- Email: [email protected]
Prolonged bleeding time induced by anticoagulant glycosaminoglycans in dogs is associated with the inhibition of thrombin-induced platelet aggregation
Thrombosis Research 112 (2003) 83 – 91
Regular Article
Prolonged bleeding time induced by anticoagulant glycosaminoglycans in dogs is associated with the inhibition of thrombin-induced platelet aggregation Kenji Kitazato *, Keiko T. Kitazato, Eiji Sasaki, Kazuhisa Minamiguchi, Hideki Nagase Cancer Research Laboratory, Hannno Research Center, Taiho Pharmaceutical, 1-27, Misugidai, Hanno, Saitama, 357-8527, Japan Received 8 October 2003; received in revised form 8 October 2003; accepted 9 October 2003
Abstract Introduction: The clinical use of unfractionated heparin (UFH) is complicated by hemorrhage. This has led to a search for safer alternatives, one of which, the recently identified depolymerized holothurian glycosaminoglycan (DHG), causes less bleeding and exhibits a better antithrombotic – hemorrhagic ratio in rats and dogs than UFH and low-molecular-weight heparin (LMWH). In contrast to UFH and LMWH, which exert their anticoagulant effects by inhibiting thrombin in the presence of antithrombin III (AT), DHG exerts its anticoagulant effect by inhibiting the intrinsic factor Xase complex and thrombin in the presence of heparin cofactor II (HCII). Materials and Methods: The hemorrhagic effect of DHG was compared with those of UFH and LMWH in healthy dogs, and the mechanism responsible for prolonging bleeding time was examined both in dogs and with human platelets. Results: DHG prolonged template-bleeding time in dogs less than UFH and LMWH do. Although the maximum noneffective concentrations of each glycosaminoglycan (GAG) that prolong the bleeding time are almost the same as the concentrations that inhibit thrombin-induced platelet aggregation, they are not related to those that inhibit ADP-induced platelet aggregation. Results of experiments on gel-filtered platelets from humans indicate that the inhibition of thrombin-induced platelet aggregation caused by UFH and LMWH in the presence of AT is more prominent than that caused by DHG with HCII. Conclusions: These results suggest that the prolongation of bleeding time caused by GAGs are associated with the inhibition of thrombin-induced platelet aggregation, and DHG may cause less bleeding than UFH and LMWH because of its different thrombin inhibition mechanism in platelet-rich plasma (PRP). D 2003 Elsevier Ltd. All rights reserved. Keywords: Glycosaminoglycan; Platelet aggregation; Thrombin; Hemorrhage; Dog
Unfractionated heparin (UFH), a complex polysulfonated glycosaminoglycan (GAG), has been used to prevent venous thrombosis for many years, but unexpected hemorrhage is a major complication that occurs during its use [1,2]. Therefore, considerable effort has been expended to develop other anticoagulants with better antithrombotic effect– bleeding risk ratios. The major anticoagulant mechanisms of UFH are its antithrombin and anti-factor Xa activities exerted through antithrombin III (AT) [3 – 6]. Consequently, synthetic pentasaccharides with high affinity for AT, which inactivate factor Xa but not thrombin, have
Abbreviations: AT, antithrombin III; HCII, heparin cofactor II; APTT, activated partial thromboplastin time; TCT, thrombin clotting time. * Corresponding author. Tel.: +81-429-72-8900; fax: +81-429-720034. E-mail address: [email protected] (K. Kitazato). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016./j.thromres.2003.10.005
been investigated [7 – 10]. These pentasaccharides exhibited an antithrombotic effect without hemorrhagic effect in animals. Both active-site-blocked factor IXa [11] and factor VIIa [12], neither of which show antithrombin activity, also exhibit antithrombotic effects without impairing extravascular hemostasis in animals. However, the specific thrombin inhibitor, hirudin, a protein used to treat coronary heart disease, causes bleeding at antithrombotic doses in animals and in humans [12 – 14]. Consequently, these results suggest that an ideal antithrombotic drug would not inhibit thrombin. Although thrombin inhibitors appear to be poor drug candidates, dermatan sulfate is an unusual polysaccharide antithrombotic agent that exerts its antithrombotic effect by indirectly inhibiting thrombin through heparin cofactor II (HCII) activation [15]. This mechanism does not cause bleeding in animals or in humans [16 –18]. Recently, one such polysaccharide, depolymerized holothurian glycosaminoglycan (DHG), was found to exert
84
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91
an antithrombotic effect with less bleeding than UFH and LMWH in rats [19,20]. DHG is also an effective anticoagulant that does not prolong bleeding time in an extracorporeal circulation system in dogs, which stands in sharp contrast to UFH and low-molecular-weight heparin (LMWH) [21,22]. DHG exerts its anticoagulant action by two mechanisms: one is the inhibition of the intrinsic factor Xase complex independent of either AT or HCII; the other is the HCII-dependent inhibition of thrombin [23,24]. In a comparative affinity chromatography study between DHG and UFH on human plasma protein binding, AT-affinity is the only difference between the two GAGs (19). Unlike UFH, the anticoagulant action of DHG is independent of AT; this characteristic has also been confirmed in vivo [25]. Indeed, DHG showed significantly improve survival in both normal and low AT-level mice with acute thromboembolism; in contrast, UFH and LMWH do not increase survival in low ATlevel mice. These observations indicate that the inhibition of thrombin mediated by AT results in an increased hemorrhagic tendency, but inhibition mediated by HCII does not. In the present study, plasma GAG levels that prolong bleeding time, changes in coagulation parameters, and changes in platelet aggregation induced by GAG were examined to test this hypothesis.
1. Materials and methods 1.1. Animals Male beagle dogs (body mass 8 –14 kg) from HazeltonLRE (Kalamazoo, MI) were used in all animal experiments. These experiments were reviewed and approved by the Animal Ethics Committee of Taiho Pharmaceutical.
Table 1 Specific activities and molecular weight of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), and depolymerized holothurian glycosaminoglycan (DHG) Molecular Antithrombin activity weight
APTT prolongation activity
(AT) (HCII) DHG (U/mg) heparin (U/mg) heparin (U/mg) UFH 15,000 LMWH 9000 DHG 15,000
205.7 58.7 0.0
184.8 21.6 101.9
3702 1033 1013
activities of GAG were obtained by the parallel line bioassay against an unfractionated heparin standard (the Japanese Pharmacopeia Standard of unfractionated heparin, 189.4 JP U/mg) distributed by the National Institute of Hygienic Sciences in Japan, or against a DHG standard (Taiho Fine Chemical, 1000 DHG U/mg). 1.3. Bleeding time measurements Template bleeding time was measured in conscious dogs 5 min after intravenous GAG administration according to Dejana et al. [29] with slight modifications. An incision was made on the inner surface of the pinna with a SimplateR template incision device (Organon Teknica, Tokyo, Japan). Bleeding time was defined as the time necessary to stop bleeding for 1 min. Bleeding times longer than 15 min were recorded as >15 min. Fifteen dogs were used in this study. First, bleeding time of all animals was measured for the control, and then the influence of GAG was examined using 15 dogs per one experiment and performed three times at regular intervals of washout period to avoid the influence of GAG. 1.4. Platelet count
1.2. GAGs The following GAGs were purchased from the companies listed: UFH (from porcine mucosa; Nacalai Tesque; Kyoto, Japan), and LMWH (H5640, from porcine mucosa; Sigma, St. Louis, MO). DHG was prepared by Taiho Phramaceutical from GAG extracted from sea cucumbers followed by depolymerization with hydrogen peroxide [26,27]. The specific activities and molecular weight of GAGs used in this study were listed in Table 1 [20,25]. Antithrombin activity in the presence of AT (human, heparin kit, Kabi Vitrum, Stockholm, Sweden) or HCII (human, purified as described by Tran et al. [28]) was measured with chromogenic substrate Boc –Val –Pro– Arg– pNA (Chisso, Chiba, Japan) and thrombin (human, Chisso). Activated partial thromboplastin time (APTT) prolongation activity was measured by use of commercially available human plasma (VerifyR 1, Organon Teknica, Durham, NC), PlatelinR (Organon Teknica) and cellite (No. 512, Wako, Osaka, Japan). The specific
The number of platelets was measured using an automatic analyzer (NE-6000; Toa Medical Electronics, Kobe, Japan). 1.5. Plasma sample for coagulation assay Venous blood was drawn from six healthy human donors after obtaining informed consent, or from beagle dogs. The blood was taken into 3.8% sodium citrate (1 volume vs. 9 volumes of blood), and plasma was obtained by centrifugation at 1870 g for 10 min at 4 jC. The plasma samples were stored at 80 jC until use. 1.6. Ex vivo and in vitro coagulation assays APTT and thrombin clotting time (TCT) were determined by adding 100 Al of a commercially available APTT reagent (PlatelinR LS, Organon Teknica) and human thrombin (1340 NIH U/mg protein, Sigma, final
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91
