European Journal of Clinical Investigation (1979) 9, 1-2
EDITORIAL
Polyunsaturated fatty acids and thrombosis The participation of polyunsaturated fatty acids (PUFA) in the process of intra-arterial thrombosis has been suspected for many years. A rationale for this empirically founded belief emerged from the recent discovery of the new metabolic pathways of arachidonic acid (eicosa-5,8, 11,144etraenoic acid, 20:4w6, AA) in the circulatory system. In blood platelets AA gives rise to an unstable product ( t M z 3 0 s at 37'C) called thromboxane A, (TXA,) [l]. TXA, and its immediate precursors, the prostaglandin endoperoxides (PGG, and PGH,), induce platelet aggregation. TXA2 is also a powerful vasoconstrictor. In blood vessels, predominantly in their endothelial layers [2], AA is converted via PGG, and PGH, to another unstable substance (t?4 N 3 min at 37"C, pH 7.4) named prostacyclin (PGI,) [3]. Prostacyclin prevents platelets from aggregating by raising their CAMP levels [2] . Furthermore, prostacyclin dis-aggregates platelet clumps both in vitro [2] and in uivo [4], and disperses circulating platelet aggregates in man [S] . Prostacyclin is also a potent vasodilator in man [5]. A balance between formation of TXAz by platelets and of prostacyclin by arteries has been proposed as a homeostatic mechanism that controls the state of platelet aggregability in uiuo [ 2 ] . What is the role of AA vis-a-vis other members of PUFA family? Unless ingested with meat and animal fat, AA is derived from dietary linoleic acid (18:2w6). In the liver, 18:2w6 is desaturated and elongated to dihomo-ylinolenic acid (20:306, DHLA). Although DHLA may be the substrate for formation of prostaglandins (PGEI, PGDI, PGF1,) most of DHLA is further desaturated to AA. AA circulates in small quantities in an albuminbound form. In tissues, AA is esterified with glycerol and cholesterol. AA is also incorporated into plasma and membrane phospholipids. The 'phospholipid reservoir' is the main source of AA for generation of TXA, and prostacyclin. Phospholipase Az liberates AA from the phospholipid pool which is then the substrate for cyclooxygenase either in platelets or in endothelial cells. Other PUFA, such as y-linolenic (18:3w3), palmitoleic (16: 1w7) and oleic (1 8: 1 w9) acids are also desaturated and elongated in the liver but they cannot be the substrates for generation o f AA. Saturated fatty acids such as palmitic (16:O) and stearic (18:O) acids cannot give rise to AA. PUFA are easily auto-oxidized or enzymically converted to the corresponding linear hydroperoxy fatty acids, e.g. AA is auto-oxidized or converted by soya bean lipoxygenase to 1 Shydroperoxy arachidonic acid (15-HPAA). We have shown that 15-HPAA is a powerful inhibitor of prostacyclin synthetase in porcine aortic microsomes [6] and in arteries. This finding was extended to other lipid hydroperoxides. Hydroperoxides of DHLA, 0014-2972/79/0200-0001$02.00 D 1979 Blackwell Scientific Publications
linoleic, a-linolenic, y-linolenic acids inhibit prostacyclin synthetase when incubated with the enzyme at range of concentrations of 1.l-2.2 /AM [4] . Interestingly, in the absence of vitamin E, which is a natural anti-oxidant, PUFA-rich diet fed to pigs causes endothelial damage and subsequent thrombosis [ 7 ] . The most obvious explanation for PUFA-induced endothelial lesions is spontaneous peroxidation of dietary PUFA to hydroperoxy lipids, followed by the inhibition of endothelial prostacyclin synthetase. Inhibition of prostacyclin synthetase will decrease the ability of the endothelium to 'protect itself against the formation of platelet thrombus. In atherosclerotic rabbits with accompanying hyperlipidaemia, the generation of prostacyclin is dramatically suppressed [8]. The above findings may have a direct impact on the attitude of physicians and nutritionists towards the preventive or therapeutic administration of PUFA to subjects with a high risk of thrombosis. Is an excess of dietary PUFA dangerous due to the formation of lipid peroxides? Is it desirable to supplement the diet with all kinds of PUFA or to increase the per cent of ingested linoleic acid, DHLA