itated

rate was

with

frozen

Amicon

ml/hr

frozen

Diaflo

filter

without

About

41%

fraction.

derived

used.

and studied

and dialyzed

latter

were

We were

the amount

However,

unable

The up-

and other

procedure.

the salt.

Some

by dialysis

main

we precippreparations

with

to dry the product

proper-

The

Sometimes

was the most efficient

to the Sephadex

was about

column

protein

yield

prothrombin

3.770.

We could

no way has been devised quite

different

in the original

we did determine

column.

cm.

the

for preservfrom the

loss of activity.

applied

because

away

procedure

in the purified

activity

x 95

ways.

concentrated

mg of prothrombin fraction

2.5

superfine

activities

in various

the average

could

to the Sephadex

The specific

For 9 preparations,

if it is something

thrombin.

and a column

column

G-100

to be the same for either

and pH,

complete

of the precursor not assay

3).

Others

This

volume

in the active

of inhibitor

(Fig.

sulfate

of the protein

from 590

yield

combined

apparatus.

almost

superfine

appeared

ammonium

controlling

state

hibitor

or less II-A

were

with

G-100

and subsequently

ing activity,

yield

15

activity

the protein

were

tein

used Sephadex

of the autoprothrombin

fractions

3

The average

was in the purified

was

complex;

mixture

to measure

hence,

was 80%

specific

was 30,000

This

was

the pro-

the overall the concentration

Furthermore,

because

that the recovery activity

mg.

not calculate

from prothrombin.

activation

22.4

in-

we

it contained

of the activity u/mg

proapplied

protein.

1148

A1J1‘01’1~OT111~OM1~TN 1-T-A

Without

exception,

a small amount of thrombin remained

this was troublesome,

in some experiments

ample,

chemical

in the physical

a reaction

of autoprothrombin

mixture consisting

brain thromboplastin, thromboplastin

C (41,

Although

significance

as, for cx-

or the assay procedure.

To measure its effects

of the following:

purified Ac-globulin,

forms autoprothrombin

purified

in just adequate

quantitatively,

we devised

prothrombin complex,

and calcium chloride.

sedimented

In this mixture,

of thromboplastin

and Ac-globulin,

converts

By design, the reagents were put

amounts to convert a given amount of prothrombin (1000

Under those conditions,

with the function of autoprothrombin

the

C from the prothrombin complex and this autopro-

the prothrombin of the prothrombin complex to thrombin.

to thrombin.

5

PROCEDURES

thrombin C, with “help” of the lipoprotein

together

1 ,No.

II-A inhibits the formation of thrombin because it is a competi-

Autoprothrombin tive inhibitor

in the preparations.

it was of negligible

measurements

ASSAY

Vol.

there is a sensitive C.

The following

units)

response to any interference

standard activation

mixture was

arranged: Prothrombin complex (Prothrombin 2000

0.5

ml

Sedimented brain thromboplastin (5 mg/ml)

0.1

ml

Ac-globulin

0.1

ml

Sample or control

0.2

ml

Calcium chloride (0.1 M) in imidazole buffer, pH 7.2

0.1

ml

This reaction

purified

u/ml)

(2150

mixture was kept for 20 minutes at 37’

sample was taken for thrombin analysis. all the prothrombin being converted to the reaction

As the amount of inhibitor

creased.

Consequently,

The activation

to thrombin (Fig.

mixture in small quantity,

reduced.

of inhibitor

u/ml)

4).

C.

Every 5 minutes,

was complete

in 20 minutes with

By adding autoprothrombin

was increased,

mixture (Fig.

II-A

the rate of thrombin formation and its yield was the yield of thrombin was further de-

the reduced thrombin yield proved to be a reflection

in the reaction

a

4).

of the amount

5

Vol.l,No.

I

AUTOPROTHROMBIN

449

II-A

FIG.

