- Email: [email protected]
Preparation of β-XIIa (Hageman factor fragment) from human plasma
TEROYiCSIS RESE%C1.C:;. 25; 307-317, 1982 !~0~9-38,8!82;040337-i~~03.~~~~ ?rinted in t:he !:Si\. Copyright (c) 1982 Pergamcn ?ress Ltd. ~11 rights reserved.
?RE?AR_~TIO:JOF 3-XIIa (HACE:lcL': FACTOR FRAQIENT) FROEI HU?lrV' . . PUS">1
Donald L. Tankersley, Barbara !l. Alving* and J. S. Finlayson Bureau of Biologics, Food and Drug Administration, Bethesda, :ID 20205, USA *Walter Reed Army Institute of Research, Washington, DC 20012, USA
(Received 14.9.1981; in revised form 14.12.1981. Accepted by Editor G.A. Jamieson) ABSTRACT B-Factor XILa (S-XIIa, !I n,30,000) was isolated from hunan plasma by a procedure which uti I* ited, as an initial step, the adsorption Activation of Factor XII and subsequent of Factor XII to celite. release of S-XIIa was brought about by the proteolytic action of Two successive chromatographic procedures co-adsorbed kallikrein. were then used to achieve a final purification of 4,420-fold over plasma and an overall yield of 2.3 mg of 8-XIIa per liter.
INTRODUCTION 8-XIIa (Hageman factor fragment; PKA) is derived from Factor XII (Hagernan It is a potent factor, !l, 80,000) by the proteolytic action of kallikrein. 80,000) activator of prekallikrein (PK) but, unlike a-Factor XIIa (cl-XIIa,& has little or no ability to activate Factor XI (for a review, see reference 1). The occurrence of B-XIIa in therapeutic protein solutions prepared from human plasma (2-4) may result in adverse reactions, most notably hypotension In the course of our studies on the pharmacologic and enzymic 2,536). properties of 8-XIIa, it became necessary to prepare rather large (i.e., 10 mg) quantities of the purified enzyme. Published procedures (T-10) for its preparation from human plasma were not entirely satisfactory, inasmuch as large-volume chromatographic procedures were usually required. This paper describes a convenient, high-yield procedure for the preparation The convenience and novelty of this of highly purified (>95X) B-XIIa. procedure lie in the initial adsorption-activation step, which affords a 40- to 200-fold purification and a S- to lo-fold concentration over plasma. The subsequent chromatographic purification steps are therefore greatly simplified.
Keywords:
Hagenan factor, Factor XII, prekallikrein. 307
For preparativ? prac?dares, h~an plss1.a ~;as obtained from the :iIiH :iaterials. 30th freshly frozen, platelet-poor .ACD plasma and outdated plasma biood bank. (stored as whole blood for 21 da:;s at 4 ‘C befor? separation of the ?laSi.la) To compare different adsorption-activation conditions, a reference retire used. plasma pool, prepared iz the fo!lowing i!an;lrr, was employed. Blood ‘Gas drawn froi.1 four liealthy subjects into plastic syringes and immediately mixed with li9 volui,le of 3.32 sodiun citrate dihydrate. After centrifugation, the plasmas were pooled, aliquoted, and stored at -70 “C. Deficient plasmas !ii;:h-purity Cl--inhibitor were obtained from George King, Overland “ark, KS. was kindly provided by 11. Gickerhauser, American Red Cross. Antithro[.lbin III was furnished by G. ilurano, Bureau of Biologics. B-XII.? inhibitor (AT III) isolated frol,l popcorn (11) izas provided by ‘i. tio jir:ia, YIILBI. The chr~ogenic substrate S-2302 (3-Pro-Phe-Arg p-nitroanilide) vas obtained from I:abi Soybean trypsin inhibitor (SBTI)(\:orthington,)) Diagnostica, Sweden. diisopropylphosphorofluoridate (DFP)(Sigr,la), celite (llyflo Supercel, Johns DEAT.