Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 3, (TA FP) , ; 1) 1, 102phenan th ro line TA FP ;

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1 ISSN CN gQ 16 3 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 3, (TAFP) 3 1) 1) 1) 3 3 (, ; 1), ) (TA FP), TA FP, TA FP ph 715, ph 610 TA FP : ST I> an tipain> SBB I> an titryp sin> TL CK> leupep tin > bacteracin> PM SF > T PCK, 1, 102phenan th ro line TA FP TA FP ; ;,, Q 51 Properties of F ibr inolytic Prote in from Tabanus am aenus W a lker (TAFP) and Its Effects on Hem orheology in Ra ts 3 YAN G X ing2yong, YAN Guang2fan 1), HU Kai2zh i 1), ZHAN G Yu2fang 1),LU X iao2feng, PE I Yan 3 3 (B iotechnology Center of S ou thw est A g ricu ltu ral U niversity, Chong qing , Ch ina) ( 1) Institu te of M ed icinal P lant P lantation of Chong qing, Chong qing , Ch ina) Abstract In v itro, on fib rin p late and p lasm inogen2free fib rin p late, the fib rino lytic p ro tein from T abanus am aenus W alker (TA FP ) show ed the sam e fib rino lytic activity as the m ix tu re of TA FP and p lasm inogen indicating that TA FP w as a fib rino lytic enzym e degrading fib rin directly, no t a p lasm inogen activato r, degrading fib rin via activating p lasm inogen. TA FP w as found also to de2 grade fib rinogen. Its op tim um ph w as Inh ib ition of TA FP activity by p ro tease inh ib ito rs show ed: soybean tryp sin inh ib ito r> an tipain> soybean Bowm an2b irk inh ib ito r> an titryp sin> N 2 P2to luenesu lfonyl2l 2lysine ch lo rom ethyl ketone > leupep tin > bacteracin > phenylm ethylsufonyl fluo ride> N 2P2to luenesu lfonyl2l 2phenylan ine ch lo rom ethyl ketone, and 1, 102phenan th ro line had no inh ib ition of TA FP activity. It w as m o st rem arkab le, versu s con tro l, that TA FP cau sed p ro2 longation of b leeding tim e, inh ib ition of p latelet aggregation, decrease of fib rinogen level in p las2 m a, w ho le b lood visco sity, p lasm a visco sity and eryth rocyte sedim en tation, thu s reducing hem al2 ocrit velocity in v ivo. Key words T abanus am aenus W alker, F ib rino lytic p ro tein, P roperty, H em o rheo logy (T abanus biv itta2 tus M artṡ ),, 3 (962103) 3 3 T el: (023) , Fax: (023) E2m ail: peiyan@ sw au. edu. cn, [ 1 ],,,,,, ,, : , : China Academic Journal Electronic Publishing House. All rights reserved.

2 3 : (TA FP) 353 (T. am eanusw alker), 116 TAFP :,, TA FP, 2 h, 15 l, 35 l, 0102 m o lgl (ph 714) T ris2hc l ( 0115% Tw een2 [ 2 ] 80) 10 l (ch rom ozym P), 28 (TA FP) 5 m in, M odel nm A,, TA FP 117 ph TAFP : J. H :, (30 m g gkg d) (15 m g : Sephadex G275 Pharm acia ; ) 9, 7 d, 1, ( ) 4 h,,, ; AD P : 015 cm, ch rom ozym P (N 2bu tyloxyoarbonyl2l 2glycyl2 L 2p ro lyl2l 2argin ine p 2n itroan ilide) PM SF (phenyl2 m ethylsufonyl fluo ride) ST I ( soybean tryp sin in2 h ib ito r ) an tipain an titryp sin 1, 102phenan th ro2 line leupep tin TL CK (N 2P2to luenesu lfonyl2l 2ly2 sine ch lo rom ethyl ketone ) T PCK (N 2P2to luene2 su lfonyl2l 2phenylan ine ch lo rom ethyl ketone) SB2 B I(soybean Bowm an2b irk inh ib ito r) Sigm a m in, rgm in 10 m in, ; tpa B radfo rd [ 7 ], (m ggdl) (N IBSC) : : W istar,, g, ( ) ( ) 114 (TAFP) : : 80 mm, [2 ] 115 TAFP [ : ] : ( 1%, 015 ggl,, 37 1 h,, 014 U gm l ), h, rgm in 10 m in,, IU gm l tpa (% ) : 300 l 0102 m o lgl ph l (20 ggl ) 50 l, 37 5 m in, (10 U g, [ 4 N o rth rop ], ch rom ozym P, TA FP 118 : 3, gkg d), ( (37 ), : [ 5 ] (PR P) (PPP) PPP PR P 41 ( 3165) 10 4 gmm, 5 m o lgl AD P,, 5 m in :, 150 mm, 112 mm 013 mm TAFP, TA FP m l) (118 U gm l) 60 l,, tpa 37 : y = x, r= (y, x,, ) TA FP, tpa ;, 5870 tpa IU m g China Academic Journal Electronic Publishing House. All rights reserved.