concentration: 5 U/ml) or dog thrombin (extracted in house [30,31], final concentration: 1 U/ml) to 200 Al of plasma. In ex vivo experiments, plasma was obtained at various times after intravenous administration of a GAG, and APTT and TCT were measured. In in vitro experiments, the effects of GAGs upon TCT were determined by adding 10 Al of GAG to the assay mixture (n = 6). 1.7. Determination of plasma levels of GAG The plasma level of GAG after intravenous administration of one of the GAGs to separate dogs was determined by measuring antithrombin activity in the plasma. A DS kit (Stago Diagnostica, Asnieres, France) was used to measure antithrombin activity in the plasma in the presence of bovine HCII by comparing the results with GAG standard curves for each GAG [32]. 1.8. ADP-, collagen-, and thrombin receptor agonist peptide-induced platelet aggregation Platelet-rich plasma (PRP) was obtained from citratetreated venous blood (from humans or dogs) by centrifugation at 170 g for 10 min at 22 jC. The number of platelets in PRP was adjusted to 2.5 1011/l by diluting with platelet-poor plasma (PPP), obtained by centrifugation at 1870 g for 10 min. In a siliconized glass cuvette, 270 Al PRP was mixed at 1000 rpm at 37 jC for 1 min, and then 15 Al of one of several concentrations of a GAG in saline was added. The resulting mixtures were incubated for 2 min. After that, 15 Al ADP (final concentration: 1 or 2 AM, Sigma), collagen (final concentration: 200 Ag/ml; Worthington Biochemical, NJ), or thrombin receptor agonist peptide (TRAP) (final concentration: 15 AM; Sawady Technology, Tokyo, Japan) in 0.9% saline solution was added [31]. Platelet aggregation was recorded using a Hematracer 801 (Nicoh Bioscience, Tokyo, Japan) by following changes in optical density. 1.9. Thrombin-induced platelet aggregation We examined two methods to study thrombin-induced platelet aggregation. One is the study using washed platelets and defibrinogenated plasma to obtain information under physiological condition that only lack fibrinogen. The other is the study using gel-filtered platelets and AT and/or HCII to elucidate the respective role of these heparin cofactors on the inhibition of thrombin-induced platelet aggregation by GAGs. PRP was obtained by centrifuging blood samples at 170 g for 10 min at 22 jC; these were recentrifuged at 1870 g for 5 min at 4 jC to obtain packed washed platelets after mixing with ice-cold Tris buffer (pH 7.35) of the same volume. PPP was defibrinogenated with Ancrod (final concentrations: 0.15 or 0.3 U/ml) at 37 jC for 10 min followed by centrifugation at 1870 g for
85
10 min at 4 jC. Packed platelets were washed once more with Tris buffer and suspended in defibrinogenated PPP to obtain defibrinogenated PRP (2.5 1011/l). The effect of GAG on thrombin-induced platelet aggregation using defibrinogenated PRP was tested according to the previously described procedure. The final thrombin concentration was 5 U/ml. In other experiments, gel-filtered platelets obtained by using the following method were also used to examine the roles of AT and HCII in GAG-mediated inhibition of platelet aggregation. Human PRP was collected as described previously and loaded onto a Sepharose 2B column pre-equilibrated with HEPES-buffered Tyrode’s solution (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5.5 mM glucose, 0.35% [w/v] bovine serum albumin, 3 mM NaH2PO4, 3.5 mM HEPES, pH 7.35). The gel-filtered platelets were pooled, and the number of platelets was adjusted to 3.5 1011/l with HEPES-buffered Tyrode’s solution. The reaction mixture consisted of 250 Al of gel-filtered platelets, 10 Al of AT (final concentration: 1 U/ml, human, Diagnostica Stago) or HCII (final concentration: 1 U/ml, human, Diagnostica Stago), 15 Al of test GAG, and 15 Al of human thrombin (final concentration: 5 U/ml, human, Sigma). 1.10. Statistical analysis All group data were expressed as mean values and S.D. unless stated otherwise. Differences between control group and GAG-treated groups in bleeding time experiment were tested statistically by the Kruskal – Wallis test. Differences between saline control and GAG-treated groups in platelet aggregation experiments were tested statistically by the Dunnet’s test.
2. Results 2.1. Effect of GAGs on ear-bleeding time A dose of 3 mg/kg UFH, 3 mg/kg LMWH, or 30 mg/kg DHG prolonged template bleeding time significantly 5 min after intravenous GAG administration (Fig. 1). However, one out of five dogs given 1 mg/kg UFH had a bleeding time of more than 15 min. Consequently, the dose that prolongs bleeding time of UFH was deemed to be 1 mg/kg on the basis of toxicology. Therefore, the threshold doses for bleeding time for these GAGs are considered to be 1 mg/kg for UFH, 3 mg/kg for LMWH, and 30 mg/kg for DHG. The results show that DHG causes less bleeding per mass administered than both LMWH and UFH in this canine model. 2.2. Changes in platelet counts in dogs Platelet counts were determined 10 and 20 min after administration of UFH, LMWH, and DHG at hemorrhagic
86
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91
min after administration of GAGs was 200 s or more for UFH, 37 s for LMWH, and 200 s or more for DHG, and APTT of plasma 20 min after administration was 136 s for UFH, 32 s for LMWH, and 200 s or more for DHG at their hemorrhagic doses when normal APTT was 14.1 s. These results were essentially the same as reported earlier in rats [20]. 2.4. TCT prolongation in human or dog plasma in vitro
Fig. 1. Effects of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), and depolymerized holothurian glycosaminoglycan (DHG) on template bleeding time in dogs. The bleeding time was measured 5 min after intravenous administration of glycosaminoglycans to conscious dogs. An incision was made with a SimplateR template incision device on the inner surface of the pinna. Bleeding time was defined as the time necessary to obtain complete cessation of bleeding for 1 min, and bleeding times longer than 15 min are recorded as >15 min. Horizontal bars indicate the mean value. *p < 0.05, **p < 0.01 compared to the control by the Kruskal – Wallis test.
dose. These GAGs did not change the platelet count (data not shown). 2.3. APTT and TCT prolongation ex vivo At their hemorrhagic doses, all GAGs prolonged TCT markedly; however, APTT prolongation differed greatly among the three GAGs . TCT of all plasma obtained from dogs 10 and 20 min after administration of UFH, 1 mg/kg, LMWH, 3 mg/kg, or DHG, 30 mg/kg, was 200 s or more when normal TCT was 14.6 s. Whereas, APTT of plasma 10
As mentioned above, the three GAGs at their hemorrhagic doses markedly prolonged TCT in dogs ex vivo. Therefore, this effect was examined in vitro using plasma from dogs and humans. For the in vitro system, 1 Ag/ml UFH, 3 Ag/ml LMWH, and 10 Ag/ml DHG prolonged TCT markedly in human plasma, and 3 Ag/ml UFH, 10 Ag/ml LMWH, and 30 Ag/ml DHG prolonged TCT markedly in dog plasma (Fig. 2). Since the magnitude of hemorrhagic effects correlates with both in vitro and ex vivo TCT prolongation, the results suggest that thrombin inhibition caused by these GAGs is one of the factors responsible for the prolongation of bleeding time during treatment. 2.5. Plasma levels of GAG administered intravenously in dogs The antithrombin activity in the presence of HCII was used as a measure of the plasma levels of each GAG (Fig. 3). The plasma level of UFH was 3 Ag/ml, of LMWH was 10 Ag/ml, and of DHG was 80 Ag/ml 10 min after administration at their maximum noneffective doses for prolonging bleeding times (0.3, 1, and 10 mg/ kg, respectively). These plasma concentrations are thought to be the concentrations related to threshold bleeding times for each GAG.
Fig. 2. Effects of unfractionated heparin (UFH, 4), low-molecular-weight heparin (LMWH, 5), and depolymerized holothurian glycosaminoglycan (DHG, .) on TCT in (A) human (n = 6) and (B) dog plasma (n = 6) in vitro. TCT was measured by adding human thrombin to human plasma or dog thrombin to dog plasma to give normal TCT 20.8 s for human, and 15.3 s for dogs.
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91
87
Fig. 3. Plasma level of (A) depolymerized holothurian glycosaminoglycan (DHG), (B) unfractionated heparin (UFH), and (C) low-molecular-weight heparin (LMWH) 10, 20, and 60 min after the intravenous injection of each glycosaminoglycan into dogs (n = 3). The antithrombin activity in the presence of bovine HCII, measured using a DS kit for each glycosaminoglycan and a standard curve, was used to determine the plasma levels of each glycosaminoglycan. Each point indicates the mean F S.D. Closed circles represent the dose at which the risk of hemorrhage increases and open circles represent the maximum nonhemorrhagic dose of each agent.
2.6. Effect of GAG on collagen-, ADP-, and thrombininduced dog platelet aggregation in vitro Collagen-induced platelet aggregation was inhibited concentration-dependently by these GAGs and inhibited significantly at 30 Ag/ml UFH, 300 Ag/ml LMWH, and 30 Ag/ml DHG (Fig. 4). These concentrations are not only completely
different from the concentrations related to threshold bleeding time for each of the three GAGs, but also completely independent of the magnitude of hemorrhagic effect for prolonging bleeding time exhibited by the GAG (i.e., severity decreases in the order of UFH, LMWH, and DHG). Next, ADP-induced platelet aggregation was only slightly inhibited by all GAGs at concentrations greater than
Fig. 4. Effects of unfractionated heparin (UFH, 4), low-molecular-weight heparin (LMWH, 5), and depolymerized holothurian glycosaminoglycan (DHG, .) on (A) collagen-, (B) ADP-, and (C) thrombin-induced platelet aggregation in dogs (n = 5 – 6). For the collagen- and ADP-induced platelet aggregation experiments, platelet-rich plasma (PRP) was obtained by centrifuging citrate-treated venous blood from dogs. The number of platelets in PRP was adjusted to 2.5 1011/l by diluting with platelet-poor plasma. For the thrombin-induced platelet aggregation experiments, defibrinogenated PRP was prepared by mixing packed platelets (obtained by centrifuging PRP), with Ancrod (0.15 – 0.3 U/ml)-treated platelet-poor plasma at 37 jC for 10 min. In a siliconized glass cuvette, 270 Al of PRP or defibrinogenated PRP and 15 Al of test GAG were mixed at 1000 rpm at 37 jC, and then 15 Al of one of the agonists was added to start the reaction. An open circle (o) signifies the mean value for the no-GAG control experiment. Vertical bars represent the S.D. *p < 0.05, **p < 0.01 compared to the saline control by the Dunnett’s test.
