Báo cáo khoa học: Dual modulation of prothrombin activation by the cyclopentapeptide plactin pptx

Dual modulation of prothrombin activation by the cyclopentapeptide plactin Tomotaka Harada*, Tomoko Tsuruta*, Kumi Yamagata, Toshiki Inoue and Keiji Hasumi Department of Applied Biological Science, Tokyo Noko University, Tokyo, Japan Plactin is a family of cyclic pentapeptides that enhance fibrinolytic activity both in vitro and in vivo [1,2]. Structure–activity relationship studies using 50 plactin congeners revealed that a sterically restricted arrange- ment of four hydrophobic amino acids and one basic amino acid is essential for their activity. The plactin- mediated increase in fibrinolytic activity accompanies an elevation in cellular urokinase-type plasminogen activator (u-PA) activity [2]. In this mechanism, the presence of plasma is an absolute requirement. u-PA, as well as tissue-type plasminogen activator, is a physiologically relevant protease that catalyzes the limited proteolysis of plasminogen to afford the fibri- nolytic enzyme plasmin [3,4]. u-PA is produced as an inactive, single-chain proenzyme (scu-PA) that binds to a cell-surface receptor in an autocrine fashion follow- ing secretion [5]. Activation of scu-PA is catalyzed by plasmin [4] and some other proteases, such as cathep- sin B [6], plasma kallikrein [7] and mast cell tryptase [8], involves cleavage at Lys158–Ile159 (numbering Keywords blood coagulation; fibrinolysis; proteolysis; prothrombin; urokinase Correspondence K. Hasumi, Department of Applied Biological Science, Tokyo Noko University, 3-5-8 Saiwaicho, Fuchu-shi, Tokyo 183 8509, Japan Fax: +81 42 367 5708 Tel: +81 42 367 5710 E-mail: hasumi@cc.tuat.ac.jp *These authors contributed equally to this work (Received 30 January 2009, revised 17 February 2009, accepted 20 February 2009) doi:10.1111/j.1742-4658.2009.06976.x Plactin, a family of cyclopentapeptides, enhances fibrinolytic activity by elevating the activity of cellular urokinase-type plasminogen activator (u-PA), a protease involved in a variety of extracellular proteolytic events. Factor(s) in the blood plasma is an absolute requirement for this plactin activity. In this study, we found that plactin promoted plasma cofactor- dependent conversion of inactive single-chain u-PA to active two-chain u-PA on U937 cells. Using plactin-affinity chromatography, we identified prothrombin as one of the plasma cofactors. In incubations of U937 cells with prothrombin and Xa, plactin increased the formation of thrombin, which cleaved single-chain u-PA to afford the inactive two-chain form. Thrombin-cleaved two-chain u-PA was alternatively activated by cellular cystatin-sensitive peptidase activity, yielding fully active two-chain u-PA. In a purified system, plactin bound to prothrombin, altered its conformation and dually modulated factor Xa-mediated proteolytic activation of pro- thrombin to a-thrombin. Plactin inhibited the activation catalyzed by Xa in complex with Va, Ca 2+ and phospholipids (prothrombinase), whereas the activations catalyzed by nonmembrane-associated Xa were enhanced markedly by plactin. Plactin inhibited in vitro plasma coagulation, which involved prothrombinase formation. Plactin did not cause prothrombin activation or thrombosis in normal mice at doses that produced a protec- tive effect in a thrombin-induced pulmonary embolism mouse model. Therefore, the dual modulation of prothrombin activation by plactin may be interpreted as leading to anticoagulation under physiological coagulat- ing conditions. Abbreviations DAPA, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide; DPP-I, dipeptidyl peptidase I; GGA-MCA, glutaryl-Gly-Arg-4-methylcoumarin-7-amide; PCPS, phospholipid vesicles composed of 75% (w ⁄ w) phosphatidylcholine and 25% (w ⁄ w) phosphatidylserine; scu-PA, single-chain u-PA; tcu-PA, two-chain u-PA; tcu-PA ⁄ T, thrombin-cleaved two-chain u-PA; u-PA, urokinase-type plasminogen activator. 2516 FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS based on the human scu-PA sequence), and yields an active two-chain form of the enzyme (tcu-PA). u-PA establishes a localized cell-surface proteolytic system through activation of plasminogen and some matrix- degrading metalloproteinases [9,10]. In this study, we investigated the plasma-dependent mechanism by which plactin increases cellular u-PA activity and identified prothrombin as one plasma component that supported the action of plactin. Pro- thrombin is a zymogen of the blood coagulation enzyme thrombin that proteolytically forms fibrin from fibrinogen [11]. At the site of vascular injury, prothrombin is rapidly activated to thrombin by coagulation factor Xa, which is assembled in a Ca 2+ - dependent manner with factor Va on acidic phospho- lipid membranes of damaged vascular endothelium or activated platelet aggregates [12–14]. Activation of pro- thrombin by the complex (prothrombinase complex) is >10 5 times faster than activation by free Xa [15]. Therefore, physiological coagulation is eventually cata- lyzed by the prothrombinase complex. In addition to promoting fibrin formation, thrombin in complex with thrombomodulin can activate protein C [16,17] and thrombin-activated fibrinolysis inhibitor [18], which modulate coagulation and fibrinolysis. Thus, thrombin plays multiple roles in hemostatic processes. In this study, we show that in a cultured cell system, plactin enhances prothrombin activation to thrombin, which cleaves cellular scu-PA to afford inactive two- chain u-PA, which is activated by cystatin-sensitive peptidase activity to yield fully active tcu-PA. In a purified system, plactin dually modulates prothrombin activation, depending on the conditions of catalysis by Xa. Under conditions where membrane-associated Xa formation is restricted, plactin enhances the formation of a-thrombin, whereas plactin inhibits prothrombin activation by membrane-associated Xa. Plactin is