- Email: [email protected]
Amidolytic assay of factor V in human plasma
THROMBOSIS RESEARCH 41; 79-88, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Press ltd. All rights reserved.
AMIDOLYTIC
ASSAY OF FACTOR V IN HUMAN PLASMA
U. Hagglund and M. Blomb;ick Dept of Clinical Chemistry and Blood Coagulation, Karolinska Hospital; S-104 01 Stockholm, Sweden.
(Received 1.7.1985; Accepted in revised form 1.10.1985 by Editor H. Suomela)
ABSTRACT An amidolytic assay was developed for measuring factor V in human plasma. Diluted plasma is incubated at 370C with lipid; Taipan Snake Venom (Oxyuranus Ca++ Scutellatus Scuteliatus) and bovine prothrombin. The reaction is stopped after 4 min with EDTA. The content of factor V in samples is then determined by measuring the amidolytic activity of formed thrombin on synthetic ~~ .JY_ substrate S-2238, H-D-Pne_Pip_ cnromogenic peptiae Arg-pNA. The assay appears to be specific for factor V as: 1) human congenitally deficient FV plasma gives no activity in the system, 2) deficiency of factor II, VII; VIII; IX, X; XI or XII does not affect the deter3) no prothrombin is activated if mination of FV and the assay is run without venom. The correlation coefficient r between this amidolytic assay and a clotting coefficient of assay was 0.91 (n=20). The intra-assay variation was 3.7% for the amidolytic method.
INTRODUCTION Several methods have been suggested for the assay of factor V. Owren (1) originally devised a one-stage method in which the accelerating effect of factor V on the transformation of partially purified prothrombin to thrombin was measured in the presence of tissue thromboplastin and calcium. Quick and KEY WORDS: factor V, assay, Taipan Venom; chromogenic peptide substrate.
79
Snake
Venom,
Tiger
Snake
80
AMIDOLYTICASSAY OF FACTOR V
Vol. 41, No. 1
Stefanini (2) suggested a one-stage method based upon the observation that factor V rapidly dissappears from stored oxalated the ability plasma. In this method and its various modifications of the test plasma to reduce the prolonged prothrombin time of plasma is determined. two-stage factor V deficient Some techniques were also early described (3,4) in which the influence of factor V on the rate of thrombin formation from partially purified prothrombin was measured in the presence of tissue thromboplastin and calcium. the kinetics of thrombin formation in an BY studying intrinsic system composed of partially purified coagulation factors; a three-stage system for the assay of factor V could be developed (5). *en using this test system and an Australian tiger snake venom, Notechis Scutatus Scutatus, known to have it was found that this snake potent clot-promoting activity; venom was an activator for factor V (6). As chromogenic substrates for measurement of thrombin are now availably, it was considered of interest to investigate whether it was possible to devise a simpie method for factor V analysis. MATERIALS
AND
METHODS
Materials Bovine prothrombin was obtained as a concentrate from Kabi Vitrum Peptide Research; MBlndal; Sweden. It was prepared according to Pepper and Prowse (7); with some modifications. The concentrate was diluted with Tris-buffer and the final solution had an absorbance of 1.4 at 280 nm. Taipan Snake Venom (Oxyuranus Scutellatus Scutellatus) was obtained from Sigma Chemical Company (St. Louis, MO.). According thi x,annm p,ay ai fhar Frnm DranrrmTl to fhn L&A_ .m>nllf>~t,,rar ..U....LUICUL_L. ,_I&.&”E .UA1.,..1 be CA, LA&b& LL V... LUrUU" taipan or Australian taipan (Oxyuranus Scutellatus Scutellatus) or a mixture of both. It was diluted to 50 ug/ml. Tiger Snake venom (Notechis Scutatus Scutatus) was obtained as a gift from Commonwealth Serum Laboratories (Parkville 3052, Victoria, Australia). It was diluted to 250 ug/ml. The li id used was Platelet Substitute from Diagnostic ReasentZY?%i&ame, Oxon, England) and it was dissolved in distilled water according to the manufacturer's instructions. The volume needed has to be determined for every batch. In optimising the final method, it was found that 5-10 parts of dissolved lipid should be mixed with 5 parts of 50 mM CaC12 and 2 parts of the venom. Synthetic chromogenic peptide substrate S-2238, H-D-PhePip-ArgqAy ~~~~ was obtained from Kabi Vitrum PeptideResearch, Molndal; Sweden. S-2238 was diluted to 3.5 mM with distilled water. Factor deficient plasmas were obtained from Helena Laboratories (Beaumont, Texas). Other solutions used were 0.15 M Tris-HCl, pH 8.4 and 1=0.20 (adjusted with NaCl), Verona1 buffer and 0.1 M Na2-EDTA dissolved in Tris-buffer.
