THROMBOSISRESEARCHVol.i2,pp.953-9G. Q Pagmon Press Lrd.1973.Printd inGreat Britain
THE MECHANISM CF THE FIBRINOGEN-THROMBIN WCTION
Desmond H. Hogg and Birger BlombPck Department of Blood Coagulation Research, Karolinska Institutet, Stockholm, Sweden
(Received 20.1.1978. Accepted hy Received by Executive Editorial
Editor Office
S. Magnusson) 1.5.1978)
ABSTRACT The kineticsof the thrombin-catalysed release of fibrinopeptide A from fibrinogen, modified fibrinogen and various fragments of fibrinogen has been investigated in an attempt to elucidate the structural elements of fibrinogen which define its interaction with thrombin. These elements have been found to be contained within the structure formed by the first 51 amino acid residues of the &-chain. It is proposed that the binding which is of fundamental importance takes place within the sequence 1-23 of this chain and may even be localised to the sequence 8-16. This binding is strengthened, either directly or indirectly, by structures within the sequence 34-44 and to a lesser extent by structures within the sequence 45-51.
INTRODUCTION Thrombin catalyses the release of fibrinopeptides A and B from fibrinogen (1,2). In so doing, the enzyme cleaves only four specific arginyl-glycyl peptide bonds in the fibrinogen dimer, whereas trypsin, a closely related serine proteinase, cleaves almost all arginyl and lysyl peptide bonds in fibrinogen (3,4). To explain the difference between the specificities exhibited by the two enzymes, it has been suggested (5,6) that thrombin has an extraordinarily hidden active site which for steric reasons can react only with the N?i2-terminal portions of the Acr- and Be-chains. In a previous report (7) we concluded that the structural elements of fibrinogen which are of major importance in defining the thrombin-catalysed release of fibrinopeptide A, are located within the architecture formed by the first 51 amino acid 957
THROMEIN-FIBRINOGEN
954
REACTION
Vo1.12,No.6
residues of the h-chain. In this communication,we report findings concerning locality cf these thrombin-bindingelements within this architecture.
MATERIALS AND METHODS Bovine thronbinwas purified to an activity of 1,800 NIH units/m8 by the procedure described previously (7). Trypsin, TPCK-treated,was obtained from WorthingtonBiochemicalCorporationand sperm whale apomyoglobinwas supplied by Beckman InstrumentsInternationalS.A.
Human plasmin, having an activity
of lo-15 caseinolytic(CTAl) units/mg, was kindly supplied by Dr. Bjarn Wiman, Dept. of Medical Chemistry, University of Umei, Sweden. Tryptic digests, two dimensionalpeptide maps and amino acid analysis were carried out as described before (8). The NH2-terminalswere analysed as described in a previous communication(7). Preparationof fragments of fibrinogenused as subsrrates.
;=
The various substratesused in this investigationare listed in Table I.
TABLE I Initial rates of the thrombin-catalysedappearanceof NH2-terminal glycine in various substrates at 370C in 25 mM Tris-Xl, 0.125 M NaCl, pH 7.2. Results for substrates l-4 have previouslybeen published elsewhere (7).
Substrate Substrate No. 1 2
3 4 5
Fibrinogen Thioredoxintreated fibrinogen N-DSK Ao(l-51) AcY(l-51) BB(f-118) An(l-44) &i(l-3312 Ao(l-23) BB(l-118)
Molecular Molarity weight uM
10 (mol NI?l Zc-1)
20 25
2.00 t 0.14 2.62 + 0.13
5,500 5,500
20 40 40
1.48 2 0.17 1.05 f 0.07
12,200 4,800 7,900 2,300 12,200
40 40 20 40 40
340,000 340,000
59,000
'Footnote: CTA = Committee on ThrombolyticAgents
0.55 + 0.01 0.42 z 0.07 0.08 _=0.01 0.06 - 0.01 0
Vol.12,No.h
THROYBI4-FIBRINOGEN
REACTIOS
955
Fibrinogen, thioredoxin-treated fibrinogen, N-DSK and Acr(l-51) were prepared as described previously (7). The B8-chain of N-DSK, B8(1-118), was isolated from reduced and S-carboxymethylated N-DSK by gel-filtration (9) and this was further purified by chromatography on CM-Cellulose (9.10). Two sub-fragments of the Pa-chain of N-DSK, h(l-44)
and pCr(l-23)were pre-
pared by digesting this chain with plasmin. To a solution of 22 mg of Pa(l51) in 1 ml of 0.2 M NH4HCO3, pH 8.2, was added 0.7 mg of human plasmin. The mixture was incubated at 37' for 2 h, 0.1 ml of glacial acetic acid was then added and the clear solution was gel-filtered on a column of Sephadex G-25 (Fig. la). The material recovered from peak 1 was chromatographed on G-100 to remove plasmin and yielded 11.5 mg of freeze-dried &(l-44).
FIG. 1
1.5-
,/‘.
: ;
1.0 -
:
-r
-,: :
Gel-filtration of plasmin digested An(l-51) on a column (82 cm x 1.8 cm*) of Sephadex G-25 in 10% acetic acid. Flow rate: 15 ml/h; fractions: 2 ml. a) aftek a 2 h digestion; b) after an 18 h digestion, inset: thin-layer chromatography of material recovered from peak 2 with arrow indicating the spot found to contain Aa(l-23).In both diagrams, the final peak of radioactivity having no absorbance at 280 ran is due to the peptide segment 45-50 (see Fig. 4).
The smaller fragment, Pa(l-23), was obtained on prolonged digestion of Acr (l-51) with plasmin. To a solution of 36 mg of Ao(l-51) in 2 ml of 0.2 M NH4HCG3, P H 8.2, wad added 0.8 mg of human plasmin. The mixture
was incuba-
ted at 37' for 18 h and after addition of 200 ul of glacial acetic acid the clear solution was gel-filtered on a column of Sephadex G-25 (Fig. lb). The second peak, which was pooled as indicated, yielded 15.5 mg of freeze-dried material. Thin-layer chromatography of this material on cellulose (Whatman CC41) in the medium l-butanol-pyridine-acetic acid-water (150:100:30:120v~v)
956
THROWBTN-FIBRISOGE3
REX-l-TON
vo1.12,so.6
produced the ninhydrin-positive pattern shown in the inset in Fig. lb. Amino acid and NH*-terminal analysis of the material eluted from the spots indicated that the arrowed spot in Fig. lb contained &(l-23).
