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Effects of low doses of thrombin on human platelet phospholipids
IHROMBOSIS RESEARCH 18; 285-290 Printed in the United States (c) Pergamon Press Ltd. 1980. OO@-3848/80/080285-06$02.00/O
BRIEF
COMMUNICATION EFFECTS OF L'IWDOSES OF THROMBIN ON HUMAN PLATELET PHOSPHOLIPIDS.
M. GUICHARDANT and M. LAGARDE Institut Pasteur, Inserm U 63, Laboratoire d'Hemobiologie, Facultg Alexis Carrel, 69372 Lyon Cedex 2, France. Accepted (Received 10.8.1979. Received in final form by Executive
by Editor Editorial
J.B. Smith. Office 19.2.1980)
INTRODUCTION Platelet synthesize prostaglandins and related compounds from arachidonic acid (1). This acid is located in platelet membrane phospholipids and released by aggregating agents such as thrombin (2,3). Radioactivity of phospholipids prelahelled with arachidonic acid is decreased by thromhin (5 U/ml) except for phosphatidyl ethanolamine in which the acid is transferred into plasmalogen form (4). Recently, it has been shown that initial steps of activation by thrombin (5 U/ml) is the breakdown of phosphatidylinositol with a simultaneous increase of phosphatidic acid. This suggests an initial activation of a phospholipase C (5,6). In this work we were interested by the action of low doses of thromhin able to induce complete aggregation. MATERIALS AND METHODS Bovine thromhin was yielded from Hoffman La Roche, Basel. [1-'4C] arachidonit acid (60 Ci/mole) was purchased by the radiochemical Center of Amersham. Human platelets from donors who had taken no drugs ten days before venous punction, were isolated according to a described technique (7). Briefly, ACD was used as anticoagulant and platelet-rich plasma was acidified to pH 6.4 with citric acid 0.15 M before centrifugation at 9OOg for 10 min. Pellets were resuspended in a calcium free Tyrode-Hepes buffer pH 7.35. Pla telets were maintained at room temperature before testing. Platelet phospholipids were lahelled with [l-'4C] arachidonic ac d. Plate-
Key words : Platelets - Thromhin - Phospholipids - Arachidonic acid 285
286
PLATELET
PHOSPHOLIPASE
ACTIVITY
Vo1.18,No.1/2
let suspensions (300.000 111-l)were incubated with 20 nCi/ml of [I-"'C] arachidonic acid (60 Ci/mole) during 2 hours at room temperature. After, this platelet suspension was incubated with or without thromhin before a double lipid extraction [a neutral then an acidic extraction (8)l. Phospholipids were purified from total lipids by thin layer chromatography with diethylether-methanol-acetic acid 90:1:2 as eluent. In this system, phospholipids have Rf = 0. This spot was scrapped off and extracted with methanol. The extract was submitted to a hidimensionnal chromatography on silicagel G. The First eluent was chloroform-methanol-water 63:27:4 and the second one was tetrahydrofuran-methylal-methanol-2N NH 4 Ok!6C:30:3C!:6(9). To obtain a better separation of different phospholipids, a third migration limited to IOcm in the second dimension was performed with the last eluent. Phospholipids were located by autoradiography and scrapped off for scintillation counting. Biosynthesis of thromhoxane B2, 12-hydroxy-heptadecatrienoic acid (HHT) and 12-hydroxy-eicosatetraenoic acid (HETE) was investigated with a described method (IO). Radioactivity of the compounds was expressed in percentage of initial radioactivity of phospholipids. RESULTS Platelet phospholipid labelling by [1-'4C] arachidonic acid represented 94.d _+ 1.8 (n = 15) of the total radioactivity. The profile of this radioactivity is shown figure I. Almost 50% of it was located in phosphatidylcholine. The rest was mainly found in phosphatidylethanolamine and inositol.
1
Z of radioactivity
90 -
80 Figure I 70.
Radioactivity rofile in phospholiP pids after [I- 'Cl arachidonic acid lahelling (n=4).
60 -
Pph : sphingomyelin ; PC,PS,PE and PI : phosphatidylcholine, serine, ethanolamine and inositol ; PA : phosphatidic acid.
sph
PC
PS
PA
PE
PI
Vo1.18,No.1/2
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PHOSPHOLIPASE
ACTIVITY
287
Less than 10 % of radioactivity was in phosphatidylserine. Radioactivity in phosphatidic acid and sphingomyelin was not detectable.
