al49-3&18/78/0601-107979.00/o
THROMBOSfSRESEMKH~ol. 1Z.p~. 1079-1036. 0 PergamonPrcssL~d.1978. PrinrcdinGreat B-i&n.
ON
IN VIVG
THE EFFECT OF ~NcO~ETHACIN ANO ASA INDUCE0 WHITE PLATELET ARTERIAL THROMBUS FCRMATION* ??? ?
R.H. Bourgain Laboratory of Physiology and Physiopathology Free University of Brussels (V.U.B.1 , Eversstr. 2 - B-1000 @russels. Belgium
(Received 10.2.1??8; in revised form 4.q.1976. Accepted by- Editor ?f. Verstraete)
ABSTRACT White platelet thrombus formation in a branch ot the mesenteric artery of the rat induced by electrical current application followed by AOP superfusion can be inhibited by Aspirin and indomethacin topically applied. This phenomenon could result from the decrease of the cyclooxygenase-like activity present in the vascular wall structures. INTRODUCTION Recent investigations clearly demonstrated the role of prostaglandins ~1.2.3.41 in the platelet aggregation phenomena. Indeed, the platelet cyclic endoperoxides PGG2 and PGH2 as well as their metabolic derivative thromboxane A2 evidence potent aggregating properties. The synthesis of these endoperoxides from arachidonic acid requires cyclooxygenase, an enzyme which is also normally present in the thrombocytic stroma. Until recently it was assumed that the platelets passed PGG2 and PGH2 to the endothelial cells and possibly the underlying vascular structures; within the vessel wall however the further biochemical pathway was not to thromhoxane A2. but to a very unstable compound PGI2 [prostacyclin), a powerful platelet anti-aggregating factor as well as a smooth muscle cell relaxant Cl). Very recently when it was evidenced that PG12 could be formed in the vascular wall tissue in rats made thrombocytopenic by the administration of antiplatelet serum IS), considerable doubt arose about the possible source of PGG2 and PGH2 within the arterial wall. Indeed, if
t
**This investigation was supported by F.G.W.O. contract No. 3.0010.76. With the technical assistance of R. Andries. '079
1080
PLATELET ARTERIAL
THFZOXBUS
Vol.l2,No.6
the lat’cer structurss and particularly the arterial microsomes are capable of generating PGI2 activity in animals with induced thrcmbocytopenia, then one has to admit that the vessel wall itsslf necessarily must contain all required snzymes and substrates involved in tha local synthesis not only of PGI2 but also of PGG2 and PGH2 in order to gear the biochemical pathways. As the cyclic endoperoxides PGG2 and PGHz demonstrate well-defined platelet aggregating properties, it could well be that the equilibrium between these endoperoxides and PGI2 determines the extent of platelet adhesion onto the vessel wall followed by platelet aggregation and local whit2 thrombus formation. Evidently thromboxane A2 intervenes latsr when the sequence of the release phenomena is started. In this investigation therefore we intend to study the effect on thrombus formation of the decrease in the synthesis of PGG2 and PGH2 through tha inhibition of the vessel wall cyclooxygenase-liks activity by local superfusion of the arterial segment with ASA or indomethacin. Indeed, in a previous investigation (61 we wer2 able to demonstrate in our "in vivo" model that superfusion of the for thrombus induction prepared arterial segment with a solution of tranylcypromine resulted in a marked enhancement of thrombus formation. This finding could be explained on the basis of an inhibition of the vessel wall PGI2 synthetase and is an argument for the hypothesis that PGI2 in the endothelial cells accounts for the non-thrombogenicity of the endothelium while the endoperoxides PGG2 and PGHz have an opposite effect. If ASA and indomethacin are wall known inhibitors of cyclooxygenase activity, their effect on the prostaglandin metabolism in the vessel wall in relation to white thrombus formation is much less known.