or AA? Will AA and its precursors stimulate preferentially PGIz formation by arteries than TXA, generation by platelets? Although many of these questions remain to be answered, the paper in this issue by Norddy, Svensson & Hoak helps to identify new areas of research. It is well known that AA causes formation of PGG2, PGH, and TXA2. Norddy e t al. have shown that AA stimulates the formation of a PG1,-like material in cultures of human endothelial cells. Furthermore, other PUFA, and especially DHLA, decrease the formation of an anti-aggregatory principle by vascular endothelium. The important point is that these albumin-bound fatty acids need a long time (20 h) to inhibit the generation of PGI,, probably due to slow incorporation into the membrane phospholipids. A different time course is observed in platelet rich plasma where antagonism to AA-induced aggregation has been observed after short-term incubation with DHLA [9]. A mechanism of the inhibitory effect of DHLA and of the stimulatory effect of AA on the generation of a PG12-material by cultured endothelial cells has been elegantly explained by Norddy e t al. It is interesting to note that no such difference in action of DHLA and AA has been found in smooth muscle cell cultures from guinea-pig aorta. Both fatty acids inhibit proliferation of the cultured cells, most likely due to their conversion to the corresponding metabolites [ l o ] . However, we should keep in mind that in an in viuo situation DHLA may behave in a different way from that in cell cultures, mainly due to its conversion to prostacyclin via AA. A single dose of DHLA (0.1-0.2 g) and a 4 week administration of DHLA to man were reported to decrease platelet aggregability in platelet rich plasma [l 11 . DHLA and y-linolenic acid were shown to inhibit platelet aggreg1
2
EDITORIAL
ability ex vivo in monkey and in man [ 121 . On the other hand a prolonged feeding of rabbits with ethyl-DHLA had no effect on their platelet aggregability [13]. So far the results with DHLA are not conclusive. This could be because, firstly, DHLA cannot be converted into a prostacyclin-like substance by the vessel wall and, second, most of the studies with DHLA have only analysed the behaviour of platelets in vitru, neglecting the important platelet-vessel wall interaction. An important lead could arise from recent work [ 141 which shows that eicosapentaenoic acid (EPA, C20: 5 w3) does not aggregate platelets (probably because it is converted to a non-active thromboxane A3) but is converted by fresh vascular tissue into an anti-aggregatory principle similar to prostacyclin-probably A1’ prostacyclin. A situation like this would shift the balance between proaggregating and anti-aggregating substances in vivo in favour of the latter. The authors suggest that high content of EPA in the diet might afford protection from thromboembolic disorders, as it apparently does in Eskimos. An understanding of the way in which arachidonic acid is incorporated and utilized by the two systems and the way in which other fatty acids affect this process in vivo becomes a subject for urgent research. In acute experiments, arachidonic acid infused in high concentrations intravenously induces sudden death due to the formation of pulmonary thrombi [ 151 . In lower doses, however, it seems to be converted mainly to PG12 which inhibits platelet aggregation [161 . Perhaps variations in the albumin binding of AA and the presence of a special type of plasma protein may also divert the pathway of AA metabolism in the circulation. Indeed, AA induces hypotension in rabbits and this effect is potentiated by heparin and by an increased haptoglobin plasma level [ 171. When this is achieved, AA at doses of 24-35 &/kg i.v. causes a 50% decrease ofmean arterial blood pressure, perhaps due to the generation of prostacyclin. Certainly at this stage the work on fatty acid metabolism is re-emerging with fresh impetus and several possibilities have to be explored. The work of Norddy et al. confirms previous results which show that fresh vascular tissue [18] or endothelial cells in culture [19] generate from AA an anti-aggregating principle identified as prostacyclin. Moreover, they have demonstrated that albumin bound fatty acids can be utilized by the vessel wall enzyme. These authors have also shown that by competing with AA, other fatty acids may decrease the availability of AA from phospholipids for generation of this anti-aggregating principle. There still remains a long way to go to reach full understanding of the significance of these findings. However, the work of Norddy and collaborators as well as other recent work is adding light to a very complex but exciting area of research. R. J. GRYGLEWSKI & S. MONCADA