To isolate this
fragment on a preparative scale, 70 )~l of an aqueous solution of 6 mg of the material obtained from the above gel-filtration were applied as a streak to a 0.5 nm thick layer of cellulose on a 20x2@ cm plate. The plate was developed in the same medium as was used above and was air dried. The band corresponding to the arrowed spot in Fig. lb was located by spraying the two vertical edges of the thin layer with a ninhydrin-collodine solution (11) and allowing this to develop for 1 h at room temperature. The appropriate band of cellulose was then scraped from the plate and the peptide material (0.8 mg) was recovered by three one-hour long extractions with 2 ml of 10% acetic acid at room temperature.
30
40
50
60
TO
80
sfacl,o!. Nurntm
FIG. 2 a) Gel-filtration cf N-bromosuccinimide treated N-DSK on a column (95 cm x 5 cm 2, of Sephadex G-50 in 10% acetic acid. Flow rate: 20 ml/h; fractions: 5 ml. b)Gelfiltration of material recovered from "a" on a column (82 cm x 5 cm2) of Bio-Gel P-10 in 10% acetic acid. Flow rate: 15 ml/h; fractions: 5 ml. Fractions in "b" were subjected to ninhydrin analysis (12).
*or
A small dimeric fragment of fibrinogen, &~(1-33)~, the two halves being joined by the symmetrical disulphide Aa28-&28, the tryptophanyl peptide bonds (h33 nimide. Amodificationof
was prepared by cleaving
and Aa44) in N-DSK with N-bromosucci-
a procedure which has been described (13) wasused.
To minimise the modification of Pa24 His, free histidine was incorporated in the reaction mixture. To a solution of 195 mg of N-DSK and 18.7 mg of histidine in 11 ml of 70% acetic acid was added 9.5 ml of. a solution of Nbromosuccinimide (18 n&ml)
in the same solvent. This was allowed to stand
Vo1.12,No.6
THROXBIN-FIBRTNOCEX
REACTIOS
95;
at room temperature for 2 h. Following addition of 1 ml of a solution of tryptophan (24.8 mg/ml) in 70% acetic acid, the solution was allowed to stand at room temperature for an additional 16 h. This was then diluted with water to 10% in acetic acid and was freeze-dried. The residue was dissolved in 5 ml of 10% acetic acid and gel-filtered through a column of Sephadex G-50 (Fig 2a). The fractions indicated were pooled and freeze-dried and the recovered material was dissolved in 4 ml of 10% acetic acid and gel-filtered through a column of Bio-Gel P-10 (Fig. 2b). On freeze-drying the fractions indicated yielded 3.5 mg of material, the amino acid composition of which was in agreement with the structure An(l-33)2; see Table II. TABLE II Amino acid analysis of substrates 6-8 obtained after 22 h hydrolysis (7). Half-cystine was determined as S-carboxymethylcysteine. Figures in parenthesis are the theoretical number of residues and those underlined were used as integer. Peptide
. Amino Acid
Aci(l-44)
Half-cystine Aspartic acid Serine Glutamic acid ProTine Glycine Alanine Valine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine
1.85(Z) 6.30(7) 3.11(4) 5.87(5) 1.79(2) 6.30(6) 2.71(3) 3.00(3) 1.52(l) 0.83(l) 1.76(2) 0.70(l) 1.94(2) 3.40(3)
Ao(l-23)
&X(1-33)2 - half molecule
2.24(2) 0.82(l) 2.62(3) 0.68(l) 5.99(6) 1.84(2) 1.46(3) 0.96(l)
0.30(l) 3.63(4) 2.02(3) 4.92(4) 1.11(l) 5.76(6) 2.88(3) 3.00(3) 1.32(l)
1.00(l) 2.58(3)
1.22(l) 0.86(l) 0.75(l) 3.48(3)
Kinetic Techniques The kinetics of the thrombin-catalysed hydrolysis of all substrates was investigated at 37O in 25 mM Tris-HCl, 0.125 M NaCl, pH 7.2. The procedures for following the hydrolysis of substrates l-4 (Table I) have been described in detail elsewhere (7). Of these procedures, "method C" was used in experiments involving substrates 5-8. The various methods consisted of periodically removing aliquots of the digest and subjecting these to NH2-terminal analysis
958
THROXBIN-FIBRIXOGEN
REACTIOS
vol.l2,~0.6
The PTU-amino acids thus obtained in each using 35S -phenyl-isothiocyanate. L aliquot were separated by paper chromatography, the radioactivity was eluted and this was estimated by liquid scintillation. The percentage hydrolysis at each time could thus be calculated by expressing the counts for PTH-Gly as a percentage of those obtained for either PTH-Tyr, in the case of substrates 1-3, or the sum of PTH-Ala and PTH-Asp in the case of substrates 4-8. Velocities at "time zero" were calculated by statistical analysis (14) of these results. As the Be-chain of N-DSK, substrate 9, has a blocked NH2-terminal (lS), it was not possible to measure the appearance of NH*-terminal Gly as a function of this substrate's NH2-terminal. Instead, aliquots of the digestionmixture were added to a known amount of freeze-dried sperm whale apomyoglobin and the NH 2-terminal glycine was measured as a function of the NH*-terminalof this protein, valine (16), according to "method C".
The concentrations in the reaction mixtures of substrates l-4 were determined as described before (7) and those of substrates S-9 were based on their amino acid analysis.
RESULTS AND DISCUSSION The Substrates Chemical data concerning substrates l-4 and substrate 9 are given elsewhere (8,10,15,17). The amino acid compositions of substrates 6-8 are provided in Table II and a two-dimensional peptide map of Ao(l-51) is shown in Fig. 3 FIG. 3 Two-dimensional tryptic peptide map of Ao(l-51). The ninhydrin-positive spots are numbered according to the topography of the corresponding peptides in the intact sequence (Fig. 4). 1:fibrinopeptide A, sequence l-16; 1':fibrinopeptide A where Ser 3 is phosporylated; 2:sequence 17-19; 3:sequence 20-23; 4:sequence24-29; S:sequence 30-44; 6:sequence 45-50; 7:homoserine arising from CNBr-modificationof Met 51. Spots 2 and 6 stained yellow with ninhydrin.