Vol. 41, No. 1
81
AMIDOLYTIC ASSAY OF FACTOR V
Venous blood samples were collected into plastic tubes containing as anticoagulant 0.13 M trisodium citrate x 2 H20 (one volume per nine volumes of blood). Plasma was immediately prepared by centrifuging the blood samples at 2200 x g for 30 min at room temperature and then frozen at -700C until assayed. Normal plasma was prepared by pooliny plasma samples from 15-20 subjectively healthy males. The spectrophotometer used was a Shimadzu model UV-240; provided with an Optional Program Unit model OPI-2; which could calculate the results in A/min. and test tubes were made of polystyrene and Cuvettes ----disposable. Final method A standard curve can be prepared for the range 0.50-1.62 the activity in 1 mL of normal pooled U/mLY where 1 unms plasma, by using the following dilutions: Dil. I. 2. 3. 4. 5.
50 50 50 50 50
iii -^--_'1 I,"llllcll plasma UL fi @I II UL I# UL " " II UL H
c 600 iiL ..----_, "cfL",lb.l buffer II + 750 UL II + 1000 UL I, + 1350 UL II + 2050 UL
= = = = =
I.62 1.31 1.00 0.75 0.50
T,/_T ",‘IIti u/mL U/mL u/mL U/mL
Store in an ice bath. Dilute 50 UL of test Test plasma: Verona1 buffer. Store in an ice bath.
plasma
with
1000
UL
Activation __---_---_- procedure _----_-_ Tris-buffer + bovine prothrombin (keep on ice until assay) Preheat 10 min at 370C
1000 UL 100 UL
+ lipid; calcium and venom mixture (preheated at least 10 min at 37oC) + diluted plasma (keep on ice until assay) Mix and incubate at 370C for exactly 240 set
120 UL
+ Na2-EDTA (room temperature) Mix immediately
1000 UL
50 UL
Use a fixed time (as short as possible) between pipetting of the venom solution and the diluted plasma. 120 UL venom solution is used when 5 parts of lipid are mixed with calcium (5 parts) and venom (2 parts). The volume of venom solution has to be changed when different parts of lipid are used. This solution can be preheated for at least 60 minutes at 370C. The volumes of the solutions can be decreased to 2/5 without significant loss of accuracy. Incubated samples can, if needed, be kept at room temperature for 1-2 hours before the amidolytic reaction.
AMIDOLYTIC ASSAY OF FACTOR V
Amidolytic reaction ----T-_-------__Tris-buffer 737oC) substrate S-2238 (37oc) aliquot of the activated sample Mix immediately Determine A/min (405 nm) at 370C
Vol. 41, No. 1
1000 UL 100 UL 50 UL
or incubate at 370C for exactly 60 set acetic acid; 50% Mix immediately Determine A (405 nm) against a reagent blank.
300 UL
For a standard curve see Fig. 1. RESULTS 0.4
”
<-
Activation conditions
and amidolytic
reaction
The conditions employed in the following optimising experiments were the same as in the final method unless otherwise specified. The venom from Oxyuranus Scutellatus Scutellatus (OSS) was used in the optimising steps.
Standard curve for endpoint assay of factor V.
Lipid concentration _- -__--_--__-_---_ The method was tested with different lipid volumes in the range O-200 UL (Fig. 2). In accordance with the result found when optimising the calcium concentration (Fig. 31, 50 UL lipid was chosen for the final method. However; when another lipid batch was used 100 UL was needed.
CaC12-concentration __-- -----r-------The influence of different Ca++-concentrations system was investigated (Fig. 3). In the final method, CaC12 is used for each sample. pH=deeendence _ -- ___--__ from 7.3 to pH was varied influence of pH on the activation well as on the reaction with S-2238 The pH-range 8.2-8.6 was found the final method pH 8.4 is chosen.
on the 2.5 mmol
The 9.6 with Tris-buffer. of prothrombin with OSS as was tested (Fig. 4). optimal in both steps and in
Ionic strength --------___The dependence upon the ionic strength for the activation of prothrombin and in the reaction with S-2238 was investigated using the ionic strength range 0.1-0.4 (adjusted with NaCl) in 0.15 M Tris-HCl, pH 8.4.
83
AMlDOLYTIC ASSAY OF FACTOR V
Vol. 41, No. 1
FIG. 2 The influence of different lipid concentrations. CaC12 as in final method. 0.5 ug venom was used for each sample. Ionic strength 0.15 and pH 8.1 in Trisbuffer. 2.0 U/mL of FV (-1 and 1.0 U/mL of FV !W! .
FIG.3 Influence of different CaC12 concentrations. 50 UL CaC12 (concentrations see Fig&) and 0.5 ug venom was used for each sample. Ionic strength 0.15 and pH 8.1 in Tris-buffer. 100 UL lipid + 2.0 U/mL of FV (-1; 50 UL lipid + 2.0 U/mL and 50 *uL limi" of FV (--4 r-L- + 1.0 u/mL of FV (o---o).
In the activation step with OSS, an optimum value was found when the ionic strength was 0.20. For the reaction with substrate; the absorbance reached the same level when the ionic strength was 0.20 or more. Buffer 0.15 M Tris-HC1; pH 8.4 and 1=0.20 were therefore chosen for the final method. Taipan Snake Venom; OSS --- ------_------_-___ Different amounts of OSS, from 0 up to 1.25 ug, were added to LX._^_^L1^.. -:..I..-C‘LC: ~e:QCl-LI"‘I 1111ALurC!. The maximal activation was obtained with 0.75 ug or more OSS and in the final method 1.0 ug is used for each sample. Activation time _---------__--Various activation times with normal plasma samples (Fig. 5).