Department o f Prostaglandin Research, Wellcome Research Laboratories, LangleY Court, Beckenham, Kent BR3 3Bs
References 1 Hamberg M., Svensson J. & Samuelsson B. (1975) Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Nut1 Acad Sci USA, 72,2994-2998. 2 Moncada S. & Vane J.R. (1978) Unstable metabolites of arachidonic acid and their role in haemostasis and thrombosis. BrMedBull 34, 129-136. 3 Moncada S., Gryglewski R.J., Bunting S. &Vane J.R. (1976) An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263,663665. 4 Gryglewski R.J., Korbut R. & Ocetkiewicz A. (1978) Deaggregatoryaction of prostacyclin in vivo and its enhancement by theophylline. Prostaglandins 15,637644. 5 Gryglewski R.J., Szczeklik A. & Nizankowski R. (1978) Antiplatelet action of intravenous infusion of prostacyclin in man. Thromb Res 13,153-163. 6 Gryglewski R.J., Bunting S., Moncada S., Flower R.J. & Vane J.R. (1976) Arterial walls are protected against deposition of platelet thrombi by a substance (prostaglandin X) which they make from prostaglandin endoperoxides. Prostaglandins 12, 6 85-7 13. 7 Nafstad J. (1974) Endothelial damage and platelet thrombosis associated with PUFA-rich, vitamin E-deficient diet fed to pigs. Thromb Res 5,251-258. 8 Dembinska-Kiec A,, Gryglewska T., Zmuda A. & Gryglewski R.J. (1977) The generation of prostacyclin by arteries and by the coronary vascular bed is reduced in experimental atherosclerosis in rabbits. Prostaglandins 14, 1025-1034. 9 Willis A.L., Vane F.M., Kuhn D.C., Scott C.G. & Petrin M. (1974) An endoperoxide aggregator (LASS), formed in platelets in response to thrombotic stimuli: Purification, identification and unique biological significance. Prostaglandins 8,453. 10 Huttner J.J., Gwebu E.G., Panganamala R.V., Milo G.E., Cornwell D.G., Sharma H.M. & Geer J.C. (1977) Fatty acids and their prostaglandin derivatives: inhibitors of proliferation in aortic smooth muscle. Science 197,289-291. 11 KernoffP.B.A.,WillisA.L.,Stone K.J., Davies J.A. & McNicol G.P. (1977) Anti-thrombotic potential of dihomo-y-linolenic acid in man. Br Med J ii, 1441-1444. 12 Sim A.K. & McCraw A.P. (1977) The activity of 7-linolenate and dihomo-y-linolenate methyl esters in vitro and in vivo on blood platelet function in non-human primates and in man. Thromb Res 10,385-397. 13 Oelz O., Seyberth H.W., Knapp H.R., Sweetman J.B. & Oates J.A. (1976) Effects of feeding ethyl dihomo-7-linolenate on prostaglandin biosynthesis and platelet aggregation in rabbits. Biochim Biophys Acta 431,268-277. 14 Dyerberg J., Bang H.O., Stoffersen E., Moncada S. & Vane J.R. (1978) Polyunsaturated fatty acids, atherosclerosis and thrombosis. Lancet ii, 117-1 19. 5 Silver M.J., Hoch W., Kocsis J.J., Ingerman C.M. & Smith J.B. (1974) Arachidonic acid causes sudden death in rabbits. Science 194,1085-1087. 6 Korbut R. & Moncada S. (1978) Prostacyclin (PGI,) and thromboxane A, interaction in vivo. Regulation by aspirin and relationship with anti-thrombotic therapy. Thromb Res 13,489-500. 7 Deby C., Van Caneghem P. & Bacq Z.M. (1978) Arachidonate induced hypotension and haptoglobin plasma level in the rabbit. Biochem Pharmacol27,613-615. 18 Bunting S., Gryglewski R.J., Moncada S. & Vane J.R. (1976) Arterial walls generate from prostaglandin endoperoxides a substance (prostaglandin X) which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation. Prostaglandins 12, 89 7-9 13. 19 Weksler B.B., Marcus A.J. & Jaffe E.A. (1977) Synthesis of prostaglandin I, (prostacyclin) by cultured human and bovine endothelial cells. Proc Natl Acad Sci USA 74,3922-3926.
