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The role of sulfatide in thrombogenesis and haemostasis
ABB Archives of Biochemistry and Biophysics 426 (2004) 157–162 www.elsevier.com/locate/yabbi
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The role of sulfatide in thrombogenesis and haemostasis Mamoru Kyogashima* Seikagaku Corporation, Central Research Laboratories, 1253 Tateno 3 chome, Higashiyamato-shi, Tokyo 207-0021, Japan Received 12 December 2003, and in revised form 5 February 2004 Available online 18 March 2004
Abstract In 1961, Wago et al. reported a potential anticoagulant role for sulfatide using animal experiments. Since then there have been many studies of sulfatide in the field of thrombogenesis/haemostasis, yielding contradictory conclusions. Some report that sulfatide has anti-thrombotic activity because it prolongs clotting time, inhibits platelet adhesion, and prolongs bleeding. Others report that sulfatide induces thrombosis in animal models. This mini-review is a chronologic review of reports examining the role of sulfatide in thrombogenesis/haemostasis together with the introduction of data from our laboratory and a discussion of the possible mechanisms underlying these curious phenomena. Ó 2004 Elsevier Inc. All rights reserved.
Sulfatide, the sulfuric ester of galactosylceramide at the C3 of the galactose residue, is a representative sulfated glycolipid. A large amount of sulfatide is contained as an important component of myelin in the nervous system. In most studies, the sulfatide used was prepared from brain. Therefore, the ceramides of the sulfatide is composed of C16–C26 fatty acids, including 2-hydroxy fatty acids, and long chain base of d18:1 (sphingenine) [1]. Sulfatide is also abundant in the gastrointestinal tract [2], pancreas [3], and kidney [4]. Various blood cells, such as leukocytes [5], platelets [6–8], and erythrocytes [8,9], contain sulfatide on their surface. Sulfatide is also present in serum as a major component of glycosphingolipids in lipoprotein [10]. The presence of sulfatide in endothelial cells has also been reported [11], although the information is limited to brain. Because these elements participate in thrombogenesis/ haemostasis, sulfatide might be involved under various conditions. In fact, many reports describe an important relation between sulfatide and thrombogenesis/haemostasis, some of which contradict each other [12]. We also demonstrated evidence of contradictory roles of sulfatide *
Present address: Division of Molecular Pathology, Aichi Cancer Center Research Institute, 1-1 Kanokoden Chikusa-ku, Nagoya 4648681, Japan. Fax: +81-42-563-5846. E-mail addresses: [email protected], miyaura@seikagaku. co.jp. 0003-9861/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.02.005
in blood coagulation, such as coagulant and anticoagulant actions in animal disease models [13–15]. This report is a chronologic review of the role of sulfatide in thrombogenesis/haemostasis. We also present thrombogenetic and antithrombogenetic results and discuss these complicated, confusing, but very interesting phenomena.
Chronology From 1961 to mid-1980s In 1961, Wago [16] reported possible anticoagulant and anti-atherosclerotic activities of sulfatide. He administered sulfatide orally or intravenously to rabbits and observed not only a prolongation of plasma clotting time but also a decreased cholesterol level with an improvement of atheromatous changes in the aorta. This report came out before the structure of sulfatide was determined [17], therefore some unknown contamination might have affected the results. In 1973, Bourgain and Six [18] supported WagoÕs finding that sulfatide-rich fraction from bovine brain prolonged clotting time, although they also reported another uncharacterized lipid fraction that exhibited unstable anti-coagulant activity. These reports suggest an anticoagulant role of sulfatide. On the contrary, in 1980, sulfatide was reported to activate blood coagulation factor XII (Hageman factor) in vitro, sug-
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c Fig. 1. (A) The effect of sulfatide on thrombogenesis in a rat deep vein thrombosis model. The thrombi 3 h after the administration of sulfatide or vehicle are presented. From [12,14] with permission. (B) Thrombus induced by continuous infusion of sulfatide. Sulfatide was continuously infused through a plastic cannula at a rate of 1.25 mg/kg for 4 h. From [12,13] with permission. Fig. 3. The possible roles of sulfatide in thrombogenesis and haemostasis with related molecules. Italics are sulfatide binding molecules. vWF, von Willebrand factor; TSP, thrombospondin.
