Vol. 90, No. 3, 1979 October
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
12, 1979
Pages
963-968
MECHANISM OF INHIBITION OF THROMBIN-INDUCED PLATELET AGGREGATION BY PYRIDOXAL PHOSPHATE Elizabeth
August
and Harold
Feinberg
Department of Pharmacology of Illinois at the Medical Chicago, Illinois 60680
University
Received
Kornecki
Center
24,1979
Summary Thrombin and ADP-induced platelet aggregation are reversibly inhibited by pyridoxal phosphate. Sodium borohydride converts Schiff bases formed between pyridoxal phosphate and amino groups to covalent bonds. When platelets treated with sodium borohydride and pyridoxal phosphate are resuspended in fresh platelet-poor plasma, they recover their response to thrombin, but not to ADP. Thus Schiff base formation between pyridoxal phosphate and platelet surface amino groups does not block thrombin aggregation. The loss of thrombin potency as an aggregating agent is due to interaction between pyridoxal phosphate and thrombin. This is evidenced by spectrophometric determination of adduct formation and loss of hydrolytic action on p-tosyl-L-arginine methyl ester. Blood
platelets
epinephrine,
are
collagen
granular
contents.
sites
interact
with
phate
(PLP),
other
There
the
form
inhibits
by acting
separate
site
or by reacting
present
report
demonstrates
on a site
face
Corollaries membrane
amino
Materials
groups
agonists of vitamin
directly
with
(l-4).
is
finding
interfere
We found
are
with
pyridoxal
(5).
On the
PLP may inhibit by acting
to inactivate
by adduct
for
formation
1) ADP and thrombin of Schiff
bases
action
with
of thrombin
phos-
of platelets
or A23187.
ADP and thrombin,
the
membrane
that
responsible
ADP,
and secrete
aggregation acid
thrombin
(e.g.,
platelet
aggregation
the mechanism
and 2) formation
does not
separate
arachidonic
aggregation to this
sites,
that
agonists
aggregate
B6, inhibits
common to both
that
by PLP of thrombin-induced
platelet
evidence
shape,
thrombin-induced
thrombin
thrombin.
is
not to epinephrine,
hand PLP also
endogenous
to change
different
the coenzyme
to ADP but
by various
and thrombin)
their
exposed
activated
it. the
The
inhibition
between act
on a
PLP and
on separate
platelet
sur-
on platelets.
and Methods
Human platelet-rich plasma was prepared as previously described (6). Platelets in plasma treated with EDTA (7.5 mM) were centrifuged at 365 xg for 0006-291X/79/190963-06$01.00/0
963
Copyright All rights
@ 1979 by Academic Press. Inc. oj’reproduction in any formreserr~ed
Vol. 90, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
15 min and resuspended either in a calcium and albumin-free Tyrodes solution (NaCl, 136; KCl, 2.7; NaH2P04, 0.4; NaHC03, 1.2; MgCl, 1.19 and glucose, 5.5; in mM) or in platelet-poor plasma obtained by centrifuging platelet-rich plasma at 2000 xg for 15 min. Platelet aggregation was measured at 370C by methods previously described (6). Pyridoxal phosphate, imipramine, and p-tosyl-L-arginine methyl ester HCl (TAME) and sodium borohydride were obtained from Sigma Chemical Co., St. Louis, Missouri. 5-hydroxy [side chain-214C] tryptamine creatinine sulfate, 58 mCi/mmole [14C]-serotonin was obtained from Amersham/Searle Corp., Arlington Heights, Illinois. Human thrombin was obtained from two sources. Commercial human thrombin was purchased from Ortho Diagnostics, Raritan, New Jersey. Highly purified thrombin (specific activity = 2,513 units/mg) was the gift of Dr. John W. Fenton, II, New York State Department of Health, Albany, New York. Thrombin and TAME were dissolved in distilled water and adjusted to pH 7.4 with phosphate was initially dissolved in a few drops of 1N NaOH, 1N NaOH. Pyridoxal made up to volume with distilled water, kept on ice and stored in the dark. The final pH was adjusted to 7.4. To determine the was added to platelet-rich mine (2 uM) was added continued for 5 min. of the total platelet fraction 3 min following
extent
of platelet release, [14C]-serotonin (0.34 uM) plasma and incubated for 1 hour at 23'C. Imiprato prevent re-incorporation of serotonin and incubation The extent o release was calculated as the percentage content of [ f4 Cl-serotonin appearing in the supernatant stimulation.