OSS were
tested
on diluted
Vol. 41, No. 1
AMIDOLYTIC ASSAY OF FACTOR V
84
FIG. 5
FIG. 4
Influence of incubation time. All reagent conditions are as in the final method. 1.62 ~*OO ll,.“T I-, I...7 r.J= -_u ..__ ..--A m-...?. ^-.....-_-I^ u,11111 u, pn \-‘I, (2.0 U/mL of FV) was activated g; ;; I-_' and 0.25 U/mL M). with 0.5 ug OSS. Dependence on pH. 0.15 M Tris-buffer with ionic strength 0.15 and different
II
wa.3
USCU.
CIQLII
$7’;
lw_l
Pa‘llplt2
“L
In the sample with the highest factor V content (1.62 U/mL), the absorbance increased linearly with the incubation time up to at least six minutes while the lowest factor V concentration (0.25 U/mL) showed a linear relationship up to fifteen minutes. time of four minutes is For practical reasons; an activation used in the final method. Substrate S-2238 -___-_____-----_ ..TL_.-
wnen
L3.-
one
__LL_-_Y
mernou
._--
was
.____r---_a
perrormea
51s hitl eii6-polnr:
---___ asscly,
IL AL
WdS ---
found sufficient to use 100 UL of a 3.5 mM S-2238 solution. The concentration of the substrate can be lowered if the method is used as a kinetic assay. Validity of the method Various dilutions were made with normal plasma and congenital factor V deficient plasma. The same activity was registered as when normal plasma was diluted with buffer. As will be seen from Table I; none of the tested deficient plasmas differed noticeably from the results obtained with the clotting method according to Kappeler (8).
Vol.
41,
No.
1
AMIDOLYTIC ASSAY
TABLE
OF FACTOR
V
85
I
Effect of coagulation factor deficient plasmas on factor V determination with the amidolytic and clotting methods Plasma
-------
Human F Human F Human F Human F Human F Human F Patient Patient Patient Patient
--.--‘-7Amidolytic Clotting method method rr/_* ,.I-",llur --~ u/m
VII deficient (congenital*) VIII deficient (congenital*) IX deficient (congenital*) X deficient (congenital*) XI deficient (congenital*) XII deficient (congenital*) with F X 0.2 U/ml(congenital) treated with coumarol; TT=68 treated with coumarol; TT=98 treated with coumarol; TT=16%
-
0.92 0;82 1.04 0.96 0.61 0.81 0.94 1.01 0.91 1.09
0.80 0.87 0.95 1.00 0.58 0.87 0.95 0.85 0.86 0.98 -----?I-* Obtained from Helena Laboratories: TT= Thrombotest(prothrombin complex assay w. reagents from Nyegaard; Norway). from sixteen In samples subjectively healthy males and females and from four congenitally FV deficient patients, the factor V activity was assayed with a clotting assay and the amidolytic assay (Fig. 6). The correlation coefficient between these two methods was 0.91. The mean level of factor V in the sixteen normal individuals was 0.93+0.14 U/mL fit-nod and wit-n the clotting 0.98+0.16 U/mL with the amidolytic meth%d. The four patients had a mean value of 0.30+0.16 U/mL with the clotting method and 0.27+0.15 U/mL with the amidolytic assay. FIG. 6 Correlation between FV amidolytic assay and FV clotting assay in sixteen normal males and females and in four congenitally deficient natientn. ~------__- r-_--__-__
The present method can be used both as a kinetic assay and as an end-point assay. A correlation coefficient of 0.97 was found (n=52). The mean value of factor V was 0.90+0.15 U/mL for both_ types of assays,
As can be deduced from Fig. 1 and 6 the curve is linear 0.5 U/ml and the lower detection limit will be near zero. An intra-assay study was made. The coefficient of variation was 3.7%; when the mean value of factor V was 0.79 U/mL (n=15). below
AMIDOLYTIC ASSAY OF FACTOR V
vol. 41, No. 1
If the method is run without activator, i.e. snake venom, no amidolytic effect will be found in the reaction with substrate S-2238. The activator itself was found to have almost no amidolytic effect on substrate S-2238. ,.+A,.:.-.:-.. -zY the m,et-/.dto It was four;d .-.rrrr.+t:-1 SJ3C;IILIaIfor th,e pL=;C.L>L"L‘ "I pipette the plasma dilution into the test-tube with an exact time interval; as short as possible in relation to the lipidcalcium-venom solution. Some samples containing less than 0.7 U/mL heparin have been tested and it seems that the result will be only weakly affected if at all. DISCUSSION A method has been developed for amidolytic assay of factor V. In the final step, the amount of activated prothrombin is allowed to act on synthetic chromogenic thrombin substrate S-2238. The method appears to be specific for factor V as human congenitally deficient FV plasma gives no activity in the system. Furthermore, dilutions of deficient FV plasma and normal pooled plasma activate prothrombin according to the content of FV in the dilution. If the assay is run without the venom from Oxyuranus Scutellatus Scutellatus; no prothrombin will