As would be expected, the NH2-terminal analysis of the three substrates listed in Table II showed the presence of Ala with approximately 202 Asp as NH2terminal, The presence of the latter is due to heterogeneity displayed by fibrinopeptide A (8), where the NH2-terminal Ala has been removed to reveal
THROXBIN-FIBRISOGEW
v01.12,~0.6
REACTTOS
959
Asp as the new NR2-terminal. A tryptic in Fig. larly 1-3.
peptide
map of Acz(l-44)
3, verifying
that
a corresponding NH2-terminal
ratio
of Ala + Asp:Gly:Val
that
the preparation
peptide
showed spots
digest
was 1:1:0.7.
the sequence
the spots
together
analysis
of
45-51.Simi-
showed that
with this
of An(l-19)
tol-5
corresponding
of A~z(l-23)
This,
an impurity
slightly
map performed
corresponding
electrophoretic less
Kinetics
to the
the low yield
fragment,
to a level
of
indicates
of
For reasons each of
of
approxi-
l-8
of
the first
four
conclusion cificity
substrates
all
of
are present
of
of AcY(l-51)) fibrinogen
to locate
of
reaction,
for
listed rate
for of re-
9 is a
B. The results
fibrinogen it
0
substrate (7),
the Ac!(l-51)
elements
that this
within
does
of
A was investigated
thespe-
those
which
interaction. the structure
and Au(l-23).
importance
for
the following fragment
which define
is clear
Ao(l-44)
the possible
fibrinopeptide
the initial
in defining
these
of v
this
Since
dimeric
by using
struc-
the isola-
Afl(l-33)2.
The contribution fibrinopeptide
of
the B8-chainof
A was investigated
lated
Act- and B&chain
v.
substrate
by inhibition
of
two sub-fragments,
is a dimer,
of
given at length
that
importance
more precisely
was assumed to corres-
fibrinopeptide
elements
an Rf value
the value
value
been discussed
of major
we isolated
in the region
of
is possible
the structural
a role
itself
ted fragment
it
the fibrinogen-thrombin play
In an attempt
supported
having
(7),
the v.
no
substrates
I is a measure
of release
was drawn: while
not contain
elesewhere
in Table
rate
spot
having
further.
from various
A. Similarly
the initial
dimension
This
and was not examined
which have been given
the substrates
3.
4 in Fig.
Aa(l-33)2
spot was observed
in the second
of fibrinopeptide
of fibrinopeptide
measure
spot
30-33
and S-carboxymethylated
An additional
but having
than that
of release
on reduced
to l-4.
mobility
pond to the sequence
lease
showed only
a tryptic
in the amino acid
contains
of
corresponding
30%.
A tryptic
of
of
the spots
was devoid
map of Pa(l-23)
Val and Glu found
ture
the peptide
analysis
Arg,
mately
showed only
an equimolar
of N-DSK, substrate
5 compared with of
N-DSK to the thrombin-catalysedrelease by using
thrombin
by the knowledge
that
for
by the isolated that
5,
The reduction
substrate BP-chain
fibrinopeptide
solution
4 is
B itself
of
most probably
This proposal
inhibits
of
the iso-
in the value
caused
fragment.
of
is
the actionof
TH-ROMBTN-FIBRISOGES
960
REACTION
Vo1.12,Xo.6
thrombin on fibrinogen (18) and that thrombin, in high concentration, causes release of this peptide from the isolated Bg-chain fragment (5). Thus, it is clear that thrombin binds to structures in the isolated Bg-chain. However, this results in an inefficient release of fibrinopeptide B. The addition of thrombin to a solution of fibrinogen gives rise to a rapid release of fibrinopeptide B following a lag phase during which release of fibrinopeptide A takes place (19). The rapid release of fibrinopeptide B in this case can not be explained by simply assuming that an inaccesible binding site on the BPchain of N-D%
becomes exposed on release of fibrinopeptide A. If this was
true, fibrinopeptide B would also be released from the isolated BB-chain. It is mre
likely that a conformational change occurs in the Bg-chain on release
of fibrinopeptide A and that this architecture is favourable for the thrombin-catalysed release of fibrinopeptide B. Alternatively, the BB-chain as it presents itself in native fibrinogen may be more susceptible to thrombin than the corresponding structure in the isolated chain. In the latter case, efficient release is not possible until the site has been exposed by the release of fibrinopeptide A. Removal of the carboxy-terminal sequence 45-51 from Ao(l-51) (see Fig. 4) to produce Au(l-44), substrate 6, caused a lowering of the v o value from 1.05 to 0.42. It would appear, therefore, that this carboxy-terminal sequence 1 2345610 9 10 11 12 13 14 15 Aio-A~-Ssr-Gly-Glu-Gly-Asp-Phe-Leu-Alo-Glu-Gly-Gly-Gly-~ol-Arg-Cly-Pro-
16
I7 18
t 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 -Arg-Vol-Vo~-Glu-Arg-His-Gln-Ser-Alo-Cyr-Lys-Asp-Ser-Asp-Trp-Pro-Phe-Cy9
I
I
35
4
37 30 39 40 41 42 43 44 45 46 47 40 49 -Ser-Asp-Glu-Asp-Trp-A~n-Tyr-Ly~-Cys-Pro-Ser-Gly-C~~-Arg-Met f FIG.
34
50
36 I
51
I 4
The amino acid sequence of the M-chain of N-DSR, Aa;(l-51). Filled arrow: peptide bond cleaved in the thrombin-catalysed release of fibrinopeptide A; unfilled arrows: carboxy-terminals of substrates 8, 7 and 6, respectively. (From Blomblck et al., see ref. 8.) plays some part in the thrombin-catalysed release of fibrinopeptide ASurthetmore, this function may be the why and wherefore this very sequence has been strongly preserved during marmnalian evolution (20,Zl). Since this segmentcon-
\-ot.
12,X0.6
THROYBIW-FTBRTliC’GEN
tains
two of
that
modification
Thus,
as
was
fibrinogen
the
rate of
by the
0.06
in
region
that of
connection, substrate
2 that
a significant
An attempt
Arg16-Gly17
i.e.
the
were
several
ded,
that
highly
sequence
some
lower
obtained as
at
the
for
a substrate
there
the
8-16)
of
has
residue
thrombin-susceptible
not
in
former
lower
nine
that
sequence
are
lower
than
may exist in
either
the or
segment,
of is
the
situated bond
both
of
the
fact
suggestion
in
and
in
the
the
that
of
the
this
and
thrombin-
were
examined,
these
out
the
was
same the
for
this
order
and
of
Furthermore,
(25)
than
In
a
se-
con-
portion(remammalian
importance since
prior Phe
con-
the
21-23.
during
residues
magni-
fibrinopeptide
carboxy-terminal
secretin
value of
to of
is
the
specificity
by much more
stable
conclu-
voobtainedfor Since
l-11
using
substrates It
to account of
structure
nine
Per se.
the
release
rather
(19,24).
for
do not,
Acz(l-23),extrinsic
that
be
this
carried
that
segments
sequence
An(l-51)
In
been
fibrinogen in
remained
substrate
both
for
this
33.
thrombin-catalysed
A has
enzyme
that
of
fibrinogen.
conceivable
elements
structure
around
value
is
thevacan
necessary
which
since it
for
for
a factorof
is
imPOr-
different
subsites
constants
than
0.08.