88
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91
300 Ag/ml. However, there were no major differences between the inhibition exerted by each GAG. Finally, thrombin-induced platelet aggregation (using defibrinogenated PRP to prevent fibrin formation by the added thrombin) was inhibited with concentration-dependent fashion and significantly inhibited at 3 Ag/ml of UFH, 10 Ag/ml of LMHW, and 100 Ag/ml of DHG. These concentrations were essentially the same as respective concentrations related to threshold bleeding time for each GAG and are also consistent with the magnitude of hemorrhagic effects to prolong the bleeding time exhibited by each GAG on the view point of dose – response curves. 2.7. Effects of GAG on collagen-induced platelet aggregation in dogs ex vivo Collagen-induced platelet aggregation was also examined in PRP from blood taken 10 or 20 min after intravenous administration of GAGs at their threshold doses for bleeding time and maximum noneffective doses for prolonging bleeding time. DHG inhibited collageninduced platelet aggregation at both doses (10 and 30 mg/kg, i.v.), whereas UFH (0.3 and 1 mg/kg, i.v.) and LMWH (1 and 3 mg/kg, i.v.) did not inhibit this aggregation (data not shown); these results suggest that inhibition of collagen-induced platelet aggregation is associated with DHG-induced prolongation of bleeding time but not the common cause of GAG-induced prolongation of bleeding time in dogs.
2.8. Effect of GAG on thrombin- and TRAP-induced human platelet aggregation in vitro First, we examined thrombin-induced human platelet aggregation (Fig. 5A). That was inhibited concentrationdependently and inhibited at 1, 3, and 30 Ag/ml of UFH, LMWH, and DHG, respectively. These dose – response curves are similar to those obtained using dog platelets. Second, we examined TRAP-induced platelet aggregation to clarify more precisely the mechanisms by which GAGs inhibit thrombin-induced platelet aggregation (Fig. 5B). The concentrations of GAGs were selected more than those used thrombin-induced human platelet aggregation. None of the three GAGs inhibited TRAP-induced platelet aggregation, although thrombin-induced platelet aggregation was inhibited. These results indicate that GAGs inhibit platelet aggregation through thrombin inactivation but not through the inhibition of post-thrombin receptor signal transduction [33]. 2.9. GAG inhibition of thrombin-induced human platelet aggregation in the presence of AT, HCII, or both in vitro Human gel-filtered platelets were used to examine the respective roles of AT and HCII in inhibiting thrombininduced platelet aggregation caused by the three GAGs (Table 2). UFH and LMWH exhibited a stronger inhibition in the presence of AT alone than in the presence of HCII alone. DHG, however, showed the opposite effect; inhibi-
Fig. 5. Effects of unfractionated heparin (UFH, 4), low-molecular-weight heparin (LMWH, 5), and depolymerized holothurian glycosaminoglycan (DHG, .) on (A) thrombin- and (B) thrombin receptor agonist peptide (TRAP)-induced human platelet aggregation. For the TRAP-induced platelet aggregation experiments, platelet-rich plasma (PRP) was obtained by centrifuging citrate-treated venous blood. The number of platelets in PRP was adjusted to 2.5 1011/l by diluting with platelet-poor plasma. For the thrombin-induced platelet aggregation experiments, defibrinogenated PRP was prepared by mixing packed platelets (obtained by centrifuging PRP) with Ancrod (0.3 U/ml)-treated platelet-poor plasma at 37 jC for 10 min. In siliconized glass cuvettes, 270 Al of PRP or defibrinogenated PRP and 15 Al of one of the GAGs were mixed at 1000 rpm at 37 jC, and then 15 Al of one of the agonists was added to start the reaction. An open circle (o) signifies the mean value for the no-GAG control experiments. Vertical bars represent the S.D. *p < 0.05, **p < 0.01 vs. saline by the Dunnett’s test.
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91 Table 2 Concentrations of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), and depolymerized holothurian glycosaminoglycan (DHG) that significantly suppress thrombin-induced platelet aggregation of human platelets Drug
UFH LMWH DHG
Gel-filtered platelet
Defibrinogenated PRP
+ AT, HCII
+ AT
+ HCII
3 3 3
1 3 100
10 10 10
1 3 30
Concentrations in micrograms per milliliter. For an experiment using gel-filtered platelet, human PRP was loaded onto a Sepharose 2B column pre-equilibrated with HEPES-buffered Tyrode’s solution to obtain gel-filtered platelets. The reaction mixture consisted of 250 Al of gel-filtered platelets, 20 Al of AT and/or HCII 1 U/ml, 15 Al of test GAG, and 15 Al of human thrombin 5 U/ml. For an experiment using defrinogenated PRP, packed washed platelets were suspended defibrinogenated PPP, treated with Ancrod, to obtain defibrinogenated PRP. The reaction mixture consisted of 270 Al of defibrinogenated PRP, 15 Al of test GAG, and 15 Al of human thrombin 5 U/ml.
tion occurred at a low DHG concentration in the presence of HCII, but a high DHG concentration was required to induce inhibition in the presence of AT. The concentrations of DHG required to inhibit thrombin-induced platelet aggregation with defibrinogenated PRP were higher than a concentration for gel-filtered platelet suspensions supplemented with AT and HCII.
3. Discussion It has been reported that in dogs, hemostasis of template ear-bleeding reflects both platelet function and a procoagulant state [34 – 36]. Thus, template ear-bleeding in dogs is a good representation of the global risk of heparin-induced hemorrhage in humans. In this study, the plasma level of UFH was 3 Ag/ml, of LMWH was 10 Ag/ml, and of DHG was 80 Ag/ml 10 min after administration at their maximum noneffective doses for prolonging bleeding time (Fig. 4), and these plasma concentrations are thought to be the threshold concentrations for hemorrhagic risk for dogs for each GAG. On the other hand, as previously reported, the plasma level of UFH was 13.7 Ag/ml, of LMWH was 32.9 Ag/ml, and of DHG was 38.2 Ag/ml at their effective doses in a dog-hemodialysis model [21]. These findings indicate that DHG exhibits a better antithrombotic – hemorrhagic ratio in dog than UFH and LMWH. To clarify the role of procoagulant activity in template ear-bleeding model, the ex vivo coagulation parameters, TCT and APTT were examined after separate administration of each of the three GAGs at doses close to their doses to prolong bleeding time in dogs. All three GAGs prolonged TCT at their hemorrhagic doses in dogs, but significant APTT prolongation did not occur after LMWH administration; this result is in contrast to results for animals given DHG or UFH. However, these results are consistent with
89
results of a previous study conducted in rats [20], and they suggest that thrombin function is associated with hemostasis in both animals. To estimate the role of platelet function in template bleeding model, in vitro thrombin-, ADP-, or collageninduced platelet aggregation were measured. Thrombininduced platelet aggregation was significantly inhibited by UFH, LMWH, and DHG at 3, 10, and 100 Ag/ml, respectively. These concentrations are very close to the threshold concentrations for hemorrhagic risk for dogs, i.e., the threshold concentration for UFH is 3 Ag/ml, for LMWH is 10 Ag/ml, and for DHG is 80 Ag/ml. These results indicate that the inhibition of thrombin-induced platelet aggregation is one cause of prolonging bleeding time induced by the three GAGs. This explanation is also consistent with results described in an earlier report [37]. In this report, LMWH affected bleeding to a less extent than UFH because of weaker inhibition of thrombin-induced platelet aggregation, using plasma defibrinogenated dogs and standardized skin flap bleeding. In contrast, it has been reported that the inhibition of collagen-induced platelet aggregation is one of the causes for bleeding induced by UFH [16,38 – 40]. However, the results of present ex vivo study show this effect is observed in animal given DHG, but not UFH and LMWH. Therefore, further study is needed to clarify whether the prolongation of bleeding time induced by GAG is primarily the result of inhibiting thrombin-induced platelet activation or not by comparing DHG with other GAGs, such as dermatan sulfate and pentosan sulfate. DHG inhibits thrombin through activated HCII, whereas both UFH and LMWH inhibit thrombin through activated AT [3– 6,23]. Gel-filtered human platelets, in which almost all of plasma proteins are depleted, were used to examine the effects of the three GAGs on the function of these heparin cofactors (Table 2). UFH and LMWH inhibited gelfiltered platelet aggregation more strongly in the presence of AT than in the presence of HCII, whereas DHG showed the opposite effect as expected from results of previous studies. With regard to UFH, since the specific activities measured by antithrombin activity in the presence of AT and HCII are almost the same (205.7 heparin U/mg with AT and 184.8 heparin U/mg with HCII in Table 1), thrombin-induced platelet aggregation may be inhibited more efficiently by AT than by HCII. However, the concentration of DHG required to inhibit thrombin-induced platelet aggregation in the presence of both AT and HCII is only threefold greater than that of UFH when using gel-filtered platelets, whereas the concentration of DHG required for inhibition is 30-fold greater than that of UFH when using defibrinogenated PRP, in which all plasma proteins are present except fibrinogen. Thus, it is thought that DHG, together with certain plasma proteins, prevents the binding of the HCII to thrombin and facilitates the approximation of thrombin toward the thrombin receptor on platelets. In summary, the prolongation of template ear-bleeding time in dogs caused by GAGs involves the inhibition of
90
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91
thrombin-induced platelet aggregation. A possible reason why bleeding associated with DHG administration is less severe than UFH and LMWH is the difference in the mechanism of thrombin inhibition; DHG inhibits thrombin in the presence of HCII, but heparins inhibit thrombin in the presence of AT. Consequently, DHG is a promising candidate anticoagulant that carries with it a lower risk of bleeding complication than currently used anticoagulants.
