In vitro effects of vincristine on arachidonic acid metabolism in human platelets and rat arterial tissue

THROMBOSIS RESEARCH 23; 225-231, 1981 0049-3848/81/150225-07$02.00/O Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.

IN VITRO EFFECTS OF VINCRISTINE ON ARACHIDONICAC10 METABOLISM -IN HUMAN PLATELETS AND RAT ARTERIAL TISSUE N. T. Shah*, C. W. Karpen** and R. V. Panganamala** *Department of Pathology and *r)epartment of Physiological Chemistry ~~ The Ohio State University, Columbus, Ohio 43210

(Received 10.2.1981; in revised form 12.6.1981. Accepted by Editor J.B. Smith)

ABSTRACT We have demonstrated that -in vitro addition of vincristine to human platelets: 1) inhibits aggregation induced by arachidonic acid, epinephrine, collagen, thrombin and ADP; 2) inhibits arachidonic acid conversion to thromboxane AZ; and 3) inhibits generation of thromboxane A2 upon stimulation by mechanical agitation, collagen, or thrombin. It is suggested that the inhibitory effect of vincristine on platelet aggregation is due to the inhibition of thromboxane Ap production. Vincristine has no demonstrable effect on the release of prostacyclin from normal rat aorta.

INTRODUCTION Vincristine (VC), a member of the vinca alkaloids, is used extensively in the treatment of acute lymphoblastic leukemia and other neoplastic diseases, thrombotic thrombocytopenic purpura (TTP), and idiopathic thrombocytopenic purpura (ITP)(l). Platelets obtained from ITP patients treated with VC failed to exhibit the second phase of ADP or epinephrine

Correspondence to: Dr. Rao V. Panganamala, Department of Physiological Chemistry, 333 W. Tenth Avenue, Ohio State University, Columbus, Ohio 43210a Key Words:

prostaglandin, arachidonate

thromboxane, 225

vincristine,

platelets,

aorta,

IN VITRO EFFECTS OF VINCRISTINE

226

vo1.23, No.3

induced aggregation (2). VC, when incubated with normal human platelets, disassembled platelet microtubules and inhibited ADP and epinephrine induced second phase aggregation (3). The present study describes -in vitro effects of VC on human platelet arachidonate metabolism, platelet aggregation, and rat arterial prostacyclin (PGI2) formation. MATERIALS

AND METHODS

Adenosine diphosphate (ADP), epinephrine bitartrate, bovine thrombin, and bovine achilles-tendon collagen were purchased from Sigma Chemical Co. Soluble calfskin collagen (used as a standard for the Lowry protein determination) was from Millipore Corporation. l-14C-arachidonic acid Sp. act.=52.7 mCi/mmol), 3H-thromboxane B2 (Sp. act.=150 Ci/mmol), and sH-6-Keto-prostaglandin Fla (Sp. act.=100 Ci/mmol) were purchased from New England Nuclear Corp. Arachidonic acid (AA) was purchased from Nu Chek Prep. Prostaglandins (PG) were a kind gift from Dr. John Pike (UpJohn). Vincristine sulfate was purchased from Eli Lilly and Co. ADP, epinephrine bitartrate, vincristine sulfate, and thrombin were dissolved in physiological saline, while thromboxane B2 (TXB2), and 6-Keto-prostaglandin Fl,(6-Keto-PGFl,) were dissolved in ethanol and stored at -7O'C. AA was used as its sodium salt for aggregation studies. TXB2 and 6-Keto-PGFl, were diluted with TRIS (50 nM)-albumin (0.1%) buffer (pH 7.6) for radioimmunoassay (RIA). Bovine achilles-tendon collagen suspension was prepared according to Nakanishi -et al. (4) and the collagen concentration in suspension determined by the method of Lowry -et al. (5) using soluble calfskin collagen as the standard. Platelet Aggregation Blood (9 parts) was collected into 3.8% sodium citrate (1 part) and platelet aggregation was performed as described earlier (6). VC in different concentrations was incubated with platelet rich plasma (PRP) for one minute at 37'C before challenging with aggregating agents. Arachidonic

Acid Oxygenation

in Washed

Platelets

Blood (9.2 parts) from healthy subjects was mixed with EOTA (0.8 parts; 77mM Na2EDTA), and washed platelets prepared as described previously (7). I-14C-AA (2.1 nmoles) was added to 0.5 ml of platelet suspension (1.25~108) and stirred for 1 min at 37'C. VC was added and incubated for two minutes at 37'C before addition of arachidonic acid. The reaction was terminated with 200~1 1N HCl. The products were extracted twice with 3 ml diethyl ether, evaporated and the residue dissolved in 200~1 methanol. The extract (25i.11) and standard PGs were spotted on thin layer chromatography (TLC) plates (Whatman LK6D), and developed in a solvent mixture of chloroform-methanol-acetic acid (18O:lO:lO) (System 1). For the separation of hydroxy fatty acids and arachidonic acid, plates were developed twice in a solvent mixture of heptane-ether-acetic acid (60:40:1) (System II). PGs were visualized with iodine vapors, and radioactive peaks located using a Packard Model 7220/21 radiochromatogram scanner. Peaks were matched with the standard PGs, scraped into scintillation counting vials, and radioactivity was counted in a liquid scintillation counter (Beckman model LS8100).

