Aust N.Z. J . Surg. 1991,61,619-625
619
SURGICAL RESEARCH EFFECTS OF FUREGRELATE (UPJOHN 63557A) ON PATENCY AND PLATELET DEPOSITION AFTER CANINE CAROTID ENDARTERECTOMY J. E. PAYNE,'M. LANGSFELD,~ M.JOSEPH,3 D.H U B E R , ~A. GRAY-WEALE,5 J. H. MEYER, T. SMITH AND R. J. LUSBY~ Department of Surgery, Repatriation General Hospital, Concord, New South Wales Platelet deposition upon endarterectomy sites is a likely cause for early postoperative thrombosis, embolism and restenosis. Platelets aggregate by the thromboxane-prostacycline mechanism. Thromboxane synthetase inhibitors which have been safely administered to humans should reduce platelet deposition after surgical therapy and therefore reduce pen-operative mortality and the prevalence of stroke. A randomized prospective controlled trial was designed to determine vessel patency and platelet deposition associated with the use of 3 mg/kg and 30 mglkg of Furegrelate (Upjohn U63557A) daily in dogs, who were to have carotid endarterectomy. The 46 treated and 46 control dogs had total carotid patency of 96% and 76% respectively ( P < 0.01). Fourteen dogs treated with 30mglkg Furegrelate had no occlusions, compared with a 19% prevalence in 13 controls (P < 0.02). Furegrelate 10 mglkg significantly lowered platelet aggregation. Platelet deposition was not significantly changed, however. The reason for this disparity was a probable persistence of vessel wall factors which promoted platelet deposition. This approach might therefore lower rates of peri-operative thrombosis but it would be very unlikely to alter the incidence of restenosis or embolism. Further research could be directed towards modifying the stimuli for platelet deposition upon the endarterectomy site.
Key words: Furegrelate, platelet deposition.
Introduction Postoperative thrombosis and stenosis are disturbing problems with obvious clinical implications. After carotid endarterectomy, for example, platelet emboli which arise from the site may cause stroke or transient neurological deficit,'*' and asymptomatic stenosis has been found early in the postoperative p e r i ~ d . ~In- ~dogs, the biological basis for the stenosis may be excessive platelet deposition with thrombus formation followed by smooth muscle cell proliferation and incorporation of the thrombus into the vessel wall.' Factors that may influence early postoperative stenosis are the subject of this study. After endarterectomy, exposed collagen is supposed to initiate platelet aggregation by stimulating synthesis of thromboxane, the action of which is unopposed by local prostacycline.8-'0 The competitive inhibition of thromboxane synthetase may therefore minimize postoperative com-
' MS, FRCSE, FRACS. FACS. MD. 'MB, BS, FRCS. MB, BS. FRACS. 5MB. BS. FRACS. 6MD, FRCS. FRACS. Correspondence: I. Payne. Department of Surgery, Univenity of Sydney, Clinical Sciences Building, Repatriation General Hospital, Concord, NSW 2139, Australia. Accepted for publication 8 February IWI.
plications by 'inhibiting platelet aggregation and deposition. Furegrelate (Upjohn U63557A) inhibits thromboxane synthetase in human platelets in vitro, in rhesus monkey platelets in vivo and in dogs with coronary artery stenosis." It was therefore proposed to determine, using autologous Indium 111 labelled platelets, whether Furegrelate had any beneficial effect on the patency of carotid arteries after endarterectomy and on quantitative platelet adherence to the endarterectomy site in the canine species. We chose this dog model because of its vessel size, and because human and canine platelet aggregation patterns are similar. I' EXPERl M ENTA L PROTOCOL
The studies were performed in the research laboratories of the Professorial Surgical Unit, Repatriation General Hospital, Concord, University of Sydney. Animal Ethical Review Committee approval was obtained. Male dogs weighing between 20 and 40 kg had carotid endarterectomy (CE), endarterectomy with patch closure (CEP), exposure of left femoral artery and right femoral arteriotomy (group I), or bilateral endartemtomy alone (group 2). Nonparametric statistical analysis of the outcome was done using Fisher's exact test and the Mann-Whitney
PAYNE ETAL..
620
U test was used to estimate the significance of comparisons between groups. The Student’s t-test was used to determine the significance of differences between means.
of luminal surfaces of endarterectomy and intact endothelium were measured for standardization within and between experiments.
ANAESTHESIA
The method described by Hawker et a / . was used.I3 Indium 1 I 1 oxine (Radiochemical Centre, Amersham) complex was incubated with platelet-rich plasma separated from 17 mL of citrated blood. The labelled platelets were then washed, resuspended, evaluated for labelling efficiency and readministered intravenously.