be activated. In the absence of calcium or lipid, only a low amidolytic activity will be found on S-2238 and these reagents are therefore necessary for the reaction. Deficiency of factor 11; VII, VIII; IX, X, XI or XII does not affect the activation of prothrombin, as was shown in a comparative study with a clotting assay (Table I). It seems as if the snake venom (OSS) acts as factor Xa on prothrombin; especially as no factor X is needed in the plasma or has to be added. Waiker (9j aiso found that the prothrombin activator from this venom and factor Xa act through similar mechanisms. Owen (10) showed that the split products of prothrombin after complete activation with this venom (OSS) are indistinguishable from the products of factor Xa-catalysed prothrombin activation. Pirkle (11) used this venom (OSS) in an assay for purified prothrombin; developed by him. Later on, Owen (10) found that the rate of activation of prothrombin with the venom from Oxyuranus Scutellatus Scutellatus in calcium solution was stimulated by phospholipid but was unaffected by factor V. Differences from our work are that Pirkle (11) and Owen (10) used more calcium and venom and a longer incubation time. A_t th_e s;rm_etime a8 our rnrel_-_-..__iminarv --~ re-lts (171 \*-, wcy~, .._-reported; Kahl& (13) gave a preliminary report on an amidolytic assay for factor V. In his system; the activator is the venom from Notechis Scutatus Scutatus. This is in agreement with our earlier results (5). We have analysed the most important properties of this venom in our present amidolytic assay and found that it behaves in the same way as Oxyuranus Scutellatus Scutellatus. An amount of 5 ug is equivalent to 1.0 ug OSS in activity.
Vol. 41, No. 1
AMIDOLYTIC ASSAY OF FACTOR V
87
In his study of coagulant and anticoagulant actions of Australian snake venoms; Marshall (14) confirmed that most Australian venoms are prothrombin activators with variable dependence on the presence of factor V; phospholipid and calcium. In agreement with our study; his results showed that factors VII; VIII and IX are not involved in the actions of the venoms (including Oxyuranus Scutellatus Scutellatus and Notechis Scutatus Scutatus) and there seems to be no evidence that factor X is necessary for the coagulant action of the venoms. ACKNOWLEDGEMENTS The authors wish to thank Miss Annika Lindblom and Mrs Airi As&n for valuable assistance with the additional analyses. This investigation was supported by funds from the Swedish Medical Research Council (19X-520). REFERENCES 1.
OWREN; P.A.: The coagulation of blood. Investigations on a new clotting factor. Acta._____ Med. Stand. Suppl. 194, l-327, ___ 1947.
2.
of the M.: The conentration QUICK; A.J. and STEFANINI; labile factor of the prothrombin complex in human; dog; an in the determianton of rabbit blood; its significance prothrombin activity. J. Lab. Clin. Med. 33; 819-826, 1948.
3.
globulin: WARE; A.G. and SEEGERS; W.H.: Plasma accelerator quantitative determination; and partial purification; properties. J. Biol. Chem. 172; 699-705, 1948.
4.
SURGENOR; D.M.; WILSON; N.A. and HENRY; A.S.: Factor V from human plasma. Thromb. Diath. Haemorrh. (Stuttg.) 2; l-20, 1961.
5.
BLOMBACK; B. and BLOMBACK; M.: A method for the assay of factor V. Stand. J. Clin. Lab. Invest. -15; 639-648, 1963.
6.
BLOMBACK, B. and BLOMBACK; M.: To the discussion on genetics and the interaction of blood clotting factors. Thromb. Diath; Haemorrh. Suppl 17; 59-62; 1965.
7.
PEPPER; D.S. and PROWSE; C.: prothrombin complex on dextran Res. 11, 687-692, 1977. --
8.
von Faktor KAPPELER, R.: Das Verhalten normalen und patologischen Bedingungen. 153; 103-113; 1955.
9.
of the prothrombin WALKER, F.J. et al: Characterization Scutellatus Oxvuranus activator from the venom of Scutellatus (Taipan venom). Biochemistry -19, 1020-1023, 1980.
Chromatography of human sulphate agarose. Thromb. V in Serum unter Zschr. Klin. Med.
88
Vol. 41, No. 1
AMIDOLVTIC ASSAY OF FACTOR V
10. OWEN; W.G. and JACKSON, C.M.: Activation of prothrombin with Oxyuranus Scutellatus Scutellatus (Taipan Snake) venom. Thromb. Res. 2, 705-714, 1973. 11; PIRKLE; H. et al: Activation of prothrombin Snake Venom. Thromb. Res. 1; 559-568, 1972.
with
Taipan
12. HAGGLUND; U. and BLOMBACK, M.: Amidolytic assay of factor v in human plasma. New Frontiers in Hemophilia Research. XVth World Federation of Hemophilia Congress.243 (Abstr.), 1983. 13. KAHLE; L.H.: A simple two stage amidolytic assay V. Thromb. Haemost. -50, 0833 (Abstr.), 1983
for
factor
14. MARSHALL, L.R. and HERRMANN R.P.: Coagulant and anticoagulant actions of Australian Snake Venoms. Thromb. Haemost. -50, 707-711, 1983.
