Analysis for Sites of Anticoagulant Action of Plancinin, a New Anticoagulant Peptide Isolated from the Starfish Acanthaster planci, in the Blood Coagulation Cascade

Gen. Pharmac. Vol. 31, No. 2, pp. 277–282, 1998 Copyright  1998 Elsevier Science Inc. Printed in the USA.

ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(97)00443-6 All rights reserved

Analysis for Sites of Anticoagulant Action of Plancinin, a New Anticoagulant Peptide Isolated from the Starfish Acanthaster planci, in the Blood Coagulation Cascade Tomoyuki Koyama,1* Katsuhiko Noguchi,1 Yoko Aniya2 and Matao Sakanashi1 1 Department of Pharmacology, School of Medicine, and Laboratory of Physiology and Pharmacology, School of Health Sciences, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japan [Tel: 181-98-895-3331 (ext. 2278); Fax: 181-98-895-3293] 2

ABSTRACT. 1. Effects of plancinin, a new anticoagulant peptide, on the human blood coagulation cascade were investigated. 2. Plancinin prolonged both activated partial thromboplastin time and prothrombin time, and it significantly inhibited factor X activation by both intrinsic (factor IXa–factor VIIIa–phospholipids–Ca21) and extrinsic (factor VIIa–tissue factor–phospholipids–Ca21) tenase complexes and prothrombin activation by prothrombinase complex (factor Xa–factor Va–phospholipids–Ca21) to 13.8%, 4.8% and 10.5% of control value, respectively. 3. Results indicate that sites of anticoagulant action of plancinin may be located in activation steps of prothrombin and factor X. gen pharmac 31;2:277–282, 1998.  1998 Elsevier Science Inc. KEY WORDS. Anticoagulant, Acanthaster planci, plancinin, tenase complex, prothrombinase complex INTRODUCTION A variety of anticoagulant proteins and peptides have been isolated from many kinds of organisms (Apitz-Castro et al., 1995; Chang, 1983; Tanabe et al., 1994; Tuszynski et al., 1987; Waxman et al., 1990). Some of them are expected to be utilized in the clinical situation and have been available for hematological research as well. However, these anticoagulants differ considerably from each other not only in their structures, but also in their sites of anticoagulant action. Recently, we reported the isolation and purification of a new anticoagulant peptide from the crown-of-thorns starfish Acanthaster planci, and this substance, named plancinin, was estimated to be a peptide with a molecular weight of 7,500 (Karasudani et al., 1996). Plancinin was found to prolong the plasma recalcification time of human plasma in vitro and the bleeding time in mice after its intravenous administration (Karasudani et al., 1996). In addition, plancinin was free of protease and phospholipase activities and showed neither inhibitory effect against platelet aggregation initiated by ADP nor enhancement of vascular permeability (Karasudani et al., 1996). However, sites of the anticoagulant action of plancinin remain to be fully elucidated. Thus, in the present study, to clarify the site(s) of action of the new anticoagulant peptide plancinin in the coagulation cascade, the effects of plancinin on clotting activities of human plasma, on activities of human blood coagulant proteases and on activation of prothrombin or factor X were examined. MATERIALS AND METHODS

Materials Plancinin was purified from Acanthaster planci, which inhabits coral reefs around the Okinawa Islands in Japan, as described previously *To whom correspondence should be addressed. Received 5 September 1997.