I N D I U M I I I O X l N E PLATELET L A B E L L I N G
Each dog was premedicated with acetyl promazine maleate 0.5 mg and atropine 0.4 mg, anaesthetized with 30 mg/kg of intravenous sodium pentobarbital, intubated and ventilated using a Midget 3 respirator (CIG Medishield, Homebush, NSW). Anaesthesia was maintained with nitrous oxide, oxygen and halothane. The dogs were given pethidine 10 mglkg intramuscularly for postoperative pain relief. DRUG THERAPY
The dogs were randomized into experimental and control subjects within each group. Experimental dogs received intravenous injections of Furegrelate I mglkg (group I ) and 10mg/kg (group 2) 8hourly. The first injection was given after carotid artery clamping, before administration of labelled platelets. The drug was provided by the Upjohn Company. PROCEDURE
The relevant blood vessels were exposed. Baseline electromagnetic flow measurements were made using Cliniflow I1 (Carolina Medical Electronics Inc.) probes IOC and 12C. Bilateral 1 cm carotid endarterectomies were performed by incision to the vessel media inner layer with blunt dissection. Closure of the arteriotomy was performed using 6/0 prolene (Ethicon). Indium I 1 1 labelled autologous platelets were infused before removal of vascular clamps. In group 2, the endarterectomy site and the vessels were placed under a scintillation counter and the heart counted as a reference point. During an initial 2-hour isotope counting period, the incisions were left open and checks made for possible haematoma formation. The incisions were then closed following confirmation of vessel patency with an EM flow probe. TERMINATION
The dogs were sacrificed 24 h after initial declamping while under general anaesthesia using sodium barbitone 0.5 mg/kg. EM flow metre estimates were obtained from each artery. The vessels were weighed and the Indium 1 I 1 emissions counted for 1 min in wells of a gamma counter. After removal of the peri-adventitial tissue the vessels were pinned to polystyrene boards and opened longitudinally. The vessels were photographed using a 35mm slide camera so that the slide could subsequently be projected onto quadrille paper and sketched. The area
AGGREGOMETRY
Platelet aggregometry was performed pre-operatively, after the administration of Furegrelate, and prior to termination using the Paton Aggregation System, Scarborough, Ontario, Canada. NinemL of blood was added to 1 mL of 3.2% sodium citrate. Platelet-rich plasma was obtained by centrifugation at 200 g for 10 min. Platelet-poor plasma was obtained by centrifugation at 1000 g for 10 min. Aggregation of this sample represented 100% aggregation. Aggregation was then induced by adding IOpL of a 1 mmoVL stock solution of adenosine diphosphate (ADP) to 0.5mL of platelet-rich suspension while stirring the platelets at 900r/min at 37°C. Serial dilutions of ADP were added to subsequent platelet suspensions until disaggregation occurred. Measurements of (i) maximal rise from baseline; and (ii) rate of aggregation after 1 min time intervals were recorded.
Results G R O U P I . ENDARTERECTOMY ( C E ) A N D E N D A R T E R E C T O M Y W I T H PATCH ( C E P )
Nine dogs were treated with Furegrelate I g/kg and there were 10 controls. Patency Six of the control vessels and two of the drug vessels were occluded by thrombus P = 0.014 (Fisher’s exact test). Deposition All platelet deposition results are given in Table I . Platelet deposition in the CE group was twice that of incision alone, when adjustments for incision length were made. Deposition was negligible in the group whose arteries were only mobilized. While the difference in mean platelet deposition between controls and drug in the CE group was 6.4 ( X lo6 mm-*) the large variation prevented significance, this factor also applied to the 2.8 ( X lo6 mm-*) difference in the CEP group. Aggregation All platelet aggregation results are given in Table 2. The difference between group
FUREGRELATE AITER CAROTID ENDARTERECTOMY
62 1
Table 1. Platelet deposition ( X 106/mm2) Furegrelate
Mean f s.d. Including occluded vessels Excluding occluded vessels
Control
CE*
CEP**
CE(Group 2)***
CE*
CEP**
CE (Group 2)***
I .7 3.7 3.9 5.1 5.2 6.0 6.3 9.2t 10.2
2.8 3.2 3.2 3.5 3.8 4.5 4.5 5.5 11.4t
0.825 0.891 1.507 2.048 2.513 2.577 3.097 3.105 3.326 3.583 3.625 3.642 3.645 4.025 4.1 I7 4.646 4.793 5.348 5.378 5.423 5.446 5.498 5.824 5.973 6.784 6.969 7.079 7.522 8.079 9.435
1.3 1.9 4.1 4.2 4.9 5.4 13.97 22.6t 23.4t 43.6t
0.8 1.6 1.7 1.7 2.4 4.2 8.5
0.755 1.165 2.497 2.781 2.911 3.145 3.157 3.197 3.418 3.638 3.649 3.783 4.270 4.789 4.875 4.983 5.710 6.123 6.568 6.571t 7.412t 8.119 8.585 9.105t 10.918t 16.867t
10.1t
24.2t
5.70 f 2.66 (n = 9)
4.71 k 2.65 (n = 9)
4.557 f 2.120 (n = 30)
12.53 f 13.64 (n = 10)
6.13 f 7.53 (n = 9)
5.346 f 3.413 (n = 26)
5.26k 2.48 (n = 8)
3.88 f 0.90 (n = 8)
4.557 k 2.120 (n = 30)
3.63 f 1.66 (n = 6)
2.99 ? 2.65 (n = 7)
4.196 f 1.996 (n = 21)
*Carotid endarterectomy. **Carotid endarterectomy and patch. ***Carotid endarterectomy (group 2 ) . t Occluded.
1 control and Furegrelate treated dogs was not significant. Flow Blood flow for group 1 vessels is not shown. Because of small numbers there were no significant changes between pre- and postoperative flow according to procedure or between CE and CEP. GROUP 2 . BILATERAL ENDARTERECTOMY
Fifteen dogs were treated with Furegrelate lOmg/ kg and there were 13 controls.
fatency Five of the controls vessels were occluded ( f = 0.017; Fisher’s exact test). Deposition It can be Seen from Table 1 that, when statistical analysis included all vessels, the controls had higher deposition. The difference in platelet deposition between controls and drug-treated vessels was 0.5 ( X lo6 mn-’). This difference was not significant. When occluded vessels were removed from the analysis, Furegrelate vessels had the greater mean platelet deposition and the 0.36 ( X lo6 mm-2)
622
PAYNE ET AL.
difference was also not significant ( t = 0.61). This latter experiment would have been able to detect a difference between means of 1.3 ( X lo6 mm-’) with 80%. For a 0.4 ( X lo6 m-’)difference in means with standard deviation (+ 2.07) a sample size of 500 in each group would be required to detect a significant difference (at a power of 80°/0). The small sample size of the experiment gave it only about 13% power.
Aggregation In Table 2 it can be seen that Furegrelate produced a significant (P< 0.01, I = 3.2) fall in aggregation in the postoperative (therapeutic) period, whereas in the controls the difference between pre- and postoperative aggregation was not significant. Four of the dogs in the control group and two in the Furegrelate group were ‘non-aggregators’ - that is, no sustained aggregation was detected at any working ADP con-
Table 2. Aggregornetry Furegrelate Pre-op.
OO /
change
Pre-op.