AMIDOLYTIC
ASSAY OF FACTOR V IN HUMAN PLASMA
U. Hagglund and M. Blomb;ick Dept of Clinical Chemistry and Blood Coagulation, Karolinska Hospital; S-104 01 Stockholm, Sweden.
(Received 1.7.1985; Accepted in revised form 1.10.1985 by Editor H. Suomela)
ABSTRACT An amidolytic assay was developed for measuring factor V in human plasma. Diluted plasma is incubated at 370C with lipid; Taipan Snake Venom (Oxyuranus Ca++ Scutellatus Scuteliatus) and bovine prothrombin. The reaction is stopped after 4 min with EDTA. The content of factor V in samples is then determined by measuring the amidolytic activity of formed thrombin on synthetic ~~ .JY_ substrate S-2238, H-D-Pne_Pip_ cnromogenic peptiae Arg-pNA. The assay appears to be specific for factor V as: 1) human congenitally deficient FV plasma gives no activity in the system, 2) deficiency of factor II, VII; VIII; IX, X; XI or XII does not affect the deter3) no prothrombin is activated if mination of FV and the assay is run without venom. The correlation coefficient r between this amidolytic assay and a clotting coefficient of assay was 0.91 (n=20). The intra-assay variation was 3.7% for the amidolytic method.
INTRODUCTION Several methods have been suggested for the assay of factor V. Owren (1) originally devised a one-stage method in which the accelerating effect of factor V on the transformation of partially purified prothrombin to thrombin was measured in the presence of tissue thromboplastin and calcium. Quick and KEY WORDS: factor V, assay, Taipan Venom; chromogenic peptide substrate.
79
Snake
Venom,
Tiger
Snake
80
AMIDOLYTICASSAY OF FACTOR V
Vol. 41, No. 1
Stefanini (2) suggested a one-stage method based upon the observation that factor V rapidly dissappears from stored oxalated the ability plasma. In this method and its various modifications of the test plasma to reduce the prolonged prothrombin time of plasma is determined. two-stage factor V deficient Some techniques were also early described (3,4) in which the influence of factor V on the rate of thrombin formation from partially purified prothrombin was measured in the presence of tissue thromboplastin and calcium. the kinetics of thrombin formation in an BY studying intrinsic system composed of partially purified coagulation factors; a three-stage system for the assay of factor V could be developed (5). *en using this test system and an Australian tiger snake venom, Notechis Scutatus Scutatus, known to have it was found that this snake potent clot-promoting activity; venom was an activator for factor V (6). As chromogenic substrates for measurement of thrombin are now availably, it was considered of interest to investigate whether it was possible to devise a simpie method for factor V analysis. MATERIALS
AND
METHODS
Materials Bovine prothrombin was obtained as a concentrate from Kabi Vitrum Peptide Research; MBlndal; Sweden. It was prepared according to Pepper and Prowse (7); with some modifications. The concentrate was diluted with Tris-buffer and the final solution had an absorbance of 1.4 at 280 nm. Taipan Snake Venom (Oxyuranus Scutellatus Scutellatus) was obtained from Sigma Chemical Company (St. Louis, MO.). According thi x,annm p,ay ai fhar Frnm DranrrmTl to fhn L&A_ .m>nllf>~t,,rar ..U....LUICUL_L. ,_I&.&”E .UA1.,..1 be CA, LA&b& LL V... LUrUU" taipan or Australian taipan (Oxyuranus Scutellatus Scutellatus) or a mixture of both. It was diluted to 50 ug/ml. Tiger Snake venom (Notechis Scutatus Scutatus) was obtained as a gift from Commonwealth Serum Laboratories (Parkville 3052, Victoria, Australia). It was diluted to 250 ug/ml. The li id used was Platelet Substitute from Diagnostic ReasentZY?%i&ame, Oxon, England) and it was dissolved in distilled water according to the manufacturer's instructions. The volume needed has to be determined for every batch. In optimising the final method, it was found that 5-10 parts of dissolved lipid should be mixed with 5 parts of 50 mM CaC12 and 2 parts of the venom. Synthetic chromogenic peptide substrate S-2238, H-D-PhePip-ArgqAy ~~~~ was obtained from Kabi Vitrum PeptideResearch, Molndal; Sweden. S-2238 was diluted to 3.5 mM with distilled water. Factor deficient plasmas were obtained from Helena Laboratories (Beaumont, Texas). Other solutions used were 0.15 M Tris-HCl, pH 8.4 and 1=0.20 (adjusted with NaCl), Verona1 buffer and 0.1 M Na2-EDTA dissolved in Tris-buffer.
Vol. 41, No. 1
81
AMIDOLYTIC ASSAY OF FACTOR V
Venous blood samples were collected into plastic tubes containing as anticoagulant 0.13 M trisodium citrate x 2 H20 (one volume per nine volumes of blood). Plasma was immediately prepared by centrifuging the blood samples at 2200 x g for 30 min at room temperature and then frozen at -700C until assayed. Normal plasma was prepared by pooliny plasma samples from 15-20 subjectively healthy males. The spectrophotometer used was a Shimadzu model UV-240; provided with an Optional Program Unit model OPI-2; which could calculate the results in A/min. and test tubes were made of polystyrene and Cuvettes ----disposable. Final method A standard curve can be prepared for the range 0.50-1.62 the activity in 1 mL of normal pooled U/mLY where 1 unms plasma, by using the following dilutions: Dil. I. 2. 3. 4. 5.
