Activation of duck prothrombin by factor Xa and thrombin

Z. Banaé-Gruazka, T. Krajewski, B. Bretsznajder ACTIVATION OF DUCK PROTHROMBIN BY FACTOR Xa

AND THROMBIN*

Prothrombin from duck aodlum cltrata plasma, was isolated according to tha method of G r a n t and S u t t 1 a [6]. Tha activation of duck prothrombin In a homologous aystam co-ntaining duck Factor X and calcium ions induced the appea-rance or the final product-thrombin and activation products of molecular ««eight In the range 21 500-52 000 as it was es-timated by polyacrylamide gel electrophoresis.

Introduction

Prothrombin la a glycoprotein« consisting of e single polype-ptide chain with molecular ««eight of about 70 000. This prote-in playa a pivotal role In the fprote-inal stages of blood coagulation by converting fibrinogen to fibrin.

Complete activation of prothrombin by activated Factor Xa,Fa-ctor Va (Fig. 1) phospholipids and calcium ions results in the formation of thrombin and two activation products (Fragment 1 end Fragment 2), ( S e e g e r s et al. [ll]i M a g n u s -e o n et al. [7]).

Factor Xa in the presence of calcium Iona is also able to activate prothrombin to thrombin. This factor is responsible for eplit of two polypeptide bonds Arfl274"Thr275 and Ar9323~ -110^24 to form Fragment 1.2 and Intermediate 2, a direct

pre-*

Ala' P R O T H R O M B I N

T\

Fig. 1. Scheme of prothrombin activation by factor Xa and throm-bin

Schemat aktywacjl protrombiny czynnlklem Xa 1 trombin*

cursor of thrombin ( O w e n at al. [10]» E a m o n at el. 12, 3]j B a n e 6-G r u a z k a at al. Ll]).

Generated thrombin cleaves the bonde A r9i56"*S*rig7 both ln tha prothrombin and Fragment 1.2 releasing Fragment 1 and Inter-mediate 1 aa «»ell as Fragment 1 and Fragment 2, raapectlvaly ( G r a n t , S u t t l e L 63* S u t t 1 e at al. ’ [12]» M a l h o t r a [9]).

Material» and methoda

Prothrombin was isolated from fresh duck sodium citrate plaama

(9 i 1) according to the method of G r a n t and S u t-

t i e [6]. To Isolate duck prothrombin we applied enzyme adeor- ption of barium citrate. The barium citrate precipitate waa die- solved in 0.2 M EOTA (pH 7.0) and then some contaminating pro-teins were precipitated by the addition of an equal volume of sa-turated aaoonium sulfate. The precipitate waa removed by cen-trifugation end prothrombin was eeparated from the euperne- tant by addition of the same volume of saturated ammonium sulfa-te. Tha precipitated prothrombin was dissolved In 0.05 M lmlda- zole-HCl. (pH 7 8) containing 0.02 M sodium citrate and 0.2 M

ammonium chloride and dlalyzed against the same buffer for 3- ••4 h. The crude prothrombin preparations were chromatographed on DEAE Sephadex A-50 using a 200 ml linear (0.2 to 0.45 M) ammonium chloride gradient in 0.02 M Tria-HCl buffer at pH 7.5. In the course of eeparatlon the main peak containing purified prothrom-bin (elution between 0.24 to 0,26 M ammonium chloride) and 2-3 peaka being most probably impurity proteins and prothrombin de-gradation products were obtained. Next the main peak «os rechro-matographed on an identical OEAE Sephadex A-50 column. Every pre-paration and purification step wee carried out In the presence of inhibitors such as: soybean tripsin inhibitor 20 jig/ml, he-parin 10 U/ml, phenylmethylsulfonylfluorlde 0.001 M and DFP

10"4M.

Factor X was also isolated from duck blocd according to the method of E s n o u f et al. [4], The conversion to the active form (Xa) was performed with Russell's viper venom pro-tease (ratio of enzyme to substrate 1 : 100) in 0.025 M Tris-HCi buffer, pH 7.2 In the presence of calcium ions (0.01 M)(F u J 1- k a v* a et al. [ 5] ).

Prothrombin activation by Factor Xa In the presence of cal-cium ion« was performed according to G r a n t and S u t- t 1 • [6]. Prothrombin samples (50 jig, 1 mg per ml) were in-cubated with Factor Xa (5 pg) In the presence of calcium ions (5 mM) for 0, 5, 15, 30, 60, 120 and 240 min. at 37°C. The rea-ction was etoppsd by adding sodium dodecylsulfate (SOS) to a fi-nal concentration of 1& «nd heating to 70°C (water bath) for 5- -10 mln.

Prothrombin activation products appearing during the conver-sion of prothrombin into thrombin were examined by SOS polyacry-lamide gal electrophoresis (7.55a) by W e b e r and 0 s- b o r n method [13].

Result» anti discussion

The preparation of duck prothrombin after purification on OEAE Sephadex A-50 according to G r a n t and S u t t 1 e [6] was chromatographlcally homogenic and moved In polyacrylami-de gel as a single bandCsee Fig. 2, 3, end of this volume,p. 191).