A (7).
has
interaction.The
it
considerable
fibrinogen
sequence
specificity
this
fibrinopeptide
prompted
association
nylalanine
the
concentration
function
may reside
evolution rapid
thrombin
than thatforfibrinogenby
is
with
sidues
of
outside
lO’.Thus
12-20,which
These
site amino
yet
linked of
event.
active
all,
being
v o obtained
of
a v.
value
of
on
In
have
of
fibrinopeptide
bond.
to
value
bonds
acid
reduction
course
the
of
the
residues.
a v.
dimeric
this
for to
Furthermore,
release
magnitude
Aa(l-23)
quence
in
from
disulphide
Kmforfibrinogen(23),
of
nection
role
have
significantly the
0
found
be of A.
A.
values
significant
to
must
v
two halves
the
nor
inferred
fibrinogen-thrombin
factor
A.
of
factors
specific
a direct
peptide
12-20,
orders
Ao(l-23)is
stant
based
not
24-33
the
map the
substrates
susceptible
tude
to
are
sequence
been
in
34-44
fibrinopeptide the
was
found
expected
half-cystine
more
Acr(l-33),
be
susceptibility
these
fibrinopeptide
symmetrical
role
(22)
synthetic
was
already
the
of
substrates
of
A?(l-23)
was
would
in
reduced
an even A:
sequence
release
plays
has
caused
of
release
of
961
it
difference from
Aa28-Aa28,
the
fibrinogen
the
the
resulted
version
the
two
neither
it
(7),
fibrinopeptide
that the
A:r(l-51),
influence
Al-chain
disulphide
these
in
carboxymethylation
a dimeric
this
for
v.
concluded
play
of
effecting
of
by
release
from
would
may have
the
and
found
suggested
of
symmetrical
tance
these
about
of
follows
lues
of
shortening
value
halfcystines
previously
brought
Further
It
four
and Aa(l-51)
thrombin
in
the
REACTION
to 9 is
for
a phethe held
to
THROMBIN-FIBRINOGEN
962
be
a
thrombin-binding
is provided tide
in
covering
thrombin
than
the
site finding
(24.26). by
8-23
the
sequence
the
Corresponding
support
Additional
Scheraga
is
~01.12,~~.6
REACTIOX
(27)
a much
sequence
that
better
for
this
PeD,
a synthetic substrate
for
X0-23.
The data presented here is consistant with the proposal that the structural elements of fibrinogen which define the thrombin-catalysed release of fibrinopeptide A are contained within pU(l-51). These elements are confirmed to three regions of this structure, namely, the sequences l-23, 34-44 and 4445, the latter being of least significance. We are not at liberty to decide whether these complete sequences or only parts of them are factors determining the release of fibrinopeptide A. For example, it is evident from the above discussion of An(l-23) that the sequence 8-16 plays a significant role in this process but it is not known if this sequence contains all of the contributing structural elements within Pa(l-23).
It is proposed here that the binding between fibrinogen and thrombin which is fundamental for the subsequent release of fibrinopeptide A, takes place within the sequence l-23. The region operative in this binding may even be confined within the sequence 8-16. This interaction is strengthened either directly or indirectly by structural elements within the sequence 34-44 and to a lesser extent by elements within the sequence 45-51. The strengthening may be due to elements tlithin these two sequences being offered as binding sites for thrombin, or it may be caused indirectly by these elements interacting with structures within the sequence l-23 to hold this in a particular conformation which is preferred by thrombin.
ACKNOWLEDGEMENTS This work was supported by grants from The Swedish Medical Research Council (No. 13X-2475-1OC) and the National Institutes of Health, Bethesda, Md. (No. HL 07379-11). One of us, DHH, wishes to acknowledge the support provided by a Wellcome Trust Travelling Research Fellowship. The excellent technical assistance of Mrss. Elvy Andersson, Helga Messel, Sonja Soderman, Lisbeth Therkildsen, Lena WikstrBm, Wolfgang Finkbeiner, Nils Grondahl and Peter Wolf is acknowledged.
Vo1.12,80.6
THHOMBTN-FIBRINOGEN
REACTTOY
943
REFERENCES 1.
BAILEY, K. and BETTELHEIM, F.R. Nature of fibrinogen-thrombin reaction. Brit. med. Bull. 11, 50, 1955.
2.
BLOMBACK, B. and YAMASHINA, I. On the N-terminal amino acids in fibrinogen and fibrin. Arkiv Kemi 12, 299, 1958.
3.
WALLiN, P. and IWANAGA, S. Differences between plasmic and tryptic digests of human S-sulfo-fibrinogen. Biochim. Biophys. Acta 154, 414, 1968.
4.
PECHET, L. and ALEXANDER, B. The effect of certain proteolytic enzymes on the thrombin-fibrinogen interaction. Biochemistry 1, 875, 1962.
5.
BLOMB&K, B., BLOMBiiCK,M., HESSEL, B. and IWANAGA, S. Structure of Nterminal fragments of fibrinogen and specificity of thrombin. Nature 215, 1445, 1967.
6.
ANDREATTA, R.H., LIEM, R.K. and SCHERAGA, H.A. Mechanism of action of thrombin on fibrinogen. Proc. Nat. Acad. Sci., U.S. 68, 253, 1971.
7.
HOGG, D.H. and BLOMBACK, B. The specificity of the fibrinogen-thrombin reaction. Thrombosis Research 5, 685, 1974.
a. BLOMBLCK, B., HESSEL, B., IWANAGA, S., REUTERBY, J. and BLOMEACK, M. Primary structure of human fibrinogen and fibrin.I.Cleavage of fibrinogen with cyanogen bromide. Isolation and characterization of NH terminal fragments of the o("A")-chain. J. Biol. Chem. 247, 1496,1972 9.
BLOMBACK, B., GRBNDAHL, N.J., HESSEL, B., IWANAGA, S. and WALLEN, I'. Primary structure of human fibrinogen and fibrin. II. Structural studies on NH2-terminal part of y-chain. J. Biol. Chem. 248, 5806,1973.
10.
HESSEL, B., MARINO, M., IWANAGA, S. and BLOMB&CK, B. Primary structure of human fibrinogen and fibrin. In preparation.
11.
LEVY, A.L. and CHUNG, D. Two-dimensional chromatography of amino acids on buffered papers. Anal. Chem. 25, 396, 1953.
12.
SJbQUIST, J., BLOMB%K, B. and WALLiN, P. Amino acid sequence of bovine fibrinopeptides. Arkiv Kemi 16, 425, 1960.
13.
RAMACHANDRAN, L.K. and WITKOP, B. N-Bromosuccinimide cleavage of peptides. In: Methods in Enzymology, Vol. XI, C.H.W. Hirs, Ed., Academic Press, New York, 1967, p. 283.