+ 20.0
75.0 25.0 71.3 72.5 21.3 77.5 31.3 67.5 37.5*
Control Group 2
Group 1
+O - 27.5 + 41.5 - 5.0 -37.5*
change
Pre-op.
- 10.0 + 5.0 - 1.2 + 13.7 - 32.7 - 33.7 -31.5 -11.2 -47.5 - 12.5 - 3.1 7.5 - 56.3 - 18.3**
80.0 90.0 12.5 50.0 40.0 80.0 91.3 85.0 62.5 66.3t
Oh
45.0 37.5 5.0 46.3 82.7 60.0 80.0 50.0 65.0 61.9 50.6 37.5 84.4 59.4**
+ 62.0 - 22.5 + 2.5
Group 1 O/O
Group 2 change
Pre-op.
- 5.0
% change
+ 9.4 + 6.3
38.1 40.0 67.5 53.8 88.8 95.0 17.5 81.3 50.6 60.6 15.0 20.6 20.6$
+O + 8.8 - 13.8 + 18.8 - 11.3 - 37.5 -7.5 - 13.8 + 33.7t
+
- 5.0 - 10.0 - 9.8 -42.5 1.3 -3.8 + 12.5 - 19.3 + 7.5 + 2.5 + 23.8$
+
P = 0.705; paired r-test. **I = 3.199. P = 0.007; paired r-test * I = 0.392,
t r = 0.443, P = 0.068; paired r-test. $1 = 0 . 4 5 5 . P = 0.657; paired r-test.
Table 3. Flow rates (mUrnm) Furegrelate dogs Left Pre
Post
I34 I70 62 176
82 80 70 144 80 135 115 119 106 79 61 66
100
I15 125 109
120 136 66 102 Ill 143 55
110
88 106
Control dogs Right
Left
Term
Pre
Post
Term
160
138 125 62 155 75 125 130 93 180 116 90 102 70
184 100 70 167 I00 89 113
70 100 120 135 81 103 65 93
148 120 120 163 89 83 113 81 105
91 102 68 10 49
160
106 105
77 56 37 94 132 60
110
120 25 78 76 65 15
Right
Pre
Post
Term
Pre
Post
Term
85
88 195 141 157 22 110 123 126 84 190 55 87
82 152 186 53 27 134 49 32
67 125 127 182 136 239 103 92 165
85
10
0
52 4 81 27 I06 I36 I09 35 53 200 95 I28 25
150
153 155 110
165 85 51
130 154 250 188 185
0 160
135 97
100
285 162 145
0
72 10
84 0
70 75
90 I50 157 I23 0
X=114.9 X=96.07 X=IOO.l X=115.8 X=99.33 X=83.73 X=143.2 X=106.8 X=85.15 X=148.31 X=80.XS X=70.46 SD=35.57 SD=25.32 SD=40.93 SD=35.M SD=39.54 SD=33.63 SD=52.I I SD=57.40 SD=63.45 SD=60.44 SD=55.32 SD=S4.72
FUREGRELATE AFTER CAROTID ENDARTERECTOMY
centration. The highest working concentration of ADP was 19.6fim. No corrections were made for the 'non-aggregators' as it was thought they were numerically too few to affect outcome. Flow Blood flow results with means and standard deviations can be seen in Table 3. In the preoperative (untreated) period there was a 30.4mU min lower flow in the group who were to receive Furegrelate. This difference was significant (P < 0.02) (independent samples). Overall mean postoperative flow and pre-termination flows (Table 4) were both significantly less than pre-operative flows within control (P < 0.03, P < 0.002 respectively; paired samples) and Furegrelate (PC 0.02 for both; paired samples) groups. This applied whether occluded control blood vessels were counted in the analysis or not. Table 4. Group 2 mean blood flow*
Re-op Postop. 24 h
Furegrelate
Control
115.3 f 35.0 97.7 ? 32.7 91.9 & 37.7
145.7 f 55.4 93.8 k 56.8 77.8 f 58.5 96.3 k 49. I**
*mUmin k s.d. **Without occluded vessels.
Discussion This study shows that an inhibitor of thromboxane synthetase, Furegrelate (Upjohn U63557A) will significantly reduce the prevalence of canine carotid artery occlusion after endarterectomy from 24% to 4% (92 vessels; P C 0.01). Both doses of Furegrelate produced a significant reduction of platelet aggregation. As thromboxane synthetase inhibitors have been given safely to humans," the results could imply that this approach may be beneficial in humans in the reduction of early postoperative thrombosis. This could be accompanied by reduction in neurological deficit which is known to occur in up to 15% of patients.lS2These serious complications are permanent in 50% of cases and attended by about a 10% mortality. It is important to record that the significant reduction in occlusion was not dose-related or accompanied by changes in deposition at the endarterectomy site. The similar differences in rates of occlusion between Furegrelate and control dogs in both groups suggest that the occlusions were not caused by technical, rheological or unknown reasons. This highlights different mechanisms which cause aggregation and deposition. l4 It is unclear whether blood flow effects played a major role in the outcome of these experiments. The model was selected to produce thrombosis and platelet aggregation because, unlike in human
623
carotid atheroma, the lumen is always slightly occluded. It is likely that the significant reductions in postoperative flow were caused by slight occlusion, the effect of which was not sufficient to obliterate the benefit of Furegrelate therapy. There may, however, be increased production of prostacycline in regions of arterial stenosis ,l5 It is always difficult to provide proof for a negative outcome, particularly when there is a wide variance within and between groups. It is our opinion that variance in these experiments would be little different even if the optimal size was used, because the relevant phenomena have exponential rates; there are wide biological differences within species (e.g. aggregators and non-aggregators). The use of dogs of unknown pedigree, which accentuates this problem,'6-1s was however, a deliberate attempt to produce the type of variation which is found in humans. While it might be accepted that the deposition mechanism is different from that of platelet aggregation, it has to be left to clinical judgement as to whether the effects of Furegrelate impinge on the deposition mechanism. Our opinion, that there is no effect, is supported by the photographs (Fig. l), which show the magnitude of mean platelet deposition and the difference between the means. Furthermore, it would be safe to presume that there might be no change in the rate of postoperative stenosis even though it is not evident from this work whether the mechanism of postoperative ~ t e n o s i s lwould ~ . ~ ~ be affected by Furegrelate.