T a b l e 1 Molecular weight of prothrombin

and prothrombin activation producte Ciężar cząsteczkowy protrombioy

i produktów Jej aktywacji

I ;" ... Protein f . Molecular weight 0 Incubation 5 15 30 t i m (»in«) 60 120 240 Prothrombin (duck) !. Intermediate 77 000 ♦ ♦ ♦ ■v ♦ form 1 52 000 - - ♦ r Fragment 1.2 45 000 - - '♦ ♦ ♦ ♦ Thrombin 33 000 mm ♦ ♦ ł ♦ ♦ ♦ Fragment 1 23 500 mm - ♦ ♦ ł ♦ ♦ ■ Fragment 2 21 500 m - m m♦m ♦ ł ♦

presence, - absence« - trace amount of fraction

T a b l e 2

Molecular weight of prothrombin and prothrombin activation products (activation in the preeence of DFP)

Ciężar cząsteczkowy protrombiny

i produktów jej aktywacji (proces aktywacji prowadzono w obecności DFP)

Protein Molecular weight Incubation 0 5 15 tin# 30 («ln») 60 120 Prothrombin (duck) 77 000 ♦ * ♦ ♦ ♦ t Fragment 1.2 45 000 *» •*♦ ♦ ♦ ♦ ♦ Thrombin 33 000 • ♦ ♦ ♦ ♦ ♦

The activation of duck prothrombin in a homologous system containing Factor Xa and calcium iona induces the appearance of degradation products. The analysis carried out by the PAGE-SDS (7,5/o) revealed the presence of an intermediate and th© final product-thrombin of molecular weight in the range of 21 500- -52 000 (Tab. 1j Fig. 4 - end of this volume, p. 192 and Fig. 5). On the basis of the results obtained we can assume that similarly to

F

• I* Prdhromb#*

T * Thrombin

Fig. 5. Densitometer scans of stained 1% SOS polyacrylamide gal electrophoresis (7.5%), 8 «A per gel, of the time course of the activation of duck prothrombin by Factor xa. Incubation times

were 0, 5, 15, 30, 60, 120, 240 min.

Wykreay denaytometryczne rozdziałów w 7,5% żelu pollekryloamido- wym, 8 «A ni rurkę, po aktywacji kaczej protrombiny czynnikiem Xa.

comraalian prothrombin. Factor Xa cleaves the eame kinds of bind-ing sites in duck prothrombin between Arg274-Thr275 and Ar9323~ -Ile^24 ( S e o g e r s et al. [Il] s M a g n u s s o n et al, [ 8 J: G r a n t et al. [6]). The appearance of Frag-ment 1 and FragFrag-ment 2 ae well as FragFrag-ment 1 and Intermedia-te 1, probably results from the fact that during the activation of duck prothrombin, generated thrombin splits the bonds be-tween Ar9156”Seri57* Addition of DFP into incubation mixture re-sulted in appearance of Fragment 1.2 and inactivated form of thrombin. Electrophoretic separation, densitometer scan« and mo-lecular weight of particular fragments are shown in Tab. 2, Fig. 6 - end of this volume, p. 192 and Fig. 7.

P - Prothrombin T - Thrombin FI-2 - Fragment 12 120 min ÔO min 30 min --9 min 0 min P FI-2 T

Fig. 7. Den3itomster scans of stained 1% SOS polyacrylamide gel electrophoresis (7.5?»), 8 mA per gel of the time course of ac-tivation duck prothrombin by Factor Xa in the presence of DFP <Inhibition of thrombin action). Incubation times were 0, 5, 15,

30, 60, 120 min.

iVykreoy densytorotryczne rozdziałów w 7,5% żelu poliakryloamido* wyfa, 8 mA na rurkę, po aktywacji kaczej protrombiny czynnikiem Xa w obecności OFP (.hamuje działanie trombiny). Czas inkubacji

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Department of Blochowi«try Instituto of Blochealatry and Blophyelco

Unlverelty of Lodz

Z. Banaś-Gruszka, T. Krajewski, B. Brotsznajdor AKYYWAC3A PROTROMBINY KACZE O CZYNNIKIEM Xa I T RO M B IT

Protroablnę otrzymywano za świeżego oaooza cytrynlanowego ka-czek (9 i 1) zgodni« z metody O r a n t a 1 S u t t i e [6], Uzyskane preparaty poddawano rechromatografll na DEAE Sep- hsdoxle A-50, uzyskujęc jednorodne pod względem elektroforotycz-nym blełko. Aktywację prowadzono czynnikiem Xa w obecności Jo-nów wapniowych. Czynnik X ektywowano enzyaea proteolitycznym z

Jadu węża (Russell e vlper venoa). Próbki prOtroablny lnkubo- wano z preparatem czynnike Xa 1 jonaal wapńIowyui w czasie 0,5, 15, 30, 60, 120, 240 aln. w 37°C. Reakcję przeryweno przez do-danie SOS do koócowego stężenia 1% 1 umieszczenie mieszaniny re-agującej w temp. 70 C na okree 5-10 min. Analizę produktów po-średnich pojawlajęcych eię podczaa konwersji protrombiny w troa- binę przeprowadzano za pomocę rozdziału w 7,5% żelu pollakryloa- midowya z SOS. Analiza przeprowadzona za poaocę PAGE-SOS wykaza-ła obecność w określonych przedziawykaza-łach czeeowych frakcji białko-wych o mesie cząsteczkowej około 21 500-52 000.