inhibitory to plasma coagulation in vitro and does not cause prothrombin activation or thrombosis in vivo. Thus, we suggest that the dual modulation of pro- thrombin activation by plactin leads to an antithrom- botic state under physiological coagulating conditions. Results and Discussion Plactin promotes cell-surface activation of scu-PA Previous experiments have demonstrated that plac- tin D promotes a plasma-dependent elevation in u-PA activity in U937 cells. The increase in u-PA activity was not associated with an increase in the total amount of u-PA [2]. Therefore, we tested whether plac- tin D increased the conversion of inactive scu-PA to active tcu-PA on cell surfaces in the plasma milieu. First, we determined the levels of total and active u-PA on U937 cells. Total u-PA activity was obtained by treating U937 cells with plasmin, which could acti- vate scu-PA to tcu-PA by cleaving at Lys158–Ile159. Taking this value as 100%, the level of cellular active u-PA, obtained without plasmin pretreatment, was as low as  1% (Fig. 1A). This implied that  99% of the total u-PA on U937 cells was in the inactive single- chain form. Treatment of U937 cells with 50 lm plac- tin D increased the level of active u-PA to  35% of scu-PA B-chain A-chain 66 45 29 (kDa) 20% plasma Plactin D + − + − No plasma B 0 1 2 3 4 5 A 706050403020100 u-PA activity (fluorescence intensity) Time of second incubation (min) 1 st plactin 2 nd PM 1 st plactin 2 nd none 1 st none 2 nd none 1 st none 2 nd PM Fig. 1. Promotion of scu-PA activation on U937 cells by plactin. (A) U937 cells were first incubated with or without plactin D in the presence of 20% (v ⁄ v) human plasma. After washing, cells were incubated in the absence or presence of 100 n M plasmin (PM) for the indicated time (second incubation). After incubation, cellular uPA activity was determined using a chromogenic u-PA substrate in the presence of aprotinin, an inhibitor of plasmin. Line indicates the average of duplicate determinations. (B) U937 cells were incu- bated with 125 I-labeled scu-PA in the absence or presence of 50 lM plactin D and 20% plasma. Aliquots of cell lysates were resolved on reduced SDS ⁄ PAGE on a 12.5% gel. The positions of molecular mass standards, as well as scu-PA, A- and B-chains of tcu-PA, are shown. T. Harada et al. Dual modulation of prothrombin activation FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS 2517 the total u-PA level (Fig. 1A). The finding that  60% of u-PA in plactin-treated cells was not activated by plasmin might be partly explained by the observation that thrombin-cleaved tcu-PA (see below for the involvement of thrombin-cleaved tcu-PA) was 500 times less sensitive to activation by plasmin when com- pared with scu-PA [19]. Next, we determined the con- version of scu-PA to tcu-PA on the cell surface. In this experiment, U937 cells equilibrated with 125 I-labeled scu-PA were treated with plactin D, followed by SDS ⁄ PAGE of the labeled protein to resolve scu-PA and tcu-PA. As shown in Fig. 1B, plactin D markedly promoted conversion of scu-PA to the two-chain form. The apparent molecular masses of the resulting poly- peptide chains were comparable with those of the A- and B-chains of tcu-PA (an A-chain doublet was caused by differential glycosylation) [20]. The plactin effect was specific in the presence of plasma (Fig. 1B), consistent with previous observations [2]. From these results, we concluded that plactin D promoted cell- surface activation of scu-PA to tcu-PA, and that the conversion (specific proteolysis) required a cofactor in the plasma. Identification of prothrombin as a plasma factor participating in plactin activity To identify the plasma cofactor required for plactin promotion of scu-PA proteolysis to tcu-PA, we attempted to develop affinity media to purify plactin- binding protein. To immobilize plactin onto a gel matrix, we first looked for plactin derivatives with a free amino group. One such candidate was plactin-14 (Fig. 2A). Although plactin-14 itself had no activity, modification of its amino group with a dansyl group converted the molecule an active form (Fig. 2B). This 0 2. 0 4 . 0 6 .0 8. 0 lortnoC D nitc a l P u-PA activity (fluorescence intensity) -41-nitcalP e sor a hpeS -eso r ahpeS B 4 o N s d a eb - 41- ni tc a l P Sephaose Sepharose-4B 6 6 54 9 2 )a D k( 502 611 4.79 Fraction E4A50 RVXXLFGKNA Prothrombin T AV Q DANVS I-A-opA V I - A -op A Sequence not obtained VRDWSSQPDD 0 2.0 4.0 6 .0 8.0 1 2.1 4.1 05040302010 D nitcalP SND-41-nitcalP 41-nitcalP Concentration (μ M ) u -PA activity (fluorescence intensity) HN HN 2 NH N H H N HN N H NH O O O O O N H O HN e s orahpe S d a eb esorahpeS-41-n it calPSND-41-nitcalP41-nitca lPD nitcalP HN HN 2 N H A C D E B N H HN HN N H NH O O O O O - D g r A -D laV ueL -D ue L eh P HN H N 2 N H NH HN HN N H N H O O O O O HN 2 syL HN HN 2 N H NH HN HN N H N H O O O O O N H S O O N S N D - s yL 0 1.0 2.0 3.0 4.0 u- PA activity (fluorescence intensity) aX + T Pa XT P lortnoC D nitcalP Fig. 2. Identification of prothrombin as a plasma cofactor required for plactin activity. (A) Structures of plactin D and its analogs. DNS, dansyl. (B) The activities of plactin D, plactin-14 and plactin-14–DNS to enhance cellular u-PA activity were measured by incubating each compound with U937 cells at the concentrations shown. (C) Human plasma (diluted to 25% v ⁄ v in buffer D) was incubated with or without Sepharose 4B or plactin-14–Sepharose at 4 °C for 20 min. After centrifugation, the resulting supernatant was assayed for scu-PA activation on U937 cells at a concentration of 10% (v ⁄ v) of original plasma in the presence or absence of plactin D (50 l M). (D) Partially purified bovine plasma fraction E4A50 was subjected to plactin-14–Sepharose chromatography. After flow-through fraction (FT) was collected and the col- umn was washed with buffer D, elution was carried out successively with buffer D containing 0.5 M NaCl or 6 M