50 50 50 50 50
iii -^--_'1 I,"llllcll plasma UL fi @I II UL I# UL " " II UL H
c 600 iiL ..----_, "cfL",lb.l buffer II + 750 UL II + 1000 UL I, + 1350 UL II + 2050 UL
= = = = =
I.62 1.31 1.00 0.75 0.50
T,/_T ",‘IIti u/mL U/mL u/mL U/mL
Store in an ice bath. Dilute 50 UL of test Test plasma: Verona1 buffer. Store in an ice bath.
plasma
with
1000
UL
Activation __---_---_- procedure _----_-_ Tris-buffer + bovine prothrombin (keep on ice until assay) Preheat 10 min at 370C
1000 UL 100 UL
+ lipid; calcium and venom mixture (preheated at least 10 min at 37oC) + diluted plasma (keep on ice until assay) Mix and incubate at 370C for exactly 240 set
120 UL
+ Na2-EDTA (room temperature) Mix immediately
1000 UL
50 UL
Use a fixed time (as short as possible) between pipetting of the venom solution and the diluted plasma. 120 UL venom solution is used when 5 parts of lipid are mixed with calcium (5 parts) and venom (2 parts). The volume of venom solution has to be changed when different parts of lipid are used. This solution can be preheated for at least 60 minutes at 370C. The volumes of the solutions can be decreased to 2/5 without significant loss of accuracy. Incubated samples can, if needed, be kept at room temperature for 1-2 hours before the amidolytic reaction.
AMIDOLYTIC ASSAY OF FACTOR V
Amidolytic reaction ----T-_-------__Tris-buffer 737oC) substrate S-2238 (37oc) aliquot of the activated sample Mix immediately Determine A/min (405 nm) at 370C
Vol. 41, No. 1
1000 UL 100 UL 50 UL
or incubate at 370C for exactly 60 set acetic acid; 50% Mix immediately Determine A (405 nm) against a reagent blank.
300 UL
For a standard curve see Fig. 1. RESULTS 0.4
”
<-
Activation conditions
and amidolytic
reaction
The conditions employed in the following optimising experiments were the same as in the final method unless otherwise specified. The venom from Oxyuranus Scutellatus Scutellatus (OSS) was used in the optimising steps.
Standard curve for endpoint assay of factor V.
Lipid concentration _- -__--_--__-_---_ The method was tested with different lipid volumes in the range O-200 UL (Fig. 2). In accordance with the result found when optimising the calcium concentration (Fig. 31, 50 UL lipid was chosen for the final method. However; when another lipid batch was used 100 UL was needed.
CaC12-concentration __-- -----r-------The influence of different Ca++-concentrations system was investigated (Fig. 3). In the final method, CaC12 is used for each sample. pH=deeendence _ -- ___--__ from 7.3 to pH was varied influence of pH on the activation well as on the reaction with S-2238 The pH-range 8.2-8.6 was found the final method pH 8.4 is chosen.
on the 2.5 mmol
The 9.6 with Tris-buffer. of prothrombin with OSS as was tested (Fig. 4). optimal in both steps and in
Ionic strength --------___The dependence upon the ionic strength for the activation of prothrombin and in the reaction with S-2238 was investigated using the ionic strength range 0.1-0.4 (adjusted with NaCl) in 0.15 M Tris-HCl, pH 8.4.
83
AMlDOLYTIC ASSAY OF FACTOR V
Vol. 41, No. 1
FIG. 2 The influence of different lipid concentrations. CaC12 as in final method. 0.5 ug venom was used for each sample. Ionic strength 0.15 and pH 8.1 in Trisbuffer. 2.0 U/mL of FV (-1 and 1.0 U/mL of FV !W! .
FIG.3 Influence of different CaC12 concentrations. 50 UL CaC12 (concentrations see Fig&) and 0.5 ug venom was used for each sample. Ionic strength 0.15 and pH 8.1 in Tris-buffer. 100 UL lipid + 2.0 U/mL of FV (-1; 50 UL lipid + 2.0 U/mL and 50 *uL limi" of FV (--4 r-L- + 1.0 u/mL of FV (o---o).
In the activation step with OSS, an optimum value was found when the ionic strength was 0.20. For the reaction with substrate; the absorbance reached the same level when the ionic strength was 0.20 or more. Buffer 0.15 M Tris-HC1; pH 8.4 and 1=0.20 were therefore chosen for the final method. Taipan Snake Venom; OSS --- ------_------_-___ Different amounts of OSS, from 0 up to 1.25 ug, were added to LX._^_^L1^.. -:..I..-C‘LC: ~e:QCl-LI"‘I 1111ALurC!. The maximal activation was obtained with 0.75 ug or more OSS and in the final method 1.0 ug is used for each sample. Activation time _---------__--Various activation times with normal plasma samples (Fig. 5).