METHODOLOGY An "in vivo" model for the study of arterial thrombosis has been described in previous publications (8.91. White Wistar male rats (200 to 350 gl were used for investigation. After induction of general narcosis with thalamonal (4.4 ml thalamonal/kg b.w.1 the animal is kept under artificial respiration. An incision is made along the lateral border of the right rectus abdominis and a loop of small intestine is gently extracted. A branch (150 to 200 pm diameter) of the mesenteric artery is dissected free from the surrounding mesenteric tissue over a distance of 2 mm under binocular operative microscopy. The dissected artery is kept under constant superfusion with isotonic Ringer solution at 37°C. Thrombus induction is performed by applying only once a DC current of small intensity (25 to 50 pA) to the arterial wall using a platinum electrode insulated except at the tip (25 pm) for sixty seconds. The polarity is inversed every five seconds in order to avoid vasospastic phenomena. The electrode is immediately removed after application of the Current. Sometimes a small white mural platelet thrombus develops at the site of application of the current but this thrombus rapidly disappears within two to three minutes. The application of the current results in a desendotheliazation of the arterial lumen over a distance of 150 to 250 um. Eventually some foci of lysis within the smooth muscle cells occur but the lamina elastica interna is never disrupted.
Yol. ‘2.X0.6
PLATELET
ARTERIAL
THROYBCS
1081
White platelet thrombi can now be induced $within the vascular lumen by superfusion of the arterial segment with a solution of ADP. The experimental set-up is so arranged that by m2ans of a gradient method the molarity of the ADP in solution is exponentially increased. As soon as a whit2 platelet thrombus becomes visible, the concentration of AOP is kept constant for an additional time interval of 30 seconds. After this period normal isotonic Ringer solution is again superfused ov2r the preparation. The arterial thrombus grows during these thirty seconds and ev2n later however the total duration of the thrombotic phenomenon normally lasts between 140 to 180 seconds following the first signs of thrombosis and the thrombotic mass disappears either by fragmentation or a complete emoolization of the whole mass. If experimental conditions such as general narcosis and operative care as well as appropriat8 superfusion of the arterial praparation are kept constant, a thrombus can be induced every 15 minutes with quit2 comparable results as estimated from the investigated paramstars and this over a period of five to six hours. REGISTRATION of the thrombotic phenomenon is performed by a microprojection technique. The investigated arterial segment is projected onto a set of thirty LOR elements [Light Oepending Resistances1 arranged in two juxtaposed columns of 15 each. No free space is left between two adjoining LOR's. Each one of these LDR's is connected into its own Wheatstone bridge and the differences in light intensity due to the appearance of the whit2 platelet thrombus which contrasts with the surrounding blood flow are recorded as potential variations. Oifferent discriminating PARAMETERS are thus used : the T(t1 curve : this curve results from the summation of the potentials registered on all thirty LOR elements during a time interval of 30 x.1/9 seconds [the TTV(t1 curve results from the integration of the T(tl x IF value in function of time]; the Ott] curve represents during each 30 x l/9 x IO'* seconds interval the greatest potential deviation on one of the LOR's covered by the projected thrombus image; the O(t) curve indicates the number of LOR's covered by the thrombus image; the tell interval is a "lag period" or the time interval from the beginning of the AOP superfusion to the first visibl e signs of thrombus formation. Because the molarity of AOP increases exponentially in function of time, this interval is a measure for the concentration of AOP required to induce thrombosis; the tcdl interval is the duration of the thrombotic phenomenon: m[Ol* m(T1 and rncOl correspond to the maximal values registered respectively on the OttI. T(t) and O(t) curves; TTM is the integration of the T(t) curve to the point where m[T] is reached.
MATERIALS Computer facilities with AD conversion and suitable programmation were available on a POP 8 computer for data acquisition and computatiorlof th2 indicated parameters. - On a direct writing device T(t), m(Ol, TTV(t1 and O(t) were registered continuously as well as the heart rate of the animal.
The thirty LDR's are displayed on a Tektronix scope (type 5542 - stcrage osciiloscops with auto-erase). registared oy mu?tipleXing at the rate of one LOR every 1/S x lO'*seconds. Aspirin is used as lysine-acetylsalicylate salt for superfusion at 11 m:! in isotonic Ringer. Indomethacin 1 mM is also dissolved in isotonic Ringer. All solutions are kept at 37'C and the superfusion rate is 1 mi/min.