TABLE I Effects of low doses of thrombin (0.1 U/ml) on radiolabelled phospholipids after 30 sec. and 4 min. at 37'C.
Phospholipids
PC PS PA PE PI total
A
B
A
37367 9057 186 19051 17657 83318(D)
36605 9284 1637 17898 11131 76555
A : Control C : Thrombin 0.1 U/ml for 4 min.
-0.91 +0.27 +I.74 -1.38 -7.83 -8.1
40182 4305 212 13220 11391 69310(D)
C
36288 3572 4520 12955 4940 62275
-5.62 -1.06 +6.2 -0.38 -16.4 -10.4
B : Thrombin 0.1 U/ml for 30 sec.
Each result, in dpm/lOg platelets, represents the mean of 3 determinations.
TABLE II Effect of 1 U/ml thrombin on radiolabelled phospholipids after 30 sec. and 4 min. at 37'C.
Phospholipids I PC PS PA PE PI total
B
A I 39985
8460 120 ~ 15684 18229 82478(D)
-4.28 -0.40 +1.92 +I.24 -6.11 -7.6
36451 8131 1710 16708 13190 76190
A: Control C : Thrombin 1 U/ml for 4 min.
B
:
41231 7233 514 20987 21214 91179(D)
32852 6373 2020 23976 9258 74479
-9.19 -0.94
+0.61 +3.28 -13.11 -18.3
Thrombin 1 U/ml for 30 sec.
Each result, in dpm/l@' platelets, represents the mean of 3 determinations.
288
PLATELET
PHOSPHOLIPASE
ACTIVITY
Vo1.18,No.1/2
Table I and II lead to different results. Either PI, PC or both may be hydrolysed after thrombin stimulation to release arachidonic acid for prostaglandin biosynthesis. 30 sec. after 0.1 U/ml of thrombin, there was a little or no decrease in PC, but a major decrease in the radioactivity of PI. Apparently, some of this radio activity reappeared in PA. 4 min. after 0.1 U/ml of thrombin, there was an even greater decrease in PI, a larger increase in PA and some radioactivity was lost from PC and to a lesser extent from PS. At either 30 sec. or 4 min. after 0.1 U/ml of thrombin, there was no incorporation of radioactivity into PE. At both 30 sec. and 4 min. after 1 U/ml of thrombin there was a much greater decrease in PC than after 0.1 U/ml of thrombin. On the other hand, the changes in PI were if anything somewhat less. There was incorporation of radioactivity into PE at both 30 sec. and 4 min. after 1 U/ml of thrombin. The breakdown of radioactivity from total phospholipids was about the same after 30 sec. of both concentrations of thrombin but increased 4 min. after I U/ml of thrombin. Lastly, the decrease of phospholipid radioactivity under thrombin 0.1 U/ml was accompanied with a synthesis 'of thromboxane B,, HHT and HETE (Table.111). These three compounds were assayed after 4 min. of treatment by thrombin 0.1 U/ml at 37'C. TXB2 Control
Thrombin
HHT
HETE
1.8 ? 0.2
0.15 + 0.04
0.9 ? 0.4
10.7 2 4.2
0.61 -I0.13
5.1 ? 2.0
TABLE III
TX%, HHT and HETE from endogenous arachidonic acid produced after 4 min. of incubation with thrombin 0.1 U/ml. Results, expressed in percentage of initial phospholipid radioactivity, were the mean of 6 determinations. DISCUSSION The method used in this work gives a good incorporation of arachidonic acid as tracer. Phosphatidic acid is weakly labelled indicating that platelets are not stimulated since this phospholipid only appears when phospholipase C is activated (5,6). Results show that phosphatidylinositol is the major platelet phospholipid breakdown by thrombin used at low concentrations. Even after 30 sec. of thrombin action, this phospholipid is decreased and phosphatidic acid is increased as well. This suggests a very rapid activation of platelet phospholipase C as previously shown (11). Stable metabolites of arachidonic acid such as TXBs, HHT and HETE are synthezised after platelet incubation with thrombin 0.1 U/ml. This indicates an effective release of arachidonic acid from phospholipids. Moreover, it was