gesting a coagulant role of sulfatide [19]. This report was subsequently confirmed by many groups in a series of investigations of sulfatide and blood coagulation factors related to intrinsic pathways involved in factor XII (contact activation) [20,21], although the physiologic importance of the pathway still remains unclear [22]. From mid-1980s to early 1990s Two lines of investigation on the involvement of sulfatide thrombogenesis/haemostasis occurred in this period. Roberts and Ginsburg [23] demonstrated an important function of sulfatide as an adhesive molecule. They then reported that sulfatide specifically binds to laminin [24], thrombospondin [6], and von Willebrand factor (vWF)1 [7], and by using these proteins as probes they demonstrated the presence of sulfatide on platelets. Binding sites of each molecule to sulfatide were identified later [25–27]. Laminin is a major component of the basement membrane [28] and the laminin receptor on platelets, identified as GPIc/IIa (a6b1), mediates platelet adhesion [29]. Thrombospondin (thrombospondin-1) is also a component of the extracellular matrix [26] and is the most abundant constituent in platelet a-granules. Thrombospondin promotes platelet adhesion [30]. vWF is produced in endothelial cells and megakaryocytes, and is stored in the Weibel–Palade bodies in endothelial cells and in the a-granules of platelets. vWF is secreted not only to the plasma, where it carries procoagulant factor VIII [31], but also to the subendothelial matrix to induce platelet aggregation through platelet receptor GPIb [32]. Thus, sulfatide binds to plasma proteins, platelets, and other components of the extracellular matrix closely involved in thrombogenesis/haemostasis. Late in the 1980s, group of Hara and Taketomi [10] began to investigate the effects of sulfatide in atherosclerosis using Watanabe hereditable hyperlipidemic rabbits, which is an animal model of human familial hypercholesterolemia. They intravenously injected 10 mg/kg sulfatide into rabbits every other day for 3 months to study whether relatively long-term treatment influences atherosclerosis and lipid metabolism, such as total cholesterol and triacylglycerols [33]. In this series of experiments, there were incidental important findings of the involvement of sulfatide in cardiovascular diseases, although sulfatide failed to lower cholesterol or to 1 Abbreviations used: vWF, von Willebrand factor; DVT, deep vein thrombosis.
suppress the progression of atherosclerosis, which opposed the findings of Wago. First, they reported that sulfatide is a major glycosphingolipid in serum (approximately 70% of all serum-glycosphingolipids) as a component of all kinds of lipoproteins such as chylomicrons, very low density lipoprotein, low density lipoprotein, and high density lipoprotein [10,34]. Second, they observed an abnormal accumulation of sulfatide in the atheromatous lesions of the aorta. Based on the analysis of the ceramide portion of the accumulated sulfatide, they hypothesized that the sulfatide originated from the serum lipoprotein [35]. Third, they confirmed WagoÕs report of prolongation of clotting time by sulfatide and, unlike the anticoagulant mechanism of heparin, this effect was unrelated to antithrombin III [36], but possibly due to binding of sulfatide to fibrinogen, thereby disturbing fibrin clotting [37].
Effects of sulfatide on an in vivo animal thrombosis model Since the information so far we had in mid-1990s supported an anticoagulant role of sulfatide, we investigated the effects of sulfatide on blood coagulation, together with molecules structurally related to sulfatide, using a rat disease of human deep vein thrombosis (DVT) that is conventionally used to evaluate the potential of anticoagulant agents [14]. Under anesthesia, rats were laporotomized and the inferior vena cava was ligated just below the branch of the renal vein with surgical thread. Sulfatide was injected into the rats through the tail vein 1 min prior to ligation. Two control groups were prepared. In one group, vehicle was injected the same way 1 min prior to ligation of the inferior vena cava and in the other group, sulfatide was injected through the tail vein without vein ligation. When sulfatide was simply injected without vein ligation, the rats appeared to be normal and no thrombus was observed. Furthermore, the bleeding times of the rats administered sulfatide were significantly prolonged, as expected [14,15,37], suggesting that simple injection of sulfatide produces an anticoagulant state in animals. In contrast, unexpectedly sulfatide remarkably enhanced thrombogenesis in the rats with vein ligation (Fig. 1A) [14,15]. This phenomenon was observed within 1 min, suggesting that probably no de novo protein synthesis is necessary for this event. In another animal experiment, continuous infusion of sulfatide into rats using a polyethylene cannula produced giant thrombi at the tip of the cannula, suggesting
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Fig. 2. (A) Effect of sulfatide on plasma coagulation time. Aliquots of 100 ll of plasma were added to the wells of a 96-well microtiter plate and coagulation was initiated by adding CaCl2 . Plasma without sulfatide began to coagulate at approximately 12 min in this experiment. Sulfatide (23.4 lg/ml) apparently accelerated this process by approximately 3 min and the accelerating activity was still observed at a dose of 0.37 lg/ml. At a high concentration (375 lg/ml), the accelerating activity was abated. From [14] with permission. (B) The relation between the sulfatide concentration and plasma coagulation onset time. Sulfatide and sulfatide 6S had similar coagulant activities, although sulfatide 6S had slightly stronger activity. Cholesterol 3-sulfate exhibited weaker coagulant activity than either of the sulfatides and GM4 exhibited no activity. From [15] with permission.