Fig. 1. Effects of Furegrelate at (a) 4.646 platelets x Id mm-* and (b) 4.983 platelets x lo6
PAYNE ET AL..
624
I n vivo thromboxane synthetase inhibitors divert prostaglandin H2 metabolism away from thromboxane and towards formation of prostacycline. lo Experiments in dogs have compared Furegrelate favourably with cyclo-oxygenase inhibitors, aspirin, ibuprofen, or indomethacin in the dog coronary artery model." This superiority was independent of the effects of intravenous infusions of prostacycline. Thromboxane synthetase inhibitors have the advantages of being more stable in solution, and having longer duration of action and oral administration than prostacycline. Furegrelate is Sodium 5-(3 pyridinylmethyl)-2-benzofurancarboxylate. A single oral dose of 3 mg/kg inhibits primate thromboxane synthesis 80% for at least I2h.l9 It does not appear to produce tachyph laxis or rebound when administered for 14 days.' It does not inhibit thrombin stimulated prostacycline biosynthesis in a variety of test systems. Under appropriate conditions the protective effects of Furegrelate could be reversed by cyclo-oxygenase inhibitors, also suggesting that its efficacy depended in part on endogenous prostacycline formation. l9 It is possible, therefore, that the lack of effect of Furegrelate upon platelet deposition was in part related to a lack of endogenous prostacycline locally. The development of intra-arterial thrombus following carotid endarterectomy; involves a complex of factors including the residual vessel wall, suture and patch material, the formed elements of the blood, blood coagulation proteins and blood flow. Comparison of carotid and femoral artery results showed that vessel wall effect was caused by both the suture line and the endarterectomy. In arterial thromus formation platelets play a major role in both the initiation and growth of thrombi. Following endartemtomy collagen, von Willebrand factor, fibronectin and other proteinsI4 in the medial layer of the vessel are exposed to circulating platelets. Collagen readily reacts with platelets resulting in adhesion and aggregation.20.2' Adhesion initiates a changes sequence of biochemical and ultrast~~ctural (release reaction), during which adenosine diphosphate (ADP) and other substances are selectively secreted, while the contents of the cytoplasm and mitochondria are retained." The release of ADP results in further platelet aggregation, with growth of the thrombus. Platelets also participate in the sequential activation of plasma coagulation proteins, converting prothrombin to thrombin which catalyses the formation of fibrin from fibrinogen finally resulting in still further platelet aggregation. The vessel wall effect and lack of prostacycline appear to have overruled the inhibition of thromboxane synthesis within the platelet in this model. It is unclear which of the mechanisms is the most powerful. However, any drugs which act on platelets by competitive inhibition of prostaglandin
J
metabolites will have less than predicted and less than optimum effects after endarterectomy. Aspirin therapy might be expected to have a better outcome. Both aspirin and heparin,22however, would be clinically associated with significant risk of bleeding and haematoma formation. Thromboxane synthetase inhibitors might provide an appropriate balance between the risks of thrombosis, recurrent stenosis and bleeding. Alternatively, a combination of thromboxane synthetase inhibition and reduction of local platelet deposition could be beneficial. The use of thromboxane synthetase inhibitors is also of theoretical interest because of their ability to increase future understanding about the role of platelets in vascular surgery.
Acknowledgement This project was supported by a grant from the CH & MRC Department of Veterans Affairs, Canberra, Australian Capital Territory.
References 1. THOMPSON J. E. (1983) Carotid endarterectomy, 1982. The state of the art. Brit. J. Surg. 70, 371-6. 2. BUERCER R., SIEGCREEN M. Y.,LAZOA. & HODAKOWSKI G. T. (1986) The silent brain infarct in carotid surgery. J. Vasc. Surg. 3, 442-7. 3. STONEY R . J., & STRING S. T. (1976) Recurrent carotid stenosis. Surgery 80, 705-10. 4. FRENCHB. H. & REWCASTLE N. B. (1977) Recurrent stenosis at site of carotid endarterectomy. Stroke 8,
597-605. 5. ZIERLENR. E., BANOYKD. F., THIELE B. L. & STRANONESSD. E. (1982) Carotid artery stenosis following endarterectomy. Arch. Surg. 117, 1408-15. 6. CLACETI. G. P.,RICHN. M., MCDONALD P. T. er ul. (1988) Etiologic factors for recurrent carotid artery stenosis. Surgery. 93,313-8. 7. Lusev R. J., FERRELLL. D., ENCLESTAD B. L., PRICE D. C., LIFTONM. J. & STONEY R. J. (1983) Vessel wall and indium-Ill-labelled platelet response to carotid endarterectomy. Surgery 93,424-32. 8. SICARD G. A., ALLENB. T., LQNCJ. A. er al. (1984) Prostoglandin production and platelet reactivity of small-diameter grafts. J. Vasc. Surg. 1, 774-81. 9. CAMPION T., LYNCHT. G.,KERRJ. C. & HOBSON R. W . (1986) Effects of prostacycline injections and infusions on canine femoral hemodynamics. J. Vasc. Surg. 3, 540-9. 10. OATESJ. A., FITZCERALD G . A., BRANCH R. A., JACKSON E. K., KNAPPH. R. & ROBERTS L. J. (1988) Clinical implications of prostaglandin and thromboxane A2 formation. (First of two parts.) N. Engl. J. Med. 319, 689-98. I I . GORMANR. R., JOHNSONR. A,, SPILMAN C. H. & AIKENJ. W. (1983) Inhibition of platelet thromboxane A2 synthese activity by sodium 5-(3-pyridinylmethyl) benzofuran-2-carboxylate. Prostuglandins 26, 325-42. 12. FEING~LD H. M., PIVACEK L. E., MELARAGNO A. 1. & VALERI C. R. (1986) Coagulation assays and platelet
FUREGRELATE A R E R CAROTID ENDARTERECTOMY
13. 14. 15. 16.