Vo1.18,No.1/2
PLATELET
PHOSPHOLIPASE
ACTIVITY
289
possible to observe a prostaglandin endoperoxides synthesis 1 min. after platelet stimulation by thrombin 0.1 U/ml (12). This fact suggests an initial release of arachidonic acid by this low dose of thrombin. Increase radioactivity in PA was previously shown with a low dose of thrombin (0.2 U/ml) and soon 50 sec. whereas ADP 5.10s7M (which does not induce platelet prostaglandin formation) failed to enhance PA radioactivity (13). Lastly, increase of radioactivity in phosphatidylethanolamine, previously described as a plasmalogen form (4), only appears with thrombin 1 U/ml. These results are agree with those previously published (2,3) but they point out the role of phosphatidyl inositol in human platelet activation by thrombin. Thus, phosphatidyl inositol could be the physiological pool of arachidonic acid rapidly available to prostaglandin biosynthesis under low doses of thrombin. ACKNOWLEDGMENTS This work was supported by grant INSERM, ASS N"5. REFERENCES 1. HAMBERG, M. and SAMUELSSON, B. Thromboxanes : a new group of biologically active compounds derived from prostaglandin endoperoxides. ?'hOC. NaL Ac.ad. SQL, 72, 2994-2998, 1975. 2. BILLS, T.K., SMITH, J.B. and SILVER, M.J. Metabolism of [l-'4C] arachidonit acid by human platelets. B~ockim. Biophyb. Acta 424, 303-314, 1976. 3. BILLS, T.K., SMITH, J.B. and SILVER, M.J. Selective release of arachidonic acid from the phospholipids of human platelets in response to thrombin. J. Cl%. lnve&?Z. 60, 1-6, 1977. 4. RITTENHOUSE-SIMl4ONS,S., RUSSEL, F.A. and DEYKIN, D. Transfert of arachidonic acid to human platelet plasmalogen in response to thrombin. G~cch~. Biophqh. R&A. Camnun. 70, 295-301, 1976. 5. MAUCO, G., CHAP, H., SIMON, M.F. and DOUSTE-BLAZY. L. Phosphatidic and lysophosphatidic acid production in phospholipase C and thrombin-treated platelets. Possible involvement of platelet lipase. Bidtiie, 60, 653-661, 1978. 6. LAPETINA, E.G., CHANDRABOSE, K.A. and CUATRECASAS, P. Ionophore A 23187 and thrombin induced platelet aggregation. Independance from cycle-oxygenase product. Phoc. Nat. Acad. Sci. 75, 818-822, 1978. 7. LAGARDE, M., BRYON, P.A., GUICHARDANT, M. and DECHAVANNE, M. A simple and efficient method for platelet isolation from their plasma. Th40m60b. RCQ. In press. 8. DITTMER, B.G. and DOUBLAS, M.G. Quantitative determination of phosphoinositides. Ann. N. Y. Acad. Sci. 165, 515-525, 1969. 9. BEREZIAT, G., CHAMBAZ, J., TRUGNAN, G., PEPIN, D. and POLONOVSKI, J. Turnover of phospholipid linoleic and arachidonic acids in human platelets from plasma lecithins. j. LiyJ-id.Ren. 19, 495-500, 1978.
PLATELET
PHOSPHOLIPASE
ACTIVITY
Vo1.18,No.1/2
IO. LAGARDE, II., GHARIB, A. and DECHAVANNE, M. A simple radiochemical assay of thromboxane Bz, IZ-hydroxy-eicosatetraenoic acid (HFTE) and 12-hydroxyheptadecatrienoic acid (HHT) synthezised by human platelets. Cl!&. whim.
Ada. 79, 255-259, 1977. 11. RITTENHOUSE-SIMMONS, S. Production of diglyceride from phosphatidylinositol in activated human platelets. J. C&n. 7nwQcSX.63, 580-587, 1979. 12. LAGARDE, M. BRYON, P.A., VARGAFTIG, B.B. and DECHAVANNC, )I.Impairment of platelet thromboxane A2 generation and of the platelet release reaction in two patients with congenital deficiency of platelet cycle-oxygenase. 8hit. _J.ffaemdtcd. 38, 251-266, 1978. 13. LLOYD, J.V. and MUSTARD, J.F. Changes in 32P-content of phosphatidic acid and the phosphoinositides of rabbit platelets during aggregation induced 26, 243-253, 1974. by collagen or thrombin. Rtif. J. Haumtat.