guanidine ⁄ HCl (Gnd-HCl). All fractions were dialyzed against buffer A before the assay. Fractions were resolved on reduced SDS ⁄ PAGE on a 10% gel. Arrowheads denote specifically enriched proteins. N-terminal sequences of such proteins, and their identifications, are shown. Apo-A, apolipoprotein A. (E) U937 cells were incubated with the indicated protein(s) at 37 °C for 30 min in the absence or presence of 50 l M plactin D. The con- centrations of prothrombin and Xa were 347 n M and 50 pM, respectively. After washing, cellular u-PA activity was measured. Error bars represent SD from triplicate determinations. In some data points, error bars are too small to be recognized. Dual modulation of prothrombin activation T. Harada et al. 2518 FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS result was consistent with the idea that a sterically restricted arrangement of four hydrophobic amino acids and a basic amino acid is essential for plactin activity [2]. Therefore, we speculated that coupling of plactin-14 to CNBr-activated Sepharose gels via its amino group should afford an active affinity matrix (Fig. 2A). Indeed, plactin cofactor activity in human plasma was successfully adsorbed to plactin-14–Sepha- rose affinity gel (Fig. 2C). Similar results were obtained when partially purified bovine plasma (frac- tion E4A50; see Experimental Procedures) was used for plactin-14–Sepharose chromatography, and cofac- tor activity was recovered in fractions eluted with 0.5 m NaCl or 6 m guanidine ⁄ HCl. Some proteins were specifically enriched in these fractions, although many protein bands were detected on reduced SDS ⁄ PAGE (Fig. 2D). No significantly adsorbed pro- tein was detected when Sepharose 4B alone was used, suggesting that the nonspecific protein binding in the plactin-14–Sepharose chromatography was caused by the hydrophobic surface provided by the coupled plactin-14. The N-terminal amino acid sequences of three specifically enriched proteins suggested that these were prothrombin, apolipoprotein A-IV and apolipo- protein A-I (Fig. 2D). We chose prothrombin for further analysis because prothrombin, but not apolipoproteins, might participate in the proteolytic cleavage of scu-PA. When prothrom- bin was used in place of plasma to determine plactin cofactor activity, it did not support plactin D-dependent enhancement of u-PA activity in U937 cells (Fig. 2E). This was not unexpected, as prothrombin itself is an inactive protease zymogen. Specific proteolysis by the coagulation factor Xa activates prothrombin to thrombin. Simultaneous incubation of U937 cells with prothrombin and factor Xa produced a plactin D-dependent increase in u-PA activity (Fig. 2E). There- fore, we suggest that prothrombin is one of the plasma cofactors participating in plactin D-promoted scu-PA activation on U937 cells. Mechanism of prothrombin- and plactin-mediated enhancement of scu-PA activation The above results suggested that prothrombin activa- tion (thrombin formation) was involved in the mecha- nism of plactin action and that plactin affected this reaction. Indeed, plactin D increased prothrombin activation in the U937 cell system (Fig. 3A), and a-thrombin alone could produce a significant increase in scu-PA activation on U937 cells (Fig. 3B,C). Plac- tin D affected a-thrombin-mediated scu-PA activation only slightly (Fig. 3B). Hirudin, a specific inhibitor of thrombin, abolished the plactin D effect on scu-PA activation by prothrombin ⁄ Xa (Fig. 3C). Thus, it seemed likely that plactin D played a role in increasing the formation of a-thrombin in prothrombin ⁄ Xa-medi- ated promotion of scu-PA activation. a-Thrombin can specifically cleave human scu-PA at Arg156–Phe157 [7], two residues proximal to the acti- vation cleavage site (Lys158–Ile159). Thrombin-cleaved tcu-PA (tcu-PA ⁄ T), however, showed < 1% activity of tcu-PA (consistent with previous reports) [7,19,21]. Plactin D failed to activate tcu-PA ⁄ T (data not shown). Nevertheless, incubation of tcu-PA ⁄ T with U937 cells resulted in the generation of u-PA activity (Fig. 3D). Thus, there was an additional, cell-associ- ated mechanism to achieve the generation of fully active u-PA. One possible candidate is dipeptidyl pep- tidase I (DPP-I), a thiol protease that could activate tcu-PA ⁄ T [19], and is expressed at high levels in cyto- toxic lymphocytes and myeloid cells, including U937 cells. Therefore, we examined the effects of cystatin, an inhibitor of DPP-I, on tcu-PA ⁄ T activation by U937 cells. Cystatin effectively inhibited tcu-PA activation by U937 cells (Fig. 3D) and prothrombin ⁄ Xa-mediated scu-PA activation on U937 cells (Fig. 3E). These results were consistent with the observation that DPP-I was able to activate tcu-PA ⁄ T by removing two amino acids (Phe157–Lys158) from the N-terminus of its B-chain [19]. The sequential mechanism leading to enhancement of scu-PA activation is shown in Fig. 3F. Dual modulation of prothrombin activation by plactin The above results suggested that plactin D affected Xa-catalyzed activation of prothrombin not only in the U937 system, but also under other conditions. To characterize the plactin action, prothrombin activation was assayed using a purified system. Consistent with the results obtained with the U937 system, prothrom- bin activation was markedly increased by plactin D when prothrombin was incubated with Xa (Fig. 4A). Xa activity, measured using a chromogenic peptide substrate (Spectrozyme Xa), was minimally affected by plactin D (Fig. 4A, inset). Thus, it appeared likely that plactin D altered prothrombin such that it was suscep- tible to activation by Xa. Under physiological coagula- tion conditions, prothrombin activation is catalyzed by the prothrombinase complex (factor Xa in complex with factor Va, phospholipids and Ca 2+ ). When pro- thrombin activation was assayed using prothrombinase complex, plactin D inhibited the reaction (Fig. 4B). Because plactin