Protein concentration was determined according to the method of Bradford (1976) (7) with the use of a BioRad Protein assay kit (BioRad Laboratories, 2200 Wright Avenue, Richmond, California 94804). Results
and Discussion In agreement
(PLP)
inhibits
with
earlier
reports
thrombin-induced
platelet
ence of 3 mM PLP became reversible 9 mM PLP completely change also
(Fig.
The effect platelets
in plasma
inhibition (Table
1).
seen with
platelets
inhibited
we confirm
aggregation.
of PLP to inhibit in
buffer
when
(Table
not
exhibited increasing
1).
phosphate
in the
pres-
by 6 mM PLP while to inhibit
thrombin-induced
in buffer
release
pyridoxal
inhibited
but appeared
aggregation,
or resuspended
that
Aggregation
and was further
resuspended
of [14C]-serotonin
(8-9)
shape
aggregation In addition,
was both
a dose-dependent amounts
of
PLP were
added
1). We investigated
on platelets.
the
Thrombin
PLP showed
a decreased
mately
times
six
same initial
direct treated
with
ability
the control
velocity
effect
of PLP on the ability PLP and then
to aggregate amount
and extent
platelets
of thrombin
of aggregation.
%4
dialyzed
of thrombin to eliminate
in plasma.
was necessary
Spectrophotometric
free
Thus,
to obtain
to act
approxithe
studies
Vol. 90, No. 3, 1979
BIOCHEMICAL
Effect of pyridoxal Fig. 1. omelets in platelet-rich concentrations shown above) added at the arrow. In the formation altered the record. of thrombin.
AND BIOPHYSICAL
phosphate (PLP) on thrombin-induced aggregation plasma. The lasma was incubated with PLP (final for 1 min at 37 g C. Thrombin (0.25 units/ml) was absence of added PLP, aggregation ensued until clot Increasing amounts of PLP inhibited the action
TABLE I. EFFECT OF PYRIDOXAL AGGREGATION AND SECRETION.'
PHOSPHATE
PLP
Platelet-rich
mM
g.
0.0
100
58 44 38 7 2
2.0
10.0
The dies
in
thrombin the
induction of
enzymes,
indicated
appearance of
this e.g.,
of peak malate
by
PLP
Platelets3
a.
% Release
(45)
100
8 2
a8
1
29 11
of
the
has
been
dehydrogenase
965
total
change maximum previously (lo),
at
L14C]-serotonin
in about
the
spectrum 325
observed and
(55)
50 25 20 9
3
percent alone.
absorption
100 100
50
a characteristic a new
PLATELET
Resuspended
0 0
'Mean of three experiments. 2Thrombin (0.25 units/ml). 3Thrombin (0.125 units/ml). Numbers in parenthesis indicate the taken up that is released by thrombin
PLP-treated
THROMBIN-INDUCED
Plasma2
100
4.0 5.0
sulting
(PLP)ON
% Release
0.5
of
RESEARCH COMMUNICATIONS
appears
in to
re-
nm (Fig.
3).
spectral involve
stuthe
Vol. 90, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
I
I
260
260
300
320
WAVELENGTH
340
(nm)
E;.
2.. The effect of pyridoxal phosphate on the absorption spectra of thromHighly purlfled thrombln (2,513 units/mg) was incubated for 30 min at 37'6 with O.lM pyridoxal phosphate (solid line) or with 0.155M NaCl (dashed line). The solutions were dialyzed at 4OC for 12 hrs against 0.155M NaCl to eliminate free pyridoxal phosphate. Protein concentration of each solution was 240 ug/ml.
formation the
by PLP of an azolidine
enzyme active
of thrombin's
site
activity
thrombin
lll/thrombin
of the
ability
methyl
ester
versible platelets
of platelets inhibition did
groups
not
4 (a-c)
previously
inhibit
(5),
sensitivity
to heparin
we observed
the
synthetic
of thrombin platelet
could
be attributed
shows
that
aggregation
thrombin-induced
in the anti-
an inhibition
PLP directly
agent.
by interacting the
inhibition
to a similar
the resuspension both
aggregation
sur-
in platelet-poor
such prior (Fig.
by PLP
platelet
PLP and NaBH4 produced
whereas
arginine
inhibits
as an aggregating
whether
by PLP
tosyl
aggregation
we tested
(11).
of figrinogen
substrate,
that
at
activity
in loss
with
residue Modification
of catalytic
indicated
treated
of ADP-induced
loss
by PLP results
ADP-induced
amino
and histidine
of enzyme activity.