17. 18.
aggregation patterns in human, baboon and canine blood. Amer. J. Vet. Res. 41, 2197-99. HAWKER R. J., HAWKER L. M. & WILKINSON A. R. (1980) Indium ("'In)-labelled human platelets: o p timal method. CIinicul Sciences 58, 243-8. COLLER B. S. (1990) Platelets and thrombolytic therapy. N . Engl. J. Med. 322,33-42. QVARFORDT P. G., REILLYL. M., LWBYR. J. ei ul. (1985) Prostacyclin production in regions of arterial stenosis. Surgery 98,484-91. KAPLAN S . , MARCOEK. F., SAWAGE L. R. ei a/. (1986) The effect of predetermined thrombotic potential of the recipient on small-caliber graft performance. J. Vusc. Surg. 3,311-21. ALLENB. T., SICARD G. A., WELCHM. J., METHIAS C. J. & CLARK R. E. (1986) Platelet deposition on vascular grafts. Ann. Surg. 203, 318-28. VALENTIN L. I., SICARD G. A., FREEMANM. B., ALLEN B. T., MCGOFF M. A. & ANDERSON C. B. (1988)
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19.
20.
21.
22.
Combined arachidonic acid and ADP platelet inhibition maximizes patency of small-diameter vascular grafts. Surgery 104, 178-84. WYNALDA M. A., L I G GW~. F.& FIIZPATIUCK F. A. ( 1983) Sodium 5-(pyridinylmethyl)beenzofuran-2carboxylate (U-63557A). a new, selective thromboxane synthetase inhibitor: intravenous and oral pharmacokinetics in dogs and correlations with ex siiu thromboxane B2 production. Prosiugludim 26, 31 1-24. BRE~SLER N. N . , BRWKMAN M. J. & MARCUSA. J. (1979) Concurrent studies of oxygen consumption and aggregation in stimulated human platelets. Blood 53, 167-8. ZUCKERM. B. (1980) The functioning of blood platelets. Sci Amer. 242, 86-93. BURNETT J., PAYNE J., GRAY-WEALE A. C. & Lusev R. J. (1988) Selective use of heparin in aortic surgery. Ausi. N.Z.J. Surg. 58, 81 1-5.
619
SURGICAL RESEARCH EFFECTS OF FUREGRELATE (UPJOHN 63557A) ON PATENCY AND PLATELET DEPOSITION AFTER CANINE CAROTID ENDARTERECTOMY J. E. PAYNE,'M. LANGSFELD,~ M.JOSEPH,3 D.H U B E R , ~A. GRAY-WEALE,5 J. H. MEYER, T. SMITH AND R. J. LUSBY~ Department of Surgery, Repatriation General Hospital, Concord, New South Wales Platelet deposition upon endarterectomy sites is a likely cause for early postoperative thrombosis, embolism and restenosis. Platelets aggregate by the thromboxane-prostacycline mechanism. Thromboxane synthetase inhibitors which have been safely administered to humans should reduce platelet deposition after surgical therapy and therefore reduce pen-operative mortality and the prevalence of stroke. A randomized prospective controlled trial was designed to determine vessel patency and platelet deposition associated with the use of 3 mg/kg and 30 mglkg of Furegrelate (Upjohn U63557A) daily in dogs, who were to have carotid endarterectomy. The 46 treated and 46 control dogs had total carotid patency of 96% and 76% respectively ( P < 0.01). Fourteen dogs treated with 30mglkg Furegrelate had no occlusions, compared with a 19% prevalence in 13 controls (P < 0.02). Furegrelate 10 mglkg significantly lowered platelet aggregation. Platelet deposition was not significantly changed, however. The reason for this disparity was a probable persistence of vessel wall factors which promoted platelet deposition. This approach might therefore lower rates of peri-operative thrombosis but it would be very unlikely to alter the incidence of restenosis or embolism. Further research could be directed towards modifying the stimuli for platelet deposition upon the endarterectomy site.
Key words: Furegrelate, platelet deposition.
Introduction Postoperative thrombosis and stenosis are disturbing problems with obvious clinical implications. After carotid endarterectomy, for example, platelet emboli which arise from the site may cause stroke or transient neurological deficit,'*' and asymptomatic stenosis has been found early in the postoperative p e r i ~ d . ~In- ~dogs, the biological basis for the stenosis may be excessive platelet deposition with thrombus formation followed by smooth muscle cell proliferation and incorporation of the thrombus into the vessel wall.' Factors that may influence early postoperative stenosis are the subject of this study. After endarterectomy, exposed collagen is supposed to initiate platelet aggregation by stimulating synthesis of thromboxane, the action of which is unopposed by local prostacycline.8-'0 The competitive inhibition of thromboxane synthetase may therefore minimize postoperative com-
' MS, FRCSE, FRACS. FACS. MD. 'MB, BS, FRCS. MB, BS. FRACS. 5MB. BS. FRACS. 6MD, FRCS. FRACS. Correspondence: I. Payne. Department of Surgery, Univenity of Sydney, Clinical Sciences Building, Repatriation General Hospital, Concord, NSW 2139, Australia. Accepted for publication 8 February IWI.
plications by 'inhibiting platelet aggregation and deposition. Furegrelate (Upjohn U63557A) inhibits thromboxane synthetase in human platelets in vitro, in rhesus monkey platelets in vivo and in dogs with coronary artery stenosis." It was therefore proposed to determine, using autologous Indium 111 labelled platelets, whether Furegrelate had any beneficial effect on the patency of carotid arteries after endarterectomy and on quantitative platelet adherence to the endarterectomy site in the canine species. We chose this dog model because of its vessel size, and because human and canine platelet aggregation patterns are similar. I' EXPERl M ENTA L PROTOCOL
The studies were performed in the research laboratories of the Professorial Surgical Unit, Repatriation General Hospital, Concord, University of Sydney. Animal Ethical Review Committee approval was obtained. Male dogs weighing between 20 and 40 kg had carotid endarterectomy (CE), endarterectomy with patch closure (CEP), exposure of left femoral artery and right femoral arteriotomy (group I), or bilateral endartemtomy alone (group 2). Nonparametric statistical analysis of the outcome was done using Fisher's exact test and the Mann-Whitney
PAYNE ETAL..