CYTROL

4

Standard reaction mixture, top curve, contained prothrombin complex, tissue thromboplastin, purified Ac-globulin, and 0.01 M calcium chloride at pH 7.2 and 37’ C. Maximum lllrombin yield was

IO

ACTIVATION

ZD

TIME

The relationships concentrations, possible. formation

h4lh!)

are projected

the yield

We defined

reduced progressively in proportion to the amount of inhibitor added; namely, 0.02, 0.04, etc. mg/ml. Note approach to equilibrium in 20 minutes.

of thrombin

2 units

in another was easily

of inhibitor

of 1 unit of thrombin

way by means reduced,

as the amount

in the range

of 50%

of Figure

but a complete which

substrate

REWCTION OF THROMBIN YIELD WITH INHIBITOR

would

In low

block

was not

stop or prevent

(prothrombin)

FIG.

5.

the

inhibition.

5

Reduction in thrombin yield by autoprothrombin II-A when added to standard prothrombin activation mixture e Compare with Fig. 4. Approach to equilibrium at 5, 10, and 20 minutes as indicated by 3 curves. ’ ’ ’ ’ ’ O/Z345 PROTEIN CONCENTRATION (mg /mU 0

As an example, Fig.

4,

mixture.

0.04

The calculation

The

respective

ber,

dilution

a little nonlinear

numbers in mixture

This

relationship.

range

in thrombin if the first

preparation amount

was then refer

to:

1000

Usually yield. one failed,

-

was diluted

the reduction factor

a second

by 500

5 times

thrombin

to make the dilutions

experience,

yield

x 2 x 5 x 1 =

In case

an interpolation

we tried With

500

the thrombin

concentration,

dilution.

units,

reduced

of inhibitor

substrate

and original

more or less than 500

unit reduction within

mg curve).

a certain

(of

in the reaction

5000 yield,

u/ml

sample.

definition

in thrombin

was used.

units

This

num-

yield

was

involved

so that there was a 500 analysis,

if required,

fell

a

4 50

ATJTOPl?OTllROMl~TN

INHIBITED

CLOTTING

To study the effect of the inhibitor thrombelastograph, prothrombin II-A 0.2

ml blood.

TT-A

AND

62,000

on blood coagulation

Within

with the use of the

A selected

u/ml and 3 u/ml thrombin.

This began to clot almost at once (Fig.

rigid as that of the control.

‘,

FIBRINOLYSIS

we mixed dog blood with the inhibitor, contained

I,No.

Vol.

6).

1 hour and 30 minutes,

solution

To 0.1

of auto-

ml, we added

The clot never became as the clot had lysed completely.

__.._-c---------__~_

3

FIG. 6 Thrombelastogram of dog blood. Curve #1 was control. Curve #2 highest concentration of autoprothrombin II-A added to dog blood. Curve #3 one-fifth the quantity of autoprothrombin II-A as compared with curve #2. Inhibitor preparation contained small amount of thrombin.

By taking one-fifth cuvette. lysed.

the amount of inhibitor,

There was very little Thus,

of concentrations,

preparation,

Evidently,

there would have been no clotting

thrombin in order to demonstrate obtained;

of having thrombin in the preparation

that the autoprothrombin

and induces fibrinolysis.

however,

lysis.

we found,

II-A is an inhibitor of

if there had been no thrombin in the and it would have been necessary

to add

With the use of human blood, the same result was This latter result is in accord-

with rabbit blood no lysis followed.

ance with the fact that it is difficult

u/ml in the

and that small amount of fibrin which formed was

working with the difficulty

by proper selection coagulation

clotting

the thrombin was reduced to 0.2

to induce substantial

fibrinolysis

in the blood of

rabbits e Next,

we wanted to see whether the inhibition

lysis might also occur in vivo.

To obtain information

of clotting and induction on this point, we selected

of fibrinoa 12 kg

AUTOPROTHROMBIYN II-A

Vol.l,No.5

He was given

male clog as test animal. devices slowly

for blood infused

preparations,

heart

respiration,

over a period 56

Throughout

pressure,

of 18

mg of inhibitor

the experiment,

rate or blood

sodium

were

which

pentobarbital

and heart

minutes

with

anesthesia.

rate were

in 18

5 hours,

Recording

attached.

use of an infusion

dissolved

lasted

451

The pump.

ml physiological

there were

From saline

no effects

the infusion,

the infusion

the beginning firm as with some fibrin

the control; after

the infusion.

with

the recorded

hours after

dency

sample

7).

slope

control

it was

182

OF was studied

characteristics

for protein

in about

12 minutes.

progressively minutes

after

concentration

PURIFIED

were

there were After

was

12

was clotted

the use of a Spinco

quite

312

than taken in accord

mg% and 4 l/2 in the values

the infusion,

a bleeding

pressure

measuring

tensites.