14.
BOOMAN, K.A. and NIEMANN, C. The empirical evaluation of the initial velocities of enzyme-catalyzed reactions. J. Amer. Chem. Sot. 78, 3642, 1956.
15.
BLOMBACK, B. and BLOMB;iCK,M. The molecular structure of fibrinogen. Ann. N.Y. Acad. Sci. 202, 77, 1972.
964
THROMEXB-FIBRISOCES
REACTION
Vol.t2,xo.6
16.
ED*MUNSON, X.B. Amino-acid sequence of sperm whale myoglobin. Nature. 205, 883, 965.
17.
BLOMBACK, B., BLOMBXCK, M., FINKBEINER, W., HOLMGREN, A. KOWALSKcZ-LOTH, B. and OLOVSON, G. Enzymatic reduction of disulfide bonds in fibrinogen by the thioredoxin system. Thrombosis Research 4, 55, 1974.
18.
BLOMBACK, B. Chemical aspects of fibrinogen and fibrin. Thronb. Diath. Haem. Suppl. 13, 29, 1964.
19.
BLOMBXCK, B. Fibrinogen to fibrin transformation. In: Blood Clotting Enzymology. W.H. Seegers (Ed.), New York and London, Academic Press, 1967, p 143.
20.
StiDERQVIST,T. and BLOMBXCK, B. Evolutionary constancy of primary strut ture in an o(A)-chain fragment of fibrinogen. Febs. Letters. 7, 321, 1970.
21.
SFDERQVIST, T. and BLOMBACK, 8. Fibrinogen structure and evolution. Naturwissenschaften 58, 16, 1971.
22.
LIEM, R.K. and SCHERAGA, H.A. Mechanism of action of thrombin on fibrinogen. IV. Further mapping of the active sites of thrombin and trypsin. Arch. Biochem. Biophys. 160, 333, 1974.
23.
BANDO, M.. MATSUSHIMA, A., HIRANO, J. and INADA, Y. Thrombin-catalyzed conversion of fibrinogen to fibrin. J. Biochem. 71, 897, 1972.
24.
BLOMBACK, B. and BLOMBiiCK, M. Primary structure of animal proteins as a guide in taxonomic studies. In: Chemotaxonomy and Serotaxonomy, J.G. Hawkes (Ed.), London, Academic Press 1968, p. 3.
25.
MUTT, V., MAGNUSSON, S., JORPES, J.E. and DAHL, E. Structure of porcine secretin. Biochemistry 4, 2358, 1965.
26.
BLOMBACK, B. The chemistry of proteases. Ann. N.Y. Acad. Sci., 146, 364, 1968.
27.
SCHERAGA,H.A. Active site mvping of thrombin. In: Chemistrv and Biologv of Thromhin. H.L. Lllndhlad, I.W. Fenton and K.G. Mann (Eds.). Ann Arbor. MI, Ann Arbor Science, 1977, p. 145.
THE MECHANISM CF THE FIBRINOGEN-THROMBIN WCTION
Desmond H. Hogg and Birger BlombPck Department of Blood Coagulation Research, Karolinska Institutet, Stockholm, Sweden
(Received 20.1.1978. Accepted hy Received by Executive Editorial
Editor Office
S. Magnusson) 1.5.1978)
ABSTRACT The kineticsof the thrombin-catalysed release of fibrinopeptide A from fibrinogen, modified fibrinogen and various fragments of fibrinogen has been investigated in an attempt to elucidate the structural elements of fibrinogen which define its interaction with thrombin. These elements have been found to be contained within the structure formed by the first 51 amino acid residues of the &-chain. It is proposed that the binding which is of fundamental importance takes place within the sequence 1-23 of this chain and may even be localised to the sequence 8-16. This binding is strengthened, either directly or indirectly, by structures within the sequence 34-44 and to a lesser extent by structures within the sequence 45-51.
INTRODUCTION Thrombin catalyses the release of fibrinopeptides A and B from fibrinogen (1,2). In so doing, the enzyme cleaves only four specific arginyl-glycyl peptide bonds in the fibrinogen dimer, whereas trypsin, a closely related serine proteinase, cleaves almost all arginyl and lysyl peptide bonds in fibrinogen (3,4). To explain the difference between the specificities exhibited by the two enzymes, it has been suggested (5,6) that thrombin has an extraordinarily hidden active site which for steric reasons can react only with the N?i2-terminal portions of the Acr- and Be-chains. In a previous report (7) we concluded that the structural elements of fibrinogen which are of major importance in defining the thrombin-catalysed release of fibrinopeptide A, are located within the architecture formed by the first 51 amino acid 957
THROMEIN-FIBRINOGEN
954
REACTION
Vo1.12,No.6
residues of the h-chain. In this communication,we report findings concerning locality cf these thrombin-bindingelements within this architecture.
MATERIALS AND METHODS Bovine thronbinwas purified to an activity of 1,800 NIH units/m8 by the procedure described previously (7). Trypsin, TPCK-treated,was obtained from WorthingtonBiochemicalCorporationand sperm whale apomyoglobinwas supplied by Beckman InstrumentsInternationalS.A.
Human plasmin, having an activity
of lo-15 caseinolytic(CTAl) units/mg, was kindly supplied by Dr. Bjarn Wiman, Dept. of Medical Chemistry, University of Umei, Sweden. Tryptic digests, two dimensionalpeptide maps and amino acid analysis were carried out as described before (8). The NH2-terminalswere analysed as described in a previous communication(7). Preparationof fragments of fibrinogenused as subsrrates.
;=
The various substratesused in this investigationare listed in Table I.
TABLE I Initial rates of the thrombin-catalysedappearanceof NH2-terminal glycine in various substrates at 370C in 25 mM Tris-Xl, 0.125 M NaCl, pH 7.2. Results for substrates l-4 have previouslybeen published elsewhere (7).