the procedure of vein ligation is not always necessary (Fig. 1B) [13]. Comparative studies of molecules structurally related to sulfatide, such as sulfatide 6S (sulfuric ester of galactosylceramide at the C6 of the galactose residue), cholesterol sulfate (5-cholesten-3b-ol sulfate), and ganglioside GM4 (I3 -a-N -acetylneuraminosylgalactoylceramide), indicate that the sulfate moiety in the sulfatides is essential for the coagulant activity and the galactose residue enhances the activity [15]. The results of these two different animal experiments are very consistent with the result of in vitro experiment, the kinetic turbidimetry of plasma coagulation: (i) sulfatide, sulfatide 6S, and cholesterol sulfate, but not ganglioside GM4, accelerate blood coagulation on plastic, and (ii) the degree of acceleration or induction of thrombogenesis is in the order of sulfatide 6S, sulfatide, and cholesterol sulfate (Fig. 2B) [13–15]. Thrombi produced in the animal experiment with continuous infusion might have the same mechanism as the in vitro plasma coagulation on plastic because the thrombi were observed at the tip of the plastic cannula. On the other hand, the mechanism underlying the DVT
animal model result remains uncertain, because simple bolus shot injection of sulfatide into the animals did not induce thrombosis, rather it prolonged plasma clotting time and bleeding time. In the absence of plasma coagulation factor XII, sulfatide does not shorten plasma clotting time on plastic, suggesting a critical role of factor XII, although generally the physiologic importance of factor XII still remains uncertain [22]. The procedure of vein ligation, which induces sudden severe vascular injury and venous congestion, is absolutely necessary for sulfatide to enhance thrombogenesis in this DVT animal model. Once such injury occurs, apparently the endothelium is heavily damaged, exposing basement membranes and other extracellular matrix components with the simultaneous activation of platelets and extravasation of fibrinogen into the matrix [38]. Binding of the sulfatide to extracellular matrix components such as laminin, thrombospondin, and vWF might present the place like a surface similar to that of plastic, which was observed in an in vitro experiment of the kinetic turbidimetry of plasma coagulation. Very incidentally, vWF, thrombospondin, P-selectin, and fibrin-
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Table 1 Chronologic findings of sulfatide in haemostasis/thrombogenesis (1961–2004 February) Findings Prolongation of clotting time by administration of sulfatide in rabbits Prolongation of clotting time by sulfatide-rich fraction from bovine brain extract Activation of blood coagulation factor XII by sulfatide Binding of sulfatide to laminin Binding of sulfatide to thrombospondin Binding of sulfatide to von Willebrand factor Detection of sulfatide on platelets by labelled thrombospondin and von Willebrand factor Occurrence of sulfatide as a major glycosphingolipid component of plasma lipoprotein Accumulation of sulfatide in atherosclerotic aorta Binding of sulfatide to P-selectin Antithrombin III independent anticoagulant activity of sulfatide Increase factor VII activity by sulfatide Binding of sulfatide to fibrinogen Generation of giant thrombi in animal model of thrombosis by administration of sulfatide Inhibition of platelet-aggregation by sulfatide Heparin cofactor II independent anticoagulant activity of sulfatide Direct inhibition of thrombin activity by sulfatide Sulfatide on platelets as a ligand for P-selectin to stabilize platelet aggregation Inhibition of platelet adhesion to vWF under flow conditions by sulfatide Annexin V mediated anticoagulant action by sulfatide *
Thrombotic
Anti-thrombotic
Modulated
Others
References
+
[16]
+
[18]
+
[19–21] + + + +
+ + +
[24] [6] [7] [6,7] +
[10]
+
[35] [5] [36]
+
[50] [37] [13–15]
+ +
[44] [15]
+
[15] [43]
+
[40]
+
[51]
+
+
Endogenous sulfatide.
ogen are stored in the a-granules of platelets [39], whereas vWF and P-selectin are also stored in the Weibel–Palade bodies of endothelial cells [31]. It is quite interesting that such molecules with the capability to bind to sulfatide are also involved in thrombogenesis and co-exist in a closely localized place. In the presence of sulfatide, if some pathologic conditions disturb the balance of the concentration of such molecules, sulfatide might modulate thrombogenesis with various ways. Indeed, it was reported that sulfatide inhibits platelet adhesion to vWF in flow blood [40]. Cancer cells are often reported to induce thrombotic diseases [41,42]. Although the major causes of such diseases have been attributed to procoagulant or coagulant activities, such as tissue factor or factor X activators, some uncharacterized factors, including membrane vesicles, which would be enormously shed from cancer cells [41] and may be sulfatide [5], have been reported.