did not affect the activity of prothrom- binase toward Spectrozyme Xa (Fig. 4B, inset), it was T. Harada et al. Dual modulation of prothrombin activation FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS 2519 also likely that plactin D altered prothrombin such that it became resistant to the activation by Xa that formed prothrombinase. To understand the mechanism for these conflicting effects of plactin D on prothrombin activation, we tested several combinations of the factors that make prothrombinase a catalyst (Fig. 4C–H). Prominent enhancement by plactin D was observed when the catalyst was Xa ⁄ phospholipids, Xa ⁄ phospholipids ⁄ Va, Xa ⁄ Va or Xa ⁄ Ca 2+ . However, plactin D led to marked inhibition when Xa ⁄ phospholipids ⁄ Ca 2+ was used. A marginal promotive effect of plactin D was observed when the catalyst was Xa ⁄ Va ⁄ Ca 2+ . Under all these conditions, plactin D did not affect Xa activity (Fig. 4, insets) or the activity of isolated a-thrombin (data not shown). In summary, the data demonstrated that plac- tin D could promote or inhibit prothrombin activation, depending on the conditions of activation (Fig. 4I). For the inhibitory plactin D effect, the presence of both phospholipids and Ca 2+ was required, whereas the pro- motive effect was seen in the absence of either phospho- lipids or Ca 2+ , irrespective of the presence or absence of factor Va. Phosphatidylserine-containing phospholipid membranes act as a scaffold for the Ca 2+ -dependent 0 20.0 40.0 60.0 0.08 0.1 A DE F B C 080604020 aX + T P )nitcalp +( aX + TP )lortnoc( lo rtnoc ni t cal p + enola TP Thrombin activity (Δ A 405 ) )nim( emiT 66 54 92 )aDk( 6 11 4.79 AP-ucs niahc-B niahc-A nibmorhT D nitc a lP + − + − aX + nibmorhtorP − ni duriH −− − +++ − u-PA activity (fluorescence intensity) α - T h r o m b i n 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 P r o t h r o m b i n α - T h r o m b i n + p l a c t i n D 0 2 .0 4 .0 6 . 0 8.0 1 2 .1 4.1 6 . 1 u-PA activity (fluorescence intensity) l ort n o C ni t at sy C T/AP-uct + − 0 20.0 4 0 . 0 6 0 . 0 8 0 . 0 1 .0 21.0 41. 0 u-PA activity (fluorescence intensity) nitatsyC + – D nitcalP niduri H ++ –––+–––+ –+ – ––+ – – ) evi tc a n i( AP- ucs ) e vi t c a ni( T / AP - uc t ) evitc a ( A P - u c t α nibmorhT-α nibmorhT- es a et orP FGE R F K I elgnir K es a et orP FGE R F K I elgnir K FGE F K I es a e t orP elgnirK R FGE F K I es a e t orP elgnirK R FGE elgnirK R es aetorP I FGE elgnirK R es aetorP I e sa d i t p e p e k i l - I -PPD e sa d i t p e p e k i l - I -PPD nitc a lPnitc a lP nibmorhtorPnibmorhtorP aXaX Fig. 3. Mechanism of plactin promotion of prothrombin-mediated scu-PA activation in U937 cells. (A) U937 cells were incubated with human prothrombin in the presence of 2 m M CaCl 2 and 0.1 mM Spectrozyme TH to determine thrombin formation. Where indicated, 0.1 nM human Xa and 25 l M plactin D were included in the incubation. (B) U937 cells were incubated with the indicated protein in the absence or presence of 50 l M plactin D. The concentrations of prothrombin and a-thrombin were 347 and 27 nM, respectively. After washing, cellular u-PA activity was measured. (C) U937 cells equilibrated with 125 I-labeled scu-PA were incubated with either prothrombin (347 nM) plus factor Xa (3 nM)or a-thrombin (10 n M) in the absence or presence of 50 lM plactin D and 30 nM hirudin. After washing, cells were lysed and subjected to reduced SDS ⁄ PAGE on a 12.5% gel, followed by autoradiography. Positions of scu-PA, A- and B-chains of tcu-PA are shown. (D) U937 cells (5.0 · 10 6 ) were equilibrated with tcu-PA ⁄ T (10 nM)at4°C for 30 min in buffer A. After washing, cells were incubated with GGA-MCA in the absence or presence of 100 n M cystatin to determine u-PA activity. (E) U937 cells were treated with prothrombin (347 nM) and factor Xa (100 p M) in the absence or presence of 25 lM plactin D. After washing, cells received GGA-MCA to determine u-PA activity in the second incubation. Where indicated, 30 n M hirudin or 100 nM cystatin was included both in the first and second incubations. Error bars represent SD from determinations carried out in triplicate. In some data points, error bars are too small to be recognized. (F) Schematic representation of prothrombin- and plactin-mediated enhancement of scu-PA activation on U937 cells. The u-PA molecule is shown schematically with each domain in a colored circle. Amino acid residues involved in proteolytic cleavages are given in white circles. A disulfide bond that connects A- and B-chains of tcu-PA is shown as red dashed line. Dual modulation of prothrombin activation T. Harada et al. 2520 FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS assembly of the protein components that form pro- thrombinase [11]. Therefore, the plactin D action can be interpreted as follows: plactin D is inhibits prothrombin activation by membrane-associated Xa, whereas it is promotive when free Xa is used as a catalyst. We next examined the possibility that plactin D induced alternative proteolytic prothrombin cleavages that resulted in increases or decreases in thrombin activity. When prothrombin activation was catalyzed by fully assembled prothrombinase complex, plactin D inhibited the formation of a-thrombin without produc- ing proteolytic fragment species other than those produced during the course of normal prothrombinase catalysis (Fig. 5A). When free Xa was used to activate prothrombin, plactin D increased the level of a-throm- bin (Fig. 5B). Thus, the plactin D effects were increas- ing or decreasing the formation of a-thrombin without the accompanying conversion of prothrombin to highly active or inactive thrombin species. Interaction between plactin and prothrombin To investigate the interaction between plactin and prothrombin, we synthesized a radiolabeled plactin analog. The analog, [ 14 C]plactin-50 [cyclo(-d-Val-l- [ 14 C]Leu-d-Leu-l-Phe-d-Lys-)], had two to three times the