(Karasudani et al., 1996). Briefly, homogenate of the spines of 20–30 starfish was filtered and made 50% saturated with ammonium sulfate. The precipitate thus obtained was used as a crude venom stock and dialyzed against 0.01 M Tris-HCl buffer (pH 8.0) and applied on a DEAE (diethylaminoethyl) cellulose (Nacalai tesque, Kyoto, Japan) column followed by a linear gradient elution of 0.0–0.6 M NaCl in the same buffer. Protein content in the eluate was monitored with ultraviolet absorbance at 280 nm. The fraction was concentrated by ultrafiltration (Amicon 10, Japan Grace, Tokyo, Japan) under nitrogen gas and then applied on a Sephadex G-50 (Pharmacia LKB Biotechnology, Uppsala, Sweden) column that was equilibrated with 0.01 M potassium phosphate buffer (pH 7.0). Thus the fraction (plancinin) obtained by chromatography was pooled, after the purity had been judged as a single band on SDSPAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) for peptide analysis, as described by Scha¨gger and Jagow (1987). Plancinin was then dialyzed against distilled and deionized water or 0.01 M Tris-HCl buffer (pH 8.0), and its amount was measured by the method of Lowry et al. (1951). All of the procedures for purification were performed in the chromatochamber at 48C. The following materials were purchased from the listed companies: heparin sodium (187.5 IU/mg), N,N,N,N-ethylenediaminetetraacetic acid sodium (EDTA) and bovine serum albumin (BSA) from Nacalai tesque; antithrombin III (ATIII), thrombin (factor IIa) and plasma kallikrein from Sigma Chemical Co. (St. Louis, MO, USA); factor Xa and heparin cofactor II (HCII) from Diagnostica Stago (Asnie´res, France); factors II (prothrombin), Va, VIIa, IXa, X, XIa and XII from Haematologic Technologies Inc. (Essex, VT, USA); tissue factor (TF) derived from placental tissues (Thromborel S) from Behringwerke AG (Marburg, Germany). All the aforelisted coagulant reagents had been purified from humans. All of the MCA (4-methylcoumaryl-7-amide) substrates were purchased from Peptide Institute (Osaka, Japan). Other reagents used were of analytical grade.

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Preparation of factor VIIIa Human factor VIIIa was prepared by activating factor VIII (250 U; CROSS EIGHT M-250, Japanese Red Cross, Tokyo, Japan) with 26.8 pmol of human factor IIa for 3 min. Before activating, a vial of CROSS EIGHT M-250 was dissolved with 2 ml of distilled water and further purified by a fast protein liquid chromatography system (FPLC, Pharmacia LKB Biotechnology). After the reaction was stopped by d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone (0.05 mM) (PPACK, Calbiochem, La Jolla, CA, USA), factor VIIIa was purified on a Mono-S column and a Superdex 75 column by using the FPLC system before use, as described by Fay et al. (1991). Finally, 0.8 ml of factor VIIIa solution was obtained through concentrating the eluted fractions.

Preparation of phospholipids suspension Phospholipids (PL) suspension, consisting of 25% phosphatidylcholine (Nacalai tesque) and 75% phosphatidylserine (Sigma Chemical Co.) at concentrations of 2 mM, was prepared in Tris-HCl buffer (50 mM, pH 8.0) containing NaCl (175 mM). This suspension was diluted to a definite concentration before use.

Effects of plancinin on clotting activities Effects of plancinin on the clotting activities of human plasma were estimated as the measurement of clotting times in three different tests such as activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin clotting time (TCT). All clotting tests were performed by using an automated coagulation meter (Coagrex-700, International Reagents Co., Kobe, Japan). Sample mixture (500 ml) was prepared by mixing 9 volumes of normal human plasma (Coagtrol N, International Reagents Co.) and 1 volume of plancinin solution at various concentrations.

Effects of plancinin on activities of coagulant proteases Protease activities of coagulant factors were estimated as the hydrolysis of the MCA substrate (50 mM) in the presence or absence of plancinin for a definite time (Kawabata et al., 1988; Morita et al., 1977). The reaction buffer used was Tris-HCl buffer (50 mM, pH 8.0) containing NaCl (175 mM) and CaCl2 (5 mM). After incubation, the reaction was stopped by the addition of 17% acetic acid (1 ml) into the reaction mixture (500 ml). Intensity of fluorescence derived from the degenerative substance of the MCA substrate was determined with a fluorescence spectrometer (RF-1500, Shimadzu, Kyoto, Japan) with excitation at 370 nm and emission at 480 nm. The final concentration and incubation time of each factor were as follows. Factor Xa (0.5 nM) was incubated with Boc-Ile-Glu-GlyArg-MCA for 20 min. Each of factors IIa (0.5 nM), VIIa (7.2 nM) and IXa (178 nM) was incubated with Boc-Val-Pro-Arg-MCA for 20 min. Factor XIa (5 nM) and plasma kallikrein (35 nM) were incubated with Boc-Glu(OBzl)-Ala-Arg-MCA and Z-Phe-Arg-MCA for 10 min, respectively. Factor XII (25 nM), which was activated to factor XIIa by contact with a glass tube during reaction, was incubated with Boc-Glu-Gly-Arg-MCA for 10 min. Time-dependence of increases in fluorescence intensity by the coagulant proteases in the absence of plancinin was confirmed in advance under the respective conditions heretofore described.