620
U test was used to estimate the significance of comparisons between groups. The Student’s t-test was used to determine the significance of differences between means.
of luminal surfaces of endarterectomy and intact endothelium were measured for standardization within and between experiments.
ANAESTHESIA
The method described by Hawker et a / . was used.I3 Indium 1 I 1 oxine (Radiochemical Centre, Amersham) complex was incubated with platelet-rich plasma separated from 17 mL of citrated blood. The labelled platelets were then washed, resuspended, evaluated for labelling efficiency and readministered intravenously.
I N D I U M I I I O X l N E PLATELET L A B E L L I N G
Each dog was premedicated with acetyl promazine maleate 0.5 mg and atropine 0.4 mg, anaesthetized with 30 mg/kg of intravenous sodium pentobarbital, intubated and ventilated using a Midget 3 respirator (CIG Medishield, Homebush, NSW). Anaesthesia was maintained with nitrous oxide, oxygen and halothane. The dogs were given pethidine 10 mglkg intramuscularly for postoperative pain relief. DRUG THERAPY
The dogs were randomized into experimental and control subjects within each group. Experimental dogs received intravenous injections of Furegrelate I mglkg (group I ) and 10mg/kg (group 2) 8hourly. The first injection was given after carotid artery clamping, before administration of labelled platelets. The drug was provided by the Upjohn Company. PROCEDURE
The relevant blood vessels were exposed. Baseline electromagnetic flow measurements were made using Cliniflow I1 (Carolina Medical Electronics Inc.) probes IOC and 12C. Bilateral 1 cm carotid endarterectomies were performed by incision to the vessel media inner layer with blunt dissection. Closure of the arteriotomy was performed using 6/0 prolene (Ethicon). Indium I 1 1 labelled autologous platelets were infused before removal of vascular clamps. In group 2, the endarterectomy site and the vessels were placed under a scintillation counter and the heart counted as a reference point. During an initial 2-hour isotope counting period, the incisions were left open and checks made for possible haematoma formation. The incisions were then closed following confirmation of vessel patency with an EM flow probe. TERMINATION
The dogs were sacrificed 24 h after initial declamping while under general anaesthesia using sodium barbitone 0.5 mg/kg. EM flow metre estimates were obtained from each artery. The vessels were weighed and the Indium 1 I 1 emissions counted for 1 min in wells of a gamma counter. After removal of the peri-adventitial tissue the vessels were pinned to polystyrene boards and opened longitudinally. The vessels were photographed using a 35mm slide camera so that the slide could subsequently be projected onto quadrille paper and sketched. The area
AGGREGOMETRY
Platelet aggregometry was performed pre-operatively, after the administration of Furegrelate, and prior to termination using the Paton Aggregation System, Scarborough, Ontario, Canada. NinemL of blood was added to 1 mL of 3.2% sodium citrate. Platelet-rich plasma was obtained by centrifugation at 200 g for 10 min. Platelet-poor plasma was obtained by centrifugation at 1000 g for 10 min. Aggregation of this sample represented 100% aggregation. Aggregation was then induced by adding IOpL of a 1 mmoVL stock solution of adenosine diphosphate (ADP) to 0.5mL of platelet-rich suspension while stirring the platelets at 900r/min at 37°C. Serial dilutions of ADP were added to subsequent platelet suspensions until disaggregation occurred. Measurements of (i) maximal rise from baseline; and (ii) rate of aggregation after 1 min time intervals were recorded.
Results G R O U P I . ENDARTERECTOMY ( C E ) A N D E N D A R T E R E C T O M Y W I T H PATCH ( C E P )
Nine dogs were treated with Furegrelate I g/kg and there were 10 controls. Patency Six of the control vessels and two of the drug vessels were occluded by thrombus P = 0.014 (Fisher’s exact test). Deposition All platelet deposition results are given in Table I . Platelet deposition in the CE group was twice that of incision alone, when adjustments for incision length were made. Deposition was negligible in the group whose arteries were only mobilized. While the difference in mean platelet deposition between controls and drug in the CE group was 6.4 ( X lo6 mm-*) the large variation prevented significance, this factor also applied to the 2.8 ( X lo6 mm-*) difference in the CEP group. Aggregation All platelet aggregation results are given in Table 2. The difference between group
FUREGRELATE AITER CAROTID ENDARTERECTOMY
62 1
Table 1. Platelet deposition ( X 106/mm2) Furegrelate
Mean f s.d. Including occluded vessels Excluding occluded vessels
Control
CE*
CEP**
CE(Group 2)***
CE*
CEP**
CE (Group 2)***
I .7 3.7 3.9 5.1 5.2 6.0 6.3 9.2t 10.2
2.8 3.2 3.2 3.5 3.8 4.5 4.5 5.5 11.4t
0.825 0.891 1.507 2.048 2.513 2.577 3.097 3.105 3.326 3.583 3.625 3.642 3.645 4.025 4.1 I7 4.646 4.793 5.348 5.378 5.423 5.446 5.498 5.824 5.973 6.784 6.969 7.079 7.522 8.079 9.435
1.3 1.9 4.1 4.2 4.9 5.4 13.97 22.6t 23.4t 43.6t
0.8 1.6 1.7 1.7 2.4 4.2 8.5
0.755 1.165 2.497 2.781 2.911 3.145 3.157 3.197 3.418 3.638 3.649 3.783 4.270 4.789 4.875 4.983 5.710 6.123 6.568 6.571t 7.412t 8.119 8.585 9.105t 10.918t 16.867t
10.1t
24.2t
5.70 f 2.66 (n = 9)
4.71 k 2.65 (n = 9)
4.557 f 2.120 (n = 30)
12.53 f 13.64 (n = 10)
6.13 f 7.53 (n = 9)
5.346 f 3.413 (n = 26)
5.26k 2.48 (n = 8)
3.88 f 0.90 (n = 8)
4.557 k 2.120 (n = 30)
3.63 f 1.66 (n = 6)
2.99 ? 2.65 (n = 7)
4.196 f 1.996 (n = 21)
*Carotid endarterectomy. **Carotid endarterectomy and patch. ***Carotid endarterectomy (group 2 ) . t Occluded.