-Sephadex A-50, SP-Sephadex C-50, Con-A Sepharose, and Sephadex :Ianville), G-150 (Pharl.lacia) i;ere purchased from the sources indicated. Other chemicals were the best available quality. Prckallikrein (PK) of >95% purity was isolated frol,l freshly frozen, titrated hur.lan plasma by sequential chromatography on DEAL-Sephadex A-50 and Con ASepharuse as described previously (9)) followed by further purification on SP-Sephadex C-50 (12) and Sephadex C-150. The product hydrolyzed S-2302 at a rate of 173 uciol~nin-l~mg-l following activation by Z-SIIa and had no detectable activity toward this substrate without prior activation. The r-XIIa used for irnmunoelectrophoretic studies ijas prepared by cilromatography of titrated human plasma on DUG-Scphadcx A-50 and C:I-Sephadex C-50 activation of Factor XII occurred during the latter procedure. Final (13); purification (~85,~:) was achieved by Seplladex G-150 chromatography. Antiserum to B-XIIa was prepared in rabbits; the 3-::IIa was inactivated with DFP, and primary i[zlmunization used 60 ug DIP-B-XIIa in complete Freund’s The rabbits were boosted after 14 and 28 days with the same adjuvant. quantity of DIP-S-XLIa in incomplete Freund’s adjuvant, and serum was Slight cross-reactivity to hur.:an albumin was noted and collected on day 35. was removed by absorption with l/10 volulle of Factor XII-deficient plasma. Antiserum (goat) to human Factor XII was furnished by J. Griffin (Scripps La Jolla, CA); this antiserum demonstrated cross-reactivity to CLinic, which was also removed by absorption with Factor XIIa27”acroglobulin, deficient plasr.la.
Protein concentrations were determined spectrophotonctrically; the Assays. absorption coefficients (a280) used were 1.21 for PK and 1.60 for 3-XILa. These values were deterl,liGed by comparison of the A280 and differential refractive index (in546) of solutions of the purified proteins. A specific refractive increl‘lcnt of 0.186 ml/g was ossuned. Similar 5280 values (1.1s for Pb, 1.55 for B-XIIa) were deterl.hined by comparing the A280 with the results of a Lowry assay in which bovine albur.lin was used as the standard. Enzyme activities of kallikrein and B-SIIa were determined with 0.5 dl S-2302 in 0.05 il Tris-HCl, 0.05 [I iiaC1 buffer (pll 8.0)(Tris-IICl buffer). The sar,ple (10 21, diluted as necessary in Tris-IICI buffer which contained 5 ng
albuninisl) , in absoroance
uas added to 4911 ~1 of substrate solution 3: 405 nn with tine was recorded. The
calculated
froi.:
the
_Aigj/K!ihute
observed
by
using
at rate
30 “C and the of hydrolysis 9,7OC
s4Qj=
change -as
for
Activities were expressed as -%oles hydrolyzed per minute. p-nitroaniline. Fixtures containing both kallikrein and 3-XIIa (i.e., celite eluates) were analyzed by measuring the rate of !:ydrolysis of S-2302 in the presence and absence of SBTI (final concentration 40 :g/:.il), which conpletely inhibits kallikrein but has only slight (
not
(concentration
in
incubate,
30
which
zginl)(ll),
inhibits
3-XIIa
kallikrein. centrifugation
;\ mall-scale, with celite
was
yield of S-XIIa. 600 ul of plasma (nias incubated at
procedure
used to iuvestigate Organic solvent
for
adsorption-activation
the effect (or buffer),
of
of various conditions 150 cl, was adnixed
plasma
on with
the
in a 1.5 ml polypropylene centrifuge tube and the mixture the desired tenperature. Cclite (25 ag) was added and the with frequent agitation, for a raeasured title. ni:;ture was incubated, The centrifuged (30 seconds) and the pellet was washed t:iice suspension was then with 1 nl portions of cold (5 OC) Tris-IICl buffer. After the second wash, buffer and allowed to stand, the pellet was resuspended in 1.2 121 of Tris-liC1 with occasional (“celite eluate”) of The
SBTI rate
as of
;.lixing, was
described activation
for 24 hours at 23 oC. The supernatant a-,XIIa bv using S-2302 then assayed for
in
liquid the presence
above. of
PK by
S-XIIa
was
deternined
as
described
previously
except that S-2302 was er.:ployed as the substrate for kallikrein. One (9)) hundred ul of PE: solution (10 :ig PK/nl in Tris-IlCl buffer containing 5 ng 50 ~1 of the S-XIIa solution albumin/ml) was incubated at 30 ‘C with (diluted in 5;: albunin). At intervals during the incubation, 10 LLL portions From these data, of the r.lixture were removed and analyzed for kallikrein. the first-order rate constant was computed and compared to that produced by of Plasma Protein Fraction naintained by the Bureau of PKA reference 2, a lot The prekallikrein-activating Biologics. 7OZ of that of PKA reference 1, a lot associated with hypotensive reactions
of
(PKA) activity Plasma Protein
of PKA reference 2 is Fraction that was
(2).