Substrate Substrate No. 1 2
3 4 5
Fibrinogen Thioredoxintreated fibrinogen N-DSK Ao(l-51) AcY(l-51) BB(f-118) An(l-44) &i(l-3312 Ao(l-23) BB(l-118)
Molecular Molarity weight uM
10 (mol NI?l Zc-1)
20 25
2.00 t 0.14 2.62 + 0.13
5,500 5,500
20 40 40
1.48 2 0.17 1.05 f 0.07
12,200 4,800 7,900 2,300 12,200
40 40 20 40 40
340,000 340,000
59,000
'Footnote: CTA = Committee on ThrombolyticAgents
0.55 + 0.01 0.42 z 0.07 0.08 _=0.01 0.06 - 0.01 0
Vol.12,No.h
THROYBI4-FIBRINOGEN
REACTIOS
955
Fibrinogen, thioredoxin-treated fibrinogen, N-DSK and Acr(l-51) were prepared as described previously (7). The B8-chain of N-DSK, B8(1-118), was isolated from reduced and S-carboxymethylated N-DSK by gel-filtration (9) and this was further purified by chromatography on CM-Cellulose (9.10). Two sub-fragments of the Pa-chain of N-DSK, h(l-44)
and pCr(l-23)were pre-
pared by digesting this chain with plasmin. To a solution of 22 mg of Pa(l51) in 1 ml of 0.2 M NH4HCO3, pH 8.2, was added 0.7 mg of human plasmin. The mixture was incubated at 37' for 2 h, 0.1 ml of glacial acetic acid was then added and the clear solution was gel-filtered on a column of Sephadex G-25 (Fig. la). The material recovered from peak 1 was chromatographed on G-100 to remove plasmin and yielded 11.5 mg of freeze-dried &(l-44).
FIG. 1
1.5-
,/‘.
: ;
1.0 -
:
-r
-,: :
Gel-filtration of plasmin digested An(l-51) on a column (82 cm x 1.8 cm*) of Sephadex G-25 in 10% acetic acid. Flow rate: 15 ml/h; fractions: 2 ml. a) aftek a 2 h digestion; b) after an 18 h digestion, inset: thin-layer chromatography of material recovered from peak 2 with arrow indicating the spot found to contain Aa(l-23).In both diagrams, the final peak of radioactivity having no absorbance at 280 ran is due to the peptide segment 45-50 (see Fig. 4).
The smaller fragment, Pa(l-23), was obtained on prolonged digestion of Acr (l-51) with plasmin. To a solution of 36 mg of Ao(l-51) in 2 ml of 0.2 M NH4HCG3, P H 8.2, wad added 0.8 mg of human plasmin. The mixture
was incuba-
ted at 37' for 18 h and after addition of 200 ul of glacial acetic acid the clear solution was gel-filtered on a column of Sephadex G-25 (Fig. lb). The second peak, which was pooled as indicated, yielded 15.5 mg of freeze-dried material. Thin-layer chromatography of this material on cellulose (Whatman CC41) in the medium l-butanol-pyridine-acetic acid-water (150:100:30:120v~v)
956
THROWBTN-FIBRISOGE3
REX-l-TON
vo1.12,so.6
produced the ninhydrin-positive pattern shown in the inset in Fig. lb. Amino acid and NH*-terminal analysis of the material eluted from the spots indicated that the arrowed spot in Fig. lb contained &(l-23).
To isolate this
fragment on a preparative scale, 70 )~l of an aqueous solution of 6 mg of the material obtained from the above gel-filtration were applied as a streak to a 0.5 nm thick layer of cellulose on a 20x2@ cm plate. The plate was developed in the same medium as was used above and was air dried. The band corresponding to the arrowed spot in Fig. lb was located by spraying the two vertical edges of the thin layer with a ninhydrin-collodine solution (11) and allowing this to develop for 1 h at room temperature. The appropriate band of cellulose was then scraped from the plate and the peptide material (0.8 mg) was recovered by three one-hour long extractions with 2 ml of 10% acetic acid at room temperature.
30
40
50
60
TO
80
sfacl,o!. Nurntm
FIG. 2 a) Gel-filtration cf N-bromosuccinimide treated N-DSK on a column (95 cm x 5 cm 2, of Sephadex G-50 in 10% acetic acid. Flow rate: 20 ml/h; fractions: 5 ml. b)Gelfiltration of material recovered from "a" on a column (82 cm x 5 cm2) of Bio-Gel P-10 in 10% acetic acid. Flow rate: 15 ml/h; fractions: 5 ml. Fractions in "b" were subjected to ninhydrin analysis (12).
*or
A small dimeric fragment of fibrinogen, &~(1-33)~, the two halves being joined by the symmetrical disulphide Aa28-&28, the tryptophanyl peptide bonds (h33 nimide. Amodificationof
was prepared by cleaving
and Aa44) in N-DSK with N-bromosucci-
a procedure which has been described (13) wasused.
To minimise the modification of Pa24 His, free histidine was incorporated in the reaction mixture. To a solution of 195 mg of N-DSK and 18.7 mg of histidine in 11 ml of 70% acetic acid was added 9.5 ml of. a solution of Nbromosuccinimide (18 n&ml)
in the same solvent. This was allowed to stand
Vo1.12,No.6
THROXBIN-FIBRTNOCEX
REACTIOS
95;
at room temperature for 2 h. Following addition of 1 ml of a solution of tryptophan (24.8 mg/ml) in 70% acetic acid, the solution was allowed to stand at room temperature for an additional 16 h. This was then diluted with water to 10% in acetic acid and was freeze-dried. The residue was dissolved in 5 ml of 10% acetic acid and gel-filtered through a column of Sephadex G-50 (Fig 2a). The fractions indicated were pooled and freeze-dried and the recovered material was dissolved in 4 ml of 10% acetic acid and gel-filtered through a column of Bio-Gel P-10 (Fig. 2b). On freeze-drying the fractions indicated yielded 3.5 mg of material, the amino acid composition of which was in agreement with the structure An(l-33)2; see Table II. TABLE II Amino acid analysis of substrates 6-8 obtained after 22 h hydrolysis (7). Half-cystine was determined as S-carboxymethylcysteine. Figures in parenthesis are the theoretical number of residues and those underlined were used as integer. Peptide
. Amino Acid
Aci(l-44)
Half-cystine Aspartic acid Serine Glutamic acid ProTine Glycine Alanine Valine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine
1.85(Z) 6.30(7) 3.11(4) 5.87(5) 1.79(2) 6.30(6) 2.71(3) 3.00(3) 1.52(l) 0.83(l) 1.76(2) 0.70(l) 1.94(2) 3.40(3)
Ao(l-23)
&X(1-33)2 - half molecule
2.24(2) 0.82(l) 2.62(3) 0.68(l) 5.99(6) 1.84(2) 1.46(3) 0.96(l)
0.30(l) 3.63(4) 2.02(3) 4.92(4) 1.11(l) 5.76(6) 2.88(3) 3.00(3) 1.32(l)
1.00(l) 2.58(3)
1.22(l) 0.86(l) 0.75(l) 3.48(3)
Kinetic Techniques The kinetics of the thrombin-catalysed hydrolysis of all substrates was investigated at 37O in 25 mM Tris-HCl, 0.125 M NaCl, pH 7.2. The procedures for following the hydrolysis of substrates l-4 (Table I) have been described in detail elsewhere (7). Of these procedures, "method C" was used in experiments involving substrates 5-8. The various methods consisted of periodically removing aliquots of the digest and subjecting these to NH2-terminal analysis
958
THROXBIN-FIBRIXOGEN
REACTIOS
vol.l2,~0.6
The PTU-amino acids thus obtained in each using 35S -phenyl-isothiocyanate. L aliquot were separated by paper chromatography, the radioactivity was eluted and this was estimated by liquid scintillation. The percentage hydrolysis at each time could thus be calculated by expressing the counts for PTH-Gly as a percentage of those obtained for either PTH-Tyr, in the case of substrates 1-3, or the sum of PTH-Ala and PTH-Asp in the case of substrates 4-8. Velocities at "time zero" were calculated by statistical analysis (14) of these results. As the Be-chain of N-DSK, substrate 9, has a blocked NH2-terminal (lS), it was not possible to measure the appearance of NH*-terminal Gly as a function of this substrate's NH2-terminal. Instead, aliquots of the digestionmixture were added to a known amount of freeze-dried sperm whale apomyoglobin and the NH 2-terminal glycine was measured as a function of the NH*-terminalof this protein, valine (16), according to "method C".