1 control and Furegrelate treated dogs was not significant. Flow Blood flow for group 1 vessels is not shown. Because of small numbers there were no significant changes between pre- and postoperative flow according to procedure or between CE and CEP. GROUP 2 . BILATERAL ENDARTERECTOMY
Fifteen dogs were treated with Furegrelate lOmg/ kg and there were 13 controls.
fatency Five of the controls vessels were occluded ( f = 0.017; Fisher’s exact test). Deposition It can be Seen from Table 1 that, when statistical analysis included all vessels, the controls had higher deposition. The difference in platelet deposition between controls and drug-treated vessels was 0.5 ( X lo6 mn-’). This difference was not significant. When occluded vessels were removed from the analysis, Furegrelate vessels had the greater mean platelet deposition and the 0.36 ( X lo6 mm-2)
622
PAYNE ET AL.
difference was also not significant ( t = 0.61). This latter experiment would have been able to detect a difference between means of 1.3 ( X lo6 mm-’) with 80%. For a 0.4 ( X lo6 m-’)difference in means with standard deviation (+ 2.07) a sample size of 500 in each group would be required to detect a significant difference (at a power of 80°/0). The small sample size of the experiment gave it only about 13% power.
Aggregation In Table 2 it can be seen that Furegrelate produced a significant (P< 0.01, I = 3.2) fall in aggregation in the postoperative (therapeutic) period, whereas in the controls the difference between pre- and postoperative aggregation was not significant. Four of the dogs in the control group and two in the Furegrelate group were ‘non-aggregators’ - that is, no sustained aggregation was detected at any working ADP con-
Table 2. Aggregornetry Furegrelate Pre-op.
OO /
change
Pre-op.
+ 20.0
75.0 25.0 71.3 72.5 21.3 77.5 31.3 67.5 37.5*
Control Group 2
Group 1
+O - 27.5 + 41.5 - 5.0 -37.5*
change
Pre-op.
- 10.0 + 5.0 - 1.2 + 13.7 - 32.7 - 33.7 -31.5 -11.2 -47.5 - 12.5 - 3.1 7.5 - 56.3 - 18.3**
80.0 90.0 12.5 50.0 40.0 80.0 91.3 85.0 62.5 66.3t
Oh
45.0 37.5 5.0 46.3 82.7 60.0 80.0 50.0 65.0 61.9 50.6 37.5 84.4 59.4**
+ 62.0 - 22.5 + 2.5
Group 1 O/O
Group 2 change
Pre-op.
- 5.0
% change
+ 9.4 + 6.3
38.1 40.0 67.5 53.8 88.8 95.0 17.5 81.3 50.6 60.6 15.0 20.6 20.6$
+O + 8.8 - 13.8 + 18.8 - 11.3 - 37.5 -7.5 - 13.8 + 33.7t
+
- 5.0 - 10.0 - 9.8 -42.5 1.3 -3.8 + 12.5 - 19.3 + 7.5 + 2.5 + 23.8$
+
P = 0.705; paired r-test. **I = 3.199. P = 0.007; paired r-test * I = 0.392,
t r = 0.443, P = 0.068; paired r-test. $1 = 0 . 4 5 5 . P = 0.657; paired r-test.
Table 3. Flow rates (mUrnm) Furegrelate dogs Left Pre
Post
I34 I70 62 176
82 80 70 144 80 135 115 119 106 79 61 66
100
I15 125 109
120 136 66 102 Ill 143 55
110
88 106
Control dogs Right
Left
Term
Pre
Post
Term
160
138 125 62 155 75 125 130 93 180 116 90 102 70
184 100 70 167 I00 89 113
70 100 120 135 81 103 65 93
148 120 120 163 89 83 113 81 105
91 102 68 10 49
160
106 105
77 56 37 94 132 60
110
120 25 78 76 65 15
Right
Pre
Post
Term
Pre
Post
Term
85
88 195 141 157 22 110 123 126 84 190 55 87
82 152 186 53 27 134 49 32
67 125 127 182 136 239 103 92 165
85
10
0
52 4 81 27 I06 I36 I09 35 53 200 95 I28 25
150
153 155 110
165 85 51
130 154 250 188 185
0 160
135 97
100
285 162 145
0
72 10
84 0
70 75
90 I50 157 I23 0
X=114.9 X=96.07 X=IOO.l X=115.8 X=99.33 X=83.73 X=143.2 X=106.8 X=85.15 X=148.31 X=80.XS X=70.46 SD=35.57 SD=25.32 SD=40.93 SD=35.M SD=39.54 SD=33.63 SD=52.I I SD=57.40 SD=63.45 SD=60.44 SD=55.32 SD=S4.72
FUREGRELATE AFTER CAROTID ENDARTERECTOMY
centration. The highest working concentration of ADP was 19.6fim. No corrections were made for the 'non-aggregators' as it was thought they were numerically too few to affect outcome. Flow Blood flow results with means and standard deviations can be seen in Table 3. In the preoperative (untreated) period there was a 30.4mU min lower flow in the group who were to receive Furegrelate. This difference was significant (P < 0.02) (independent samples). Overall mean postoperative flow and pre-termination flows (Table 4) were both significantly less than pre-operative flows within control (P < 0.03, P < 0.002 respectively; paired samples) and Furegrelate (PC 0.02 for both; paired samples) groups. This applied whether occluded control blood vessels were counted in the analysis or not. Table 4. Group 2 mean blood flow*
Re-op Postop. 24 h
Furegrelate
Control
115.3 f 35.0 97.7 ? 32.7 91.9 & 37.7
145.7 f 55.4 93.8 k 56.8 77.8 f 58.5 96.3 k 49. I**
*mUmin k s.d. **Without occluded vessels.