activity of plactin D. Binding of [ 14 C]plactin-50 to prothrombin gave a curve that appeared to become sigmoidal (Fig. 6A), although maximum binding was not obtained because of the low solubility of [ 14 C]plactin-50. This observation was consistent with the promotion and inhibition of prothrombin activa- tion by plactin, which gave sigmoidal or bell-shaped dose–response curves (Fig. 4). These properties of the plactin–prothrombin interaction suggested a change in the conformation of prothrombin after plactin bind- ing. We measured the intrinsic fluorescence of prothrombin to assess any conformational change. When prothrombin was incubated with plactin D in the absence of Ca 2+ , the intrinsic fluorescence increased by 4.6% (P < 0.01) (Fig. 6B). Prothrombin has several Ca 2+ -binding sites, and Ca 2+ binding alters its conformation. Accordingly, the intrinsic fluo- rescence of prothrombin in the presence of Ca 2+ was significantly lower (6.6%, P < 0.01) than in the absence of Ca 2+ . Plactin D increased the internal fluorescence by 7.7% (P < 0.001), even under these conditions (Fig. 6B). Therefore, it was likely that plactin–prothrombin binding altered the conformation of prothrombin and resulted in dual modulation of prothrombin activation, depending on the conditions of factor Xa catalysis. 0 05 001 051 002 052 0504 0 3020 1 0 0 002 004 0 0 6 0 0 8 0001 0 02 1 0 5 0 40302010 0 0 2 04 06 08 0 01 0 504 030201 0 Xa activity 0 1 2 3 0 5 0 4 0302 0 10 0 05 0 01 0 51 002 05 2 003 053 05040302010 0 05 0 01 051 0 02 0 52 003 0 5 0 4 0302010 0 001 0 02 003 004 005 05040302010 0 05 001 051 002 052 003 05040302010 0 01 0 2 0 3 04 05 06 050403020 1 0 0 1 2 3 05 04 03 0 2 0 1 0 Xa activity 0 1 2 3 0 5040302 0 10 Xa activity 0 1 2 3 0 504 0 3 0 2 010 Xa activity 0 1 2 3 0 50 4 030 2 010 Xa activity 0 1 2 3 05040302010 Xa activity 0 1 2 3 050 40 30 2 010 Xa activity 0 1 2 3 05040302010 Xa activity a X aX LP /aX LP /aX aC /aX +2 aC /aX + 2 a V / aX a V / a X aC / LP /aX + 2 aC / LP / aX +2 aC / LP /aX +2 aV / aC / L P /aX + 2 aV / aV / LP /a X aV / LP / aX aC /aX +2 aV / aC /aX +2 aV / Generation of thrombin activity (Δ A 405 · h –2 × 10 3 ) Plactin D (μM) A B C D G EF H I Maximal response (%) 001– 05 – 0 0 001 4121% 0002 LP aC aV ––––++++ ––++++–– –+–+–+–+ Fig. 4. Dual modulation of prothrombin activation by plactin D. (A–H) Factor Xa-catalyzed activation of human prothrombin was determined by measuring the generation of thrombin using the chromogenic substrate Spectrozyme TH, in the presence of the indicated concentrations of plactin D. Where indicated, factor Va (4 p M in panel B and 2 nM in the other panels), PCPS (PL) (50 lM) or CaCl 2 (2 mM) were included. The concentration of Xa was 1 p M in (B) and 0.5 nM in the other panels. Inset shows the effect of plactin D on factor Xa activity in each con- dition. The Xa concentration was 0.5 n M for all incubations and the Va concentration was 2 nM when added. Ordinate denotes Xa activity as expressed in A 405 Æmin )1 · 10 3 , and abscissa plactin D concentration in lM. Each value represents the mean ± SD from determinations performed in triplicate. (I) Summary of the plactin D effects on prothrombin activation. Maximal response values are plotted. T. Harada et al. Dual modulation of prothrombin activation FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS 2521 Can plactin be a procoagulant or an anticoagulant? We asked whether plactin inhibited or stimulated the blood coagulation system, because plactin dually modulated prothrombin activation. As shown in Fig. 7A, plactin D showed anticoagulant activity in experimental coagulation tests: plactin D significantly prolonged both activated partial thromboplastin time (fibrin clot formation time after Ca 2+ addition to phospholipid-supplemented, contact-phase-activated plasma) and prothrombin time (clot formation time after the addition of tissue factor–phospholipids com- plex and Ca 2+ to plasma). In these measurements, the enzyme that catalyzed prothrombin activation was in situ-generated, membrane-associated Xa. How- ever, thrombin time (clot formation time after the addition of a-thrombin to plasma), which did not involve prothrombin activation, was not affected by plactin D (data not shown). These observations appeared consistent with results obtained using puri- fied systems. Finally, the effect of plactin D on prothrombin activation was examined in vivo using normal mice. In one experiment, prothrombin activation was evalu- ated as the formation of a thrombin ⁄ antithrombin III complex. When mice were treated with plactin D at 0.1 and 1 mgÆkg )1 , the level of the complex was not elevated significantly (Fig. 7B). In another experiment, the fate of intravenously injected 125 I-labeled pro- thrombin was determined. Forty minutes after plac- tin D treatment, 125 I-labeled prothrombin ⁄ thrombin species in the blood were immunopurified and resolved by SDS ⁄ PAGE. We did not detect the formation of thrombin or its complex with antithrombin III in plac- tin D-treated mice (Fig. 7C). The dose of plactin D used in these experiments (0.1 or 1 mgÆkg )1 ) was suffi- cient for plactin D to produce a protective effect in a thrombin-induced pulmonary embolism model. In this model, plactin D improved the survival of thrombin- treated mice. A plactin D dose of 0.1 mgÆkg )1 increased the survival rate to levels comparable with that produced by 0.01 UÆkg )1 of the fibrinolytic enzyme plasmin (Fig. 7D). Furthermore, plactin D did not show acute toxicity after intravenous injection at 25 mgÆkg )1 . These results may exclude the idea that plactin D is a procoagulant. It is possible that plactin inhibits physiological coagulation, which proceeds via membrane-associated processes [11], and that plactin does not behave as a procoagulant under normal circulation conditions. 5.00 6 6 5 4 92 )aDk( 611 4 .7 9 54 35. 2 25. 