Acknowledgements The authors thank Tomio Hirota, Safety Research Laboratory, Taiho Pharmaceutical, for his skillful assistance and animal care, Professor Hidehiko Saito, First Department of Internal Medicine, Nagoya University School of Medicine, for his invaluable advice, and Mr. Steven E Johnson for editing the manuscript.
References [1] Mant MJ, O’Brien BD, Thong KL, Hammond GW, Birtwhistle RV, Grace GC. Haemorrhagic complications of heparin therapy. Lancet 1997;1:1133 – 5. [2] Salzman EW, Deykin D, Shapiro RM, Rosenberg RD. Management of heparin therapy—controlled prospective trial. N Engl J Med 1975;292:1046 – 50. [3] Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973;248:6490 – 7. [4] Rosenberg RD, Rosenberg JS. Natural anticoagulant mechanism. J Clin Invest 1984;74:1 – 6. [5] Be´guin S, Lindhout T, Hemker HC. The mode of action of heparin in plasma. Thromb Haemost 1988;60:457 – 62. [6] Ofosu FA, Sie´ P, Modi GJ, Fernandes F, Buchanan MR, Blajchman MA, et al. The inhibition of thrombin-dependent positive feedback reaction is critical to the expression of the anticoagulant effect of heparin. Biochem J 1987;243:579 – 88. [7] Van Boeckel CAA, Petitou M. The unique antithrombin III binding domain of heparin: a lead to new synthetic antithrombotics. Angew Chem Int Ed Engl 1993;32:1671 – 90. [8] Hobbelen PMJ, Van Dinther TG, Vogel GMT, Van Boeckel CAA, Moelker HCT, Meuleman DG. Pharmacological profile of the chemically synthetized antithrombin III binding fragment of heparin (pentasaccharide) in rats. Thromb Haemost 1990;63:265 – 72. [9] Cadroy Y, Hanson SR, Harker LA. Antithrombotic effects of synthetic pentasaccharide with high affinity for plasma antithrombin III in nonhuman primates. Thromb Haemost 1993;70:631 – 5. [10] Herbert JM, He´rault JP, Bernat A, Van Amsterdam RGM, Vogel GMT, Lormeau JC, et al. Biochemical and pharmacological properties of SANORG 32701—comparison with the ‘synthetic pentasaccharide’ (SR90107/ORG31540) and standard heparin. Circ Res 1996;79:590 – 600. [11] Benedict CR, Ryan J, Wolitzky B, Ramos R, Gerlach M, Tijburg P, et al. Active site-blocked factor IXa prevents intravascular thrombus formation in the coronary vasculature without inhibiting extravascular coagulation in a canine thrombosis model. J Clin Invest 1991;88:1760 – 5. [12] Harker LA, Hanson SR, Kelly AB. Antithrombotic benefits and hemorrhagic risks of direct thrombin antagonists. Thromb Haemost 1995;74:464 – 72.
[13] Antman EM. TIMI9A Investigators. Hirudin in acute myocardial infarction: safety reports from the thrombolysis and thrombin inhibition in myocardial infarction (TIMI) 9A trial. Circulation 1994;90:1624 – 30. [14] GUSTO IIa Investigators. Randomized trial of intravenous heparin versus recombinant hirudin for acute coronary syndromes. Circulation 1994;90:1631 – 7. [15] Tollefsen DM, Perska CA, Monafo WJ. Activation of heparin cofactor II by dermatan sulfate. J Biol Chem 1983;258:6713 – 6. [16] Fernandez F, Van Ryn J, Ofosu FA, Hirsh J, Buchanan MR. The haemorrhagic and antithrombotic effects of dermatan sulphate. Br J Haematol 1986;64:309 – 17. [17] Agnelli G, Cosmi B, Filippo PD, Ranucci V, Veschi F, Longetti M, et al. A randomized double-blind, placebo-controlled trial of dermatan sulfate for prevention of deep vein thrombosis in hip fracture. Thromb Haemost 1992;67:203 – 8. [18] Cohen AT, Phillips MJ, Edmondson RA, Skinner JA, Das SK, Cooper DJ, et al. A dose ranging study to evaluate dermatan sulphate in preventing deep vein thrombosis following total hip arthroplasty. Thromb Haemost 1994;72:793 – 8. [19] Minamiguchi K, Nagase H, Kitazato KT, Kitazato K. Interaction of a depolymerized holothurian glycosaminoglycan with proteins in human plasma. Thromb Res 1996;83:253 – 64. [20] Kitazato K, Kitazato KT, Nagase H, Minamiguchi K. DHG, a new depolymerized holothurian glycosaminoglycan, exerts an antithrombotic effect with less bleeding than unfractionated or low molecular weight heparin, in rats. Thromb Res 1996;84:111 – 20. [21] Minamiguchi K, Kitazato KT, Sasaki E, Nagase H, Kitazato K. The anticoagulant and hemorrhagic effects of DHG, a new depolymerized holothurian glycosaminoglycan, on experimental hemodialysis in dogs. Thromb Haemost 1997;77:1148 – 53. [22] Minamiguchi K, Kitazato KT, Nagase H, Sasaki E, Ohwada K, Kitazato K. Depolymerized holothurian glycosaminoglycan (DHG), a novel alternative anticoagulant for hemodialysis, is safe and effective in a dog renal failure model. Kidney Int 2003;63:1548 – 55. [23] Nagase H, Enjyoji K, Minamiguchi K, Kitazato KT, Kitazato K, Saito H, et al. Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions: antithrombin III- and heparin cofactor II-independent inhibition of factor X activation by factor IXa-factor VIIIa complex and heparin cofactor II-dependent inhibition of thrombin. Blood 1995;85:1527 – 34. [24] Nagase HH, Enjyoji K, Shima M, Kitazato K, Yoshioka A, Saito H, et al. Effect of depolymerized holothurian glycosaminoglycan (DHG) on the activation of factor VIII and factor V by thrombin. J Biochem 1996;119:63 – 9. [25] Nagase H, Kitazato KT, Sasaki E, Hattori M, Kitazato K, Saito H. Antithrombin III-independent effect of depolymerized holothurian glycosaminoglycan (DHG) on acute thromboembolism in mice. Thromb Haemost 1997;77:399 – 402. [26] Suzuki N, Kitazato K, Takamatsu J, Saito H. Antithrombotic and anticoagulant activity of depolymerized fragment of the glycosaminoglycan extracted from Stichopus japonicus selenka. Thromb Haemost 1991;65:369 – 73. [27] Yoshida K, Minami Y, Nemoto H, Numata K, Yamanaka E. Structure of DHG, a depolymerized glycosaminoglycan from sea cucumber, Stichopus japonicus. Tetrahedron Lett 1992;33:4959 – 62. [28] Tran TH, Lammle B, Zbinden B, Duckert F. Heparin cofactor II: purification and antibody production. Thromb Haemost 1986; 55:19 – 23. [29] Dejana E, Villa S, De Gaetano G. Bleeding time in rats: a comparison of different experimental conditions. Thromb Haemost 1982;48:108 – 11. [30] Hashimoto N, Morita T, Iwanaga S. A method for systematic purification from bovine plasma of six vitamin-K-dependent coagulation factors: prothrombin, factor X, factor IX, protein S, protein C, and protein Z. J Biochem 1985;97:1347 – 55. [31] Owen WG, Esmon CT, Jackson CM. The conversion of prothrombin to thrombin. J Biol Chem 1974;249:594 – 605.
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91 [32] Dupouy D, Sie´ P, Dol F, Boneu B. A simple method to measure dermatan sulfate at a sub-microgram concentrations in plasma. Thromb Haemost 1988;60:236 – 9. [33] Vu T-KH, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991;64:1057 – 68. [34] Brassard JA, Meyers KM. Evaluation of the buccal bleeding time and platelet glass bead retention as assays of hemostasis in the dog: the effects of acetylsalicylic acid, warfarin and von Willebrand factor deficiency. Thromb Haemost 1991;65:191 – 5. [35] Giles AR, Tinlin S, Greenwood R. A canine model of hemophilic (factor VIII: C deficiency) bleeding. Blood 1982;60:727 – 30. [36] Graham JB, Buckwalter JA, Hartley LJ, Brinkhous KM. Canine hemophilia—observations on the course, the clotting anomaly, and the effect of blood transfusion. J. Exp. Med. 1949;90:97 – 111.
91
[37] Ljungberg B, Johnsson H. In vivo effects of a low molecular weight heparin fragment on platelet aggregation and platelet dependent hemostasis in dogs. Thromb Haemost 1988;60:232 – 5. [38] Carter CJ, Kelton JG, Hirsh J, Cerskus A, Santos AV, Gent M. The relationship between the hemorrhagic and antithrombotic properties of low molecular weight heparin in rabbits. Blood 1982;59:1239 – 45. [39] Fernandez F, N’guyen P, Van Ryn J, Ofosu FA, Hirsh J, Buchanan MR. Hemorrhagic doses of heparin and other glycosaminoglycans induce a platelet defect. Thromb Res 1986;43:491 – 5. [40] Messmore Jr HL, Griffin B, Fareed J, Coyne E, Seghatchian J. In vitro studies of the interaction of heparin, low molecular weight heparin and heparinoids with platelets. Ann N Y Acad Sci 1989;556:217 – 32.