Effects of plancinin on ATIII activity and HCII activity Activities of ATIII and HCII were estimated as residual factor IIa activities after incubation of factor IIa with ATIII and HCII, respectively, in the presence of plancinin or heparin for a definite time.

T. Koyama et al. To initiate the degenerative reaction, factor IIa (5 nM) was added to a reaction mixture containing plancinin (5.7 ng/ml–57 mg/ml) or heparin (10 ng/ml–100 mg/ml) and ATIII (5.7 nM) or HCII (7.6 nM) in Tris-HCl buffer (50 mM, pH 8.0) including NaCl (150 mM). After 10 min, Boc-Val-Pro-Arg-MCA (50 mM), a fluorescent substrate for factor IIa, was added to the reaction mixture and incubated for 20 min. Intensity of fluorescence was measured as heretofore described.

Effects of plancinin on activation of factors II and X Factor X activation by intrinsic (factor IXa–factor VIIIa–PL–Ca21) or extrinsic (factor VIIa–TF–PL–Ca21) tenase complex and factor II activation by prothrombinase complex (factor Xa–factor Va–PL– Ca21) were measured as respective protease activities of generated factors Xa and IIa according to the method of Apitz-Castro et al. (1995) and Nagase et al. (1995) with minor modification. Briefly, with regard to the intrinsic tenase complex, factor VIIIa (10 ml) was mixed with factor IXa (200 pM), PL (200 nM), CaCl2 (5 mM) and various amounts of plancinin in 500 ml of Tris-HCl buffer (50 mM, pH 8.0) containing NaCl (175 mM) and BSA (0.5%). Similarly, with regard to the extrinsic tenase complex, TF (1.67 nM) was mixed with factor VIIa (72 pM), PL (20 nM) and CaCl2 (5 mM). The reaction was initiated by adding factor X (42.5 nM for the intrinsic tenase complex or 20 nM for the extrinsic tenase complex) to the mixtures. On the other hand, the effect of plancinin on the activity of the prothrombinase complex was estimated by using factor Xa (2.2 pM), factor Va (1.67 nM), PL (20 nM) and factor II (50 nM). Finally, protease activities of factors Xa and IIa generated during a 10-min incubation were measured by adding Boc-Ile-Glu-GlyArg-MCA (50 mM) and Boc-Val-Pro-Arg-MCA (50 mM), respectively, containing EDTA (10 mM).

Effects of plancinin on factor X activation by factor X activator isolated from Russell’s viper venom Russell’s viper venom (RVV-X; Haematologic Technologies Inc.) can activate factor X in the presence of Ca21 without PL. Factor X activation by this RVV-X was measured as the protease activity of generated factor Xa. Tris-HCl buffer (50 mM, pH 8.0), including RVV-X (20 nM), Ca21 (5 mM), factor X (25 nM) and plancinin (0– 186 mg/ml), was incubated for 5 min in advance. Then, factor Xa generated during a 15-min incubation after adding Boc-Ile-GluGly-Arg-MCA (50 mM) was measured.

Comparison of plancinin and annexin in their binding abilities to phosphatidylserine in the presence or absence of Ca21 The binding ability of plancinin to phosphatidylserine was estimated according to the method of Schlaepfer and Haigler (1987) and was compared with that of annexin V (Sigma Chemical Co.) isolated from human placenta. Plancinin or annexin V (50 mg/ml) was incubated in 100 ml of Tris-HCl buffer (10 mM, pH 8.0) containing NaCl (175 mM) in the presence or absence of Ca21 (5 mM) with or without phosphatidylserine (430 mM) suspension for 1 hr at room temperature. After a centrifugation (20 min, 124,000g), the supernatants and precipitates were analyzed by electrophoresis followed by Coomassie Brilliant Blue staining (Laemmli, 1970) for annexin or by Alcian Blue staining (Cowman et al., 1984) for plancinin. Because plancinin has been found to be more effectively detected by Alcian Blue staining than by Coomassie Brilliant Blue staining in preliminary experiments, we employed the Alcian Blue staining method. Because phosphatidylserine precipitates completely