1 15.005435.22 5.11 ) 1 Fs e d (T P A-2,1F B T P 2, 1 F A nim ni tcalPl ort noC A B noitavitca dezylatac-esanibmorhtorP nitcalP α nibm or hT - dr a d n a ts 66 5 4 9 2 )aD k ( lo r tnoC noitavitca dezylatac-aX Fig. 5. Analysis of thrombin species formed in the presence of plactin D. (A) Human pro- thrombin was activated by prothrombinase complex in the absence or presence of 50 l M plactin D. At the indicated times, aliquots of the incubation mixtures were withdrawn to analyze by reduced SDS ⁄ PAGE on a 10% gel. Proteins were visualized by Coomassie Brilliant Blue R-250. The positions of prothrombin (PT), prothrombin(desF1) [prothrombin without fragment 1; PT(desF1)] and fragment 1 + 2 + A-chain (F1,2-A), as well as A-chain and B-chain of a-thrombin B, are shown. (B) Human prothrombin was activated by free factor Xa in buffer F containing 2 m M CaCl 2 in the absence or presence of 25 lM plactin D. Proteolytically active molecular species were visualized by casein zymo- graphy after resolving on nonreduced SDS ⁄ PAGE on a 10% gel. Human a-thrombin (0.3 lg) was used as a standard. Dual modulation of prothrombin activation T. Harada et al. 2522 FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS Conclusion Our studies demonstrate plactin-mediated modulation of prothrombin activation. Plactin binds to prothrom- bin and dually modulates its activation, depending on the form of the catalyst, factor Xa. Under physiologi- cal conditions, the coagulation reaction proceeds via membrane-associated processes. Plactin inhibits pro- thrombin activation catalyzed by membrane-associated Xa. This is consistent with the observation that plactin inhibits the coagulation of plasma in activated partial thromboplastin time tests and prothrombin time tests. However, plactin enhances prothrombin activation when the catalyst is nonmembrane-bound Xa. This mechanism may participate in the enhancement of fibrinolytic activity in the U937 cell system, in which plactin enhances prothrombin activation and the for- mation of inactive tcu-PA ⁄ T, which is subsequently converted to fully active tcu-PA by cellular cystatin- sensitive, DPP-I-like peptidase. The specificity of prothrombinase for prothrombin is mediated by exosites, which are physically separated from the catalytic site, on the surfaces of the catalytic domains. It is postulated that substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the Xa catalytic site [22]. Thus, prothrombin activation is a conforma- tionally regulated process. This may partly explain the plactin-mediated dual modulation of prothrombin activation. The pharmacological application of dual thrombin modulation would be an intriguing approach to intervention in thromboembolic diseases. Experimental procedures Plactins Plactin D [cyclo(-d-Val-l-Leu-d-Leu-l-Phe-d-Arg-)] and plactin-14 [cyclo(-d-Val-l-Lys-d-Leu-l-Phe-d-Arg-)] were synthesized according to Fmoc chemistry, as described pre- viously [1,2]. Dansylplactin-14 (plactin-14-DNS) was syn- thesized by mixing 1 mL of plactin-14 (1 mgÆmL )1 in water), 1 mL of dansyl chloride (4 mgÆ mL )1 in acetone) and 90 mg of NaCO 3 overnight at ambient temperature. Plactin-14–Sepharose was prepared by reacting 35 mL of 0.7 mgÆmL )1 plactin-14 with 1.5 g of CNBr-activated Sepharose 4B (GE Healthcare Biosciences, Tokyo, Japan) in 0.1 m sodium bicarbonate, pH 9.0, and 0.5 m NaCl, fol- lowed by blocking with 1 m ethanolamine. The amount of plactin-14 immobilized was 7.0 lmolÆmL )1 of gel. [ 14 C]Plac- tin-50 [cyclo(-d-Val-l-Leu-d-Leu-l-Phe-d-Lys-)] was synthe- sized using Fmoc-l-Leu (1- 14 C) (American Radiolabeled Chemicals Inc, St Louis, MO, USA). The specific radioac- tivity was 1.02 BqÆ pmol )1 . For assays, plactins dissolved in dimethylsulfoxide were used at a solvent concentration of 1% (v ⁄ v). Other materials Human scu-PA was provided by Mitsubishi Tanabe Pharma Corporation (Osaka, Japan). Other proteins and chemicals were from the following sources: human tcu-PA from JCR Pharmaceutical (Kobe, Japan); human plasmin and aprotinin from Wako (Osaka, Japan); human pro- thrombin, human coagulation factor Xa, the thrombin 1.8 1.5 1.2 0.9 0.6 0.3 0 86 4 2 0 [ 14 C]Plactin-50 (µM) [ 14 C]Plactin-50 binding to prothrombin (pmol bound per pmol prothrombin) * * 21 20 19 18 22 B A Ca 2+ Control Plactin D (50 µ M ) Intrinsic fluorescence + − Fig. 6. Interaction between plactin and prothrombin. (A) The bind- ing of [ 14 C]plactin-50 to human prothrombin was determined in the presence of the indicated concentrations of [ 14 C]plactin-50. Specific binding data are shown. (B) The intrinsic fluorescence of human prothrombin was measured in the absence or presence of CaCl 2 (2 mM) and plactin D (50 lM). *P < 0.01 by Student’s t-test, com- pared with control. Error bars represent SD from determinations performed in triplicate. T. Harada et al. Dual modulation of prothrombin activation FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS 2523 inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and polyclonal anti-(human thrombin) sheep IgG from Haematologic Technologies (Essex Junction, VT, USA); human coagulation factor V from Serbio (Paris, France); human a-thrombin, BSA, cystatin and l-a-phos- phatidylcholine (egg yolk) from Sigma (St Louis, MO, USA); l-a-phosphatidylserine (porcine brain) from Avanti Polar Lipids (Alabaster, AL, USA); glutaryl-Gly-Arg-4- 00.0 02.0 04.0 06.0 08 . 0 00 . 1 nitcalP * * 51 / 3 41/31 5 1 / 21 Fraction survived n im s a l Plort n oC TP )1Fsed(TP α nibmor h T- 66 54 92 )a D k ( 4. 