II-A

Model

component

was positive,

were

hours

by add-

sample

no differences

AUTOPROTHROMBIN

dependence

clot was not as

but even after

times

and blood

that of a single

The

the first

concentration

at the infusion

71, we observed

in the thrombelastograph

clotting

analysis.

5 min-

(Fig.

the infusion

as with

Lee-White

mg% while

with

weaker,

less amplitude

fibrinogen

by the two-stage

from the wounds

The preparation

The

The

with

7

The next one was drawn

the thrombelastograph

but not as extensive!y

Our observations results.

30

produced

was observed,

PROPERTIES

sedimentation

drawn

The clot

measured

The

and became

A sample

was observed

was taken.

sample

the infusion,

for prothrombin

sample

of the latter

remained. (Fig.

a blood

With

the control

lysis

solution,

Thrombelastogram of blood taken from doy given Intravenous infusion of autoprothrombin II-A. Curve #1, top left, was control and is incompletely shown. Curve #2 blood sample 5 minutes after infusion. Curve #3 blood sample 30 minutes after infusion and clotted by adding thrombin. Center and bottom recordings are continuations of #2 and #3.

was completed.

of clotting

ing thrombin

three

pressure.

‘20

utes after

was

on respiration,

FIG.

Before

inhibitor

with

E ultracentrifuge. (Fig.

8).

the equation

being

452

AUTOPROTHROMBIN

FIG.

II-A

Vol.l,No.5

8

Ultracentrifuge schlieren patterns of autoprothrombin II-A at 8.0 mg/ml in 0.1 M potassium chloride at pH 7.0 and at a rotor speed of 59,780 rpm. Time in minutes given on each frame. Sedimentation from left to right.

$0 w =

3.9

+ 0.0114X

(Fig.

It is interesting

tratidn in mg/ml. centration

dependence

slope (151,

described

in an accompanying

9).

In this equation,

that 3.7s

X stands for protein concen-

thrombin also had a positive

and the same was true for the prothrombin preparation

article.

FIG.

s

0

5 M/ML.

/o

15

In our amino acid analysis with an analysis All indications

of prothrombin,

20

work, we were especially

for amino acid analysis, namely,

3800

interested

in a comparison

because the latter might be the precursor of the inhibitor

are in favor of that conclusion,

paper, an element of reservation

laboratory;

9

Concentration dependence of sedimentation constants of autoprothrombin II-A in 0.1 M potassium chloride and a rotor speed of 59,780 rpm.

40

g 3.5 (/, 3.0

protein con-

but even with the work presented

needs to be maintained.

had the highest specific u/mg dry weight.

activity

in this

Our prothrombin sample,

used

of any prepared to date in this

ATJTOPl~OTW?OMBIN II-A

Vol.l,No.5

TABLE Amino

Acid

Composition

1 of Bovine

and Autoprothrombin

Amino

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine T yros i ne Phenylalanine Tryptophan NH3

(4.94) (1.80) (8.79) (9.66) (4.27) (4.66) (13.15) (4.82) (4.18) (3.59) (3.06) (5.11) (1.10) (2.87) (6.82) (3.82) (3.62) (3.61)

1.30

(1.46)

66.28

TOTAL

**

4.86 1.55 4.88 6.45 2.86 2.85 9.83 2.97 2,85 2.28 2.84 4.41 1.05 2.94 4.62 2.79 3.25 1.70

Figures Ratio

each

of appearance

of two separate

we found acids

518

alone.

imately

(91.42)

in parentheses

Our analysis

represent

of prothrombin hydrolyses. acid

of the molecular

appearance

and used a multiple

either

or insufficient

extra

acid,

numbers

a molecular

amino

sugars

weight

Based

amino

(24) (9) (38) (55) (27) (31) (68) (32) (42) (31) (19) (33) (6) (17) (40) (16) (17) (13)

424

357

(518)

weight

from duplicates

value

of 59,744

sugars

amounted

on several

acid for calculating

than acceptable

for

range for prothrombin

One less or an additional acids

29 8 24 42 20 23 51 22 32 22 20 32 6 19 30 13 17 8

;: 17 20 48 19 27 18 17 27 5 16 25 11 14 7

an average

and neutral

of prothrombin.

of amino

24 7

prothrombin.

molecular

giving

of 6 residues.