OSS were
tested
on diluted
Vol. 41, No. 1
AMIDOLYTIC ASSAY OF FACTOR V
84
FIG. 5
FIG. 4
Influence of incubation time. All reagent conditions are as in the final method. 1.62 ~*OO ll,.“T I-, I...7 r.J= -_u ..__ ..--A m-...?. ^-.....-_-I^ u,11111 u, pn \-‘I, (2.0 U/mL of FV) was activated g; ;; I-_' and 0.25 U/mL M). with 0.5 ug OSS. Dependence on pH. 0.15 M Tris-buffer with ionic strength 0.15 and different
II
wa.3
USCU.
CIQLII
$7’;
lw_l
Pa‘llplt2
“L
In the sample with the highest factor V content (1.62 U/mL), the absorbance increased linearly with the incubation time up to at least six minutes while the lowest factor V concentration (0.25 U/mL) showed a linear relationship up to fifteen minutes. time of four minutes is For practical reasons; an activation used in the final method. Substrate S-2238 -___-_____-----_ ..TL_.-
wnen
L3.-
one
__LL_-_Y
mernou
._--
was
.____r---_a
perrormea
51s hitl eii6-polnr:
---___ asscly,
IL AL
WdS ---
found sufficient to use 100 UL of a 3.5 mM S-2238 solution. The concentration of the substrate can be lowered if the method is used as a kinetic assay. Validity of the method Various dilutions were made with normal plasma and congenital factor V deficient plasma. The same activity was registered as when normal plasma was diluted with buffer. As will be seen from Table I; none of the tested deficient plasmas differed noticeably from the results obtained with the clotting method according to Kappeler (8).
Vol.
41,
No.
1
AMIDOLYTIC ASSAY
TABLE
OF FACTOR
V
85
I
Effect of coagulation factor deficient plasmas on factor V determination with the amidolytic and clotting methods Plasma
-------
Human F Human F Human F Human F Human F Human F Patient Patient Patient Patient
--.--‘-7Amidolytic Clotting method method rr/_* ,.I-",llur --~ u/m
VII deficient (congenital*) VIII deficient (congenital*) IX deficient (congenital*) X deficient (congenital*) XI deficient (congenital*) XII deficient (congenital*) with F X 0.2 U/ml(congenital) treated with coumarol; TT=68 treated with coumarol; TT=98 treated with coumarol; TT=16%
-
0.92 0;82 1.04 0.96 0.61 0.81 0.94 1.01 0.91 1.09
0.80 0.87 0.95 1.00 0.58 0.87 0.95 0.85 0.86 0.98 -----?I-* Obtained from Helena Laboratories: TT= Thrombotest(prothrombin complex assay w. reagents from Nyegaard; Norway). from sixteen In samples subjectively healthy males and females and from four congenitally FV deficient patients, the factor V activity was assayed with a clotting assay and the amidolytic assay (Fig. 6). The correlation coefficient between these two methods was 0.91. The mean level of factor V in the sixteen normal individuals was 0.93+0.14 U/mL fit-nod and wit-n the clotting 0.98+0.16 U/mL with the amidolytic meth%d. The four patients had a mean value of 0.30+0.16 U/mL with the clotting method and 0.27+0.15 U/mL with the amidolytic assay. FIG. 6 Correlation between FV amidolytic assay and FV clotting assay in sixteen normal males and females and in four congenitally deficient natientn. ~------__- r-_--__-__
The present method can be used both as a kinetic assay and as an end-point assay. A correlation coefficient of 0.97 was found (n=52). The mean value of factor V was 0.90+0.15 U/mL for both_ types of assays,
As can be deduced from Fig. 1 and 6 the curve is linear 0.5 U/ml and the lower detection limit will be near zero. An intra-assay study was made. The coefficient of variation was 3.7%; when the mean value of factor V was 0.79 U/mL (n=15). below
AMIDOLYTIC ASSAY OF FACTOR V
vol. 41, No. 1
If the method is run without activator, i.e. snake venom, no amidolytic effect will be found in the reaction with substrate S-2238. The activator itself was found to have almost no amidolytic effect on substrate S-2238. ,.+A,.:.-.:-.. -zY the m,et-/.dto It was four;d .-.rrrr.+t:-1 SJ3C;IILIaIfor th,e pL=;C.L>L"L‘ "I pipette the plasma dilution into the test-tube with an exact time interval; as short as possible in relation to the lipidcalcium-venom solution. Some samples containing less than 0.7 U/mL heparin have been tested and it seems that the result will be only weakly affected if at all. DISCUSSION A method has been developed for amidolytic assay of factor V. In the final step, the amount of activated prothrombin is allowed to act on synthetic chromogenic thrombin substrate S-2238. The method appears to be specific for factor V as human congenitally deficient FV plasma gives no activity in the system. Furthermore, dilutions of deficient FV plasma and normal pooled plasma activate prothrombin according to the content of FV in the dilution. If the assay is run without the venom from Oxyuranus Scutellatus Scutellatus; no prothrombin will