A New Anticoagulant, Plancinin

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FIGURE 1. Effects of plancinin on (A) APTT (B) PT and (C) TCT of human plasma. Values are given as means6SD (n53). *P,0.05, **P,0.01, ***P,0.001 versus control values (without plancinin). Note that many SD bars are hidden by symbols because of small SD.

in this centrifugal condition, whereas dissolved proteins do not, detection of proteins in the precipitate show that the proteins bind to phosphatidylserine. The temperature of all incubations in this study was set at 378C unless otherwise noted in the text. All of the concentrations in parentheses mean final concentrations. Statistical analysis was performed by using one-way analysis of variance with Dunnett’s test. Statistical significance was accepted for P,0.05. All values are presented as means6SD. RESULTS

Effects of plancinin on clotting activities The effects of various concentrations of plancinin on the clotting times are shown in Figure 1. APTT (Fig. 1A), PT (Fig. 1B) and TCT (Fig. 1C) without plancinin were 31.260.4 sec, 11.760.1 sec and 9.660.3 sec, respectively. In the presence of plancinin, both APTT and PT were prolonged, depending on its concentrations. Significant prolongations (P,0.05) of APTT and PT were observed with plancinin at concentrations exceeding 28.5 and 143 mg/ml in plasma, respectively. On the other hand, plancinin at 200 mg/ml in plasma did not significantly affect TCT.

Effects of plancinin on protease activities of purified blood coagulant factors Protease activities of factors IIa (thrombin), VIIa, IXa, Xa, XIa, XIIa and plasma kallikrein were measured with or without plancinin. Plancinin at concentrations of 0–171 mg/ml did not significantly inhibit any protease activities of all coagulant factors under the conditions of these experiments. Effects of plancinin on factor IIa degeneration by ATIII or HCII were compared with those of heparin, as shown in Figure 2. Heparin significantly (P,0.001) accelerated factor IIa degeneration by ATIII (Fig. 2A) at concentrations between 0.1 and 1 mg/ml and by HCII (Fig. 2B) at those greater than 1 mg/ml, respectively. In contrast with heparin, plancinin did not significantly inhibit factor IIa degeneration by HCII or ATIII at the range of these concentrations.

Effects of plancinin on activation of factors II and X Protease activities of factor Xa that was generated from factor X by the intrinsic tenase complex (factor IXa–factor VIIIa–PL–Ca21) or

by the extrinsic tenase complex (factor VIIa–TF–PL–Ca21) were measured in the presence of various concentrations of plancinin. Similarly, the protease activity of factor IIa that was generated from factor II by the prothrombinase complex (factor Xa–factor Va–PL– Ca21) was measured in the presence of plancinin. As shown in Figure 3, plancinin inhibited all three activations by coagulant complexes in a concentration-dependent manner. At concentrations exceeding 0.057 mg/ml of plancinin, factor X activation by intrinsic (Fig. 3A) or extrinsic (Fig. 3B) tenase complexes was significantly inhibited (P,0.001). Factor II activation by the prothrombinase complex also was significantly (P,0.001) inhibited at concentrations greater than 0.114 mg/ml of plancinin (Fig. 3C). The inhibitory effects of plancinin on these activations were observed at considerably lower concentrations than those needed for prolongation of APTT and PT (Fig. 1).

Effects of plancinin on factor X activation by RVV-X Plancinin (0–57 mg/ml) did not significantly affect RVV-X-induced factor Xa generation (101–106% of the control generation, n53) in the presence of Ca21 (5 mM). In addition, generation of factor Xa in the presence of Ca21 (0.5 or 1 mM) was not inhibited by 186 mg/ ml of plancinin.

Comparison of plancinin and annexin in their binding abilities to phosphatidylserine in the presence or absence of Ca21 The binding abilities of plancinin and annexin V to phosphatidylserine in the presence or absence of Ca21 are shown in Table 1. Annexin V precipitated only when both phosphatidylserine and Ca21 coexisted, whereas plancinin did so in the presence of Ca21 but independently of the presence of phosphatidylserine. In the absence of both Ca21 and phosphatidylserine, neither plancinin nor annexin V precipitated. DISCUSSION It is well known that human blood coagulation is brought about by enzymatic cascade consisting of two different pathways; that is, intrinsic and extrinsic pathways. These two pathways join at the step of factor IX or factor X activation, and then the coagulation process eventually terminates in fibrinogenesis. In the present study, plan-