3 TA FP, TA FP 212 TAFP TA FP, tpa, (F ig. 1A ), m in, (P < 01001), T ab le 1.,,,, tpa TA FP, tpa tpa,, ; TA FP TA FP,, 37 3 m in, (F ig. 1B ), TA FP, ; TA FP,,, ; (P > 0105), tpa,, T ab le 2.,, TA FP Table 1 F ibrino lytic activity fo TA FP on clo t lysis in v itro T reatm ent t (clo t lysis tim e) gm in P TA FP+ P lasm inogen F ig. 1 A nalysis of the fibrino lytic activity of TA FP on fibrin agar p late heated at 85 fo r 0 m in (A ) and 30 m in (B) 11Contro l (buffer) ; 2. TA FP + p lasm inogen; 3. TA FP; 4. tpa + p lasm inogen; 5. tpa, Table 2 A ntith rom bo tic activity of TA FP in v itro TA FP P > 0105 U K+ P lasm inogen U K P < tpa + P lasm inogen T PA P < n= 5, x ṡ U K: U rok inase; tpa : T issue p lasm inogen activato r N um ber T reatm ent C lo t fo rm ation Result t (clo t lysis tim e) gm in 1 Fg+ TA FP (37, 3 m in) + T b Pg Fg+ Pg (37, 3 m in) + T b TA FP Fg+ TA FP (37, 3 m in) + T b Fg (37, 3 m in) + T b TA FP Fg+ U K (37, 3 m in) + T b Pg Fg+ Pg (37, 3 m in) + T b U K Fg+ U K (37, 3 m in) + T b Fg (37, 3 m in) + T b U K n= 5, x ṡ Fg: F ibrinogen; T b: T h rom bin; Pg: P lasm inogen; U K: U rok inase; + : C lo t fo rm ation; - : N o clo t fo rm ation 213 TAFP TA FP T ab le 3. ST I an tipain SBB I ; PM SF TA FP ; leupep tin an ti2 TA FP 1, 102phenan th ro line, tryp sin TL CK TA FP ; TA FP ; TA FP T PCK, T PCK China Academic Journal Electronic Publishing House. All rights reserved.

4 3 : (TA FP) mm o lgl ( 015 mm o lgl ) TA FP Table 3 Effects of inh ibito rs on TA FP Inhibito r Bacteracin (1 ggl ) ST I(1 ggl ) PM SF (5 mmo lgl ) L eupep tin (1 ggl ) A ntipain (1 ggl ) A ntitryp sin (1 ggl ) Residual activities of TA FP (% ) F ig. 2 Effects of ph on TA FP activity ph stability; Op tim um ph 1, 102Phenanthro line (1 ggl ) 100 TL CK (1 mmo lgl ) T PCK (1 mmo lgl ) SBB I(1 ggl ) A ctivity w as x s from five rep licates ph TAFP F ig. 2, TA FP ph 715, ph , TA FP ph , ph 610 ( , P < 0101; , P < 0105) ; ( , , P < 0105) ; ( , P < 0101; , P < 0105), ( , , P < 0105) ( , , P < 0105) ; 215 TAFP ( , d, 517, P < 0105), ( ,, T ab le 4 TA FP , P < 0105) Table 4 Effects of TA FP on hemo rheo logy of rats Item Do se 30 m g kg - 1 d m g kg - 1 d B leeding tim egs P latelet aggregation (% ) P lasm a fibrinogen contentgm g dl - 1 ) W ho le blood visco sity P lasm a visco sity ES (% ) H em alocritgmm h ES: E ryth rocyte sedim entation. n= 9, x s 216 TA FP, TA FP ;,,, ;,,, [ 6 ], ;,,, China Academic Journal Electronic Publishing House. All rights reserved.

5 356 16, 3,, (Yang X ing2yong, L u X iao2feng, Pei Yan. Purification and characterization component of T abanus quinquecinctus R i2 (References) cardo. A cta E ntom o S in), 1998, 41: N o rth rop J H, Kunitz R M. C ry stalline enzym e. N ew Yo rk: 1,, (L iu D a2you, Xu Co lum bia U niversity P ress, 1948 L i, L i L i. A dvances in tabanids. Chin T rad itional H erbal 5, : D rug s), 1997, 28: (L i Yi2kui, W ang Q ing2m ao ed. P harm acolog ical 2,,,, (T abanus a2 M ethods in Chinese T rad itional D rugṡ Shanghai: Shanghai Sci2 m aenus W alker) ence and T echno logy P ress), 1991: (Yang X ing2yong, Cheng J ing2qiu, Pei 6 B radfo rd M. A rap id and sensitive m ethod fo r the quantitation Yan, Yan Guang2fan, Zhang Yu2fang. Purification and bioac2 of m icrogram quantities of p ro tein utilizing the p rincip le of p ro2 tivity assay of the fibrino lytic p ro tein of T abanus am aenus tein dye binding. A nal B iochem, 1976, 72: W alkeṙ Ch in J B iochem M ol B iol), 1999, 15 (4) : : Steven Hou ( N IH ) ( 2) : 13 ) : 11 : G T GF2Β W n t 101 ( ) : : : : , 800 : 2714, : :, E2m ail: isst@b ilong. com ) : ) : : : : :, 8 31 : D ṙ Steven Hou, Investigato r N C I2F rederick Cancer R esearch and D evelop2 m en t Cen ter N IH,M D 21702,U SA T el: , Fax: E2m ail: shou@ncifcrf. gov : : , : W eb: www. ciccsṫ o rg. cngisst (, China Academic Journal Electronic Publishing House. All rights reserved.