SDS-PAGE (14)) alkaline disc PACE (15), and imunoelectroOther methods. SDS-PAGE gels phoresis (IEP)(lG) were performed on the purified proteins. and IEP plates were stained with Coonassie Blue; alkaline disc PAGE gels, with
Amide
Black
10B.
EXPERIilEi;TAL This procedure has been Preparation of S-SIIa. with starting plasma volumes of 0.9 laboratory, ranged from 1.1 to 2.7 r.lg E-XIIa/liter of plama. outdated) plasna have been and recovered (i.e., A typical preparation is described below.
carried out four times in our Yields have to 5.04 liters. Both freshly frozen plasma used with equivalent results.
Frozen recovered plasma (5.04 liters) was thawed, warned to (1) Adsorption. each portion was processed 23 ‘C and divided into five “-1 liter portions; separately. A 1 liter portion was stirred as 250 ml of acetone was added The suspension was stirred during l-2 minutes, followed by 25 g of celite. The for 3 ninutes and then filtered through a coarse sintered-glass funnel.
.
zetite
tilter-cafke
(5
Tris-:iCl
OC)
(i)?
Activa:ion.
The
at 23 OC for periodically filtered and conbined (3) was
.Las buffer
2P t:ours. tlonitored the celite
filtrate
and
DEAE-Se?hadex applied LO a
immediately and
then
suspensions
washed
on
the
in
151: ml
suspended from
step
‘ii t h 5 120 m1 $0f
funnel
1 were
of
the
saze
combined
and
the supernatant In some experiments for GXIIa and kallikrein content. Gjas washed vith 300 ml of Iris-!lCl wash
(1,lOC
ml)
A-50 chromatography. 5 :c 12 cm column of
was
used
in
The solution DEXE-Sephadex
the
cold
buffer. stirred
s low1 v
liquid Gas The sus>ensi on was The buffer.
next
step.
from step 2 A-50 equilibrated
(1,100 at
ml)
23 ‘C buffer
with Tris-!!Ci buffer. The column was washed with 250 ml Tris-IICl and then eluted with a linear gradient from n.05 11 Tris-IICl, ‘1.05 i! to O.C!5 II Tris-hC1, 0.6 :I :;aCl, pii 8.0 (1 liter). LaCl , pi1 8.0 (1 liter) The elution profile is shown in Figure 1. Kallikrein was not bound by the column and was found in fractions were pooled and concentrated P:l-10
25-140. Fractions to a volume of
2.9
203-230, ml by
containing ultrafiltration
c-XIIa, (.Amicon
membrane).
r c
5
4
0.4
-‘
I
03
0.5
.