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FIGURE 2. Effects of plancinin (open circles) and heparin (solid circles) on activities of (A) ATIII and (B) HCII. The residual factor IIa activity after degeneration by ATIII or HCII in the absence of plancinin or heparin was taken as 100%. Values are given as means6SD (n53–6). ***P,0.001 versus control values (without plancinin and heparin).

cinin prolonged APTT and PT without any change in TCT. Because APTT and PT mean total coagulant activities of intrinsic and extrinsic pathways, respectively, and TCT is regarded as an index of the blood coagulant activity after the factor IIa generation step, these data imply that plancinin affected a certain step(s) in both the intrinsic and the extrinsic coagulation pathways or in the downstream steps below the point at which the two pathways are combined, except for those below factor IIa generation. However, plancinin did not inhibit protease activities of these coagulant factors themselves such as factors IIa, VIIa, IXa, Xa, XIa, XIIa and plasma kallikrein. These results indicate that plancinin is not a simple protease inhibitor for these coagulant factors. Thus, plancinin may be dissimilar from previously known anticoagulants that have been isolated from other animal sources, such as Russell’s viper venom inhibitor II (Iwanaga et al., 1976), hirudin (Chang, 1983), antistasin (Tuszynski et al., 1987), tick anticoagulant peptide (Waxman et al., 1990), Streptoverticillium anticoagulant (Tanabe et al., 1994) and draculin (Apitz-Castro et al., 1995). In the physiological blood coagulation system, the protease activities of factors IXa, Xa and VIIa are known to be extremely accelerated by forming complexes with factor VIIIa, factor Va and TF, respectively, on the phospholipid bilayer surface in the presence of Ca21 (Dieijen et al., 1981; Nemerson and Gentry, 1986; Rosing et al., 1980). In this study, plancinin inhibited factor X activations by the intrinsic (factor IXa-VII–factor VIIIa–PL–Ca21) and extrinsic (factor VIIa–TF–PL–Ca21) tenase complexes and factor II activation by the prothrombinase (factor Xa–factor Va–PL–Ca21) complex. It is unlikely that these inhibitory effects might be derived

TABLE 1. Comparison of plancinin and annexin in their binding abilities to phosphatidylserine in the presence or absence of Ca21 Plancinin

Annexin V

Phosphatidylserine Ca21

2 1

1 1

1 2

2 1 1 1 1 2

Detection

P

P

S

S

P

S

The letters ‘‘S’’ and ‘‘P’’ show that plancinin and annexin V were detected in the supernatant and the precipitate, respectively; 1 and 2 show the presence and absence of phosphatidylserine or Ca21 in the reaction mixture.

from degenerating PLs or proteins that are essential to the formation of these complexes or both, because plancinin itself has been shown to lack phospholipase and protease activities (Karasudani et al., 1996). It is therefore possible that plancinin might inhibit these activations through blocking formation of the complexes or through binding to one or several components of the complexes. Some glycosaminoglycans such as heparin and DHG (depolymerized holothurian glycosaminoglycan) have been reported to inhibit blood coagulation through accelerating the activity of ATIII or HCII, through inhibiting factor X activation by the intrinsic tenase complex and partly through inhibiting factor II activation by the prothrombinase complex (Barrow et al., 1994; Nagase et al., 1995). In this study, plancinin but not heparin failed to accelerate the factor IIa-degenerative activities of antithrombin proteases, indicating that plancinin lacks the heparin-like anticoagulant action related to ATIII or HCII. In addition, anticoagulant effects of plancinin were different from the ATIII- or HCII-independent inhibitory activities of glycosaminoglycans, because they have not been found to inhibit the factor X activation by the extrinsic tenase complex (Nagase et al., 1995). After all, these results suggest that anticoagulant activity of plancinin may be primarily due to the inhibitory action on activation steps of factors II and X rather than the heparin-like action. As plancinin-like substances that can inhibit selectively at the activation steps by these blood coagulant complexes, annexins and factor IX- and factor X-binding proteins are known so far. Activities of annexins are reported to be derived from their binding to PL in the presence of Ca21 (Crumpton and Dedman, 1990; Maki et al., 1989). Factor IX- and factor X-binding proteins inhibit to form blood coagulant complexes through binding to factor X or factor IX or both (Atoda and Morita, 1989; Cox, 1993; Teng and Seegers, 1981). In addition to plancinin, annexins and factor IX- and factor X-binding proteins have been reported to possess no phospholipase activity, no protease activity and no inhibitory activity against blood coagulant proteases alone (Atoda and Morita, 1989; Maki et al., 1989; Teng and Seegers, 1981). Thus, the characteristics of the anticoagulant action of these substances and plancinin seem to be similar in that they can selectively inhibit at activation steps of factors II and X. On the other hand, plancinin precipitated in the presence of Ca21, whereas annexin precipitated in the presence of both Ca21 and phosphatidylserine. These results suggest that plancinin may