Vo1.23, No.3

Formation

of Endogenous

IN VITRO EFFECTS OF VINCRISTINE

227

TxB7

The platelet pellet as described above as suspended in calcium-free Krebs-Henseleit buffer. Platelets (1.0 x 108 /D.5 ml volume) were stirred at 37°C for 10 minutes with and without collagen or thrombin after a 5 minute preincubation with various concentrations of VC at 4°C. TxB2 was extracted with 3 ml diethyl ether. The residue after evaporation was dissolved in TRIS-albumin buffer and TxB2 was estimated by RIA. Cross reactivity of the anti-TxB2 antibody with other prostaglandins was as follows: PGF2,=0.26%; PGE2=.021%; PGO2=1.6%; and arachidonic acid=O.ODl%. Estimation

of the Release of 6-Keto-PGFl,

From Rat Aorta

Rat aorta was separated and cleaned by the method of Panganamala et al. (8). Incubations were carried out at room temperature in TRIS-buffer CH8.0) containing VC and aliquots were withdrawn at 30, 60 and 90 minutes for the estimation of 6-Keto-PGFl, by RIA. The validity of the RIA was confirmed using a bioassay measuring the inhibitory effects of PGI2 on Cross-reactivity of the antibody against platelet aggregation. 6-Keto-PGFl, for different prostaglandins was as follows: PGE2 = 0.15%; PGD2 = 0.02%; and PGF2, = 0.1%. RESULTS Inhibition of Platelet Aggregation The concentration of aggregating agents tested was adjusted for each subject in order to obtain the same percent aggregation for all the subjects. The dose-dependent inhibitory effect of VC on platelet aggregation is shown in Table 1. VC at 3DOpM concentration inhibited ADP and thrombin induced aggregations by 33% and 48% respectively. The same concentration of VC inhibited the aggregation induced by collagen, epinephrine (2nd phase) and arachidonic acid by over 80%. Alteration

of Arachidonic

Acid Transformation

into Oxygenated

Products

Conversion of l-l4 C-AA to TxB2 was complete after two minutes incubation, at which time most of the substrate was consumed. One minute incubations were chosen because substrate was still detectable, and conversion to TxB2 was nearly complete. Recovery of radioactivity with diethyl ether extraction was over 90%. Solvent system 1 separated TxB2 (Rf: 0.3) from PGE2 (Rf:0.36), PGD2 (Rf:0.44), and PGF2, (Rf:0.23). Solvent system II separated arachidonic acid (Rf:U.58) from HHT (Rf:0.30), HETE (Rf:0.43) and TxB2 + PG (Rf:O). The peak with Rf 0.30 was designated HHT since its formation disappeared when indomethacin (0.3mM) (Merck and Co., Inc.) was included in the incubation. Formation of all the products disappeared when ETYA (0.31Wl) (HoffmannLaRoche, Nutley, NJ) was included in the incubation. Table II shows the inhibitory effect of VC on TxB2 formation from arachidonic acid. There was a progressive increment of PGE2 and PGD2 production with increasing concentrations of VC. The increase of PGE2 and PGD2 production in the presence of VC was proportionately greater than the inhibition of TxB2 formation. For example, VC (1 ti) inhibited TxB2 formation by less than 5D%, while PGE2 and PGD2 formation were increased The same concentration of VC decreased sevenfold and fourfold respectively. the consumption of arachidonic acid by 10% and caused a slight increment in HETE formation. PGF2a, estimated in the range of 0.5-0.7%, was unaltered by VC.

IN VITRO EFFECTS OF VINCRISTINE

228

'~01.23,No.3

TABLE 1 Percent

Inhibitiona of Human Platelet Aggreqation

by Vincristine

Arachidonic iYl;;$;ine (A

;;;!;;e;g,m,)

$yy,“I;”

ADP

%-,.5ti) unit/ml)

(2-511~)

50

11.0+2.8(3)c

51.8*14.7(3)

29.6+ 1.0(3)

100

25.8+9.2(3)

80.6+ 9.3(4)

67.0+10.2(4)

29.0+6.4(3)

11.4+4.9(4)

200

62.0+12.5(6)

95.2? 9.7(4)

90.32 5.5(6)

45.4+7.6(3)

14.9+1.4(4)

300

81.5+10.1(4)

100

48.0+2.5(3)

33.2+5.8(4)

100

(3)

(3)

a Percent Inhibition = Maximal Slope of Aggregat ion without VC - Maximal Slope of Aggreg,ation with VC X 100 Maximal Slope of Aggregation without VC b Because thrombin was found to clot PRP, thromb in induced aggregation was performed in washed platelets prepared as described under "Formation of Endogenous TxB2". The washed platelet suspension contained 1 part platelet-free plasma in 9 parts Krebs buffer. c Mean ?;SD. Numbers in parentheses are number of subjects tested. to "Materials and Methods" for details of procedures.