0.3 E
2 2
0.2
1
0.1
T Y ZA
FRACTION NO. (10 ml) FIG. Chromatography in polypropylene S-2302 activity
of
1
celite eluate on DEAE-Sephadex. Fractions tubes and assayed for protein (A2SO; (-a----O-). i:aCl concentration (-).
were collected ) and
(4) Sephadex G-15D chromatography. The concentrate from step 3 was applied to a 1.5 x 90 cm column of Seohadex C-150 equilibrated at 4 OC with 0.05 II sodium citrate, 0.20 ;I XaCl buffer (ptl 5.0): The elution profile is shown in Figure 2. Fractions 29-33 (constant specific activity) were pooled and exhaustively dialyzed at 23 OC against Tris-tIC1 buffer, then concentrated to a final volume of 6.0 nl. The concent rate was stored at -70 ‘C in 0.25 ml aliquots.
A summary of the preparation is given in Table of the (anticoagulated) plasma was assumed to
XII content The Factor 1. be 23 q/ml (17), :-Jhich upon
complete
zg
conversion
to
S-XILa
would
yield
8.6
?-XIIa/nl.
30, .L
E
-4
!
I 30 NO. (4.2 ml)
20 FRACTION FIG. Sephadex flow rate
C-150 was
chromatography 15 ml/hour.
of A280
the
2 6-XIIa );
( TABLE
Purification
of Total
Step
B-XIIa Protein
(A280.ml)
PlaSW
302,000
Celite
Eluate
from
Human Plasrla
).
678(c)
(5.04
liters)
Purification Factor
Yield (X)
0.0055
( .)
(100)
0.329
60
Specific Activity(b)
1,663
2,060
The
I
Recovered
Total Activity(a)
concentrate from step 3. S-2302 activity (I) ---a
41
DEAE-Sephadex
45.5
612
13.45
2,44
37
Sephadex
18.6
452
24*3(d)
4,420
27
(a) (b) (c) SBTI (d) (a?80
C-150
Total activity as mol.min-1. Specific activity as umol~min-1.A2~~-1.m1-1. The activity at this step was assayed with to inhibit kallikrein. See .&says. This corresponds to a specific activity of = 1.6).
S-2302 38.9
in
the
presence
uc~ol*ain-l.og-l
of
-./ Lilr rnte (5 tep 2)
01_ +XIIa (ah.! was deter:.iineJ
ka tlikrein) release in sonr e:cperil.lents
substantially complete in 18 hours, ‘were occasionally used. ::aXj, 0.?5 z..~hrn tii.ics 1,2-t ilours ::ere ei.iployed.
of
Activation
Activation Solvent AC e t ohr Ethanol ;ietttanol l-Proparlol(h) ?-Prupanol(b) Tris-!ICL buffer
23 23 23
Acetone Acetonc‘ ;\cetone
2 11, 37
Acetone Acetone Acetone
23 23 23 23
Acetone (a) the (b)
Assessed presence Sorle
after 24 of SbTI.
precipitation
Conditions
hours
tines in the
upon
the
Recovery
of
Tir.ie
(-!i n)
Eluate
of (“),
occurred
23
‘C;
with
i:as
<-KITa 3-XIIa
Celite
-i.101 *nib-l*nl-1
0.080 0.070 Q.004 0.034 0.032
3 3 3 1 10 20
0.001 0 *0 6 4 0.1132
45
0.063
O.OSl o.v3a 0.076
S-2302 both
in
0. 080
3 3 3 3 3 3
at
step release
(up to 75 hours) suspension buffer
Activitv
(0:) 73 23 23
acrivatlon :-XII3
II
Conditions Temp.
(20,:)
tile 3).
although longer :, :Kas included
Th3LE Effect
durihg (Figure
l-
hydrolysis and
was
determined
in
2-propanol.
FIG.
3
Release of kallikrein and B-XIIa from celite during the activation step. The celite eluate was assayed at variolus tines for 5-XIIa and for c-> kallikrein ( CD- - - - - -0 by the procedures described in Abssavs.
INCUBATION
TIME
ih)
)
roi.2S,
7.