A New Anticoagulant, Plancinin

281

FIGURE 3. Effects of plancinin on activation of factors II and X by (A) intrinsic (factor IXa–factor VIIIa–PL–Ca21) and (B) extrinsic (factor VIIa–tissue factor–PL–Ca21) tenase complexes and by (C) prothrombinase complex (factor Xa–factor Va–PL–Ca21). The activity of factor Xa or IIa generated in the absence of plancinin was taken as 100%. Values are given as means6SD (n53–6). ***P,0.001 versus control values (without plancinin).

possess Ca21-binding ability and may thereby inhibit activation of factors II and X by blood coagulant complexes. But exhaustion of free Ca21 by its Ca21-binding ability would be insufficient to explain its anticoagulant activity, because plancinin at quite low concentrations selectively inhibited activation of factors II and X by blood coagulant complexes in the presence of 5 mM Ca21. In addition, it was demonstrated that factor X activation by RVV-X, whose activity is known to depend exclusively on Ca21 rather than PL, was not inhibited by plancinin in the presence of various Ca21 concentrations. If plancinin inhibited factor X activation by tenase complexes through binding to factor X as well as factor IX- and factor X-binding proteins (Takeya et al., 1992) or through exhaustion of Ca21, plancinin should inhibit factor X activation by RVV-X. Thus, the present results showed that this is not the case. Alternatively, it is likely that plancinin may disrupt some interactions related to Ca21 that are indispensable to form blood coagulant complexes. The molecular weight of plancinin has been estimated to be less than that of annexins. In addition, amino acid components and partial sequence analysis of plancinin (our unpublished data) revealed that plancinin would be different from factor IX- and factor X-binding proteins or annexins in structure. E-F hand, which is a Ca21binding sequence existing in the structure of calcium-binding proteins such as troponin, calmodulin and parvalbumin (Kretsinger and Barry, 1975), also could not be found in plancinin. Thus, these findings indicate that plancinin may be classified as a novel type of anticoagulant substance. A detail study on the inhibitory mechanism of action of plancinin may provide further interesting information concerning the interactions between components of the blood coagulant complexes. CONCLUSION The present findings suggest that the sites of anticoagulant action of plancinin may be located in factor X activation steps of the intrinsic and extrinsic tenase complexes and in the factor II activation step of the prothrombinase complex, which have been estimated as the sites of action of annexins and factor IX- and factor X-binding proteins, but the characteristics of plancinin might differ from theirs.

SUMMARY The effects of plancinin, which is a new anticoagulant peptide isolated from the starfish Acanthaster planci, on the clotting activity of human plasma and on the activities of purified human coagulant factors were examined to clarify the site(s) of its anticoagulant action in the human blood coagulation cascade. Plancinin prolonged activated partial thromboplastin time and prothrombin time but did not prolong thrombin clotting time. Plancinin did not inhibit protease activities of factors IIa, VIIa, IXa, Xa, XIa, XIIa and plasma kallikrein and did not affect degeneration of thrombin by antithrombin III or heparin cofactor II. On the other hand, plancinin significantly inhibited prothrombin activation by the prothrombinase complex (factor Xa–factor Va–phospholipids–Ca21) and factor X activation by both the intrinsic (factor IXa–factor VIIIa–phospholipids–Ca21) and the extrinsic (factor VIIa–tissue factor–phospholipids–Ca21) tenase complexes. Results indicate that the sites of anticoagulant action of plancinin may be located in factors II and X activation steps by blood coagulant complexes. The authors are grateful to the Sesoko Stations of the Tropical Biosphere Research Center of University of the Ryukyus, for collaboration in the capture of starfish, and to the Department of Clinical Laboratory of the University of the Ryukyus Hospital and to PhD. Kiichi Teruya, Tropical Technology Center Ltd., for their technical advice.

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