(32) (12) (50) (73) (36) (41) (89) (42) (56) (40) (25) (44) (8) (22) (52) (21) (22) (18)

prothrombin.

1) represents

as the reference

Residues RA x 5** RA x 6**

(683)

for purified

In the accepted

weight

we took methionine

496

6 for purified

(Table

residues,

sialic

33 10 28 48 24 27 67 26 38 26 24 38 7 22 35 15 20 8

(86.3)

analysis with

Residues per 105 gm

(4.0) (1.5) (6.4) (9.2) (4.5) (5.2) (11.3) (5.3) (7.0) (5.1) (3.2) (5.5) (1.0) (2.8) (6.6) (2.7) (2.8) (2.2)

70.7

x 5 or 6,

The total

10%

culations,

amino

4.7 1.4 4.0 6.9 3.4 3.9 9.5 3.7 5.4 3.6 3.4 5.3 1.0 3.2 5.0 2.2 2.8 1.3

Prothrombin

II-A

Ratio of Appearance

% Recovery

Acid*

453

for amino to approx-

exploratory

cal-

each

of

ratio

one involved

for the molecular

454

AUTOPROTHROMBIN

weight-of

prothrombin.

Additionally,

after treatment with bromcyanogen chain occurs at the methionine and thus, by this analysis,

II-A

Vol.l,No.5

we made a preliminary under conditions

where cleavage

residue of the sequence.

we can also conclude

estimate

of fragment numbers of the poiypeptide

There appeared to be 7 fragments

that there are 6 methionine

residues

in

prothrombin. When we assumed that the inhibitor evident

might have 6 methionine

that it could not be derived from prothrombin.

discarded to 357

in a degradation

process,

and the corresponding

our stated assumption

no lysine discarded Thr, Ser,

molecular

weight was 41,305

There is no evidence

and only a little histidine.

Glu, Pro, Gly, Ala,

however,

it was

that one was

our figure for total amino acid residues was reduced

as a working hypothesis,

rived from prothrombin.

Assuming,

residues,

and Leu (Table

for amino acids only.

the inhibitor

could theoretically

to the contrary.

With

be de-

It follows that there was

The main alterations

were with Arg, Asp,

11, each one being removed in substantial

amounts o In the N-terminal preparation

amino acid analysis,

for more extensive

sugars in proportions

isoleucine

work, we found sialic

found in prothrombin.

and glycine were found.

acid,

neutral sugars,

In

and amino

We hope to extend this work on carbohydrates

and present a report.

FIG.

10

Polyacrylamide gel electrophoresis of autoprothrombin II-A with 15.0 and 7.5 pg applied. Photograph at 20 minutes.

In the analysis ug of material material together,

by polyacrylamide

above the gel.

gel electrophoresis,

we applied

We observed mainly a single band (Fig.

was also studied and usually and just distinguishable.

7.5

10).

Freeze-dried

there were two bands of the same size, Likewise,

and 15.0

when we studied the freeze-dried

close protein

AUTOPROTMROMRIN

Vol.l,No.5

by I~licrozonal

ccllulosc

acchlc

there was a second

component

an isoelectric

of 3.9,

point

clcclrophorcsis with

!I55

(plrosplinLc

an isoelectric

and we presume

TI-A

buffers,

point of 5.8.

this corresponds

strcngLIi

ionic

0.04),

The main component to the isoelectric

hacl

point

of

the inhibitor. Our immunological

work

is considered

the use of rabbits,

we made an antibody

thrombin

according

to the procedures

inhibitor

cross-reacted

tation

band between

identity

with

investigation with

with

these

tests

is planned,

which

(6).

antiserum

Ouchtertony

(Fig.

11).