Conclusion and perspective I have reviewed the reports from the last 40 years on sulfatide in the field of thrombogenesis/haemostasis together with current data from my laboratory using an
animal disease model. These data are summarized in Table 1 and Fig. 3. There are more reports supporting a role for sulfatide in anti-thrombosis than in thrombosis, although sulfatide can induce thrombosis under specific conditions. Although these contradictory results are probably based on differences in the methods employed, sulfatide is potentially involved in both thrombosis and anti-thrombosis. It should be noted that in most of the reports, exogenous sulfatide was added to the experimental systems. Merten and Thiagarajan [43] reported that sulfatide on the cell surface of platelets serves as a ligand for P-selectin and stabilizes platelet aggregation, which may be related with the report that exogenous sulfatide inhibited platelet function [44]. Thus, studies of the role of endogenous sulfatide are necessary. Galactosylceramide sulfotransferase contributes to build sulfatide and was cloned [45] and recently its null mice were generated [46]. These mice will be a useful tool for this line of investigation. Meanwhile, studies using exogenous sulfatide might also provide critical information for drug discovery based on sulfatide-mediated antithrombotic and/or haemostatic agents. As seen in heparin and low molecular weight heparin, sulfate glycoconjugates are well-known anticoagulant therapeutics. An unstudied area of sulfatide in thrombogenesis/
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haemostasis is the fibrinolytic system. In fact, the role of factor XII in fibrinolytic system is known [47]. Furthermore, the importance of other sulfate glycoconjugates in the fibrinolytic system has already been reported [48,49]. Sulfatide in these areas also remains to be investigated. Acknowledgment This paper is dedicated to the memory of Dr. Victor Ginsburg.
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[24] D.D. Roberts, C.N. Rao, J.L. Magnani, S.L. Spitalnik, L.A. Liotta, V. Ginsburg, Proc. Natl. Acad. Sci. USA 82 (1985) 1306– 1310. [25] N.H. Guo, H.C. Krutzsch, T. Vogel, D.D. Roberts, J. Biol. Chem. 267 (1992) 17743–17747. [26] D.D. Roberts, FASEB J. 10 (1996) 1183–1191. [27] O. Christophe, B. Obert, D. Meyer, J.P. Girma, Blood 78 (1991) 2310–2317. [28] J.C. Jones, G.W. Dehart, M. Gonzales, L.E. Golfinger, Microsc. Res. Tech. 51 (2000) 211–213. [29] J.S. Benett, Semin. Hematol. 27 (1990) 186–204. [30] T. Browder, M. Rupnick, in: J. Loscalzo, A.I. Schafer (Eds.), Thrombosis and Hemorrhage, third ed, Lippincott Williams & Watkins, Philadelphia, 2003, pp. 220–235. [31] Z.M. Ruggeri, J. Ware, in: J. Loscalzo, A.I. Schafer (Eds.), Thrombosis and Hemorrhage, third ed, Lippincott Williams & Watkins, Philadelphia, 2003, pp. 246–265. [32] Z.M. Ruggeri, Thromb. Haemost. 74 (1995) 460–463. [33] T. Taketomi, A. Hara, K. Uemura, N. Kyogashima, Jpn. J. Exp. Med. 59 (1989) 221–231. [34] X.H. Zhu, A. Hara, T. Taketomi, J. Biochem. (Tokyo) 110 (1991) 241–245. [35] A. Hara, T. Taketomi, J. Biochem. (Tokyo) 109 (1991) 904–908. [36] A. Hara, Y. Kutsukake, K. Uemura, T. Taketomi, J. Biochem. (Tokyo) 110 (1993) 781–785. [37] A. Hara, K.I. Uemura, T. Taketomi, Glycoconj. J. 13 (1996) 187– 194. [38] B.C. Berk, in: J. Loscalzo, A.I. Schafer (Eds.), Thrombosis and Hemorrhage, third ed, Lippincott Williams & Watkins, Philadelphia, 2003, pp. 236–245. [39] J.H. Hartwig, in: J. Loscalzo, A.I. Schafer (Eds.), Thrombosis and Hemorrhage, third ed, Lippincott Williams & Watkins, Philadelphia, 2003, pp. 140–160. [40] G. Borthakur, M.A. Cruz, J.F. Dong, L. McIntire, F. Li, J.A. Lopez, P. Thiagarajan, J. Thromb. Haemost. 1 (2003) 1288–1295. [41] R.L. Edwards, F.R. Rickles, in: G.A. Levy, E.H. Cole (Eds.), Role of Procoagulant Activity in Health and Disease, CRC Press, Boca Raton, 1993, pp. 153–176. [42] K.B. Green, R.L. Silverstein, Hematol. Oncol. Clin. North Am. 10 (1996) 499–530. [43] M. Merten, P. Thiagarajan, Circulation 104 (2001) 2955–2960. [44] A.K. Devaiah, T.J. Raife, J.A. Barton, J.D. Olson, Blood Coagul. Fibrinol. 