RESULTS After the registration of the control thrombi superfusion of the arterial segment with the solution containing Aspirin or indomethacin is started 10 minutes before and during thrombus induction with AOP. No vasospastic phenomena were observed and superfusions of the artery did not modify to any extent the basic conditions required for registration. Sixteen male Mistar rats were divided randomly in two groups of each eight animals. Each animal of course serves as its own control. In the 0 results observed before (81 and immediately tables 1 and 2 are given th_ [fO minutes) after (Al topical superfusion with ASA or indomethacin. Once superfusion with these substances is discontinued, normal control values again are found within 30 minutes (these findings are not presented in the tables). The ValUeS of t[lj, t(dJ and m(G) before and after superfusion with Aspirin were statistically different at P < 0.01 [paired t-test]. For the m[Tl, TTM and m(G) values a Wilcoxon test was statistically significant. These findings clearly evidence that superfusion of the arterial segment with indomethacin or Aspirin results in a decrease of arterial thrombus formation induced by AGP.
DISCUSSION AND CONCLUSION The role of the prostaglandin metabolism in platelets has already been clearly established: the results published by Moncada et al. c2.31 demonstrated that arterial microsomes are capable of converting the cyclic endoperoxides PGG2 and PGH2 known for their platelet aggregating properties Into a potent anti-aggregating compound PG12 fprostacyclinl. The presence of PGI2 within the endothelium as well as in other vascular structures could well be the predominating factor in preventing platelet-vessel wall interaction (71 and subsequent platelet aggregation. More recently Villa et al. (51 demonstrated that PG12 could be synthetized within the vessel wall when thrombocytopenia was induced in rats. This finding implies that most likely these cyclic endoperoxides are synthetized within the vessel wall.
x-01.12,No.i;
PLATELFT
ARTERIAL
TAELE Values and
of after
Rats
NC.
No.
No.
No.
No.
No.
Na.
No.
tne
Parameters
[Al
Tapical
t
t[l),
t[dl,
Super-fusion
1087
1 m(5),
of
THROYBC5
th s
m(T): TTM and mIOl before (3) Arterial Segment with Aspirin
TTM
1
0
65
94
92
255
A
65
00
49
126
2.252
B
61
76
78
140
2.93s
A
60
75
64
92
1.707
0
63
103
A
61
102
0
56
A
1
2
3.332
169.613
0
450
2.049
160.703
7
307
763
3.926
541.090
10
71
270
468
2.634
307.532
9
B
67
112
79
198
5.704
4
A
116
104
73
140
3.404
3
0
01
101
02
154
4.021
5
A
102
105
81
100
2.335
3
0
67
104
343
135.604
10
A
03
176
70
2.911
5
0
43
173
440
1.101
44.701
6
A
43
118
336
009
10.470
5
1.031
3
4
5
6
1.713
7 149
6
TAELE 'JalueS
and after
the ?xmT&erS t(l), [Al Tcpical Superfusion
of
Rats
NC.
No.
No.
No.
No.
No.
No.
No.
Vol.
PLATELET .-\RTERI_XL THZOHBIJS
‘084
t(d],
12,Xo.h
2 lTID~.
of the
r~(~),
Arterial
TTM and mCO1 before [a) Segment with Indomethacin
TTM
“(11
t(dl
mm1
m(T1
a
62
190
637
2.275
130.696
a
A
51
183
559
T.SGO
91.628
8
8
85
99
50
61
1.812
2
A
89
89
29
38
a20
2
a
4s
281
1.056
7.457
968.829
77
A
61
311
071
7.093
755.756
14
B
82
287
301
1.258
a8.751
9
A
80
Ii0
187
567
21.535
6
a
32
153
379
792
24.057
6
A
72
151
343
456
12.684
4
a
16
117
a54
2.269
71.614
6
A
16
100
603
2.081
59.376
6
38
246
615
1.421
38.728
7
72
135
529
1.298
18.613
5
27
190
559
I.@40
00.352
a
26
147
240
629
22.936
4
mfO1
1
2
3
4
5
6
7
8
vo1.12,50.~
PLXTELET
t\RTERI.AL THRO>fFjT?S
1085