With

chromatographed

pro-

There

There

to thank

study.

plates

were

used.

was a single

The

precipi-

was a band of immunological

of the prothrombin-antiserum

and we wish

are being

only an introductory

DEAE-cellulose

and the antiserum.

one of the components of this

to twice

described

the rabbit

the inhibitor

as being

Glenn

interaction. R. Van

Further

Noord for his help

continued.

FIG.

11

Ouchterlony plates. Center contained full-strength rabbit prothrombin antiserum. Well #l prothrombin at 16.7 mg/ml. #4 prothrombin at 8.5 mg/ml . #2 and #6 autoprothrombin II-A at 0.5 mg/ml. #3 and #5 autoprothrombin 11-A at 0.25 mg/ml. Photograph at 48 hours.

DISCUSSION Autoprothrombin presented that

the first

it might

sive, Another

II-A

description

be an agent

are consistent possibility

one non-prothrombin

was first

with

which

found

in 1960

of its purification induces

(1);

and have obtained

fibrinolysis.

The facts

the idea that an inhibitor-activator

is that there precursor

however,

are two non-prothrombin for the two activities.

we believe the first

presented,

is derived precursors

we have

indication if not conclu-

from prothrombin.

for each activity

or

456

AUTOPROTHROMBIN

PROTHROMBIN

II-A

Vol.l,No.5

COMPLEX

PRETHkXtBlN AN

AlJTOPf?OTHROMBlN

III

co ions Tissue Eatmct Plotekt Foctol ,3 Plosmo Focta R Other Factors

1 ;-,$$~in* Co ions*

I

AUTOPROTHROMBIN + Peptide

??

I

C

JOLYSIN

THROMBIN + Peptidc Is)

??These occclemtei Autopmthmbin C alone is sufficknt . *In voriis sequences ond combinotiis. **Nomad AutopFothromMn II-A; pmbobly on8 molecule with two disttnct activities.

FIG.

12

Possible molecular basis for holding blood coagulation at level of latent coagulation and fibrinolysis through a negative feedback system. Under conditions favoring formation of large amounts of autoprothrombin C, prothrombin conversion to thrombin would occur, without the formation of much prethrombin or autoprothrombin II-A. A dependence of fibrinolysis on the prothrombin system does not conflict with other pathways for fibrinolysis.

By adopting a conservative interesting

interpretation

ideas can be developed

and staying nearest the facts,

as avenues for further experimental

Small amounts of thrombin are not readily

removed by antithrombin

amounts of thrombin arise in our circulation,

the high affinity

as prothrombin (17);

be for thrombin formation to be retarded. hibitor of autoprothrombin The induced fibrinolysis of fibrinolysis minogen). dimension

11-A (Fig.

or possibly

the concept of latent coagulation

added which is outlined

in terms of molecular

12).

in-

for that reason also. The acceleration

of profibrinolysin

and fibrinolysis mechanics.

Prethrom.

is a competitive

in the removal of fibrin. by direct activation

When small

the tendency would

II-A

C, thrombin formation would be inhibited

could be indirect

In this way,

III(16).

therefore,

Since autoprothrombin

would be operational

exploration.

for prothrombin would re-

sult in the formation of prethrombin as well as autoprothrombin bin does not form thrombin as readily

some

(plas-

has another

This is essentially

considered

a negative

its own formation

feedback

system,

and to lyse the first

In case of perceptible autoprothrombin

C would

of prothrombin

to thrombin

18).

Autoprothrombin

the fibrinogen

when

often

Whenever thrombin

II-A

in 1939

when

complex

form fibrin,

mentioned,

fact that platelets, autoprothrombin

II-A

when

for the fact that platelet extensive ops,

intravascular

accelerated

coagulation.

deals

with

lated

factor

phenomena

In many respects, and extrinsic

or compartmentalization

for

that autopro-

was historically

(15,

to a prothrombin

has frequently

Most

interesting

and platelet complex.

can be infused

II-A

coagulation, and is subsumed

producing devel-

is to function

to explore

or latent

because

form

activity

membranes

is more meaningful

is the

that accounts

without

be useful

been

membranes

Possibly

3 and platelet

noticed

state

20).