Refer

TABLE 2 Effect of Vincristine on Arachidonate Vincristine (Ii+!)

Subjects

AA

TxB?

Metabolism

&&

in Platelets HtJ

_ HETE

% 14C-Conversion/108platelets None

A* B

10.3 9.9

29.4 29.8

1.2 1.1

1.4 1.3

37.2 35.5

20.0 21.1

0.25

A B

16.7 14.4

23.8 24.7

4.7 4.3

2.9 3.0

30.1 29.7

21.1 22.1

0.5

A B

18.6 16.5

22.1 22.0

5.3 5.3

2.9 3.4

27.6 27.1

2U.7 23.4

1.0

A B

20.3 18.5

18.4 18.6

7.2 7.2

3.6 4.0

24.6 24.9

23.9 24.9

*A, B are different subjects. Refer to "Materials and Methods" for details of procedures.

Vo1.23, No.3

IN VITRO EFFECTS OF VINCRISTINE

229

TABLE 3 Inhibition

of Endogenous

TxB7 Production

by Vincristine

% Inhibition Vincristine (ti)

Collagen (80 ug/ml)

Thrombin (2 Units/ml)

31+5* 4555 7322 95&l

17a7 31*7 62*6 88+4

:o" 160 400 *Mean + SEM from 4 different details of procedure.

Inhibition of Generation

subjects.

of Endogenous

Refer to "Material and Methods"

for

TxB2

Endogenously formed thromboxane A2 (TxA2) was estimated as TxB2 by RIA. In a typical incubation, 600 and 150 pmoles TxB2 was formed when platelets were stimulated with thrombin and collagen respectively. Without stimulators, platelets produced 20 pmoles TxB2 when stirred at 37°C for 10 min. Table III shows the effect of different concentrations of VC on thrombin and collagen induced TxB2 generation. A concentration of 4OOpM VC inhibited mechanically induced TxB2 formation by 80% while the same concentration of VC inhibited thrombin and collagen induced TxB2 formation by 90%. Clearly these results show that VC is a potent inhibitor of TxA2 formation. Release of 6-Keto-PGFl,

From Rat Aorta

The validity of the 6-Keto-PGFl, RIA was confirmed using bioassay of the anti-platelet aggregatory effects of PG12. In four 30 minute aortic incubations, bioassay measured 19.4, 19.7, 22.9, and 24.7 pmoles PG12 released/mg tissue, while RIA measured 19,21,24 and 28 pmoles 6-Keto-PGFl,/mg tissue. VC in the concentration range of 0.1-1.0 r&i had no effect on the production of 6-Keto-PGFl, at 30,60 or 90 min. incubation. DISCUSSION White (3) has suggested that the effects of vinca alkaloids on platelets were due to damage to the platelet surface or alteration of energy metabolism rather than merely the disassembly of microtubules. The present -in vitro study demonstrates that VC inhibits human platelet aggregation and TxA2 synthesis from exogenous and endogenous arachidonic acid. Collagen, thrombin, and epinephrine have been implicated in the activation of platelet phospholipase-A2 (10). The mechanism of arachidonic acid, epinephrine, and collagen induced aggregation involves PG endoperoxide and TxA2 formation (lO,ll), and aggregation induced by these agents was very sensitive to VC. VC only partially inhibited thrombin induced aggregation, since PG endoperoxide formation is not the only mechanism by which thrombin induces aggregation (12). Similarly, AOP induced aggregation, which does not involve endoperoxide formation as the main mechanism, was also less sensitive to VC.