It^ the presence of FGG2 and PC82 within the vessel wall is the result of an active local intramural synthesis, then of course cyclooxygenase activity must be available to gear in and control this important metabolic pathway. Weksler et al. (101 could demonstrate that endothelial cells in culture not only have the capacity to generate PG12 from exogenous sodium arachidonats via an intracellular cyclooxygenase but are also able to convert externally formed prostaglandin endoperoxides to PGI2. In our experimental "in vivo" model we were able to demonstrate that superfusion of the for thrombus induction prepared arterial segment with ASA or indomethacin resulted in a marked inhibition of thrombus formation and development induced by ADP. Experimental data [Ill clearly indicate that this effect does not result from the passage of these drugs through the vessel wall into the bloodstream, thus directly affecting the platelets. A likely explanation for this inhibitory effect on thrombus formation could be the decreased production of PGG2 and PGH2 as a direct result of the inhibition by ASA and indomethacin of the vessel wall cyclooxygenase activity. Necessity for the use of high concentrations of ASA and indomethacin in the superfusion liquid is due to the fact that these substances at the administered rate of flow have to penetrate the vessel wall in the direction adventitia-endothelium, a distance which is not negligible. Apparently penetration into the vessel wall only occurs when the electrical current has been previously applied. One could wonder why PGG2 -PGH2 inhibition does not result in an enhancement of the thrombotic phenomenon, as a decrease in the synthesis of PGG2 and PGH2 must result ultimately in less PG12 production. The observations made with tranylcypromine 161 as well as the results obtained with ASA or indomethacin suggest that local thrombus formation in an arterial segment depends very probably on the PGG2-PGH2/PGI2 ratio, so that a decrease in the synthesis of these endoperoxides results in a decrease in local thrombus formation while a decrease in PGI2 has the opposite effect. The respective half-lives of the different prostaglandin derivatives involved very probably play an inportant role in determining the equilibrium constant. in conclusion, the decrease of the induced arterial white platelet thrombi as compared to the control values can be explained on the basis of an inhibition of a cyclooxygenase activity in the vessel wall by the topical superfusion of the arterial segment by ASA or indomethacin and emphasizes the importance of the arterial wall - platelet interactions.
REFERENCES 1. BUNTING. S.. GRYGLEWSKI, R., MONCAOA, S., and VANE, J.R. Arterial walls generate from prostaglandin endoperoxides a substance [prostaglandin Xl which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation. Prostaglandins. 12. 097, 1976. 2
RONCAOA. S.. GRYGLEWSKI. R., BUNTING, S.. and VANE, J.R. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature. 263, 663. 1976.
PL.ATFLET
1 OYh
3.
IY~CxlA, ioe
S . , GRY2i_EWSiii,
inhibits
the
prostaglandin vents platelet 4.
SEST,
enzyme
6.
S.,
CALLIO?iI,
activity
in
II,
701,
2377.
BOURGAIN.
R.H.
terial wall Haemostasis.
7.
MONCAOA,
S.,
formation
of
An
8.
9.
EIOURGAIN,
and
Acad.
BOURGAIN.
de
Inhibition
GAETANO,
from
of
A.G.,
prostacyclin for
Thromb.
the
R.H.
PGI2
and
Sci.
SIX.
R.H.
White
and
in The 6,
74,
Unpublished
from
Xl whicn 1376.
pre-
Prostacyc-
F.E. activity
in plate-
prostacyclin-like rats.
Thromb.
synthesis
platelet
VANE,
perox-
thrombi
the
"in
ar-
viva".
Oifferential
J.R. the the
of of
in
Res.
arterial vasculsr
Wall. endo-
1977.
A continuous
F.
Res.
USA.
Normal
lipi
generates
PRESTON,
[prostacyclinl
E.A.,
323.
715,
thrombocytopenic
HIGGS,
11.
Res.
Thromb.
G.
A
J.R. that
cyclase
[PGX or PGI21 by layers anti-thrombotic properties
WEKSLER, @.3., MARCUS, A.J., 12 [prostacyclinl by cultured Natl.
11.
1977.
BOURGAIN, R.k!. and SIX, F. imental arterial thrombosis
formation. 10.
350.
and
adenylate
Yol.12.~0.6
VANE,
i--0somes m,,,
R.G.G.,
and
enhances the formation of for publication. Accepted
explanation
thelium.
levels
tissues
HERMAN,
and
S.,
vessei
RUSSELL,
AMP
A.,
vascular
THRO.‘fBITS
[prostaglmdin the substance Prostaglandins. 12,
T.J.,
lin incres ses cyclic lets. 'iatLi?e. 267. 5. VILLA,
3UP:TI~G. blood
endsperoxides aggregation.
rARTIPJ,
L.Z.,
R., in
ARTERI.aL
the
effect
195,
data.
of
defibrase
on
method in axper4, 599, 1374. arterial
thrombus
1975.
and JAFFE, human and
3922,
registration Thromb. Res.
rat.