2
313
f0.i
To deline the role, if any, of tieneracion of 8-XIIa in deficient plasmas. other contact factors (PI:, Factor XI, and high ?lr kininogen) in the generation of &XIIa, i:e examined various deficient plasmas by the smallAcetone (202) and a 3-minute contact time at scale centrifugation technique. 23 'C were employed in these experiments, and the celite eluates were assayed for 8-XIIa and for kallikrein aEter 1, 6, and 18 hours of incubation. PK is required for rapid (i.e., 1 hour) release of 3-XIIa (Table III). High lir kininogen also accelerates 3-XIIa generation, perhaps by increasing the amount of PK initially adsorbed to the celite (1).
TABLE III Ceneration
of 8-XIIa and Kallikrein in Celite Eluates Prepared from Deficient Plasmas
Kallikrein
B-XIIa Activity(a)
Activity(a)
Plasma
lh
Rormal
0.069
0.079
0.113
0.134
0.161
0.173
Factor XII-def.
u. 006
0.031
o.oio
0
0
0
Factor XI-def.
0.027
0.048
0.089
0.112
0.124
0.126
Iligh ii, kininogen-def.
0.005
0.011
0.031
0.030
0.101
0.143
PK-def.
0
0.001
0.007
0.001
0.058
0.083
6h
lh
18 h
6h
18 h
(a) As nnol~uin-l~(ml plasma)-1. 8-XIIa activity was measured in the presence of SBTI to inhibit kallikrein; kallikrein activity was computed as total S-2302 activity (in the absence of SBTI) minus 8-XIIa activity.
Assessment of purity. The specific activities of four preparations of f3-XIIa ranged from 37.0 to 38.9 unolwmin -1 *mg -1 . Disc PAGE revealed three closely-spaced prealbumin bands characteristic of this enzyme (1X) (Figure 4, left). SDS-PACE (61 gels) of 8-XIIa gave a single band corresponding to an apparent Ill:of 30,000. Preincubation of the enzyme (3 Vg) with Cl-inhibitor (15 ng) or AT III (8 ug) before application to SDS gels resulted in the disappearance of the 30,000 I& band and the appearance of new bands corresponding to the 6-XIIa-inhibitor complexes (Figure 4, right). Inuunoelectrophoresis of o-XIIa and B-XIIa was performed with anti-(DIPB-XIIa) serum (rabbit) and anti-(Factor XII) serum (goat). B-XIIa produced anodal arcs against both antisera, with a mote intense precipitin arc against anti-(DIP-8-XIIa) than against anti-(Factor XII). By contrast, o-XIIa produced arcs near the well and reacted more strongly with anti-(Factor XII) serum (Figure 5). Both the relative electrophoretic mobilities and the pattern of inmunoreactivity are consistent with the respective properties of a-XIIa and 5-XIIa reported by other investigators (17).
FIG. PAGE of
r, Gel
3-XIIa.
1 (left):
alkaline
SDS-PAGE gels of S-PIIa, Ci-inhibitor, AT III, and 3-XIIa + 5-XIIa + CT-inhibitor, AT III are shown as gels 2-6 (left to right). disc
PAGE of
15
s
SXLIa.
ACtivation of PK. In the foregoing experiments, all measurements of 5-SIIa To establish that activity were made with the synthetic substrate S-2302. 3-XIIa prepared by the present method retains enzyr.lic activity toward its (presumed) natural substrate and to quantitate this activity, the isolated enzyme was incubated with purified PK. For this study B-XIIa was diluted in 59 albumin (Na+ concentration, 150 meq/liter; PKA activity, 0) to a concentration of 45 ng/ml and the PK-activating ability of this solution was determined as described in Assays. The progress curve for the activation is the first-order rate constant for this reaction was ?~!~5ir~i~t~u;Pn~ht, Figure 6). In a nur.tber of recent investigations (3,5,11,23) concentrations of PKA have been reported in terms of the activity of a reference preparation (2). The availability of highly purified PKA and the knowledge of its activity toward PK provided the opportunity to determine the actual a-XIIa content of this reference. an analysis similar to that described To make this determination, in the preceding paragraph and Figure 6 was performed with a sample of PKA reference 2 that had been adjusted (with 5 !l I;aCl) to a !{a+ concentration of 150 meq/liter.** The first-order rate constant obtained was 0.0039 Din-l. By comparison of this value with that determined for purified PKA, the PKactivating activity of PKA reference 2 was computed to be equivalent to a concentration of 26 ng B-XIIa/r.Il.