Regular Article
Prolonged bleeding time induced by anticoagulant glycosaminoglycans in dogs is associated with the inhibition of thrombin-induced platelet aggregation Kenji Kitazato *, Keiko T. Kitazato, Eiji Sasaki, Kazuhisa Minamiguchi, Hideki Nagase Cancer Research Laboratory, Hannno Research Center, Taiho Pharmaceutical, 1-27, Misugidai, Hanno, Saitama, 357-8527, Japan Received 8 October 2003; received in revised form 8 October 2003; accepted 9 October 2003
Abstract Introduction: The clinical use of unfractionated heparin (UFH) is complicated by hemorrhage. This has led to a search for safer alternatives, one of which, the recently identified depolymerized holothurian glycosaminoglycan (DHG), causes less bleeding and exhibits a better antithrombotic – hemorrhagic ratio in rats and dogs than UFH and low-molecular-weight heparin (LMWH). In contrast to UFH and LMWH, which exert their anticoagulant effects by inhibiting thrombin in the presence of antithrombin III (AT), DHG exerts its anticoagulant effect by inhibiting the intrinsic factor Xase complex and thrombin in the presence of heparin cofactor II (HCII). Materials and Methods: The hemorrhagic effect of DHG was compared with those of UFH and LMWH in healthy dogs, and the mechanism responsible for prolonging bleeding time was examined both in dogs and with human platelets. Results: DHG prolonged template-bleeding time in dogs less than UFH and LMWH do. Although the maximum noneffective concentrations of each glycosaminoglycan (GAG) that prolong the bleeding time are almost the same as the concentrations that inhibit thrombin-induced platelet aggregation, they are not related to those that inhibit ADP-induced platelet aggregation. Results of experiments on gel-filtered platelets from humans indicate that the inhibition of thrombin-induced platelet aggregation caused by UFH and LMWH in the presence of AT is more prominent than that caused by DHG with HCII. Conclusions: These results suggest that the prolongation of bleeding time caused by GAGs are associated with the inhibition of thrombin-induced platelet aggregation, and DHG may cause less bleeding than UFH and LMWH because of its different thrombin inhibition mechanism in platelet-rich plasma (PRP). D 2003 Elsevier Ltd. All rights reserved. Keywords: Glycosaminoglycan; Platelet aggregation; Thrombin; Hemorrhage; Dog
Unfractionated heparin (UFH), a complex polysulfonated glycosaminoglycan (GAG), has been used to prevent venous thrombosis for many years, but unexpected hemorrhage is a major complication that occurs during its use [1,2]. Therefore, considerable effort has been expended to develop other anticoagulants with better antithrombotic effect– bleeding risk ratios. The major anticoagulant mechanisms of UFH are its antithrombin and anti-factor Xa activities exerted through antithrombin III (AT) [3 – 6]. Consequently, synthetic pentasaccharides with high affinity for AT, which inactivate factor Xa but not thrombin, have
Abbreviations: AT, antithrombin III; HCII, heparin cofactor II; APTT, activated partial thromboplastin time; TCT, thrombin clotting time. * Corresponding author. Tel.: +81-429-72-8900; fax: +81-429-720034. E-mail address: [email protected] (K. Kitazato). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016./j.thromres.2003.10.005
been investigated [7 – 10]. These pentasaccharides exhibited an antithrombotic effect without hemorrhagic effect in animals. Both active-site-blocked factor IXa [11] and factor VIIa [12], neither of which show antithrombin activity, also exhibit antithrombotic effects without impairing extravascular hemostasis in animals. However, the specific thrombin inhibitor, hirudin, a protein used to treat coronary heart disease, causes bleeding at antithrombotic doses in animals and in humans [12 – 14]. Consequently, these results suggest that an ideal antithrombotic drug would not inhibit thrombin. Although thrombin inhibitors appear to be poor drug candidates, dermatan sulfate is an unusual polysaccharide antithrombotic agent that exerts its antithrombotic effect by indirectly inhibiting thrombin through heparin cofactor II (HCII) activation [15]. This mechanism does not cause bleeding in animals or in humans [16 –18]. Recently, one such polysaccharide, depolymerized holothurian glycosaminoglycan (DHG), was found to exert
84
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91
an antithrombotic effect with less bleeding than UFH and LMWH in rats [19,20]. DHG is also an effective anticoagulant that does not prolong bleeding time in an extracorporeal circulation system in dogs, which stands in sharp contrast to UFH and low-molecular-weight heparin (LMWH) [21,22]. DHG exerts its anticoagulant action by two mechanisms: one is the inhibition of the intrinsic factor Xase complex independent of either AT or HCII; the other is the HCII-dependent inhibition of thrombin [23,24]. In a comparative affinity chromatography study between DHG and UFH on human plasma protein binding, AT-affinity is the only difference between the two GAGs (19). Unlike UFH, the anticoagulant action of DHG is independent of AT; this characteristic has also been confirmed in vivo [25]. Indeed, DHG showed significantly improve survival in both normal and low AT-level mice with acute thromboembolism; in contrast, UFH and LMWH do not increase survival in low ATlevel mice. These observations indicate that the inhibition of thrombin mediated by AT results in an increased hemorrhagic tendency, but inhibition mediated by HCII does not. In the present study, plasma GAG levels that prolong bleeding time, changes in coagulation parameters, and changes in platelet aggregation induced by GAG were examined to test this hypothesis.
1. Materials and methods 1.1. Animals Male beagle dogs (body mass 8 –14 kg) from HazeltonLRE (Kalamazoo, MI) were used in all animal experiments. These experiments were reviewed and approved by the Animal Ethics Committee of Taiho Pharmaceutical.
Table 1 Specific activities and molecular weight of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), and depolymerized holothurian glycosaminoglycan (DHG) Molecular Antithrombin activity weight
APTT prolongation activity
(AT) (HCII) DHG (U/mg) heparin (U/mg) heparin (U/mg) UFH 15,000 LMWH 9000 DHG 15,000
205.7 58.7 0.0
184.8 21.6 101.9
3702 1033 1013
activities of GAG were obtained by the parallel line bioassay against an unfractionated heparin standard (the Japanese Pharmacopeia Standard of unfractionated heparin, 189.4 JP U/mg) distributed by the National Institute of Hygienic Sciences in Japan, or against a DHG standard (Taiho Fine Chemical, 1000 DHG U/mg). 1.3. Bleeding time measurements Template bleeding time was measured in conscious dogs 5 min after intravenous GAG administration according to Dejana et al. [29] with slight modifications. An incision was made on the inner surface of the pinna with a SimplateR template incision device (Organon Teknica, Tokyo, Japan). Bleeding time was defined as the time necessary to stop bleeding for 1 min. Bleeding times longer than 15 min were recorded as >15 min. Fifteen dogs were used in this study. First, bleeding time of all animals was measured for the control, and then the influence of GAG was examined using 15 dogs per one experiment and performed three times at regular intervals of washout period to avoid the influence of GAG. 1.4. Platelet count
1.2. GAGs The following GAGs were purchased from the companies listed: UFH (from porcine mucosa; Nacalai Tesque; Kyoto, Japan), and LMWH (H5640, from porcine mucosa; Sigma, St. Louis, MO). DHG was prepared by Taiho Phramaceutical from GAG extracted from sea cucumbers followed by depolymerization with hydrogen peroxide [26,27]. The specific activities and molecular weight of GAGs used in this study were listed in Table 1 [20,25]. Antithrombin activity in the presence of AT (human, heparin kit, Kabi Vitrum, Stockholm, Sweden) or HCII (human, purified as described by Tran et al. [28]) was measured with chromogenic substrate Boc –Val –Pro– Arg– pNA (Chisso, Chiba, Japan) and thrombin (human, Chisso). Activated partial thromboplastin time (APTT) prolongation activity was measured by use of commercially available human plasma (VerifyR 1, Organon Teknica, Durham, NC), PlatelinR (Organon Teknica) and cellite (No. 512, Wako, Osaka, Japan). The specific
The number of platelets was measured using an automatic analyzer (NE-6000; Toa Medical Electronics, Kobe, Japan). 1.5. Plasma sample for coagulation assay Venous blood was drawn from six healthy human donors after obtaining informed consent, or from beagle dogs. The blood was taken into 3.8% sodium citrate (1 volume vs. 9 volumes of blood), and plasma was obtained by centrifugation at 1870 g for 10 min at 4 jC. The plasma samples were stored at 80 jC until use. 1.6. Ex vivo and in vitro coagulation assays APTT and thrombin clotting time (TCT) were determined by adding 100 Al of a commercially available APTT reagent (PlatelinR LS, Organon Teknica) and human thrombin (1340 NIH U/mg protein, Sigma, final
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91