1977.
Synthesis of E.A. bovine endothelial
prostaglandin cells. Proc.
THROMBOSfSRESEMKH~ol. 1Z.p~. 1079-1036. 0 PergamonPrcssL~d.1978. PrinrcdinGreat B-i&n.
ON
IN VIVG
THE EFFECT OF ~NcO~ETHACIN ANO ASA INDUCE0 WHITE PLATELET ARTERIAL THROMBUS FCRMATION* ??? ?
R.H. Bourgain Laboratory of Physiology and Physiopathology Free University of Brussels (V.U.B.1 , Eversstr. 2 - B-1000 @russels. Belgium
(Received 10.2.1??8; in revised form 4.q.1976. Accepted by- Editor ?f. Verstraete)
ABSTRACT White platelet thrombus formation in a branch ot the mesenteric artery of the rat induced by electrical current application followed by AOP superfusion can be inhibited by Aspirin and indomethacin topically applied. This phenomenon could result from the decrease of the cyclooxygenase-like activity present in the vascular wall structures. INTRODUCTION Recent investigations clearly demonstrated the role of prostaglandins ~1.2.3.41 in the platelet aggregation phenomena. Indeed, the platelet cyclic endoperoxides PGG2 and PGH2 as well as their metabolic derivative thromboxane A2 evidence potent aggregating properties. The synthesis of these endoperoxides from arachidonic acid requires cyclooxygenase, an enzyme which is also normally present in the thrombocytic stroma. Until recently it was assumed that the platelets passed PGG2 and PGH2 to the endothelial cells and possibly the underlying vascular structures; within the vessel wall however the further biochemical pathway was not to thromhoxane A2. but to a very unstable compound PGI2 [prostacyclin), a powerful platelet anti-aggregating factor as well as a smooth muscle cell relaxant Cl). Very recently when it was evidenced that PG12 could be formed in the vascular wall tissue in rats made thrombocytopenic by the administration of antiplatelet serum IS), considerable doubt arose about the possible source of PGG2 and PGH2 within the arterial wall. Indeed, if
t
**This investigation was supported by F.G.W.O. contract No. 3.0010.76. With the technical assistance of R. Andries. '079
1080
PLATELET ARTERIAL
THFZOXBUS
Vol.l2,No.6
the lat’cer structurss and particularly the arterial microsomes are capable of generating PGI2 activity in animals with induced thrcmbocytopenia, then one has to admit that the vessel wall itsslf necessarily must contain all required snzymes and substrates involved in tha local synthesis not only of PGI2 but also of PGG2 and PGH2 in order to gear the biochemical pathways. As the cyclic endoperoxides PGG2 and PGHz demonstrate well-defined platelet aggregating properties, it could well be that the equilibrium between these endoperoxides and PGI2 determines the extent of platelet adhesion onto the vessel wall followed by platelet aggregation and local whit2 thrombus formation. Evidently thromboxane A2 intervenes latsr when the sequence of the release phenomena is started. In this investigation therefore we intend to study the effect on thrombus formation of the decrease in the synthesis of PGG2 and PGH2 through tha inhibition of the vessel wall cyclooxygenase-liks activity by local superfusion of the arterial segment with ASA or indomethacin. Indeed, in a previous investigation (61 we wer2 able to demonstrate in our "in vivo" model that superfusion of the for thrombus induction prepared arterial segment with a solution of tranylcypromine resulted in a marked enhancement of thrombus formation. This finding could be explained on the basis of an inhibition of the vessel wall PGI2 synthetase and is an argument for the hypothesis that PGI2 in the endothelial cells accounts for the non-thrombogenicity of the endothelium while the endoperoxides PGG2 and PGHz have an opposite effect. If ASA and indomethacin are wall known inhibitors of cyclooxygenase activity, their effect on the prostaglandin metabolism in the vessel wall in relation to white thrombus formation is much less known.