7 9 11.0 Coagulating blood Plactin D (mg·kg –1 ) Control Plactin D (mg·kg –1 ) 10.1 Control Plasma TAT level (ng·mL –1 ) 4 6 40 30 20 10 2 0 31.8 Coagu- lating blood 0 35 AB CD 30 25 20 15 10 5 060504030201 0 Prothrombin time Activated partial thromboplastin time Plactin D (μ M) Percent change * ** ** ** ** ** ** ** ** Fig. 7. Effects of plactin D on plasma coagulation in vitro and prothrombin activation in vivo. (A) Activated partial thromboplastin time and prothrombin time were measured using normal human plasma. Plactin D was added 5 min before the initiation of each reaction. The clotting times in the absence of plactin D were 26.9 ± 0.2 s for activated partial thromboplastin time and 15.1 ± 0.7 s for prothrombin time. Error bars represent SD from triplicate determinations. *P < 0.05 and **P < 0.01 by Dunnett’s test, compared with control. (B) Plactin D, at the indicated dose, was given intravenously to mice (n = 5 for each group), and blood was drawn in a mixture of protease inhibitors, 40 min after the treat- ment. The level of thrombin ⁄ antithrombin III complex in the resulting plasma was determined by enzyme immunoassay. Serum obtained from blood drawn without anticoagulants from normal mice (Coagulating blood) was used as a standard. There were no statistical differences among control, 0.1 mgÆkg )1 plactin D and 1 mgÆkg )1 plactin D groups by Dunnett’s test. (C) Plactin D and human 125 I-labeled prothrombin were successively given intravenously to mice (n = 3 for each group). Blood was drawn in a mixture of protease inhibitors, 40 min after treatment. Labeled proteins were purified from plasma with anti-(human thrombin) IgG–Sepharose and resolved on nonreduced SDS ⁄ PAGE on a 10% gel. Serum from control mouse blood was similarly processed as a standard to detect prothrombin activation (Coagulating blood). Data shown are representative. Essentially the same results were obtained in each group. The positions of prothrombin (PT), and prothrombin(desF1) [PT(desF1)] and a-thrombin are shown. (D) Effect of plactin D on thrombin-induced pulmonary embolism in mice. Mice received intravenous injection with saline (Control) plactin D (0.1 mgÆkg )1 ) or plasmin (0.01 UÆkg )1 ). After 15 min, human a-thrombin was injected intravenously to induce pulmonary thromboembolism. Next day, the number of surviving animals was counted. Numbers above bars denote the number of survived ⁄ total animals in each group. *P < 0.01 by Fisher’s exact test, compared with control. Dual modulation of prothrombin activation T. Harada et al. 2524 FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS methylcoumarin-7-amide (GGA-MCA) from Peptide Institute (Osaka, Japan); Spectrozyme TH (H-d-hexahydro- tyrosyl-Ala-Arg-p-nitroanilide), Spectrozyme Xa (methoxy- carbonyl-d-hexahydrotyrosyl-Ala-Arg-p-nitroanilide) and recombinant hirudin from American Diagnostica (Green- wich, CT, USA). Factor V (300 nm) was activated to Va by incubating with a-thrombin (3 nm)at37°C for 10 min. Thrombin- cleaved tcu-PA (tcu-PA ⁄ T) was prepared by incubating scu-PA (1 lm) with a-thrombin (10 nm)at37°C for 22 h, followed by the addition of 30 nm hirudin to neutralize thrombin. Phospholipid vesicles (PCPS) composed of 75% (w ⁄ w) phosphatidylcholine and 25% (w ⁄ w) phosphatidyl- serine were prepared as described previously [23]. Radio- iodination of scu-PA and prothrombin was performed by the IODO-GEN method [24], using carrier-free Na 125 Itoa specific activity of 2000–3000 cpmÆ ng )1 of protein. Buffers used were: buffer A, 20 mm sodium phosphate, pH 7.4, and 150 mm NaCl; buffer B, 50 mm Tris ⁄ HCl, pH 7.4, and 100 mm NaCl; buffer C, 50 mm sodium phos- phate, pH 7.4, and 80 mm NaCl; buffer D, 50 mm sodium phosphate, pH 7.4; buffer E, 50 mm Tris ⁄ HCl, pH 7.4, 100 mm NaCl and 0.01% (w ⁄ v) Tween 80; buffer F, 20 mm Tris ⁄ HCl, pH 7.4, 150 mm NaCl and 0.1% (w ⁄ v) Tween 80; buffer G, 62.5 mm Tris ⁄ HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercapthoethanol and 0.002% bromo- phenol blue. Cell culture Human monocytoid line U937 cells (obtained from the Japanese Cancer Research Resources Bank, Tokyo) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS, USA), 100 UÆmL )1 penicillin G and 100 lgÆmL )1 streptomycin. For assays, cells were seeded at 2 · 10 5 cellsÆmL )1 in 15 mL of the medium and grown for 2 days. Prior to use in experi- ments, exponentially growing cells were harvested, washed twice and suspended with buffer A. Assay for cellular scu-PA activation U937 cells were suspended with buffer A at a density of 5.0 · 10 6 cellsÆmL )1 . Cells were incubated in the absence or presence of 20% (v ⁄ v) human plasma and plactin at 37 °C for 30 min, with shaking. After washing with buffer B, cells were resuspended in buffer B containing 0.1 mm GGA- MCA, a chromogenic peptide substrate for u-PA. After incubation at 22 °C for 1 h, the supernatant was removed and acetic acid was added to 10% to stop the reaction. The fluorescence of 7-amino-4-methylcoumarine liberated from GGA-MCA by the u-PA cleavage was measured (excitation at 380 nm and emission at 480 nm). In the experiment shown in Fig. 1A, cells treated in the first incubation were further incubated with plasmin (100 nm)at22°C for the indicated time in buffer B con- taining BSA (10 mgÆmL )1 ). After addition of aprotinin (40 kallikrein inhibitor unitsÆmL )1 to neutralize plasmin) and washing, cells were processed to determine u-PA activity as described above. In some experiments, scu-PA activation on U937 cells was also determined as the proteolytic cleavage of 125 I- labeled scu-PA. In this experiment, 125 I-labeled scu-PA (5.6 nm, 3000 cpmÆng )1 ) was included in the first incuba- tion. After washing twice with buffer B, cells were lysed with buffer G. An aliquot of the lysate was subjected to SDS ⁄ PAGE on a 12.5% gel. After fixing and drying, the gel was exposed to an X-ray film at )80 °C for 16 h. In the experiment shown in Fig. 3C, 125 I-labeled scu-PA was bound to cell surface at 4 °C for 30 min in RPMI-1640 medium supplemented with 10% fetal bovine serum and 20 mm Hepes, pH 7.4. The labeled cells were used for incubations, as described in the legend to Fig. 3. Partial purification of plactin cofactor from bovine plasma Citrated bovine platelet-poor plasma (490 mL) was frac- tionated using the method described by Cohn et al. [25]. Most of the cofactor activity to support plactin-dependent