concentration: 5 U/ml) or dog thrombin (extracted in house [30,31], final concentration: 1 U/ml) to 200 Al of plasma. In ex vivo experiments, plasma was obtained at various times after intravenous administration of a GAG, and APTT and TCT were measured. In in vitro experiments, the effects of GAGs upon TCT were determined by adding 10 Al of GAG to the assay mixture (n = 6). 1.7. Determination of plasma levels of GAG The plasma level of GAG after intravenous administration of one of the GAGs to separate dogs was determined by measuring antithrombin activity in the plasma. A DS kit (Stago Diagnostica, Asnieres, France) was used to measure antithrombin activity in the plasma in the presence of bovine HCII by comparing the results with GAG standard curves for each GAG [32]. 1.8. ADP-, collagen-, and thrombin receptor agonist peptide-induced platelet aggregation Platelet-rich plasma (PRP) was obtained from citratetreated venous blood (from humans or dogs) by centrifugation at 170 g for 10 min at 22 jC. The number of platelets in PRP was adjusted to 2.5 1011/l by diluting with platelet-poor plasma (PPP), obtained by centrifugation at 1870 g for 10 min. In a siliconized glass cuvette, 270 Al PRP was mixed at 1000 rpm at 37 jC for 1 min, and then 15 Al of one of several concentrations of a GAG in saline was added. The resulting mixtures were incubated for 2 min. After that, 15 Al ADP (final concentration: 1 or 2 AM, Sigma), collagen (final concentration: 200 Ag/ml; Worthington Biochemical, NJ), or thrombin receptor agonist peptide (TRAP) (final concentration: 15 AM; Sawady Technology, Tokyo, Japan) in 0.9% saline solution was added [31]. Platelet aggregation was recorded using a Hematracer 801 (Nicoh Bioscience, Tokyo, Japan) by following changes in optical density. 1.9. Thrombin-induced platelet aggregation We examined two methods to study thrombin-induced platelet aggregation. One is the study using washed platelets and defibrinogenated plasma to obtain information under physiological condition that only lack fibrinogen. The other is the study using gel-filtered platelets and AT and/or HCII to elucidate the respective role of these heparin cofactors on the inhibition of thrombin-induced platelet aggregation by GAGs. PRP was obtained by centrifuging blood samples at 170 g for 10 min at 22 jC; these were recentrifuged at 1870 g for 5 min at 4 jC to obtain packed washed platelets after mixing with ice-cold Tris buffer (pH 7.35) of the same volume. PPP was defibrinogenated with Ancrod (final concentrations: 0.15 or 0.3 U/ml) at 37 jC for 10 min followed by centrifugation at 1870 g for
85
10 min at 4 jC. Packed platelets were washed once more with Tris buffer and suspended in defibrinogenated PPP to obtain defibrinogenated PRP (2.5 1011/l). The effect of GAG on thrombin-induced platelet aggregation using defibrinogenated PRP was tested according to the previously described procedure. The final thrombin concentration was 5 U/ml. In other experiments, gel-filtered platelets obtained by using the following method were also used to examine the roles of AT and HCII in GAG-mediated inhibition of platelet aggregation. Human PRP was collected as described previously and loaded onto a Sepharose 2B column pre-equilibrated with HEPES-buffered Tyrode’s solution (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5.5 mM glucose, 0.35% [w/v] bovine serum albumin, 3 mM NaH2PO4, 3.5 mM HEPES, pH 7.35). The gel-filtered platelets were pooled, and the number of platelets was adjusted to 3.5 1011/l with HEPES-buffered Tyrode’s solution. The reaction mixture consisted of 250 Al of gel-filtered platelets, 10 Al of AT (final concentration: 1 U/ml, human, Diagnostica Stago) or HCII (final concentration: 1 U/ml, human, Diagnostica Stago), 15 Al of test GAG, and 15 Al of human thrombin (final concentration: 5 U/ml, human, Sigma). 1.10. Statistical analysis All group data were expressed as mean values and S.D. unless stated otherwise. Differences between control group and GAG-treated groups in bleeding time experiment were tested statistically by the Kruskal – Wallis test. Differences between saline control and GAG-treated groups in platelet aggregation experiments were tested statistically by the Dunnet’s test.
2. Results 2.1. Effect of GAGs on ear-bleeding time A dose of 3 mg/kg UFH, 3 mg/kg LMWH, or 30 mg/kg DHG prolonged template bleeding time significantly 5 min after intravenous GAG administration (Fig. 1). However, one out of five dogs given 1 mg/kg UFH had a bleeding time of more than 15 min. Consequently, the dose that prolongs bleeding time of UFH was deemed to be 1 mg/kg on the basis of toxicology. Therefore, the threshold doses for bleeding time for these GAGs are considered to be 1 mg/kg for UFH, 3 mg/kg for LMWH, and 30 mg/kg for DHG. The results show that DHG causes less bleeding per mass administered than both LMWH and UFH in this canine model. 2.2. Changes in platelet counts in dogs Platelet counts were determined 10 and 20 min after administration of UFH, LMWH, and DHG at hemorrhagic
86
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91
min after administration of GAGs was 200 s or more for UFH, 37 s for LMWH, and 200 s or more for DHG, and APTT of plasma 20 min after administration was 136 s for UFH, 32 s for LMWH, and 200 s or more for DHG at their hemorrhagic doses when normal APTT was 14.1 s. These results were essentially the same as reported earlier in rats [20]. 2.4. TCT prolongation in human or dog plasma in vitro
Fig. 1. Effects of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), and depolymerized holothurian glycosaminoglycan (DHG) on template bleeding time in dogs. The bleeding time was measured 5 min after intravenous administration of glycosaminoglycans to conscious dogs. An incision was made with a SimplateR template incision device on the inner surface of the pinna. Bleeding time was defined as the time necessary to obtain complete cessation of bleeding for 1 min, and bleeding times longer than 15 min are recorded as >15 min. Horizontal bars indicate the mean value. *p < 0.05, **p < 0.01 compared to the control by the Kruskal – Wallis test.
dose. These GAGs did not change the platelet count (data not shown). 2.3. APTT and TCT prolongation ex vivo At their hemorrhagic doses, all GAGs prolonged TCT markedly; however, APTT prolongation differed greatly among the three GAGs . TCT of all plasma obtained from dogs 10 and 20 min after administration of UFH, 1 mg/kg, LMWH, 3 mg/kg, or DHG, 30 mg/kg, was 200 s or more when normal TCT was 14.6 s. Whereas, APTT of plasma 10
As mentioned above, the three GAGs at their hemorrhagic doses markedly prolonged TCT in dogs ex vivo. Therefore, this effect was examined in vitro using plasma from dogs and humans. For the in vitro system, 1 Ag/ml UFH, 3 Ag/ml LMWH, and 10 Ag/ml DHG prolonged TCT markedly in human plasma, and 3 Ag/ml UFH, 10 Ag/ml LMWH, and 30 Ag/ml DHG prolonged TCT markedly in dog plasma (Fig. 2). Since the magnitude of hemorrhagic effects correlates with both in vitro and ex vivo TCT prolongation, the results suggest that thrombin inhibition caused by these GAGs is one of the factors responsible for the prolongation of bleeding time during treatment. 2.5. Plasma levels of GAG administered intravenously in dogs The antithrombin activity in the presence of HCII was used as a measure of the plasma levels of each GAG (Fig. 3). The plasma level of UFH was 3 Ag/ml, of LMWH was 10 Ag/ml, and of DHG was 80 Ag/ml 10 min after administration at their maximum noneffective doses for prolonging bleeding times (0.3, 1, and 10 mg/ kg, respectively). These plasma concentrations are thought to be the concentrations related to threshold bleeding times for each GAG.
Fig. 2. Effects of unfractionated heparin (UFH, 4), low-molecular-weight heparin (LMWH, 5), and depolymerized holothurian glycosaminoglycan (DHG, .) on TCT in (A) human (n = 6) and (B) dog plasma (n = 6) in vitro. TCT was measured by adding human thrombin to human plasma or dog thrombin to dog plasma to give normal TCT 20.8 s for human, and 15.3 s for dogs.
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91
87
Fig. 3. Plasma level of (A) depolymerized holothurian glycosaminoglycan (DHG), (B) unfractionated heparin (UFH), and (C) low-molecular-weight heparin (LMWH) 10, 20, and 60 min after the intravenous injection of each glycosaminoglycan into dogs (n = 3). The antithrombin activity in the presence of bovine HCII, measured using a DS kit for each glycosaminoglycan and a standard curve, was used to determine the plasma levels of each glycosaminoglycan. Each point indicates the mean F S.D. Closed circles represent the dose at which the risk of hemorrhage increases and open circles represent the maximum nonhemorrhagic dose of each agent.
2.6. Effect of GAG on collagen-, ADP-, and thrombininduced dog platelet aggregation in vitro Collagen-induced platelet aggregation was inhibited concentration-dependently by these GAGs and inhibited significantly at 30 Ag/ml UFH, 300 Ag/ml LMWH, and 30 Ag/ml DHG (Fig. 4). These concentrations are not only completely
different from the concentrations related to threshold bleeding time for each of the three GAGs, but also completely independent of the magnitude of hemorrhagic effect for prolonging bleeding time exhibited by the GAG (i.e., severity decreases in the order of UFH, LMWH, and DHG). Next, ADP-induced platelet aggregation was only slightly inhibited by all GAGs at concentrations greater than
Fig. 4. Effects of unfractionated heparin (UFH, 4), low-molecular-weight heparin (LMWH, 5), and depolymerized holothurian glycosaminoglycan (DHG, .) on (A) collagen-, (B) ADP-, and (C) thrombin-induced platelet aggregation in dogs (n = 5 – 6). For the collagen- and ADP-induced platelet aggregation experiments, platelet-rich plasma (PRP) was obtained by centrifuging citrate-treated venous blood from dogs. The number of platelets in PRP was adjusted to 2.5 1011/l by diluting with platelet-poor plasma. For the thrombin-induced platelet aggregation experiments, defibrinogenated PRP was prepared by mixing packed platelets (obtained by centrifuging PRP), with Ancrod (0.15 – 0.3 U/ml)-treated platelet-poor plasma at 37 jC for 10 min. In a siliconized glass cuvette, 270 Al of PRP or defibrinogenated PRP and 15 Al of test GAG were mixed at 1000 rpm at 37 jC, and then 15 Al of one of the agonists was added to start the reaction. An open circle (o) signifies the mean value for the no-GAG control experiment. Vertical bars represent the S.D. *p < 0.05, **p < 0.01 compared to the saline control by the Dunnett’s test.