METHODOLOGY An "in vivo" model for the study of arterial thrombosis has been described in previous publications (8.91. White Wistar male rats (200 to 350 gl were used for investigation. After induction of general narcosis with thalamonal (4.4 ml thalamonal/kg b.w.1 the animal is kept under artificial respiration. An incision is made along the lateral border of the right rectus abdominis and a loop of small intestine is gently extracted. A branch (150 to 200 pm diameter) of the mesenteric artery is dissected free from the surrounding mesenteric tissue over a distance of 2 mm under binocular operative microscopy. The dissected artery is kept under constant superfusion with isotonic Ringer solution at 37°C. Thrombus induction is performed by applying only once a DC current of small intensity (25 to 50 pA) to the arterial wall using a platinum electrode insulated except at the tip (25 pm) for sixty seconds. The polarity is inversed every five seconds in order to avoid vasospastic phenomena. The electrode is immediately removed after application of the Current. Sometimes a small white mural platelet thrombus develops at the site of application of the current but this thrombus rapidly disappears within two to three minutes. The application of the current results in a desendotheliazation of the arterial lumen over a distance of 150 to 250 um. Eventually some foci of lysis within the smooth muscle cells occur but the lamina elastica interna is never disrupted.
Yol. ‘2.X0.6
PLATELET
ARTERIAL
THROYBCS
1081
White platelet thrombi can now be induced $within the vascular lumen by superfusion of the arterial segment with a solution of ADP. The experimental set-up is so arranged that by m2ans of a gradient method the molarity of the ADP in solution is exponentially increased. As soon as a whit2 platelet thrombus becomes visible, the concentration of AOP is kept constant for an additional time interval of 30 seconds. After this period normal isotonic Ringer solution is again superfused ov2r the preparation. The arterial thrombus grows during these thirty seconds and ev2n later however the total duration of the thrombotic phenomenon normally lasts between 140 to 180 seconds following the first signs of thrombosis and the thrombotic mass disappears either by fragmentation or a complete emoolization of the whole mass. If experimental conditions such as general narcosis and operative care as well as appropriat8 superfusion of the arterial praparation are kept constant, a thrombus can be induced every 15 minutes with quit2 comparable results as estimated from the investigated paramstars and this over a period of five to six hours. REGISTRATION of the thrombotic phenomenon is performed by a microprojection technique. The investigated arterial segment is projected onto a set of thirty LOR elements [Light Oepending Resistances1 arranged in two juxtaposed columns of 15 each. No free space is left between two adjoining LOR's. Each one of these LDR's is connected into its own Wheatstone bridge and the differences in light intensity due to the appearance of the whit2 platelet thrombus which contrasts with the surrounding blood flow are recorded as potential variations. Oifferent discriminating PARAMETERS are thus used : the T(t1 curve : this curve results from the summation of the potentials registered on all thirty LOR elements during a time interval of 30 x.1/9 seconds [the TTV(t1 curve results from the integration of the T(tl x IF value in function of time]; the Ott] curve represents during each 30 x l/9 x IO'* seconds interval the greatest potential deviation on one of the LOR's covered by the projected thrombus image; the O(t) curve indicates the number of LOR's covered by the thrombus image; the tell interval is a "lag period" or the time interval from the beginning of the AOP superfusion to the first visibl e signs of thrombus formation. Because the molarity of AOP increases exponentially in function of time, this interval is a measure for the concentration of AOP required to induce thrombosis; the tcdl interval is the duration of the thrombotic phenomenon: m[Ol* m(T1 and rncOl correspond to the maximal values registered respectively on the OttI. T(t) and O(t) curves; TTM is the integration of the T(t) curve to the point where m[T] is reached.
MATERIALS Computer facilities with AD conversion and suitable programmation were available on a POP 8 computer for data acquisition and computatiorlof th2 indicated parameters. - On a direct writing device T(t), m(Ol, TTV(t1 and O(t) were registered continuously as well as the heart rate of the animal.
The thirty LDR's are displayed on a Tektronix scope (type 5542 - stcrage osciiloscops with auto-erase). registared oy mu?tipleXing at the rate of one LOR every 1/S x lO'*seconds. Aspirin is used as lysine-acetylsalicylate salt for superfusion at 11 m:! in isotonic Ringer. Indomethacin 1 mM is also dissolved in isotonic Ringer. All solutions are kept at 37'C and the superfusion rate is 1 mi/min.