11 (2000) 543–550. [45] K. Honke, M. Tsuda, Y. Hirahara, A. Ishii, A. Makita, Y. Wada, J. Biol. Chem. 272 (1997) 4864–4868. [46] K. Honke, Y. Hirahara, J. Dupree, K. Suzuki, B. Popko, K. Fukushima, J. Fukushima, T. Nagasawa, N. Yoshida, Y. Wada, N. Taniguchi, Proc. Natl. Acad. Sci. USA 99 (2002) 4227– 4232. [47] P.M. Mannucci, in: J. Loscalzo, A.I. Schafer (Eds.), Thrombosis and Hemorrhage, third ed, Lippincott Williams & Watkins, Philadelphia, 2003, pp. 220–235, see also pp. 915–928. [48] M. Kyogashima, J. Onaya, S. Miyauchi, M. Arai, Y. Shibata, A. Suda, T. Sakai, Y. Takada, A. Takada, Thromb. Res. 96 (1999) 557–565. [49] T. Sakai, M. Kyogashima, Y. Kariya, T. Urano, Y. Takada, A. Takada, Thromb. Res. 100 (2000) 459–465. [50] K.A. Mitropoulos, B.E. Reeves, G.J. Miller, Blood Coagul. Fibrinol. 4 (1993) 943–951. [51] M. Ida, A. Satoh, K. Aikawa-Kojima, I. Matsumoto, J. Biochem. (Tokyo) (2004) (in press).
Minireview
The role of sulfatide in thrombogenesis and haemostasis Mamoru Kyogashima* Seikagaku Corporation, Central Research Laboratories, 1253 Tateno 3 chome, Higashiyamato-shi, Tokyo 207-0021, Japan Received 12 December 2003, and in revised form 5 February 2004 Available online 18 March 2004
Abstract In 1961, Wago et al. reported a potential anticoagulant role for sulfatide using animal experiments. Since then there have been many studies of sulfatide in the field of thrombogenesis/haemostasis, yielding contradictory conclusions. Some report that sulfatide has anti-thrombotic activity because it prolongs clotting time, inhibits platelet adhesion, and prolongs bleeding. Others report that sulfatide induces thrombosis in animal models. This mini-review is a chronologic review of reports examining the role of sulfatide in thrombogenesis/haemostasis together with the introduction of data from our laboratory and a discussion of the possible mechanisms underlying these curious phenomena. Ó 2004 Elsevier Inc. All rights reserved.
Sulfatide, the sulfuric ester of galactosylceramide at the C3 of the galactose residue, is a representative sulfated glycolipid. A large amount of sulfatide is contained as an important component of myelin in the nervous system. In most studies, the sulfatide used was prepared from brain. Therefore, the ceramides of the sulfatide is composed of C16–C26 fatty acids, including 2-hydroxy fatty acids, and long chain base of d18:1 (sphingenine) [1]. Sulfatide is also abundant in the gastrointestinal tract [2], pancreas [3], and kidney [4]. Various blood cells, such as leukocytes [5], platelets [6–8], and erythrocytes [8,9], contain sulfatide on their surface. Sulfatide is also present in serum as a major component of glycosphingolipids in lipoprotein [10]. The presence of sulfatide in endothelial cells has also been reported [11], although the information is limited to brain. Because these elements participate in thrombogenesis/ haemostasis, sulfatide might be involved under various conditions. In fact, many reports describe an important relation between sulfatide and thrombogenesis/haemostasis, some of which contradict each other [12]. We also demonstrated evidence of contradictory roles of sulfatide *
Present address: Division of Molecular Pathology, Aichi Cancer Center Research Institute, 1-1 Kanokoden Chikusa-ku, Nagoya 4648681, Japan. Fax: +81-42-563-5846. E-mail addresses: [email protected], miyaura@seikagaku. co.jp. 0003-9861/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.02.005
in blood coagulation, such as coagulant and anticoagulant actions in animal disease models [13–15]. This report is a chronologic review of the role of sulfatide in thrombogenesis/haemostasis. We also present thrombogenetic and antithrombogenetic results and discuss these complicated, confusing, but very interesting phenomena.