It might

first

thrombin

into the imperceptible this

coagu-

thus accounting

we can assume

refractory

platelets

intravascular

It may be that autoprothrombin

of platelet

coagulation

it was shown

and that more arises anticoagulants

thrombin

3 or “lysed”

physiologi-

coagulation.

purified

prothrombin

of

further

aspects

and

than the division that arbitrary

of

division

by the idea of latent

and

coagulation,

Recently plasma

to purified

(15, Mosl

Under

fibrinolysis,

membranes,

than as a procoagulant.

into intrinsic

anatomy

accelerated

effect

rather

of blood

coagulation

added

red cell

coagulation,

the division

blood

3,

added

laboratory

of

quantities

conditions.

not lyse.

develops,

the two-stage

from this

factor

factor

and thus the initial

as an anticoagulant

that time,

induce

(19)

amounts

of appreciable

disseminated

the inhibitor

and Smith

in papers

platelet

state

large

under those

intravascular

refractory

is to retard

play a role in autocatalysis

with

would

disseminated

Seegers,

but mostly

would

not form extensively

that perspective,

Since

relatively

the conversion

Thrombin

11-A formation

From

preparation.

force

of thrombin

that might appear,

formation,

be a time associated

follows

Mertz,

fibrin

effect

and in a test tube it would

the two-stage

forms.

the initial

strands

would

12).

would

autoprothrombin

the fact that lysis

fibrin

This

(Fig.

there would

because

or massive

form.

II-A

would

cal conditions, lation

1-I 57

AUTOPROTIIROMBIN Tl--A

Vol.l,No.5

II-A

when

Since

use of purified

prothrombin

Although

metabolically

from plasma.

its function

(21).

that much two-stage

derived

direct

and quantitative

under physiological

synthesis

conditions.

the formation methods,

thrombin

is stopped

proof is lacking,

from the prothrombin

we have demonstrated

proteins

refractory

this

complex

zymogen

with could

we can attempt

Such a substance

Coumadin

or re-

be due to autopro-

as the latter

of autoprothrombin

is in

disappears

II-A

to accurately

with

the

imagine

has been postulated

by

458

AUTOPROTHROMBIN

still another approach by Hemker, kinetic

study,

deficiency. synthesis

they postulated

Veltkamp,

There is only limited due to complications

refractory

On the basis of a

inhibitor arises

They believed

in plasma in vitamin K

that vitamin K deficiency

or nonidentity

information

of autoprothrombin

II-A

to make advances

based on degradation

(23-25)

on that account.

Additionally,

and that is presumably

beyond the point of maximum inhibitor

producing autoprothrombin

II-A

C produces the two-stage

refractory

activity.

state,

destruction

Multiple

For instance,

ways of when puri-

one cannot be certain The courage to

reduce these problems is bound to be rewarded and perhaps with practical is known to function as a lytic agent (26) and esterase (27,

II-A

of the protein,

due to inhibitor

magnify chances for confusion.

shown to produce lysis in blood vessels

and pivka.

the two-stage

whether this is a direct effect or whether thrombin is first produced.

Thrombin

induced

on the maximum yi.eld of autoprothrombin

such as the formation of prethrombin and the lability

state is reversed

fied autoprothrombin

(22).

Was it derived from prothrombin ? That question remains as well as

many others regarding the identity

It will be difficult

Vol.l,No.5

and Loeliger

that a competitive

This was called pivka. of pivka.

II-A

applications.

thrombin also has been

28).

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MAMMEN, E.F., THOMAS, W.R., and SEEGERS, W.H. Activation of purified prothrombin to autoprothrombin I or autoprothrombin II (platelet cofactor II) or autoprothrombin II-A. Thromb. Diath. Haemorrh.: 5, 218, 1960. =

2.

SEEGERS, prethrombin

3.

SEEGERS, W.H., HEENE, D.L., and MARCINIAK, E. Activation of purified Purification of autoprothrombin C. prothrombin in ammonium sulfate solutions: Thromb. Diath. Haemorrh.: 15, 1, 1966,

4.

MARCINIAK, E., MURANO, G., and SEEGERS, W.H. Inhibitor of blood clotting derived from prothrombin, Thromb. Diath. Haemorrh.: =18, 161, 1967.

5.

MARCINIAI<, 1970.

6.

MCCOY, L .E. mci SEEGERS, W .H. and purified prothrombin, Thrombosis

7.