**The
i;a+
without for_:he
min
concentration
adjustment activation
.
first-order previously
The effect rate (9,lO).
of of
of the
PKA reference NaCl
concentration,
2 is
116 meq/liter. the
firsr-order
I/hen rate
assayed constant
PK by Pl:A reEerence 2 was determined to be 0.0408 ionic strength upon the Km (and hence upon the constant, V/?&I) of this reaction has been reported of
Innunoelectrophoresis (top
well)
well). contained and the :i-XIIa)
of
3-XIIa
and r-XIIa (botton The top and bottom troltghs
anti-(Factor center trough, Anode seruc.
XII) serun, an:i-(DIPis at right.
1.0
0.8 0.6 0.4 0.2 0.2
1
INCUBATION TIME (mid I
1
I
I
I
20
40
60
80
100
INCUBATION TIME (mid FIG. Activation
of
PK by S-XLIa.
PK (final
6 concentration,
15 ng/n?) in incubated at 30 OC with B-SIIa (final, containing albuxin (final, 22). At neasured tines, was assayed for kallikrein as described in U Assavs. of the early phase of the reaction.
6.67
pg/nl)
was
Tris-HCl buffer 10 ~1 of the incubate Inset: first-order plot
DISCUSSIOG The preparative binding properties
procedure for 6-XIIa of Factor XII in
described a manner
above utilizes the surfacethat significantly increases the Numerous investigators have yield and the simplicity of the operation. employed kaolin or celite as an activator of Factor XII in plasi.>a, particu0 ‘C) which prevent or retard inhibition larly at low temperatures (i.e., This inhibition requires the prior forrlation of by plasr.la inhibitors (19). which occurs upon cleavage of the zynogen nolecule. a serine active center, Thus by removing the celite (with adsorbed contact-activation factors) from the plasr.la milieu before substantial cleavage of the Factor XII has occurred, Al though it is possible to separate the activation and inhibitory processes. of the total plasma ‘kallikrein (or Pt:) is only a small proportion (<5X)
:;01.25,
so.4
adsorbed under the conditions we employ, this amount is sufficient to cleave the adsorbed Factor XII (and probably o-XIIa as well ) with the format ion of 3-SIIa. The latter lacks the surface binding regions of Factor XII or o-XIIa Once it is present in (20) and is :herefore released into the fluid phase. this phase, ;-XIIa can be separated from other plasma proteins by conventional chromatographic methods. B-XIIa prepared by this method has been used for pharmacologic (3) and kinetic (cf. 9,lO) investigations, and for the development of an assay for plasma prekallikrein (21). The convenience of the procedure and the continuing clinical importance of PK4 (5,6,,o2,23) suggest that it could be used to provide reference preparations for standardizing laboratory analyses, as well as naterial for sequence and other structural studies. REFEREICCES 1.
GRIFFI:;, J.H. and COCHRAKE, C.G. Recent advances in the understanding of Sen. Thromb. Ilemostas., 2, 254-273, 1979. contact activation reactions.
2.
ALVI?;G, 3.:1., KiOJItlA,Y., PISANO, J.J., :lASO?Z,B.L., BUCKIKGI1Aff,R.E., H.11. and FI!:LAYSO?<,J. S. ftypotension associated with preJK., ilOZEl:, kallikrein activator (Hagenan factor fragments) in plasma protein fraction. S. Engl. J. fled., 299, 66-70, 1978.
3.
B-L., ROSSI, F., ARONSON, D.L. and ALVING, B.?l., Tx(KERSLEY, D.L., ilASON, FIKLAYSON, J.S. Contact activated factors: contaminants of immunoglobulin preparations with coagulant and vasoactive properties. J. Lab. Clin. iled., -96, 334-346, 1980.
4.
KUWAILARA, S.S. Prekallikrein activator (Hagenan factor fragment) Transfusion, _?_(I, 433-439, 1980. plasma fractions.