EDITORIAL
Polyunsaturated fatty acids and thrombosis The participation of polyunsaturated fatty acids (PUFA) in the process of intra-arterial thrombosis has been suspected for many years. A rationale for this empirically founded belief emerged from the recent discovery of the new metabolic pathways of arachidonic acid (eicosa-5,8, 11,144etraenoic acid, 20:4w6, AA) in the circulatory system. In blood platelets AA gives rise to an unstable product ( t M z 3 0 s at 37'C) called thromboxane A, (TXA,) [l]. TXA, and its immediate precursors, the prostaglandin endoperoxides (PGG, and PGH,), induce platelet aggregation. TXA2 is also a powerful vasoconstrictor. In blood vessels, predominantly in their endothelial layers [2], AA is converted via PGG, and PGH, to another unstable substance (t?4 N 3 min at 37"C, pH 7.4) named prostacyclin (PGI,) [3]. Prostacyclin prevents platelets from aggregating by raising their CAMP levels [2] . Furthermore, prostacyclin dis-aggregates platelet clumps both in vitro [2] and in uivo [4], and disperses circulating platelet aggregates in man [S] . Prostacyclin is also a potent vasodilator in man [5]. A balance between formation of TXAz by platelets and of prostacyclin by arteries has been proposed as a homeostatic mechanism that controls the state of platelet aggregability in uiuo [ 2 ] . What is the role of AA vis-a-vis other members of PUFA family? Unless ingested with meat and animal fat, AA is derived from dietary linoleic acid (18:2w6). In the liver, 18:2w6 is desaturated and elongated to dihomo-ylinolenic acid (20:306, DHLA). Although DHLA may be the substrate for formation of prostaglandins (PGEI, PGDI, PGF1,) most of DHLA is further desaturated to AA. AA circulates in small quantities in an albuminbound form. In tissues, AA is esterified with glycerol and cholesterol. AA is also incorporated into plasma and membrane phospholipids. The 'phospholipid reservoir' is the main source of AA for generation of TXA, and prostacyclin. Phospholipase Az liberates AA from the phospholipid pool which is then the substrate for cyclooxygenase either in platelets or in endothelial cells. Other PUFA, such as y-linolenic (18:3w3), palmitoleic (16: 1w7) and oleic (1 8: 1 w9) acids are also desaturated and elongated in the liver but they cannot be the substrates for generation o f AA. Saturated fatty acids such as palmitic (16:O) and stearic (18:O) acids cannot give rise to AA. PUFA are easily auto-oxidized or enzymically converted to the corresponding linear hydroperoxy fatty acids, e.g. AA is auto-oxidized or converted by soya bean lipoxygenase to 1 Shydroperoxy arachidonic acid (15-HPAA). We have shown that 15-HPAA is a powerful inhibitor of prostacyclin synthetase in porcine aortic microsomes [6] and in arteries. This finding was extended to other lipid hydroperoxides. Hydroperoxides of DHLA, 0014-2972/79/0200-0001$02.00 D 1979 Blackwell Scientific Publications
linoleic, a-linolenic, y-linolenic acids inhibit prostacyclin synthetase when incubated with the enzyme at range of concentrations of 1.l-2.2 /AM [4] . Interestingly, in the absence of vitamin E, which is a natural anti-oxidant, PUFA-rich diet fed to pigs causes endothelial damage and subsequent thrombosis [ 7 ] . The most obvious explanation for PUFA-induced endothelial lesions is spontaneous peroxidation of dietary PUFA to hydroperoxy lipids, followed by the inhibition of endothelial prostacyclin synthetase. Inhibition of prostacyclin synthetase will decrease the ability of the endothelium to 'protect itself against the formation of platelet thrombus. In atherosclerotic rabbits with accompanying hyperlipidaemia, the generation of prostacyclin is dramatically suppressed [8]. The above findings may have a direct impact on the attitude of physicians and nutritionists towards the preventive or therapeutic administration of PUFA to subjects with a high risk of thrombosis. Is an excess of dietary PUFA dangerous due to the formation of lipid peroxides? Is it desirable to supplement the diet with all kinds of PUFA or to increase the per cent of ingested linoleic acid, DHLA or AA? Will AA and its precursors