88
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91
300 Ag/ml. However, there were no major differences between the inhibition exerted by each GAG. Finally, thrombin-induced platelet aggregation (using defibrinogenated PRP to prevent fibrin formation by the added thrombin) was inhibited with concentration-dependent fashion and significantly inhibited at 3 Ag/ml of UFH, 10 Ag/ml of LMHW, and 100 Ag/ml of DHG. These concentrations were essentially the same as respective concentrations related to threshold bleeding time for each GAG and are also consistent with the magnitude of hemorrhagic effects to prolong the bleeding time exhibited by each GAG on the view point of dose – response curves. 2.7. Effects of GAG on collagen-induced platelet aggregation in dogs ex vivo Collagen-induced platelet aggregation was also examined in PRP from blood taken 10 or 20 min after intravenous administration of GAGs at their threshold doses for bleeding time and maximum noneffective doses for prolonging bleeding time. DHG inhibited collageninduced platelet aggregation at both doses (10 and 30 mg/kg, i.v.), whereas UFH (0.3 and 1 mg/kg, i.v.) and LMWH (1 and 3 mg/kg, i.v.) did not inhibit this aggregation (data not shown); these results suggest that inhibition of collagen-induced platelet aggregation is associated with DHG-induced prolongation of bleeding time but not the common cause of GAG-induced prolongation of bleeding time in dogs.
2.8. Effect of GAG on thrombin- and TRAP-induced human platelet aggregation in vitro First, we examined thrombin-induced human platelet aggregation (Fig. 5A). That was inhibited concentrationdependently and inhibited at 1, 3, and 30 Ag/ml of UFH, LMWH, and DHG, respectively. These dose – response curves are similar to those obtained using dog platelets. Second, we examined TRAP-induced platelet aggregation to clarify more precisely the mechanisms by which GAGs inhibit thrombin-induced platelet aggregation (Fig. 5B). The concentrations of GAGs were selected more than those used thrombin-induced human platelet aggregation. None of the three GAGs inhibited TRAP-induced platelet aggregation, although thrombin-induced platelet aggregation was inhibited. These results indicate that GAGs inhibit platelet aggregation through thrombin inactivation but not through the inhibition of post-thrombin receptor signal transduction [33]. 2.9. GAG inhibition of thrombin-induced human platelet aggregation in the presence of AT, HCII, or both in vitro Human gel-filtered platelets were used to examine the respective roles of AT and HCII in inhibiting thrombininduced platelet aggregation caused by the three GAGs (Table 2). UFH and LMWH exhibited a stronger inhibition in the presence of AT alone than in the presence of HCII alone. DHG, however, showed the opposite effect; inhibi-
Fig. 5. Effects of unfractionated heparin (UFH, 4), low-molecular-weight heparin (LMWH, 5), and depolymerized holothurian glycosaminoglycan (DHG, .) on (A) thrombin- and (B) thrombin receptor agonist peptide (TRAP)-induced human platelet aggregation. For the TRAP-induced platelet aggregation experiments, platelet-rich plasma (PRP) was obtained by centrifuging citrate-treated venous blood. The number of platelets in PRP was adjusted to 2.5 1011/l by diluting with platelet-poor plasma. For the thrombin-induced platelet aggregation experiments, defibrinogenated PRP was prepared by mixing packed platelets (obtained by centrifuging PRP) with Ancrod (0.3 U/ml)-treated platelet-poor plasma at 37 jC for 10 min. In siliconized glass cuvettes, 270 Al of PRP or defibrinogenated PRP and 15 Al of one of the GAGs were mixed at 1000 rpm at 37 jC, and then 15 Al of one of the agonists was added to start the reaction. An open circle (o) signifies the mean value for the no-GAG control experiments. Vertical bars represent the S.D. *p < 0.05, **p < 0.01 vs. saline by the Dunnett’s test.
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91 Table 2 Concentrations of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), and depolymerized holothurian glycosaminoglycan (DHG) that significantly suppress thrombin-induced platelet aggregation of human platelets Drug
UFH LMWH DHG
Gel-filtered platelet
Defibrinogenated PRP
+ AT, HCII
+ AT
+ HCII
3 3 3
1 3 100
10 10 10
1 3 30
Concentrations in micrograms per milliliter. For an experiment using gel-filtered platelet, human PRP was loaded onto a Sepharose 2B column pre-equilibrated with HEPES-buffered Tyrode’s solution to obtain gel-filtered platelets. The reaction mixture consisted of 250 Al of gel-filtered platelets, 20 Al of AT and/or HCII 1 U/ml, 15 Al of test GAG, and 15 Al of human thrombin 5 U/ml. For an experiment using defrinogenated PRP, packed washed platelets were suspended defibrinogenated PPP, treated with Ancrod, to obtain defibrinogenated PRP. The reaction mixture consisted of 270 Al of defibrinogenated PRP, 15 Al of test GAG, and 15 Al of human thrombin 5 U/ml.
tion occurred at a low DHG concentration in the presence of HCII, but a high DHG concentration was required to induce inhibition in the presence of AT. The concentrations of DHG required to inhibit thrombin-induced platelet aggregation with defibrinogenated PRP were higher than a concentration for gel-filtered platelet suspensions supplemented with AT and HCII.
3. Discussion It has been reported that in dogs, hemostasis of template ear-bleeding reflects both platelet function and a procoagulant state [34 – 36]. Thus, template ear-bleeding in dogs is a good representation of the global risk of heparin-induced hemorrhage in humans. In this study, the plasma level of UFH was 3 Ag/ml, of LMWH was 10 Ag/ml, and of DHG was 80 Ag/ml 10 min after administration at their maximum noneffective doses for prolonging bleeding time (Fig. 4), and these plasma concentrations are thought to be the threshold concentrations for hemorrhagic risk for dogs for each GAG. On the other hand, as previously reported, the plasma level of UFH was 13.7 Ag/ml, of LMWH was 32.9 Ag/ml, and of DHG was 38.2 Ag/ml at their effective doses in a dog-hemodialysis model [21]. These findings indicate that DHG exhibits a better antithrombotic – hemorrhagic ratio in dog than UFH and LMWH. To clarify the role of procoagulant activity in template ear-bleeding model, the ex vivo coagulation parameters, TCT and APTT were examined after separate administration of each of the three GAGs at doses close to their doses to prolong bleeding time in dogs. All three GAGs prolonged TCT at their hemorrhagic doses in dogs, but significant APTT prolongation did not occur after LMWH administration; this result is in contrast to results for animals given DHG or UFH. However, these results are consistent with
89
results of a previous study conducted in rats [20], and they suggest that thrombin function is associated with hemostasis in both animals. To estimate the role of platelet function in template bleeding model, in vitro thrombin-, ADP-, or collageninduced platelet aggregation were measured. Thrombininduced platelet aggregation was significantly inhibited by UFH, LMWH, and DHG at 3, 10, and 100 Ag/ml, respectively. These concentrations are very close to the threshold concentrations for hemorrhagic risk for dogs, i.e., the threshold concentration for UFH is 3 Ag/ml, for LMWH is 10 Ag/ml, and for DHG is 80 Ag/ml. These results indicate that the inhibition of thrombin-induced platelet aggregation is one cause of prolonging bleeding time induced by the three GAGs. This explanation is also consistent with results described in an earlier report [37]. In this report, LMWH affected bleeding to a less extent than UFH because of weaker inhibition of thrombin-induced platelet aggregation, using plasma defibrinogenated dogs and standardized skin flap bleeding. In contrast, it has been reported that the inhibition of collagen-induced platelet aggregation is one of the causes for bleeding induced by UFH [16,38 – 40]. However, the results of present ex vivo study show this effect is observed in animal given DHG, but not UFH and LMWH. Therefore, further study is needed to clarify whether the prolongation of bleeding time induced by GAG is primarily the result of inhibiting thrombin-induced platelet activation or not by comparing DHG with other GAGs, such as dermatan sulfate and pentosan sulfate. DHG inhibits thrombin through activated HCII, whereas both UFH and LMWH inhibit thrombin through activated AT [3– 6,23]. Gel-filtered human platelets, in which almost all of plasma proteins are depleted, were used to examine the effects of the three GAGs on the function of these heparin cofactors (Table 2). UFH and LMWH inhibited gelfiltered platelet aggregation more strongly in the presence of AT than in the presence of HCII, whereas DHG showed the opposite effect as expected from results of previous studies. With regard to UFH, since the specific activities measured by antithrombin activity in the presence of AT and HCII are almost the same (205.7 heparin U/mg with AT and 184.8 heparin U/mg with HCII in Table 1), thrombin-induced platelet aggregation may be inhibited more efficiently by AT than by HCII. However, the concentration of DHG required to inhibit thrombin-induced platelet aggregation in the presence of both AT and HCII is only threefold greater than that of UFH when using gel-filtered platelets, whereas the concentration of DHG required for inhibition is 30-fold greater than that of UFH when using defibrinogenated PRP, in which all plasma proteins are present except fibrinogen. Thus, it is thought that DHG, together with certain plasma proteins, prevents the binding of the HCII to thrombin and facilitates the approximation of thrombin toward the thrombin receptor on platelets. In summary, the prolongation of template ear-bleeding time in dogs caused by GAGs involves the inhibition of
90
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91
thrombin-induced platelet aggregation. A possible reason why bleeding associated with DHG administration is less severe than UFH and LMWH is the difference in the mechanism of thrombin inhibition; DHG inhibits thrombin in the presence of HCII, but heparins inhibit thrombin in the presence of AT. Consequently, DHG is a promising candidate anticoagulant that carries with it a lower risk of bleeding complication than currently used anticoagulants.