Chronology From 1961 to mid-1980s In 1961, Wago [16] reported possible anticoagulant and anti-atherosclerotic activities of sulfatide. He administered sulfatide orally or intravenously to rabbits and observed not only a prolongation of plasma clotting time but also a decreased cholesterol level with an improvement of atheromatous changes in the aorta. This report came out before the structure of sulfatide was determined [17], therefore some unknown contamination might have affected the results. In 1973, Bourgain and Six [18] supported WagoÕs finding that sulfatide-rich fraction from bovine brain prolonged clotting time, although they also reported another uncharacterized lipid fraction that exhibited unstable anti-coagulant activity. These reports suggest an anticoagulant role of sulfatide. On the contrary, in 1980, sulfatide was reported to activate blood coagulation factor XII (Hageman factor) in vitro, sug-
158
M. Kyogashima / Archives of Biochemistry and Biophysics 426 (2004) 157–162
c Fig. 1. (A) The effect of sulfatide on thrombogenesis in a rat deep vein thrombosis model. The thrombi 3 h after the administration of sulfatide or vehicle are presented. From [12,14] with permission. (B) Thrombus induced by continuous infusion of sulfatide. Sulfatide was continuously infused through a plastic cannula at a rate of 1.25 mg/kg for 4 h. From [12,13] with permission. Fig. 3. The possible roles of sulfatide in thrombogenesis and haemostasis with related molecules. Italics are sulfatide binding molecules. vWF, von Willebrand factor; TSP, thrombospondin.
gesting a coagulant role of sulfatide [19]. This report was subsequently confirmed by many groups in a series of investigations of sulfatide and blood coagulation factors related to intrinsic pathways involved in factor XII (contact activation) [20,21], although the physiologic importance of the pathway still remains unclear [22]. From mid-1980s to early 1990s Two lines of investigation on the involvement of sulfatide thrombogenesis/haemostasis occurred in this period. Roberts and Ginsburg [23] demonstrated an important function of sulfatide as an adhesive molecule. They then reported that sulfatide specifically binds to laminin [24], thrombospondin [6], and von Willebrand factor (vWF)1 [7], and by using these proteins as probes they demonstrated the presence of sulfatide on platelets. Binding sites of each molecule to sulfatide were identified later [25–27]. Laminin is a major component of the basement membrane [28] and the laminin receptor on platelets, identified as GPIc/IIa (a6b1), mediates platelet adhesion [29]. Thrombospondin (thrombospondin-1) is also a component of the extracellular matrix [26] and is the most abundant constituent in platelet a-granules. Thrombospondin promotes platelet adhesion [30]. vWF is produced in endothelial cells and megakaryocytes, and is stored in the Weibel–Palade bodies in endothelial cells and in the a-granules of platelets. vWF is secreted not only to the plasma, where it carries procoagulant factor VIII [31], but also to the subendothelial matrix to induce platelet aggregation through platelet receptor GPIb [32]. Thus, sulfatide binds to plasma proteins, platelets, and other components of the extracellular matrix closely involved in thrombogenesis/haemostasis. Late in the 1980s, group of Hara and Taketomi [10] began to investigate the effects of sulfatide in atherosclerosis using Watanabe hereditable hyperlipidemic rabbits, which is an animal model of human familial hypercholesterolemia. They intravenously injected 10 mg/kg sulfatide into rabbits every other day for 3 months to study whether relatively long-term treatment influences atherosclerosis and lipid metabolism, such as total cholesterol and triacylglycerols [33]. In this series of experiments, there were incidental important findings of the involvement of sulfatide in cardiovascular diseases, although sulfatide failed to lower cholesterol or to 1 Abbreviations used: vWF, von Willebrand factor; DVT, deep vein thrombosis.
suppress the progression of atherosclerosis, which opposed the findings of Wago. First, they reported that sulfatide is a major glycosphingolipid in serum (approximately 70% of all serum-glycosphingolipids) as a component of all kinds of lipoproteins such as chylomicrons, very low density lipoprotein, low density lipoprotein, and high density lipoprotein [10,34]. Second, they observed an abnormal accumulation of sulfatide in the atheromatous lesions of the aorta. Based on the analysis of the ceramide portion of the accumulated sulfatide, they hypothesized that the sulfatide originated from the serum lipoprotein [35]. Third, they confirmed WagoÕs report of prolongation of clotting time by sulfatide and, unlike the anticoagulant mechanism of heparin, this effect was unrelated to antithrombin III [36], but possibly due to binding of sulfatide to fibrinogen, thereby disturbing fibrin clotting [37].