BRIEF
COMMUNICATION EFFECTS OF L'IWDOSES OF THROMBIN ON HUMAN PLATELET PHOSPHOLIPIDS.
M. GUICHARDANT and M. LAGARDE Institut Pasteur, Inserm U 63, Laboratoire d'Hemobiologie, Facultg Alexis Carrel, 69372 Lyon Cedex 2, France. Accepted (Received 10.8.1979. Received in final form by Executive
by Editor Editorial
J.B. Smith. Office 19.2.1980)
INTRODUCTION Platelet synthesize prostaglandins and related compounds from arachidonic acid (1). This acid is located in platelet membrane phospholipids and released by aggregating agents such as thrombin (2,3). Radioactivity of phospholipids prelahelled with arachidonic acid is decreased by thromhin (5 U/ml) except for phosphatidyl ethanolamine in which the acid is transferred into plasmalogen form (4). Recently, it has been shown that initial steps of activation by thrombin (5 U/ml) is the breakdown of phosphatidylinositol with a simultaneous increase of phosphatidic acid. This suggests an initial activation of a phospholipase C (5,6). In this work we were interested by the action of low doses of thromhin able to induce complete aggregation. MATERIALS AND METHODS Bovine thromhin was yielded from Hoffman La Roche, Basel. [1-'4C] arachidonit acid (60 Ci/mole) was purchased by the radiochemical Center of Amersham. Human platelets from donors who had taken no drugs ten days before venous punction, were isolated according to a described technique (7). Briefly, ACD was used as anticoagulant and platelet-rich plasma was acidified to pH 6.4 with citric acid 0.15 M before centrifugation at 9OOg for 10 min. Pellets were resuspended in a calcium free Tyrode-Hepes buffer pH 7.35. Pla telets were maintained at room temperature before testing. Platelet phospholipids were lahelled with [l-'4C] arachidonic ac d. Plate-
Key words : Platelets - Thromhin - Phospholipids - Arachidonic acid 285
286
PLATELET
PHOSPHOLIPASE
ACTIVITY
Vo1.18,No.1/2
let suspensions (300.000 111-l)were incubated with 20 nCi/ml of [I-"'C] arachidonic acid (60 Ci/mole) during 2 hours at room temperature. After, this platelet suspension was incubated with or without thromhin before a double lipid extraction [a neutral then an acidic extraction (8)l. Phospholipids were purified from total lipids by thin layer chromatography with diethylether-methanol-acetic acid 90:1:2 as eluent. In this system, phospholipids have Rf = 0. This spot was scrapped off and extracted with methanol. The extract was submitted to a hidimensionnal chromatography on silicagel G. The First eluent was chloroform-methanol-water 63:27:4 and the second one was tetrahydrofuran-methylal-methanol-2N NH 4 Ok!6C:30:3C!:6(9). To obtain a better separation of different phospholipids, a third migration limited to IOcm in the second dimension was performed with the last eluent. Phospholipids were located by autoradiography and scrapped off for scintillation counting. Biosynthesis of thromhoxane B2, 12-hydroxy-heptadecatrienoic acid (HHT) and 12-hydroxy-eicosatetraenoic acid (HETE) was investigated with a described method (IO). Radioactivity of the compounds was expressed in percentage of initial radioactivity of phospholipids. RESULTS Platelet phospholipid labelling by [1-'4C] arachidonic acid represented 94.d _+ 1.8 (n = 15) of the total radioactivity. The profile of this radioactivity is shown figure I. Almost 50% of it was located in phosphatidylcholine. The rest was mainly found in phosphatidylethanolamine and inositol.
1
Z of radioactivity
90 -
80 Figure I 70.
Radioactivity rofile in phospholiP pids after [I- 'Cl arachidonic acid lahelling (n=4).
60 -
Pph : sphingomyelin ; PC,PS,PE and PI : phosphatidylcholine, serine, ethanolamine and inositol ; PA : phosphatidic acid.
sph
PC
PS
PA
PE
PI
Vo1.18,No.1/2
PLATELET
PHOSPHOLIPASE
ACTIVITY
287
Less than 10 % of radioactivity was in phosphatidylserine. Radioactivity in phosphatidic acid and sphingomyelin was not detectable.