Acknowledgements The authors thank Tomio Hirota, Safety Research Laboratory, Taiho Pharmaceutical, for his skillful assistance and animal care, Professor Hidehiko Saito, First Department of Internal Medicine, Nagoya University School of Medicine, for his invaluable advice, and Mr. Steven E Johnson for editing the manuscript.
References [1] Mant MJ, O’Brien BD, Thong KL, Hammond GW, Birtwhistle RV, Grace GC. Haemorrhagic complications of heparin therapy. Lancet 1997;1:1133 – 5. [2] Salzman EW, Deykin D, Shapiro RM, Rosenberg RD. Management of heparin therapy—controlled prospective trial. N Engl J Med 1975;292:1046 – 50. [3] Rosenberg RD, Damus PS. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem 1973;248:6490 – 7. [4] Rosenberg RD, Rosenberg JS. Natural anticoagulant mechanism. J Clin Invest 1984;74:1 – 6. [5] Be´guin S, Lindhout T, Hemker HC. The mode of action of heparin in plasma. Thromb Haemost 1988;60:457 – 62. [6] Ofosu FA, Sie´ P, Modi GJ, Fernandes F, Buchanan MR, Blajchman MA, et al. The inhibition of thrombin-dependent positive feedback reaction is critical to the expression of the anticoagulant effect of heparin. Biochem J 1987;243:579 – 88. [7] Van Boeckel CAA, Petitou M. The unique antithrombin III binding domain of heparin: a lead to new synthetic antithrombotics. Angew Chem Int Ed Engl 1993;32:1671 – 90. [8] Hobbelen PMJ, Van Dinther TG, Vogel GMT, Van Boeckel CAA, Moelker HCT, Meuleman DG. Pharmacological profile of the chemically synthetized antithrombin III binding fragment of heparin (pentasaccharide) in rats. Thromb Haemost 1990;63:265 – 72. [9] Cadroy Y, Hanson SR, Harker LA. Antithrombotic effects of synthetic pentasaccharide with high affinity for plasma antithrombin III in nonhuman primates. Thromb Haemost 1993;70:631 – 5. [10] Herbert JM, He´rault JP, Bernat A, Van Amsterdam RGM, Vogel GMT, Lormeau JC, et al. Biochemical and pharmacological properties of SANORG 32701—comparison with the ‘synthetic pentasaccharide’ (SR90107/ORG31540) and standard heparin. Circ Res 1996;79:590 – 600. [11] Benedict CR, Ryan J, Wolitzky B, Ramos R, Gerlach M, Tijburg P, et al. Active site-blocked factor IXa prevents intravascular thrombus formation in the coronary vasculature without inhibiting extravascular coagulation in a canine thrombosis model. J Clin Invest 1991;88:1760 – 5. [12] Harker LA, Hanson SR, Kelly AB. Antithrombotic benefits and hemorrhagic risks of direct thrombin antagonists. Thromb Haemost 1995;74:464 – 72.
[13] Antman EM. TIMI9A Investigators. Hirudin in acute myocardial infarction: safety reports from the thrombolysis and thrombin inhibition in myocardial infarction (TIMI) 9A trial. Circulation 1994;90:1624 – 30. [14] GUSTO IIa Investigators. Randomized trial of intravenous heparin versus recombinant hirudin for acute coronary syndromes. Circulation 1994;90:1631 – 7. [15] Tollefsen DM, Perska CA, Monafo WJ. Activation of heparin cofactor II by dermatan sulfate. J Biol Chem 1983;258:6713 – 6. [16] Fernandez F, Van Ryn J, Ofosu FA, Hirsh J, Buchanan MR. The haemorrhagic and antithrombotic effects of dermatan sulphate. Br J Haematol 1986;64:309 – 17. [17] Agnelli G, Cosmi B, Filippo PD, Ranucci V, Veschi F, Longetti M, et al. A randomized double-blind, placebo-controlled trial of dermatan sulfate for prevention of deep vein thrombosis in hip fracture. Thromb Haemost 1992;67:203 – 8. [18] Cohen AT, Phillips MJ, Edmondson RA, Skinner JA, Das SK, Cooper DJ, et al. A dose ranging study to evaluate dermatan sulphate in preventing deep vein thrombosis following total hip arthroplasty. Thromb Haemost 1994;72:793 – 8. [19] Minamiguchi K, Nagase H, Kitazato KT, Kitazato K. Interaction of a depolymerized holothurian glycosaminoglycan with proteins in human plasma. Thromb Res 1996;83:253 – 64. [20] Kitazato K, Kitazato KT, Nagase H, Minamiguchi K. DHG, a new depolymerized holothurian glycosaminoglycan, exerts an antithrombotic effect with less bleeding than unfractionated or low molecular weight heparin, in rats. Thromb Res 1996;84:111 – 20. [21] Minamiguchi K, Kitazato KT, Sasaki E, Nagase H, Kitazato K. The anticoagulant and hemorrhagic effects of DHG, a new depolymerized holothurian glycosaminoglycan, on experimental hemodialysis in dogs. Thromb Haemost 1997;77:1148 – 53. [22] Minamiguchi K, Kitazato KT, Nagase H, Sasaki E, Ohwada K, Kitazato K. Depolymerized holothurian glycosaminoglycan (DHG), a novel alternative anticoagulant for hemodialysis, is safe and effective in a dog renal failure model. Kidney Int 2003;63:1548 – 55. [23] Nagase H, Enjyoji K, Minamiguchi K, Kitazato KT, Kitazato K, Saito H, et al. Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions: antithrombin III- and heparin cofactor II-independent inhibition of factor X activation by factor IXa-factor VIIIa complex and heparin cofactor II-dependent inhibition of thrombin. Blood 1995;85:1527 – 34. [24] Nagase HH, Enjyoji K, Shima M, Kitazato K, Yoshioka A, Saito H, et al. Effect of depolymerized holothurian glycosaminoglycan (DHG) on the activation of factor VIII and factor V by thrombin. J Biochem 1996;119:63 – 9. [25] Nagase H, Kitazato KT, Sasaki E, Hattori M, Kitazato K, Saito H. Antithrombin III-independent effect of depolymerized holothurian glycosaminoglycan (DHG) on acute thromboembolism in mice. Thromb Haemost 1997;77:399 – 402. [26] Suzuki N, Kitazato K, Takamatsu J, Saito H. Antithrombotic and anticoagulant activity of depolymerized fragment of the glycosaminoglycan extracted from Stichopus japonicus selenka. Thromb Haemost 1991;65:369 – 73. [27] Yoshida K, Minami Y, Nemoto H, Numata K, Yamanaka E. Structure of DHG, a depolymerized glycosaminoglycan from sea cucumber, Stichopus japonicus. Tetrahedron Lett 1992;33:4959 – 62. [28] Tran TH, Lammle B, Zbinden B, Duckert F. Heparin cofactor II: purification and antibody production. Thromb Haemost 1986; 55:19 – 23. [29] Dejana E, Villa S, De Gaetano G. Bleeding time in rats: a comparison of different experimental conditions. Thromb Haemost 1982;48:108 – 11. [30] Hashimoto N, Morita T, Iwanaga S. A method for systematic purification from bovine plasma of six vitamin-K-dependent coagulation factors: prothrombin, factor X, factor IX, protein S, protein C, and protein Z. J Biochem 1985;97:1347 – 55. [31] Owen WG, Esmon CT, Jackson CM. The conversion of prothrombin to thrombin. J Biol Chem 1974;249:594 – 605.
K. Kitazato et al. / Thrombosis Research 112 (2003) 83–91 [32] Dupouy D, Sie´ P, Dol F, Boneu B. A simple method to measure dermatan sulfate at a sub-microgram concentrations in plasma. Thromb Haemost 1988;60:236 – 9. [33] Vu T-KH, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991;64:1057 – 68. [34] Brassard JA, Meyers KM. Evaluation of the buccal bleeding time and platelet glass bead retention as assays of hemostasis in the dog: the effects of acetylsalicylic acid, warfarin and von Willebrand factor deficiency. Thromb Haemost 1991;65:191 – 5. [35] Giles AR, Tinlin S, Greenwood R. A canine model of hemophilic (factor VIII: C deficiency) bleeding. Blood 1982;60:727 – 30. [36] Graham JB, Buckwalter JA, Hartley LJ, Brinkhous KM. Canine hemophilia—observations on the course, the clotting anomaly, and the effect of blood transfusion. J. Exp. Med. 1949;90:97 – 111.
91
[37] Ljungberg B, Johnsson H. In vivo effects of a low molecular weight heparin fragment on platelet aggregation and platelet dependent hemostasis in dogs. Thromb Haemost 1988;60:232 – 5. [38] Carter CJ, Kelton JG, Hirsh J, Cerskus A, Santos AV, Gent M. The relationship between the hemorrhagic and antithrombotic properties of low molecular weight heparin in rabbits. Blood 1982;59:1239 – 45. [39] Fernandez F, N’guyen P, Van Ryn J, Ofosu FA, Hirsh J, Buchanan MR. Hemorrhagic doses of heparin and other glycosaminoglycans induce a platelet defect. Thromb Res 1986;43:491 – 5. [40] Messmore Jr HL, Griffin B, Fareed J, Coyne E, Seghatchian J. In vitro studies of the interaction of heparin, low molecular weight heparin and heparinoids with platelets. Ann N Y Acad Sci 1989;556:217 – 32.