RESULTS After the registration of the control thrombi superfusion of the arterial segment with the solution containing Aspirin or indomethacin is started 10 minutes before and during thrombus induction with AOP. No vasospastic phenomena were observed and superfusions of the artery did not modify to any extent the basic conditions required for registration. Sixteen male Mistar rats were divided randomly in two groups of each eight animals. Each animal of course serves as its own control. In the 0 results observed before (81 and immediately tables 1 and 2 are given th_ [fO minutes) after (Al topical superfusion with ASA or indomethacin. Once superfusion with these substances is discontinued, normal control values again are found within 30 minutes (these findings are not presented in the tables). The ValUeS of t[lj, t(dJ and m(G) before and after superfusion with Aspirin were statistically different at P < 0.01 [paired t-test]. For the m[Tl, TTM and m(G) values a Wilcoxon test was statistically significant. These findings clearly evidence that superfusion of the arterial segment with indomethacin or Aspirin results in a decrease of arterial thrombus formation induced by AGP.
DISCUSSION AND CONCLUSION The role of the prostaglandin metabolism in platelets has already been clearly established: the results published by Moncada et al. c2.31 demonstrated that arterial microsomes are capable of converting the cyclic endoperoxides PGG2 and PGH2 known for their platelet aggregating properties Into a potent anti-aggregating compound PG12 fprostacyclinl. The presence of PGI2 within the endothelium as well as in other vascular structures could well be the predominating factor in preventing platelet-vessel wall interaction (71 and subsequent platelet aggregation. More recently Villa et al. (51 demonstrated that PG12 could be synthetized within the vessel wall when thrombocytopenia was induced in rats. This finding implies that most likely these cyclic endoperoxides are synthetized within the vessel wall.
x-01.12,No.i;
PLATELFT
ARTERIAL
TAELE Values and
of after
Rats
NC.
No.
No.
No.
No.
No.
Na.
No.
tne
Parameters
[Al
Tapical
t
t[l),
t[dl,
Super-fusion
1087
1 m(5),
of
THROYBC5
th s
m(T): TTM and mIOl before (3) Arterial Segment with Aspirin
TTM
1
0
65
94
92
255
A
65
00
49
126
2.252
B
61
76
78
140
2.93s
A
60
75
64
92
1.707
0
63
103
A
61
102
0
56
A
1
2
3.332
169.613
0
450
2.049
160.703
7
307
763
3.926
541.090
10
71
270
468
2.634
307.532
9
B
67
112
79
198
5.704
4
A
116
104
73
140
3.404
3
0
01
101
02
154
4.021
5
A
102
105
81
100
2.335
3
0
67
104
343
135.604
10
A
03
176
70
2.911
5
0
43
173
440
1.101
44.701
6
A
43
118
336
009
10.470
5
1.031
3
4
5
6
1.713
7 149
6
TAELE 'JalueS
and after
the ?xmT&erS t(l), [Al Tcpical Superfusion
of
Rats
NC.
No.
No.
No.
No.
No.
No.
No.
Vol.
PLATELET .-\RTERI_XL THZOHBIJS
‘084
t(d],
12,Xo.h
2 lTID~.