The concentrations in the reaction mixtures of substrates l-4 were determined as described before (7) and those of substrates S-9 were based on their amino acid analysis.
RESULTS AND DISCUSSION The Substrates Chemical data concerning substrates l-4 and substrate 9 are given elsewhere (8,10,15,17). The amino acid compositions of substrates 6-8 are provided in Table II and a two-dimensional peptide map of Ao(l-51) is shown in Fig. 3 FIG. 3 Two-dimensional tryptic peptide map of Ao(l-51). The ninhydrin-positive spots are numbered according to the topography of the corresponding peptides in the intact sequence (Fig. 4). 1:fibrinopeptide A, sequence l-16; 1':fibrinopeptide A where Ser 3 is phosporylated; 2:sequence 17-19; 3:sequence 20-23; 4:sequence24-29; S:sequence 30-44; 6:sequence 45-50; 7:homoserine arising from CNBr-modificationof Met 51. Spots 2 and 6 stained yellow with ninhydrin.
As would be expected, the NH2-terminal analysis of the three substrates listed in Table II showed the presence of Ala with approximately 202 Asp as NH2terminal, The presence of the latter is due to heterogeneity displayed by fibrinopeptide A (8), where the NH2-terminal Ala has been removed to reveal
THROXBIN-FIBRISOGEW
v01.12,~0.6
REACTTOS
959
Asp as the new NR2-terminal. A tryptic in Fig. larly 1-3.
peptide
map of Acz(l-44)
3, verifying
that
a corresponding NH2-terminal
ratio
of Ala + Asp:Gly:Val
that
the preparation
peptide
showed spots
digest
was 1:1:0.7.
the sequence
the spots
together
analysis
of
45-51.Simi-
showed that
with this
of An(l-19)
tol-5
corresponding
of A~z(l-23)
This,
an impurity
slightly
map performed
corresponding
electrophoretic less
Kinetics
to the
the low yield
fragment,
to a level
of
indicates
of
For reasons each of
of
approxi-
l-8
of
the first
four
conclusion cificity
substrates
all
of
are present
of
of AcY(l-51)) fibrinogen
to locate
of
reaction,
for
listed rate
for of re-
9 is a
B. The results
fibrinogen it
0
substrate (7),
the Ac!(l-51)
elements
that this
within
does
of
A was investigated
thespe-
those
which
interaction. the structure
and Au(l-23).
importance
for
the following fragment
which define
is clear
Ao(l-44)
the possible
fibrinopeptide
the initial
in defining
these
of v
this
Since
dimeric
by using
struc-
the isola-
Afl(l-33)2.
The contribution fibrinopeptide
of
the B8-chainof
A was investigated
lated
Act- and B&chain
v.
substrate
by inhibition
of
two sub-fragments,
is a dimer,
of
given at length
that
importance
more precisely
was assumed to corres-
fibrinopeptide
elements
an Rf value
the value
value
been discussed
of major
we isolated
in the region
of
is possible
the structural
a role
itself
ted fragment
it
the fibrinogen-thrombin play
In an attempt
supported
having
(7),
the v.
no
substrates
I is a measure
of release
was drawn: while
not contain
elesewhere
in Table
rate
spot
having
further.
from various
A. Similarly
the initial
dimension
This
and was not examined
which have been given
the substrates
3.
4 in Fig.
Aa(l-33)2
spot was observed
in the second
of fibrinopeptide
of fibrinopeptide
measure
spot
30-33
and S-carboxymethylated
An additional
but having
than that
of release
on reduced
to l-4.
mobility
pond to the sequence
lease
showed only
a tryptic
in the amino acid
contains
of
corresponding
30%.
A tryptic
of
of
the spots
was devoid
map of Pa(l-23)
Val and Glu found
ture
the peptide
analysis
Arg,
mately
showed only
an equimolar
of N-DSK, substrate
5 compared with of
N-DSK to the thrombin-catalysedrelease by using
thrombin
by the knowledge
that
for
by the isolated that
5,
The reduction
substrate BP-chain
fibrinopeptide
solution
4 is
B itself
of
most probably
This proposal
inhibits
of
the iso-
in the value
caused
fragment.
of
is
the actionof
TH-ROMBTN-FIBRISOGES
960
REACTION
Vo1.12,Xo.6
thrombin on fibrinogen (18) and that thrombin, in high concentration, causes release of this peptide from the isolated Bg-chain fragment (5). Thus, it is clear that thrombin binds to structures in the isolated Bg-chain. However, this results in an inefficient release of fibrinopeptide B. The addition of thrombin to a solution of fibrinogen gives rise to a rapid release of fibrinopeptide B following a lag phase during which release of fibrinopeptide A takes place (19). The rapid release of fibrinopeptide B in this case can not be explained by simply assuming that an inaccesible binding site on the BPchain of N-D%
becomes exposed on release of fibrinopeptide A. If this was
true, fibrinopeptide B would also be released from the isolated BB-chain. It is mre
likely that a conformational change occurs in the Bg-chain on release
of fibrinopeptide A and that this architecture is favourable for the thrombin-catalysed release of fibrinopeptide B. Alternatively, the BB-chain as it presents itself in native fibrinogen may be more susceptible to thrombin than the corresponding structure in the isolated chain. In the latter case, efficient release is not possible until the site has been exposed by the release of fibrinopeptide A. Removal of the carboxy-terminal sequence 45-51 from Ao(l-51) (see Fig. 4) to produce Au(l-44), substrate 6, caused a lowering of the v o value from 1.05 to 0.42. It would appear, therefore, that this carboxy-terminal sequence 1 2345610 9 10 11 12 13 14 15 Aio-A~-Ssr-Gly-Glu-Gly-Asp-Phe-Leu-Alo-Glu-Gly-Gly-Gly-~ol-Arg-Cly-Pro-
16
I7 18
t 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 -Arg-Vol-Vo~-Glu-Arg-His-Gln-Ser-Alo-Cyr-Lys-Asp-Ser-Asp-Trp-Pro-Phe-Cy9
I
I
35
4
37 30 39 40 41 42 43 44 45 46 47 40 49 -Ser-Asp-Glu-Asp-Trp-A~n-Tyr-Ly~-Cys-Pro-Ser-Gly-C~~-Arg-Met f FIG.