TABLE I Effects of low doses of thrombin (0.1 U/ml) on radiolabelled phospholipids after 30 sec. and 4 min. at 37'C.
Phospholipids
PC PS PA PE PI total
A
B
A
37367 9057 186 19051 17657 83318(D)
36605 9284 1637 17898 11131 76555
A : Control C : Thrombin 0.1 U/ml for 4 min.
-0.91 +0.27 +I.74 -1.38 -7.83 -8.1
40182 4305 212 13220 11391 69310(D)
C
36288 3572 4520 12955 4940 62275
-5.62 -1.06 +6.2 -0.38 -16.4 -10.4
B : Thrombin 0.1 U/ml for 30 sec.
Each result, in dpm/lOg platelets, represents the mean of 3 determinations.
TABLE II Effect of 1 U/ml thrombin on radiolabelled phospholipids after 30 sec. and 4 min. at 37'C.
Phospholipids I PC PS PA PE PI total
B
A I 39985
8460 120 ~ 15684 18229 82478(D)
-4.28 -0.40 +1.92 +I.24 -6.11 -7.6
36451 8131 1710 16708 13190 76190
A: Control C : Thrombin 1 U/ml for 4 min.
B
:
41231 7233 514 20987 21214 91179(D)
32852 6373 2020 23976 9258 74479
-9.19 -0.94
+0.61 +3.28 -13.11 -18.3
Thrombin 1 U/ml for 30 sec.
Each result, in dpm/l@' platelets, represents the mean of 3 determinations.
288
PLATELET
PHOSPHOLIPASE
ACTIVITY
Vo1.18,No.1/2
Table I and II lead to different results. Either PI, PC or both may be hydrolysed after thrombin stimulation to release arachidonic acid for prostaglandin biosynthesis. 30 sec. after 0.1 U/ml of thrombin, there was a little or no decrease in PC, but a major decrease in the radioactivity of PI. Apparently, some of this radio activity reappeared in PA. 4 min. after 0.1 U/ml of thrombin, there was an even greater decrease in PI, a larger increase in PA and some radioactivity was lost from PC and to a lesser extent from PS. At either 30 sec. or 4 min. after 0.1 U/ml of thrombin, there was no incorporation of radioactivity into PE. At both 30 sec. and 4 min. after 1 U/ml of thrombin there was a much greater decrease in PC than after 0.1 U/ml of thrombin. On the other hand, the changes in PI were if anything somewhat less. There was incorporation of radioactivity into PE at both 30 sec. and 4 min. after 1 U/ml of thrombin. The breakdown of radioactivity from total phospholipids was about the same after 30 sec. of both concentrations of thrombin but increased 4 min. after I U/ml of thrombin. Lastly, the decrease of phospholipid radioactivity under thrombin 0.1 U/ml was accompanied with a synthesis 'of thromboxane B,, HHT and HETE (Table.111). These three compounds were assayed after 4 min. of treatment by thrombin 0.1 U/ml at 37'C. TXB2 Control
Thrombin
HHT
HETE
1.8 ? 0.2
0.15 + 0.04
0.9 ? 0.4
10.7 2 4.2
0.61 -I0.13
5.1 ? 2.0
TABLE III
TX%, HHT and HETE from endogenous arachidonic acid produced after 4 min. of incubation with thrombin 0.1 U/ml. Results, expressed in percentage of initial phospholipid radioactivity, were the mean of 6 determinations. DISCUSSION The method used in this work gives a good incorporation of arachidonic acid as tracer. Phosphatidic acid is weakly labelled indicating that platelets are not stimulated since this phospholipid only appears when phospholipase C is activated (5,6). Results show that phosphatidylinositol is the major platelet phospholipid breakdown by thrombin used at low concentrations. Even after 30 sec. of thrombin action, this phospholipid is decreased and phosphatidic acid is increased as well. This suggests a very rapid activation of platelet phospholipase C as previously shown (11). Stable metabolites of arachidonic acid such as TXBs, HHT and HETE are synthezised after platelet incubation with thrombin 0.1 U/ml. This indicates an effective release of arachidonic acid from phospholipids. Moreover, it was