Discussion This study shows that an inhibitor of thromboxane synthetase, Furegrelate (Upjohn U63557A) will significantly reduce the prevalence of canine carotid artery occlusion after endarterectomy from 24% to 4% (92 vessels; P C 0.01). Both doses of Furegrelate produced a significant reduction of platelet aggregation. As thromboxane synthetase inhibitors have been given safely to humans," the results could imply that this approach may be beneficial in humans in the reduction of early postoperative thrombosis. This could be accompanied by reduction in neurological deficit which is known to occur in up to 15% of patients.lS2These serious complications are permanent in 50% of cases and attended by about a 10% mortality. It is important to record that the significant reduction in occlusion was not dose-related or accompanied by changes in deposition at the endarterectomy site. The similar differences in rates of occlusion between Furegrelate and control dogs in both groups suggest that the occlusions were not caused by technical, rheological or unknown reasons. This highlights different mechanisms which cause aggregation and deposition. l4 It is unclear whether blood flow effects played a major role in the outcome of these experiments. The model was selected to produce thrombosis and platelet aggregation because, unlike in human
623
carotid atheroma, the lumen is always slightly occluded. It is likely that the significant reductions in postoperative flow were caused by slight occlusion, the effect of which was not sufficient to obliterate the benefit of Furegrelate therapy. There may, however, be increased production of prostacycline in regions of arterial stenosis ,l5 It is always difficult to provide proof for a negative outcome, particularly when there is a wide variance within and between groups. It is our opinion that variance in these experiments would be little different even if the optimal size was used, because the relevant phenomena have exponential rates; there are wide biological differences within species (e.g. aggregators and non-aggregators). The use of dogs of unknown pedigree, which accentuates this problem,'6-1s was however, a deliberate attempt to produce the type of variation which is found in humans. While it might be accepted that the deposition mechanism is different from that of platelet aggregation, it has to be left to clinical judgement as to whether the effects of Furegrelate impinge on the deposition mechanism. Our opinion, that there is no effect, is supported by the photographs (Fig. l), which show the magnitude of mean platelet deposition and the difference between the means. Furthermore, it would be safe to presume that there might be no change in the rate of postoperative stenosis even though it is not evident from this work whether the mechanism of postoperative ~ t e n o s i s lwould ~ . ~ ~ be affected by Furegrelate.
Fig. 1. Effects of Furegrelate at (a) 4.646 platelets x Id mm-* and (b) 4.983 platelets x lo6
PAYNE ET AL..
624
I n vivo thromboxane synthetase inhibitors divert prostaglandin H2 metabolism away from thromboxane and towards formation of prostacycline. lo Experiments in dogs have compared Furegrelate favourably with cyclo-oxygenase inhibitors, aspirin, ibuprofen, or indomethacin in the dog coronary artery model." This superiority was independent of the effects of intravenous infusions of prostacycline. Thromboxane synthetase inhibitors have the advantages of being more stable in solution, and having longer duration of action and oral administration than prostacycline. Furegrelate is Sodium 5-(3 pyridinylmethyl)-2-benzofurancarboxylate. A single oral dose of 3 mg/kg inhibits primate thromboxane synthesis 80% for at least I2h.l9 It does not appear to produce tachyph laxis or rebound when administered for 14 days.' It does not inhibit thrombin stimulated prostacycline biosynthesis in a variety of test systems. Under appropriate conditions the protective effects of Furegrelate could be reversed by cyclo-oxygenase inhibitors, also suggesting that its efficacy depended in part on endogenous prostacycline formation. l9 It is possible, therefore, that the lack of effect of Furegrelate upon platelet deposition was in part related to a lack of endogenous prostacycline locally. The development of intra-arterial thrombus following carotid endarterectomy; involves a complex of factors including the residual vessel wall, suture and patch material, the formed elements of the blood, blood coagulation proteins and blood flow. Comparison of carotid and femoral artery results showed that vessel wall effect was caused by both the suture line and the endarterectomy. In arterial thromus formation platelets play a major role in both the initiation and growth of thrombi. Following endartemtomy collagen, von Willebrand factor, fibronectin and other proteinsI4 in the medial layer of the vessel are exposed to circulating platelets. Collagen readily reacts with platelets resulting in adhesion and aggregation.20.2' Adhesion initiates a changes sequence of biochemical and ultrast~~ctural (release reaction), during which adenosine diphosphate (ADP) and other substances are selectively secreted, while the contents of the cytoplasm and mitochondria are retained." The release of ADP results in further platelet aggregation, with growth of the thrombus. Platelets also participate in the sequential activation of plasma coagulation proteins, converting prothrombin to thrombin which catalyses the formation of fibrin from fibrinogen finally resulting in still further platelet aggregation. The vessel wall effect and lack of prostacycline appear to have overruled the inhibition of thromboxane synthesis within the platelet in this model. It is unclear which of the mechanisms is the most powerful. However, any drugs which act on platelets by competitive inhibition of prostaglandin
J
metabolites will have less than predicted and less than optimum effects after endarterectomy. Aspirin therapy might be expected to have a better outcome. Both aspirin and heparin,22however, would be clinically associated with significant risk of bleeding and haematoma formation. Thromboxane synthetase inhibitors might provide an appropriate balance between the risks of thrombosis, recurrent stenosis and bleeding. Alternatively, a combination of thromboxane synthetase inhibition and reduction of local platelet deposition could be beneficial. The use of thromboxane synthetase inhibitors is also of theoretical interest because of their ability to increase future understanding about the role of platelets in vascular surgery.
Acknowledgement This project was supported by a grant from the CH & MRC Department of Veterans Affairs, Canberra, Australian Capital Territory.
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FUREGRELATE A R E R CAROTID ENDARTERECTOMY
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