34
50
36 I
51
I 4
The amino acid sequence of the M-chain of N-DSR, Aa;(l-51). Filled arrow: peptide bond cleaved in the thrombin-catalysed release of fibrinopeptide A; unfilled arrows: carboxy-terminals of substrates 8, 7 and 6, respectively. (From Blomblck et al., see ref. 8.) plays some part in the thrombin-catalysed release of fibrinopeptide ASurthetmore, this function may be the why and wherefore this very sequence has been strongly preserved during marmnalian evolution (20,Zl). Since this segmentcon-
\-ot.
12,X0.6
THROYBIW-FTBRTliC’GEN
tains
two of
that
modification
Thus,
as
was
fibrinogen
the
rate of
by the
0.06
in
region
that of
connection, substrate
2 that
a significant
An attempt
Arg16-Gly17
i.e.
the
were
several
ded,
that
highly
sequence
some
lower
obtained as
at
the
for
a substrate
there
the
8-16)
of
has
residue
thrombin-susceptible
not
in
former
lower
nine
that
sequence
are
lower
than
may exist in
either
the or
segment,
of is
the
situated bond
both
of
the
fact
suggestion
in
and
in
the
the
that
of
the
this
and
thrombin-
were
examined,
these
out
the
was
same the
for
this
order
and
of
Furthermore,
(25)
than
In
a
se-
con-
portion(remammalian
importance since
prior Phe
con-
the
21-23.
during
residues
magni-
fibrinopeptide
carboxy-terminal
secretin
value of
to of
is
the
specificity
by much more
stable
conclu-
voobtainedfor Since
l-11
using
substrates It
to account of
structure
nine
Per se.
the
release
rather
(19,24).
for
do not,
Acz(l-23),extrinsic
that
be
this
carried
that
segments
sequence
An(l-51)
In
been
fibrinogen in
remained
substrate
both
for
this
33.
thrombin-catalysed
A has
enzyme
that
of
fibrinogen.
conceivable
elements
structure
around
value
is
thevacan
necessary
which
since it
for
for
a factorof
is
imPOr-
different
subsites
constants
than
0.08.
A (7).
has
interaction.The
it
considerable
fibrinogen
sequence
specificity
this
fibrinopeptide
prompted
association
nylalanine
the
concentration
function
may reside
evolution rapid
thrombin
than thatforfibrinogenby
is
with
sidues
of
outside
lO’.Thus
12-20,which
These
site amino
yet
linked of
event.
active
all,
being
v o obtained
of
a v.
value
of
on
In
have
of
fibrinopeptide
bond.
to
value
bonds
acid
reduction
course
the
of
the
residues.
a v.
dimeric
this
for to
Furthermore,
release
magnitude
Aa(l-23)
quence
in
from
disulphide
Kmforfibrinogen(23),
of
nection
role
have
significantly the
0
found
be of A.
A.
values
significant
to
must
v
two halves
the
nor
inferred
fibrinogen-thrombin
factor
A.
of
factors
specific
a direct
peptide
12-20,
orders
Ao(l-23)is
stant
based
not
24-33
the
map the
substrates
susceptible
tude
to
are
sequence
been
in
34-44
fibrinopeptide the
was
found
expected
half-cystine
more
Acr(l-33),
be
susceptibility
these
fibrinopeptide
symmetrical
role
(22)
synthetic
was
already
the
of
substrates
of
A?(l-23)
was
would
in
reduced
an even A:
sequence
release
plays
has
caused
of
release
of
961
it
difference from
Aa28-Aa28,
the
fibrinogen
the
the
resulted
version
the
two
neither
it
(7),
fibrinopeptide
that the
A:r(l-51),
influence
Al-chain
disulphide
these
in
carboxymethylation
a dimeric
this
for
v.
concluded
play
of
effecting
of
by
release
from
would
may have
the
and
found
suggested
of
symmetrical
tance
these
about
of
follows
lues
of
shortening
value
halfcystines
previously
brought
Further
It
four
and Aa(l-51)
thrombin
in
the
REACTION
to 9 is
for
a phethe held
to
THROMBIN-FIBRINOGEN
962
be
a
thrombin-binding
is provided tide
in
covering
thrombin
than
the
site finding
(24.26). by
8-23
the
sequence
the
Corresponding
support
Additional
Scheraga
is
~01.12,~~.6
REACTIOX
(27)
a much
sequence
that
better
for
this
PeD,
a synthetic substrate
for
X0-23.
The data presented here is consistant with the proposal that the structural elements of fibrinogen which define the thrombin-catalysed release of fibrinopeptide A are contained within pU(l-51). These elements are confirmed to three regions of this structure, namely, the sequences l-23, 34-44 and 4445, the latter being of least significance. We are not at liberty to decide whether these complete sequences or only parts of them are factors determining the release of fibrinopeptide A. For example, it is evident from the above discussion of An(l-23) that the sequence 8-16 plays a significant role in this process but it is not known if this sequence contains all of the contributing structural elements within Pa(l-23).
It is proposed here that the binding between fibrinogen and thrombin which is fundamental for the subsequent release of fibrinopeptide A, takes place within the sequence l-23. The region operative in this binding may even be confined within the sequence 8-16. This interaction is strengthened either directly or indirectly by structural elements within the sequence 34-44 and to a lesser extent by elements within the sequence 45-51. The strengthening may be due to elements tlithin these two sequences being offered as binding sites for thrombin, or it may be caused indirectly by these elements interacting with structures within the sequence l-23 to hold this in a particular conformation which is preferred by thrombin.
ACKNOWLEDGEMENTS This work was supported by grants from The Swedish Medical Research Council (No. 13X-2475-1OC) and the National Institutes of Health, Bethesda, Md. (No. HL 07379-11). One of us, DHH, wishes to acknowledge the support provided by a Wellcome Trust Travelling Research Fellowship. The excellent technical assistance of Mrss. Elvy Andersson, Helga Messel, Sonja Soderman, Lisbeth Therkildsen, Lena WikstrBm, Wolfgang Finkbeiner, Nils Grondahl and Peter Wolf is acknowledged.
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