5.
HEIROKEi:, J., PELTOLA, K., HIllBERG, J.J. and SUOMELA, H. Ilypotensive effect of prekallikrein activator (Pf:A) in plasma protein fraction (PPF). Develop. Biol. Standard., 4J, 129-130, 1981.
6.
VAN ROSEVELT, R.F., BLEEKER, W.K., BAKKER, J.C., UFKES, J.G.R., SINCLAIR, D., VAN IlOURIG, J.A. and VAI; AKEN, W.C. Bradykinin mediated hypotension after infusion of prekallikrein activator containing plasma 259, 1981. Thrombos. tiaemostas., %, protein fraction.
7.
A prealbunin KAPLAR, A.P. and AIJSTEN, K.F. J. Imnunol., 105, 802-811, 1970.
8.
VEidKEKOD, A.11. and LAME, K. Isolation and characterization of a prekallikrein activator from acetone-activated hur!an plasma. Thromb. Res., 6, 103-118, 1974.
9.
TAM
activator
in human
of prekallikrein.
ILILLER-ANDERSSOK, Il., PIERCE, J.V. and D *L \ 10. HOJI:LA, Y., TANI'ERSLEY PISABO J .J . Enzymatii prop:rties of human liagenan factor fragment with Thromb. Res., 18, 417-430, plasma'prekallikrein and synthetic substrates. 1980.
i:.
iiO_iI::A,
Y.,
ifihibitor
2, 1.z.
?I,::CZ, in
ii9-162,
I;IGCI”S*I , r~o?rcular
j.!“‘.
corn
.X:3
?ISXX,
ib$e;;;an
J.;.
purification
seeds:
and
factor
BOi;l.-\,
R.C., wei:qht
a.:;.
,
‘Aininoden
COCt!RX;E, in
C.i;.
a;ld
surface-binding
GRIFFI:;,
prekallikrein.
Cil.i:X, J. Y. C. h;;D :!O?‘hT, 11.2. Purification 0E Factor Thror.!b. Res., 5, 337-349, 1976. fr0i.i I\ur.lan plasrla.
14.
WEBER,
‘K.
The reliability OSBOR:;, :!. J. b] dodecyl-sulfate-polyacr:.*la?ide
and
deter_iinations
15.
serum 16.
Chen.,
D,\VIS,
B.J.
4406-G&12,
244,
r)isc
Ann.
;;.Y.
P . iii;D !:ILLI:GIS
propriit& protiines. 193-194,
;!etllod
.icad. C. :i.
Glcctrophori;iques Application au 1953.
Sci.,
131,
:lGthods
et s&urn
18.
c:OCH&\;;t,
20.
Ai;D \:UEPPER,
R.D.
in hul.lan and XII (ilagernan
rabbit factur).
I;.
plaslia r.lethod.
witi arginine proesterase Thrornb. Res., 2, 409-421,
KEVX,
S. If.
Invest., ALVI:;C,
4i;D
23.
COCIlRA;;E,
in inman activities j7, R.LL. ,
I’. ,
of
Clesterase
inhibitor. J.T.,
the
LSh,
factor)
application
to
hur.an
conjugge
des
1964. I’Gtudc
de hcta,
2,
A. R. and !II:GLI, T.E. Structural of human liagenan factor.
first
conponent
plasm. J.
of
Its relationship 134, Exp. Lied., of
Factor
(prekallikrein). 1973.
II.
The
relationship The association regions of the
T.,
ILASON, B.L.,
SI;;CLAIR,
hypotensive J.
of
plasma Clin. D.,
the
kinin-
to 986-1004,
1971.
SII in huctan Studies on the
of
structure of enzynatic J. molecule.
Thorac.
TAXKEKSLEY,
prekallikrein Res., 2, 323h,
Drl;lEK,
effect
of
J. plasm
Cardiovasc.
STEIN, L.D., REYNOLDS, five-year irmune globulin: reactions in imunodeficiency. and Uses of Intravenous DllilS Publication J. S. Finlayson (Lds.) U.S. Governnent Printing Office, 1980,
Intravenous and adverse Characteristics
higil
and and Clin.
