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.

968

Mechanism of inhibition of thrombin-induced platelet aggregation by pyridoxal phosphate.

Vol. 90, No. 3, 1979 October BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 12, 1979 Pages 963-968 MECHANISM OF INHIBITION OF THROMBIN-IND...
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