Effects of sulfatide on an in vivo animal thrombosis model Since the information so far we had in mid-1990s supported an anticoagulant role of sulfatide, we investigated the effects of sulfatide on blood coagulation, together with molecules structurally related to sulfatide, using a rat disease of human deep vein thrombosis (DVT) that is conventionally used to evaluate the potential of anticoagulant agents [14]. Under anesthesia, rats were laporotomized and the inferior vena cava was ligated just below the branch of the renal vein with surgical thread. Sulfatide was injected into the rats through the tail vein 1 min prior to ligation. Two control groups were prepared. In one group, vehicle was injected the same way 1 min prior to ligation of the inferior vena cava and in the other group, sulfatide was injected through the tail vein without vein ligation. When sulfatide was simply injected without vein ligation, the rats appeared to be normal and no thrombus was observed. Furthermore, the bleeding times of the rats administered sulfatide were significantly prolonged, as expected [14,15,37], suggesting that simple injection of sulfatide produces an anticoagulant state in animals. In contrast, unexpectedly sulfatide remarkably enhanced thrombogenesis in the rats with vein ligation (Fig. 1A) [14,15]. This phenomenon was observed within 1 min, suggesting that probably no de novo protein synthesis is necessary for this event. In another animal experiment, continuous infusion of sulfatide into rats using a polyethylene cannula produced giant thrombi at the tip of the cannula, suggesting
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Fig. 2. (A) Effect of sulfatide on plasma coagulation time. Aliquots of 100 ll of plasma were added to the wells of a 96-well microtiter plate and coagulation was initiated by adding CaCl2 . Plasma without sulfatide began to coagulate at approximately 12 min in this experiment. Sulfatide (23.4 lg/ml) apparently accelerated this process by approximately 3 min and the accelerating activity was still observed at a dose of 0.37 lg/ml. At a high concentration (375 lg/ml), the accelerating activity was abated. From [14] with permission. (B) The relation between the sulfatide concentration and plasma coagulation onset time. Sulfatide and sulfatide 6S had similar coagulant activities, although sulfatide 6S had slightly stronger activity. Cholesterol 3-sulfate exhibited weaker coagulant activity than either of the sulfatides and GM4 exhibited no activity. From [15] with permission.
the procedure of vein ligation is not always necessary (Fig. 1B) [13]. Comparative studies of molecules structurally related to sulfatide, such as sulfatide 6S (sulfuric ester of galactosylceramide at the C6 of the galactose residue), cholesterol sulfate (5-cholesten-3b-ol sulfate), and ganglioside GM4 (I3 -a-N -acetylneuraminosylgalactoylceramide), indicate that the sulfate moiety in the sulfatides is essential for the coagulant activity and the galactose residue enhances the activity [15]. The results of these two different animal experiments are very consistent with the result of in vitro experiment, the kinetic turbidimetry of plasma coagulation: (i) sulfatide, sulfatide 6S, and cholesterol sulfate, but not ganglioside GM4, accelerate blood coagulation on plastic, and (ii) the degree of acceleration or induction of thrombogenesis is in the order of sulfatide 6S, sulfatide, and cholesterol sulfate (Fig. 2B) [13–15]. Thrombi produced in the animal experiment with continuous infusion might have the same mechanism as the in vitro plasma coagulation on plastic because the thrombi were observed at the tip of the plastic cannula. On the other hand, the mechanism underlying the DVT
animal model result remains uncertain, because simple bolus shot injection of sulfatide into the animals did not induce thrombosis, rather it prolonged plasma clotting time and bleeding time. In the absence of plasma coagulation factor XII, sulfatide does not shorten plasma clotting time on plastic, suggesting a critical role of factor XII, although generally the physiologic importance of factor XII still remains uncertain [22]. The procedure of vein ligation, which induces sudden severe vascular injury and venous congestion, is absolutely necessary for sulfatide to enhance thrombogenesis in this DVT animal model. Once such injury occurs, apparently the endothelium is heavily damaged, exposing basement membranes and other extracellular matrix components with the simultaneous activation of platelets and extravasation of fibrinogen into the matrix [38]. Binding of the sulfatide to extracellular matrix components such as laminin, thrombospondin, and vWF might present the place like a surface similar to that of plastic, which was observed in an in vitro experiment of the kinetic turbidimetry of plasma coagulation. Very incidentally, vWF, thrombospondin, P-selectin, and fibrin-