Vo1.18,No.1/2
PLATELET
PHOSPHOLIPASE
ACTIVITY
289
possible to observe a prostaglandin endoperoxides synthesis 1 min. after platelet stimulation by thrombin 0.1 U/ml (12). This fact suggests an initial release of arachidonic acid by this low dose of thrombin. Increase radioactivity in PA was previously shown with a low dose of thrombin (0.2 U/ml) and soon 50 sec. whereas ADP 5.10s7M (which does not induce platelet prostaglandin formation) failed to enhance PA radioactivity (13). Lastly, increase of radioactivity in phosphatidylethanolamine, previously described as a plasmalogen form (4), only appears with thrombin 1 U/ml. These results are agree with those previously published (2,3) but they point out the role of phosphatidyl inositol in human platelet activation by thrombin. Thus, phosphatidyl inositol could be the physiological pool of arachidonic acid rapidly available to prostaglandin biosynthesis under low doses of thrombin. ACKNOWLEDGMENTS This work was supported by grant INSERM, ASS N"5. REFERENCES 1. HAMBERG, M. and SAMUELSSON, B. Thromboxanes : a new group of biologically active compounds derived from prostaglandin endoperoxides. ?'hOC. NaL Ac.ad. SQL, 72, 2994-2998, 1975. 2. BILLS, T.K., SMITH, J.B. and SILVER, M.J. Metabolism of [l-'4C] arachidonit acid by human platelets. B~ockim. Biophyb. Acta 424, 303-314, 1976. 3. BILLS, T.K., SMITH, J.B. and SILVER, M.J. Selective release of arachidonic acid from the phospholipids of human platelets in response to thrombin. J. Cl%. lnve&?Z. 60, 1-6, 1977. 4. RITTENHOUSE-SIMl4ONS,S., RUSSEL, F.A. and DEYKIN, D. Transfert of arachidonic acid to human platelet plasmalogen in response to thrombin. G~cch~. Biophqh. R&A. Camnun. 70, 295-301, 1976. 5. MAUCO, G., CHAP, H., SIMON, M.F. and DOUSTE-BLAZY. L. Phosphatidic and lysophosphatidic acid production in phospholipase C and thrombin-treated platelets. Possible involvement of platelet lipase. Bidtiie, 60, 653-661, 1978. 6. LAPETINA, E.G., CHANDRABOSE, K.A. and CUATRECASAS, P. Ionophore A 23187 and thrombin induced platelet aggregation. Independance from cycle-oxygenase product. Phoc. Nat. Acad. Sci. 75, 818-822, 1978. 7. LAGARDE, M., BRYON, P.A., GUICHARDANT, M. and DECHAVANNE, M. A simple and efficient method for platelet isolation from their plasma. Th40m60b. RCQ. In press. 8. DITTMER, B.G. and DOUBLAS, M.G. Quantitative determination of phosphoinositides. Ann. N. Y. Acad. Sci. 165, 515-525, 1969. 9. BEREZIAT, G., CHAMBAZ, J., TRUGNAN, G., PEPIN, D. and POLONOVSKI, J. Turnover of phospholipid linoleic and arachidonic acids in human platelets from plasma lecithins. j. LiyJ-id.Ren. 19, 495-500, 1978.
PLATELET
PHOSPHOLIPASE
ACTIVITY
Vo1.18,No.1/2
IO. LAGARDE, II., GHARIB, A. and DECHAVANNE, M. A simple radiochemical assay of thromboxane Bz, IZ-hydroxy-eicosatetraenoic acid (HFTE) and 12-hydroxyheptadecatrienoic acid (HHT) synthezised by human platelets. Cl!&. whim.
Ada. 79, 255-259, 1977. 11. RITTENHOUSE-SIMMONS, S. Production of diglyceride from phosphatidylinositol in activated human platelets. J. C&n. 7nwQcSX.63, 580-587, 1979. 12. LAGARDE, M. BRYON, P.A., VARGAFTIG, B.B. and DECHAVANNC, )I.Impairment of platelet thromboxane A2 generation and of the platelet release reaction in two patients with congenital deficiency of platelet cycle-oxygenase. 8hit. _J.ffaemdtcd. 38, 251-266, 1978. 13. LLOYD, J.V. and MUSTARD, J.F. Changes in 32P-content of phosphatidic acid and the phosphoinositides of rabbit platelets during aggregation induced 26, 243-253, 1974. by collagen or thrombin. Rtif. J. Haumtat.