stimulate preferentially PGIz formation by arteries than TXA, generation by platelets? Although many of these questions remain to be answered, the paper in this issue by Norddy, Svensson & Hoak helps to identify new areas of research. It is well known that AA causes formation of PGG2, PGH, and TXA2. Norddy e t al. have shown that AA stimulates the formation of a PG1,-like material in cultures of human endothelial cells. Furthermore, other PUFA, and especially DHLA, decrease the formation of an anti-aggregatory principle by vascular endothelium. The important point is that these albumin-bound fatty acids need a long time (20 h) to inhibit the generation of PGI,, probably due to slow incorporation into the membrane phospholipids. A different time course is observed in platelet rich plasma where antagonism to AA-induced aggregation has been observed after short-term incubation with DHLA [9]. A mechanism of the inhibitory effect of DHLA and of the stimulatory effect of AA on the generation of a PG12-material by cultured endothelial cells has been elegantly explained by Norddy e t al. It is interesting to note that no such difference in action of DHLA and AA has been found in smooth muscle cell cultures from guinea-pig aorta. Both fatty acids inhibit proliferation of the cultured cells, most likely due to their conversion to the corresponding metabolites [ l o ] . However, we should keep in mind that in an in viuo situation DHLA may behave in a different way from that in cell cultures, mainly due to its conversion to prostacyclin via AA. A single dose of DHLA (0.1-0.2 g) and a 4 week administration of DHLA to man were reported to decrease platelet aggregability in platelet rich plasma [l 11 . DHLA and y-linolenic acid were shown to inhibit platelet aggreg1
2
EDITORIAL
ability ex vivo in monkey and in man [ 121 . On the other hand a prolonged feeding of rabbits with ethyl-DHLA had no effect on their platelet aggregability [13]. So far the results with DHLA are not conclusive. This could be because, firstly, DHLA cannot be converted into a prostacyclin-like substance by the vessel wall and, second, most of the studies with DHLA have only analysed the behaviour of platelets in vitru, neglecting the important platelet-vessel wall interaction. An important lead could arise from recent work [ 141 which shows that eicosapentaenoic acid (EPA, C20: 5 w3) does not aggregate platelets (probably because it is converted to a non-active thromboxane A3) but is converted by fresh vascular tissue into an anti-aggregatory principle similar to prostacyclin-probably A1’ prostacyclin. A situation like this would shift the balance between proaggregating and anti-aggregating substances in vivo in favour of the latter. The authors suggest that high content of EPA in the diet might afford protection from thromboembolic disorders, as it apparently does in Eskimos. An understanding of the way in which arachidonic acid is incorporated and utilized by the two systems and the way in which other fatty acids affect this process in vivo becomes a subject for urgent research. In acute experiments, arachidonic acid infused in high concentrations intravenously induces sudden death due to the formation of pulmonary thrombi [ 151 . In lower doses, however, it seems to be converted mainly to PG12 which inhibits platelet aggregation [161 . Perhaps variations in the albumin binding of AA and the presence of a special type of plasma protein may also divert the pathway of AA metabolism in the circulation. Indeed, AA induces hypotension in rabbits and this effect is potentiated by heparin and by an increased haptoglobin plasma level [ 171. When this is achieved, AA at doses of 24-35 &/kg i.v. causes a 50% decrease ofmean arterial blood pressure, perhaps due to the generation of prostacyclin. Certainly at this stage the work on fatty acid metabolism is re-emerging with fresh impetus and several possibilities have to be explored. The work of Norddy et al. confirms previous results which show that fresh vascular tissue [18] or endothelial cells in culture [19] generate from AA an anti-aggregating principle identified as prostacyclin. Moreover, they have demonstrated that albumin bound fatty acids can be utilized by the vessel wall enzyme. These authors have also shown that by competing with AA, other fatty acids may decrease the availability of AA from phospholipids for generation of this anti-aggregating principle. There still remains a long way to go to reach full understanding of the significance of these findings. However, the work of Norddy and collaborators as well as other recent work is adding light to a very complex but exciting area of research. R. J. GRYGLEWSKI & S. MONCADA