be activated. In the absence of calcium or lipid, only a low amidolytic activity will be found on S-2238 and these reagents are therefore necessary for the reaction. Deficiency of factor 11; VII, VIII; IX, X, XI or XII does not affect the activation of prothrombin, as was shown in a comparative study with a clotting assay (Table I). It seems as if the snake venom (OSS) acts as factor Xa on prothrombin; especially as no factor X is needed in the plasma or has to be added. Waiker (9j aiso found that the prothrombin activator from this venom and factor Xa act through similar mechanisms. Owen (10) showed that the split products of prothrombin after complete activation with this venom (OSS) are indistinguishable from the products of factor Xa-catalysed prothrombin activation. Pirkle (11) used this venom (OSS) in an assay for purified prothrombin; developed by him. Later on, Owen (10) found that the rate of activation of prothrombin with the venom from Oxyuranus Scutellatus Scutellatus in calcium solution was stimulated by phospholipid but was unaffected by factor V. Differences from our work are that Pirkle (11) and Owen (10) used more calcium and venom and a longer incubation time. A_t th_e s;rm_etime a8 our rnrel_-_-..__iminarv --~ re-lts (171 \*-, wcy~, .._-reported; Kahl& (13) gave a preliminary report on an amidolytic assay for factor V. In his system; the activator is the venom from Notechis Scutatus Scutatus. This is in agreement with our earlier results (5). We have analysed the most important properties of this venom in our present amidolytic assay and found that it behaves in the same way as Oxyuranus Scutellatus Scutellatus. An amount of 5 ug is equivalent to 1.0 ug OSS in activity.
Vol. 41, No. 1
AMIDOLYTIC ASSAY OF FACTOR V
87
In his study of coagulant and anticoagulant actions of Australian snake venoms; Marshall (14) confirmed that most Australian venoms are prothrombin activators with variable dependence on the presence of factor V; phospholipid and calcium. In agreement with our study; his results showed that factors VII; VIII and IX are not involved in the actions of the venoms (including Oxyuranus Scutellatus Scutellatus and Notechis Scutatus Scutatus) and there seems to be no evidence that factor X is necessary for the coagulant action of the venoms. ACKNOWLEDGEMENTS The authors wish to thank Miss Annika Lindblom and Mrs Airi As&n for valuable assistance with the additional analyses. This investigation was supported by funds from the Swedish Medical Research Council (19X-520). REFERENCES 1.
OWREN; P.A.: The coagulation of blood. Investigations on a new clotting factor. Acta._____ Med. Stand. Suppl. 194, l-327, ___ 1947.
2.
of the M.: The conentration QUICK; A.J. and STEFANINI; labile factor of the prothrombin complex in human; dog; an in the determianton of rabbit blood; its significance prothrombin activity. J. Lab. Clin. Med. 33; 819-826, 1948.
3.
globulin: WARE; A.G. and SEEGERS; W.H.: Plasma accelerator quantitative determination; and partial purification; properties. J. Biol. Chem. 172; 699-705, 1948.
4.
SURGENOR; D.M.; WILSON; N.A. and HENRY; A.S.: Factor V from human plasma. Thromb. Diath. Haemorrh. (Stuttg.) 2; l-20, 1961.
5.
BLOMBACK; B. and BLOMBACK; M.: A method for the assay of factor V. Stand. J. Clin. Lab. Invest. -15; 639-648, 1963.
6.
BLOMBACK, B. and BLOMBACK; M.: To the discussion on genetics and the interaction of blood clotting factors. Thromb. Diath; Haemorrh. Suppl 17; 59-62; 1965.
7.
PEPPER; D.S. and PROWSE; C.: prothrombin complex on dextran Res. 11, 687-692, 1977. --
8.
von Faktor KAPPELER, R.: Das Verhalten normalen und patologischen Bedingungen. 153; 103-113; 1955.
9.
of the prothrombin WALKER, F.J. et al: Characterization Scutellatus Oxvuranus activator from the venom of Scutellatus (Taipan venom). Biochemistry -19, 1020-1023, 1980.
Chromatography of human sulphate agarose. Thromb. V in Serum unter Zschr. Klin. Med.
88
Vol. 41, No. 1
AMIDOLVTIC ASSAY OF FACTOR V
10. OWEN; W.G. and JACKSON, C.M.: Activation of prothrombin with Oxyuranus Scutellatus Scutellatus (Taipan Snake) venom. Thromb. Res. 2, 705-714, 1973. 11; PIRKLE; H. et al: Activation of prothrombin Snake Venom. Thromb. Res. 1; 559-568, 1972.
with
Taipan
12. HAGGLUND; U. and BLOMBACK, M.: Amidolytic assay of factor v in human plasma. New Frontiers in Hemophilia Research. XVth World Federation of Hemophilia Congress.243 (Abstr.), 1983. 13. KAHLE; L.H.: A simple two stage amidolytic assay V. Thromb. Haemost. -50, 0833 (Abstr.), 1983
for
factor
14. MARSHALL, L.R. and HERRMANN R.P.: Coagulant and anticoagulant actions of Australian Snake Venoms. Thromb. Haemost. -50, 707-711, 1983.