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Table 1 Chronologic findings of sulfatide in haemostasis/thrombogenesis (1961–2004 February) Findings Prolongation of clotting time by administration of sulfatide in rabbits Prolongation of clotting time by sulfatide-rich fraction from bovine brain extract Activation of blood coagulation factor XII by sulfatide Binding of sulfatide to laminin Binding of sulfatide to thrombospondin Binding of sulfatide to von Willebrand factor Detection of sulfatide on platelets by labelled thrombospondin and von Willebrand factor Occurrence of sulfatide as a major glycosphingolipid component of plasma lipoprotein Accumulation of sulfatide in atherosclerotic aorta Binding of sulfatide to P-selectin Antithrombin III independent anticoagulant activity of sulfatide Increase factor VII activity by sulfatide Binding of sulfatide to fibrinogen Generation of giant thrombi in animal model of thrombosis by administration of sulfatide Inhibition of platelet-aggregation by sulfatide Heparin cofactor II independent anticoagulant activity of sulfatide Direct inhibition of thrombin activity by sulfatide Sulfatide on platelets as a ligand for P-selectin to stabilize platelet aggregation Inhibition of platelet adhesion to vWF under flow conditions by sulfatide Annexin V mediated anticoagulant action by sulfatide *
Thrombotic
Anti-thrombotic
Modulated
Others
References
+
[16]
+
[18]
+
[19–21] + + + +
+ + +
[24] [6] [7] [6,7] +
[10]
+
[35] [5] [36]
+
[50] [37] [13–15]
+ +
[44] [15]
+
[15] [43]
+
[40]
+
[51]
+
+
Endogenous sulfatide.
ogen are stored in the a-granules of platelets [39], whereas vWF and P-selectin are also stored in the Weibel–Palade bodies of endothelial cells [31]. It is quite interesting that such molecules with the capability to bind to sulfatide are also involved in thrombogenesis and co-exist in a closely localized place. In the presence of sulfatide, if some pathologic conditions disturb the balance of the concentration of such molecules, sulfatide might modulate thrombogenesis with various ways. Indeed, it was reported that sulfatide inhibits platelet adhesion to vWF in flow blood [40]. Cancer cells are often reported to induce thrombotic diseases [41,42]. Although the major causes of such diseases have been attributed to procoagulant or coagulant activities, such as tissue factor or factor X activators, some uncharacterized factors, including membrane vesicles, which would be enormously shed from cancer cells [41] and may be sulfatide [5], have been reported.
Conclusion and perspective I have reviewed the reports from the last 40 years on sulfatide in the field of thrombogenesis/haemostasis together with current data from my laboratory using an
animal disease model. These data are summarized in Table 1 and Fig. 3. There are more reports supporting a role for sulfatide in anti-thrombosis than in thrombosis, although sulfatide can induce thrombosis under specific conditions. Although these contradictory results are probably based on differences in the methods employed, sulfatide is potentially involved in both thrombosis and anti-thrombosis. It should be noted that in most of the reports, exogenous sulfatide was added to the experimental systems. Merten and Thiagarajan [43] reported that sulfatide on the cell surface of platelets serves as a ligand for P-selectin and stabilizes platelet aggregation, which may be related with the report that exogenous sulfatide inhibited platelet function [44]. Thus, studies of the role of endogenous sulfatide are necessary. Galactosylceramide sulfotransferase contributes to build sulfatide and was cloned [45] and recently its null mice were generated [46]. These mice will be a useful tool for this line of investigation. Meanwhile, studies using exogenous sulfatide might also provide critical information for drug discovery based on sulfatide-mediated antithrombotic and/or haemostatic agents. As seen in heparin and low molecular weight heparin, sulfate glycoconjugates are well-known anticoagulant therapeutics. An unstudied area of sulfatide in thrombogenesis/
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haemostasis is the fibrinolytic system. In fact, the role of factor XII in fibrinolytic system is known [47]. Furthermore, the importance of other sulfate glycoconjugates in the fibrinolytic system has already been reported [48,49]. Sulfatide in these areas also remains to be investigated. Acknowledgment This paper is dedicated to the memory of Dr. Victor Ginsburg.
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