Indeed,
the potency
inhibit
Fig.
in
results
aggregation
interaction.
results
(12).
These
surface
loss
to hydrolyze
decreasing
PLP could
platelet
plasma
3).
a lysine
in the
groups
reaction
(Fig.
with
as a decreased
of thrombin
of thrombin-induced face
lysine
as well
thereby
Since with
residues
of thrombin
clotting
thrombin
resulting
histidine
Modification
ring
4 d-f).
an irre-
treatment
of
Our find-
Vol. 90, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
02 -G zi - 01 B P
24mM
L!tl 5 TIME
PLP
IO ( M In )
Fig. 3. Effect of pyridoxal phosphate (PLP) on the hydrolytic (esterase) activity of thrombin. Esterase activity was determined accordinq to Sherry and Troil (1954) (13). TAME (p-tosyl-LLarginine methyl ester HCI) (40 mM)and saline (8) or 2.4 mM PLP (A) was incubated at 40°C at pH 7.4. The addition of thrombin, at a final concentration of 2 units/ml,'initiated the reaction. The extent of hydrolysis of TAME was followed on a recording pH Stat by measuring the addition of microequivalents (uEq) of 0.005N NaOH to maintain constant pH. The total volume was 4.05 ml.
Fig. 4. Effect of ADP and thrombin on resuspended platelets previously treated myridoxal phosphate plus sodium borohydride. The curves show the aggregation induced by lo-5M ADP (a,b,c) or thrombin (0.1 unit/ml) (d,e,f) after treatment of platelet-rich plasma followed by resuspension of platelets in platelet-poor plasma. Curves (a) and (d) indicate treatment with 0.15M NaCl; curves (b) and (e) indicate treatment with 4 mM pyridoxal phosphate; and curves (c) and (f) indicate treatment with 4 mM pyridoxal phosphate plus 5 mM sodium borohydride.
967
Vol. 90, No. 3, 1979
ings
BIOCHEMICAL
therefore
demonstrate
that
sites.
In addition
the
ceptor involving thrombin
platelet
surface
amino
AND BIOPHYSICAL
thrombin results
and ADP act show that
groups
does not
the
RESEARCH COMMUNICATIONS
on separate formation
interfere
with
platelet of Schiff the action
rebases of
on platelets.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Macfarlane, 0. E. and Mills, D. C. B. (1975) Blood, 46, 309-320. Adler, J. R. and Handin, R. I. (1979) J. Biol, Chem. 254, 3866-3872. Ganguly, P. and Gould, N. L. (1979) Brit. J. Haematology, 42, 137-145. Alexander, R. W., Cooper, B. and Handin, R. I. (1978) J. Clin. Invest. 61, 1136-1144. Kornecki, E. and Feinberg, H. (1979) Am. J. Physiol. (in press). Feinberg, H., Sandler, W. C., Scorer, M., Le Breton, G. C., Grossman, B. and Born, G. V. R. (1977) Biochim. Biophys. Acta, 470, 317-324. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. Subbarao, K., Kuchibhotla, J., Kakkar, V. V. (1979) Biochem. Pharm. 28, 531-534. Pollard, H. B., Tack-Goldman, K., Pazoles, C. J., Creutz, C. E., and Shulman,N.(1977) Proc. Nat. Acad. Sci. 74, 5295-5299. Wimmer, M. J., MO, T., Sawyers, D. L. and Harrison, J. H. (1975) J. Biol. Chem. 250, 710-715. Glover, G. and Shaw, E. (1971) J. Biol. Chem. 246, 4594-4601. Griffith, M. J. (1979) J. Biol. Chem. 254, 3401-3406. Sherry, S. and Troll, W. (1954) J. Biol. Chem. 208, 95-105.
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