activation of cellular scu-PA was recovered in the ‘precipi- tate IV-1¢ fraction. The fraction was subjected to ammo- nium sulfate fractionation at 4 °C, and precipitates obtained from 25–50% saturation were dialyzed against buffer C, yielding 2.3 g of partially purified cofactor preparation (fraction E4A50). The specific activity of the preparation was 24 times that of the original plasma. Plactin-14–Sepharose chromatography A column containing 0.5 mL of plactin-14–Sepharose was equilibrated with buffer D at room temperature, and 0.6 mL of fraction E4A50 (11 mg protein) was applied to the column. After washing with 2.5 mL of buffer D, the column was developed with 2.5 mL of buffer D containing 0.5 m NaCl, followed by 2.5 mL of buffer D containing 6 m guanidine ⁄ HCl. Each eluate was dialyzed overnight against buffer A before SDS ⁄ PAGE and assay for plactin- dependent promotion of scu-PA activation on U937 cells. Assay for prothrombin activation The activation of prothrombin was assayed by incubating human prothrombin (20 nm) and Spectrozyme TH (0.1 mm) in the presence of factor Xa in buffer F with or without factor Va (4 pm or 2 nm), PCPS (50 lm) or CaCl 2 (2 mm). The concentration of Xa was 1 pm when the incuba- tion contained Va (4 pm), PCPS and CaCl 2 to assemble pro- thrombinase complex. In other assays, the Xa concentration T. Harada et al. Dual modulation of prothrombin activation FEBS Journal 276 (2009) 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS 2525 [...].. .Dual modulation of prothrombin activation T Harada et al was 0.5 nm, and the concentration of Va was 2 nm when it was included in the mixture, other than that for prothrombinase formation The reaction was started by adding Xa, and the change in absorbance at 405 nm was monitored kinetically at 37 °C From the slope of the plots of A405 versus t2 [26], the initial velocity of thrombin generation... calculated In the SDS ⁄ PAGE assay for the prothrombinase-catalyzed reaction, 1.4 lm prothrombin was incubated with 30 lm PCPS, 2 mm CaCl2, 3 lm DAPA, 1 nm Xa and 5 nm Va at 37 °C for the indicated times in buffer F (DAPA was included to inhibit the feedback proteolysis of prothrombin by the generated thrombin) The reaction was stopped by the addition of an equal volume of acetic acid, and the resulting... a spin column (prepared by centrifuging 500 lL of a 15.2% w ⁄ v suspension of Sephadex G-25 in buffer E at 2000 g for 1 min), and the column was centrifuged at 2000 g for 1 min The radioactivity in the eluate was counted for 3 min in a liquid scintillation counter The amount of bound [14C ]plactin- 50 was calculated by subtracting the radioactivity obtained in the absence of prothrombin from that obtained... (1987) Activation of human prothrombin by human prothrombinase Influence of factor Va on the reaction mechanism J Biol Chem 262, 3291–3299 Mann KG, Nesheim ME, Church WR, Haley P & Krishnaswamy S (1990) Surface-dependent reactions of the vitamin K-dependent enzyme complexes Blood 76, 1–16 Rosing J, Tans G, Govers-Riemslag JW, Zwaal RF & Hemker HC (1980) The role of phospholipids and factor Va in the prothrombinase... at 37 °C for the times indicated in Fig 3A After centrifugation, the absorbance at 405 nm in the supernatant was measured [14C ]Plactin- 50 binding to prothrombin Human prothrombin (0.95 lm) was incubated with [14C ]plactin- 50 in buffer E at 37 °C for 1 h, followed by standing on ice for 15 min Bound and free [14C ]plactin- 50 was separated by spin column chromatography We applied 20 lL of the reaction... saturation of pro-urokinase receptors on human A431 cells Cell 45, 675–684 6 Kobayashi H, Moniwa N, Sugimura M, Shinohara H, Ohi H & Terao T (1993) Effects of membrane-associated cathepsin B on the activation of receptor-bound prourokinase and subsequent invasion of reconstituted basement membranes Biochim Biophys Acta 1178, 55– 62 7 Ichinose A, Fujikawa K & Suyama T (1986) The activation of pro-urokinase by. .. Triton X-100 to remove SDS, followed by overnight incubation at 37 °C with buffer F containing 2 mm CaCl2 After staining with Coomassie Brilliant Blue R-250 the proteolytically active position appeared as a colorless band on dark blue background The activation of prothrombin in the presence of U937 cells was assayed by incubating 1.0 · 106 cellsÆmL)1 in buffer A containing prothrombin (20 nm), 0.1 nm Xa,... unnecessarily at any stage of an experiment The protocol was approved by the Animal Experiment Committee of Tokyo Noko University To determine the level of thrombin ⁄ antithrombin III complex in plasma, male ICR mice ( 30 g; Japan SLC, Hamamatsu) were anesthetized with intraperitoneal urethane ⁄ a-chlorarose (750 and 60 mgÆkg)1, respectively) Plactin D dissolved in saline was given to the mice intravenously... 2516–2528 ª 2009 The Authors Journal compilation ª 2009 FEBS 2527 Dual modulation of prothrombin activation T Harada et al 25 Cohn EJ, Strog LE, Hughes WL, Mulford DJ, Ashworth JN, Melin M & Taylor HL (1946) Preparation and properties of serum and plasma proteins IV A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluid J Am Chem Soc 68, 459–475 26... assistance This work was supported by a grant from the Japan Society for the Promotion of Science 14 15 References 1 Inoue T, Hasumi K, Kuniyasu T & Endo A (1996) Isolation of plactins A, B, C and D, novel cyclic pentapeptides that stimulate cellular fibrinolytic activity J Antibiot (Tokyo) 49, 45–49 2 Inoue T, Hasumi K, Sugimoto M & Endo A (1998) Enhancement of fibrinolysis by plactins: structure–activity . Fig. 3F. Dual modulation of prothrombin activation by plactin The above results suggested that plactin D affected Xa-catalyzed activation of prothrombin. (%) 001– 05 – 0 0 001 4121% 0002 LP aC aV ––––++++ ––++++–– –+–+–+–+ Fig. 4. Dual modulation of prothrombin activation by plactin D. (A–H) Factor Xa-catalyzed activation of human prothrombin was determined by measuring the generation