SEEGERS, W.H., MCCOY, L., KIPFER, R.K., and MURANO, G. Preparation and properties of thrombin. Arch. Biochem. Biophys.: 128, 194, 1968.

W .H, and MARCINIAK, subunit of prothrombin.

E.

Coagulation

E. Some activation Life Sci.: 2, 1721,

inhibitor

elicited

characteristics 1965.

by thrombin.

Science:

of the

170,

452,

Preparation of bovine prothrombin complex Res, =I , 40 I, 197:~.

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3. ‘DOMBROSE,

F.A., YASUI, T., ROUBAL, W .H, Ac-globulin (factor V): Preparation Biochem.: In press.

9.

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Z., ROUBAL, A., and SEEGERS, of a practical product. Preparative

HECHT, E.R., CHO, M.H., and SEEGERS, W.H. ture and preparation of protein-free material different activator. Amer. J. Physiol.: 193, 584, 1958.

Thromboplastin: Nomenclafrom platelet factor 3 or lipid

10,

SEEGERS, W .H . and SMITH, H.P. Factors fied thrombin. Amer. J. Physiol.: 137, 348,

11.

WARE, A.G. determination 1949.

12.

SEEGERS, W.H., MCCOY, L.E., REUTERBY, J., SAKURAGAWA, N., MURANO, G., and AGRAWAL, B.B.L. Further observations on the purification and properties of autoprothrombin III (factor X1. Thrombosis Res .: 1, 209, = 1972.

13.

S . The spectrophotofluorometric DUGGAN, D.E. and UDENFRIEND, tion of tryptophan in plasma and of tryptophan and tyrosine in protein J. Biol. Chem.: 223, 313, 1956.

14.

EDMAN, P. Sequence S .B. Needleman (Ed.)

and SEEGERS, of prothrombin

which influence 1942.

the activity

of puri-

W .H. Two-stage procedure for the quantitative concentration. Amer. J. Clin. Path.: 2, 471,

determinations. In: Berlin-Heidelberg-New

determinahydrolysates.

Protein Sequence Determinations. York: Springer-Verlag, 1970,

p. 211. 15.

SEEGERS,

W .H.

Prothrombin.

Cambridge:

Harvard

University

Press,

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16.

DOMBROSE, F.A., SEEGERS, W.H., and SEDENSKY, J.A. Antithrombin: Inhibition of thrombin and autoprothrombin C (F-Xa) as a mutual depletion system, Thromb. Diath. Haemorrh.: 26, 103, 1971. =

17.

SEEGERS, W.H., DOMBROSE, F.A. and autoprothrombin

18.

SEEGERS, Three basic

19.

MERTZ, E.T., SEEGERS, W.H., and SMITH, by purified thrombin solutions. Proc. Sot. Exp.

20.

SEEGERS, W.H., MURANO, G., and MCCOY, thrombin during activation: A theory. Thromb.

21.

OUYANG, C., SEEGERS, W.H., MCCOY, L.E., and MiLLER-BERGHAUS, Metabolic formation of a prothrombin derivative. Thromb. Diath. Haemorrh.: 332, 1971.

SAKURAGAWA, N., MCCOY, L.E., SEDENSKY, J.A., and Prothrombin activation: Ac-globulin, lipid, platelet membrane, C (factor X,1 requirements. Thrombosis Res.: In press.

W.H., MCCOY, reactions. Clin.

L., and MARCINIAK, E. Chem.: 14, 97, 1968. = H.P. Biol.

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L. Structural Diath. Haemorrh

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changes in pro.: 23, 26, 1970. i G. 25, _

h6c-l

.A~JTC)l’R0Tlll?OMIITN

TT-A

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H.EMI
23.

WARE, A.G. and SEEGERS, W.H. tivation with thrombin, and activation Chem.: 174, 565, 1948.

24.

SEEGERS, W.H., ANDREWS, E.B., and McCLAUGHRY, R.I. Formation prothrombin derivatives from purified prothrombin. Amer. J. Physiol.: 164, 1951.

25.

CHO, M.H. and SEEGERS, Exp. Biol. Med.: z, 642,

26.

BRAMBEL, C.E. 68, 67, 1957.

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28.

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Proteases

W.H. 1958.

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Prothrombin

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