of the
r~(~),
Arterial
TTM and mCO1 before [a) Segment with Indomethacin
TTM
“(11
t(dl
mm1
m(T1
a
62
190
637
2.275
130.696
a
A
51
183
559
T.SGO
91.628
8
8
85
99
50
61
1.812
2
A
89
89
29
38
a20
2
a
4s
281
1.056
7.457
968.829
77
A
61
311
071
7.093
755.756
14
B
82
287
301
1.258
a8.751
9
A
80
Ii0
187
567
21.535
6
a
32
153
379
792
24.057
6
A
72
151
343
456
12.684
4
a
16
117
a54
2.269
71.614
6
A
16
100
603
2.081
59.376
6
38
246
615
1.421
38.728
7
72
135
529
1.298
18.613
5
27
190
559
I.@40
00.352
a
26
147
240
629
22.936
4
mfO1
1
2
3
4
5
6
7
8
vo1.12,50.~
PLXTELET
t\RTERI.AL THRO>fFjT?S
1085
It^ the presence of FGG2 and PC82 within the vessel wall is the result of an active local intramural synthesis, then of course cyclooxygenase activity must be available to gear in and control this important metabolic pathway. Weksler et al. (101 could demonstrate that endothelial cells in culture not only have the capacity to generate PG12 from exogenous sodium arachidonats via an intracellular cyclooxygenase but are also able to convert externally formed prostaglandin endoperoxides to PGI2. In our experimental "in vivo" model we were able to demonstrate that superfusion of the for thrombus induction prepared arterial segment with ASA or indomethacin resulted in a marked inhibition of thrombus formation and development induced by ADP. Experimental data [Ill clearly indicate that this effect does not result from the passage of these drugs through the vessel wall into the bloodstream, thus directly affecting the platelets. A likely explanation for this inhibitory effect on thrombus formation could be the decreased production of PGG2 and PGH2 as a direct result of the inhibition by ASA and indomethacin of the vessel wall cyclooxygenase activity. Necessity for the use of high concentrations of ASA and indomethacin in the superfusion liquid is due to the fact that these substances at the administered rate of flow have to penetrate the vessel wall in the direction adventitia-endothelium, a distance which is not negligible. Apparently penetration into the vessel wall only occurs when the electrical current has been previously applied. One could wonder why PGG2 -PGH2 inhibition does not result in an enhancement of the thrombotic phenomenon, as a decrease in the synthesis of PGG2 and PGH2 must result ultimately in less PG12 production. The observations made with tranylcypromine 161 as well as the results obtained with ASA or indomethacin suggest that local thrombus formation in an arterial segment depends very probably on the PGG2-PGH2/PGI2 ratio, so that a decrease in the synthesis of these endoperoxides results in a decrease in local thrombus formation while a decrease in PGI2 has the opposite effect. The respective half-lives of the different prostaglandin derivatives involved very probably play an inportant role in determining the equilibrium constant. in conclusion, the decrease of the induced arterial white platelet thrombi as compared to the control values can be explained on the basis of an inhibition of a cyclooxygenase activity in the vessel wall by the topical superfusion of the arterial segment by ASA or indomethacin and emphasizes the importance of the arterial wall - platelet interactions.
REFERENCES 1. BUNTING. S.. GRYGLEWSKI, R., MONCAOA, S., and VANE, J.R. Arterial walls generate from prostaglandin endoperoxides a substance [prostaglandin Xl which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation. Prostaglandins. 12. 097, 1976. 2
RONCAOA. S.. GRYGLEWSKI. R., BUNTING, S.. and VANE, J.R. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature. 263, 663. 1976.
PL.ATFLET
1 OYh
3.
IY~CxlA, ioe
S . , GRY2i_EWSiii,
inhibits
the
prostaglandin vents platelet 4.
SEST,
enzyme
6.
S.,
CALLIO?iI,
activity
in
II,
701,
2377.
BOURGAIN.
R.H.
terial wall Haemostasis.
7.
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formation
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An
8.
9.
EIOURGAIN,
and
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BOURGAIN.
de
Inhibition
GAETANO,
from
of
A.G.,
prostacyclin for
Thromb.
the
R.H.
PGI2
and
Sci.
SIX.
R.H.
White
and
in The 6,
74,
Unpublished
from
Xl whicn 1376.
pre-
Prostacyc-
F.E. activity
in plate-
prostacyclin-like rats.
Thromb.
synthesis
platelet
VANE,
perox-
thrombi
the
"in
ar-
viva".
Oifferential
J.R. the the
of of
in
Res.
arterial vasculsr
Wall. endo-
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Normal
lipi
generates
PRESTON,
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E.A.,
323.
715,
thrombocytopenic
HIGGS,
11.
Res.
Thromb.
G.
A
J.R. that
cyclase
[PGX or PGI21 by layers anti-thrombotic properties
WEKSLER, @.3., MARCUS, A.J., 12 [prostacyclinl by cultured Natl.
11.
1977.
BOURGAIN, R.k!. and SIX, F. imental arterial thrombosis
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350.
and
adenylate
Yol.12.~0.6
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i--0somes m,,,
R.G.G.,
and
enhances the formation of for publication. Accepted
explanation
thelium.
levels
tissues
HERMAN,
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S.,
vessei
RUSSELL,
AMP
A.,
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THRO.‘fBITS
[prostaglmdin the substance Prostaglandins. 12,
T.J.,
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3UP:TI~G. blood
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rARTIPJ,
L.Z.,
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ARTERI.aL
the
effect
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of
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