Department o f Prostaglandin Research, Wellcome Research Laboratories, LangleY Court, Beckenham, Kent BR3 3Bs
References 1 Hamberg M., Svensson J. & Samuelsson B. (1975) Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Nut1 Acad Sci USA, 72,2994-2998. 2 Moncada S. & Vane J.R. (1978) Unstable metabolites of arachidonic acid and their role in haemostasis and thrombosis. BrMedBull 34, 129-136. 3 Moncada S., Gryglewski R.J., Bunting S. &Vane J.R. (1976) An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263,663665. 4 Gryglewski R.J., Korbut R. & Ocetkiewicz A. (1978) Deaggregatoryaction of prostacyclin in vivo and its enhancement by theophylline. Prostaglandins 15,637644. 5 Gryglewski R.J., Szczeklik A. & Nizankowski R. (1978) Antiplatelet action of intravenous infusion of prostacyclin in man. Thromb Res 13,153-163. 6 Gryglewski R.J., Bunting S., Moncada S., Flower R.J. & Vane J.R. (1976) Arterial walls are protected against deposition of platelet thrombi by a substance (prostaglandin X) which they make from prostaglandin endoperoxides. Prostaglandins 12, 6 85-7 13. 7 Nafstad J. (1974) Endothelial damage and platelet thrombosis associated with PUFA-rich, vitamin E-deficient diet fed to pigs. Thromb Res 5,251-258. 8 Dembinska-Kiec A,, Gryglewska T., Zmuda A. & Gryglewski R.J. (1977) The generation of prostacyclin by arteries and by the coronary vascular bed is reduced in experimental atherosclerosis in rabbits. Prostaglandins 14, 1025-1034. 9 Willis A.L., Vane F.M., Kuhn D.C., Scott C.G. & Petrin M. (1974) An endoperoxide aggregator (LASS), formed in platelets in response to thrombotic stimuli: Purification, identification and unique biological significance. Prostaglandins 8,453. 10 Huttner J.J., Gwebu E.G., Panganamala R.V., Milo G.E., Cornwell D.G., Sharma H.M. & Geer J.C. (1977) Fatty acids and their prostaglandin derivatives: inhibitors of proliferation in aortic smooth muscle. Science 197,289-291. 11 KernoffP.B.A.,WillisA.L.,Stone K.J., Davies J.A. & McNicol G.P. (1977) Anti-thrombotic potential of dihomo-y-linolenic acid in man. Br Med J ii, 1441-1444. 12 Sim A.K. & McCraw A.P. (1977) The activity of 7-linolenate and dihomo-y-linolenate methyl esters in vitro and in vivo on blood platelet function in non-human primates and in man. Thromb Res 10,385-397. 13 Oelz O., Seyberth H.W., Knapp H.R., Sweetman J.B. & Oates J.A. (1976) Effects of feeding ethyl dihomo-7-linolenate on prostaglandin biosynthesis and platelet aggregation in rabbits. Biochim Biophys Acta 431,268-277. 14 Dyerberg J., Bang H.O., Stoffersen E., Moncada S. & Vane J.R. (1978) Polyunsaturated fatty acids, atherosclerosis and thrombosis. Lancet ii, 117-1 19. 5 Silver M.J., Hoch W., Kocsis J.J., Ingerman C.M. & Smith J.B. (1974) Arachidonic acid causes sudden death in rabbits. Science 194,1085-1087. 6 Korbut R. & Moncada S. (1978) Prostacyclin (PGI,) and thromboxane A, interaction in vivo. Regulation by aspirin and relationship with anti-thrombotic therapy. Thromb Res 13,489-500. 7 Deby C., Van Caneghem P. & Bacq Z.M. (1978) Arachidonate induced hypotension and haptoglobin plasma level in the rabbit. Biochem Pharmacol27,613-615. 18 Bunting S., Gryglewski R.J., Moncada S. & Vane J.R. (1976) Arterial walls generate from prostaglandin endoperoxides a substance (prostaglandin X) which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation. Prostaglandins 12, 89 7-9 13. 19 Weksler B.B., Marcus A.J. & Jaffe E.A. (1977) Synthesis of prostaglandin I, (prostacyclin) by cultured human and bovine endothelial cells. Proc Natl Acad Sci USA 74,3922-3926.