230

IN VITRO EFFECTS OF VINCRISTINE

Vo1.23, No.3

We next studied the effects of VC on the conversion of l-I4C-AA to its VC inhibited TxB2 and HHT formation to approximately oxygenated products. the same extent, while there was an increment in PGE2 and PGD2 formation. These findings suggest that VC inhibits thromboxane synthetase, since both TxB2 and HHT may arise from PGH2 (9). Although less arachidonic acid was consumed in the presence of VC, it is unlikely that VC inhibited cyclosxygenase since there was no decrease in PGE2 and PGD2 formation. Decreased consumption of arachidonic acid and increased PGE2 and PGD2 production may be explained by PG endoperoxide accumulation due to inhibition of thromboxane synthetase. We further confirmed the VC effect on thromboxane production by measuring its release by mechanical stimulation such as stirring or by stimulation with collagen or thrombin. These results suggest that VC, in addition to its effect on the modulation between thromboxane and prostaglandin synthesis, may inhibit the release of arachidonic acid. Such an inhibition of release of arachidonic acid was demonstrated by a related alkaloid, colchicine (13). We investigated whether VC has any influence on PG12 release from arterial tissue. VC in a concentration range of 0.1 n+l to l.On+i had no effect on the release of PG12 from rat aorta as measured by RIA of 6-Keto-PGFl,. This observation does not, however, rule out a VC effect on PG12 synthesis and release in ITP patients. Our data with platelet aggregation and arachidonate metabolism suggest that VC inhibits platelet aggregation and TxA2 formation, which may be partially responsible for the tendency towards normalization of thrombocytes in ITP patients treated with vincristine. Further -in viva studies in patients treated with VC are warranted. ACKNOWLEDGEMENTS We thank Dr. Lawrence Levine for supplying us the antibodies against TxB2 and 6-Keto-PGFl,, Dr. A. J. Merola for helpful suggestions, and L. Gonzalez for expert technical assistance. We also wish to thank Drs. Stevenson and Senhauser for their encouragement. This work was supported part by grants HL-23439-02 and OSU 221372.

in

REFERENCES 1.

AHN, Y.S., HARRINGTON, W.J., SEELMAN, R.C., and EYSTEL, C.S. Vincrist ine therapy for idiopathic and secondary thrombocytopenia. New Engl. J. Med., 291, 376-380, 1974.

2.

STEINHERZ, P.G., MILLER, D.R., HILGARTNER, M.W., and SCHMALZER, E.A. Platelet dysfunction in vincristine treated patients. Br. J. Haematol., 32, 439-450, 1976.

3.

WHITE, J.G. Effect of colchicine and vinca alkaloids on human platelets. III. Influence on primary internal contraction and secondary Am. J. Pathol., 54, 467-473, 1969. aggregation.

4.

NAKANISHI, M., IMAMURA, H., and GOTO, K. Potentiation of the ADP-induced platelet aggregation by collagen and its inhibition by a tetrahydrothienopyridine derivative (Y-3642). Biochem. Pharmacol., 20, 2116-2118, 1971.

vo1.23, No.3

231

IN VITRO EFFECTS OF VINCRISTINE

5.

LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L., and RANDALL, R.J. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 163-175, 1951.

6.

PANGANAMALA, R.V., MILLER, J.S., GWEBU, E., SHARMA, H.M., and CORNWELL, D.G. Differential inhibitory effects of vitamin E and other antioxidants on prostaglandin synthetase, platelet aggregation and lipoxidase. Prostaglandins, 14, 261-271, 1977.

7.

GWEBU, E.T., TREWYN, R.W., CORNWELL, D.G., and PANGANAMALA, R.V. Vitamin E and the inhibition of platelet lipoxygenase. Res. Comm. Chem. Pathol. Pharmacol., 28, 361-376, 1980.

8.

GILLESPIE, A., and MEROLA, A.J. Assay of prostacyclin PANGANAMALA, R.V., synthesis in intact aorta by aqueous sampling. Prostaglandins, 21, l-7, 1981.

9.

DICZFALUSY, U., FALAARDEAU, P., and HAMMARSTROM, S. Conversion of prostaglandin endoperoxide to Cl7-hydroxyfatty acids catalyzed by human platelet thromboxane synthase. FEBS Letters, 84, 271-274, 1977.

10.

INGERMAN, J., KOCSIS, J., and SILVER, M.J. Formation of an SMITH, J.B., intermediate in prostaglandin biosynthesis and its association with the platelet release reaction. J. Clin. Invest., 53, 1468-1472, 1974.

11.

HAMBERG, M., SVENSSON, J., AND SAMUELSSON, B. Thromboxanes: of biologically active compounds derived from prostaglandin endoperoxides. Proc. Nat. Acad. Sci., 72, 2994-2998, 1975.

12.

PACKHAM, M.A., MUSTARD, J.F. diphosphate. Smith (Eds.).

13.

RITTENHOUSE-SIMMONS, S., and DEYKIN, D. Effect of colchicine and ATP deprivation on release of arachidonic acid from platelets exposed to thrombin or ionphore A 23187. Prostaqlandins, 13, 1011, 1977.

A new group

KINLOUGH-RATHBONE, R.L., REIMERS, H.J., SCOTT, S., and Mechanism of platelet aggregation independent of adenosine In: Prostaqlandins in Hematology. M.J. Silver and J.B. 1976, pp. 262-265. New York: Spectrum Publication, Inc.,