1976.
I:lA:;ARI,
Inhibition
CASSIDY,
C.G.
Deternination factor fragl.lent.
VA:; DEI: STARIX,
and 404-427,
Determination
I!agman factor. with separate
852-860,
PISAKO, J.J. with Hager.~an 22.
VEi
The
LhXE,
function binding
21.
C.G. systel.1 Factor and
of
nolecular weight gel electrophoresis.
pet%ettant
REV%, S.D. , COCllICAi;I:, C.G. , JOIIKSTO:;, changes accor.qanying enzymatic activation J. Clin. Invest., 2, 619-627, 1974.
19.
(!1a7e.an ~ :?
il.i~.;unvclricliques d’un clglange sa2guin. Biochin. Biophys.
17.
forming clotting
of
:
of Sci.
1969.
electropiioresis-II.
proteins.
C i&\B‘ii’\
Zes.,
?,ole
J. il.
ani! activation. Proc. :;atl. Acad.
13.
Viol.
Thronb.
1980.
coagulation Factor X1 ani! i636-GG&G, 1977. 74 _’
J.
fragzent
characterization.
and
D.L.
and
by direct 1381.
BI:L’:EIELHUIS, protein
Surg.,
activation
ii.
solutions
2,
738-740,
by 1980.
R.T. and :lAGILAVY, D.B. clinical experience with efficacy In: Inuunoglobulins : Preparations. B. 11. .Uving and i;o. (F&X)-80-9005, Washington: pp. 161-166.
?RE?AR_~TIO:JOF 3-XIIa (HACE:lcL': FACTOR FRAQIENT) FROEI HU?lrV' . . PUS">1
Donald L. Tankersley, Barbara !l. Alving* and J. S. Finlayson Bureau of Biologics, Food and Drug Administration, Bethesda, :ID 20205, USA *Walter Reed Army Institute of Research, Washington, DC 20012, USA
(Received 14.9.1981; in revised form 14.12.1981. Accepted by Editor G.A. Jamieson) ABSTRACT B-Factor XILa (S-XIIa, !I n,30,000) was isolated from hunan plasma by a procedure which uti I* ited, as an initial step, the adsorption Activation of Factor XII and subsequent of Factor XII to celite. release of S-XIIa was brought about by the proteolytic action of Two successive chromatographic procedures co-adsorbed kallikrein. were then used to achieve a final purification of 4,420-fold over plasma and an overall yield of 2.3 mg of 8-XIIa per liter.
INTRODUCTION 8-XIIa (Hageman factor fragment; PKA) is derived from Factor XII (Hagernan It is a potent factor, !l, 80,000) by the proteolytic action of kallikrein. 80,000) activator of prekallikrein (PK) but, unlike a-Factor XIIa (cl-XIIa,& has little or no ability to activate Factor XI (for a review, see reference 1). The occurrence of B-XIIa in therapeutic protein solutions prepared from human plasma (2-4) may result in adverse reactions, most notably hypotension In the course of our studies on the pharmacologic and enzymic 2,536). properties of 8-XIIa, it became necessary to prepare rather large (i.e., 10 mg) quantities of the purified enzyme. Published procedures (T-10) for its preparation from human plasma were not entirely satisfactory, inasmuch as large-volume chromatographic procedures were usually required. This paper describes a convenient, high-yield procedure for the preparation The convenience and novelty of this of highly purified (>95X) B-XIIa. procedure lie in the initial adsorption-activation step, which affords a 40- to 200-fold purification and a S- to lo-fold concentration over plasma. The subsequent chromatographic purification steps are therefore greatly simplified.
Keywords:
Hagenan factor, Factor XII, prekallikrein. 307
For preparativ? prac?dares, h~an plss1.a ~;as obtained from the :iIiH :iaterials. 30th freshly frozen, platelet-poor .ACD plasma and outdated plasma biood bank. (stored as whole blood for 21 da:;s at 4 ‘C befor? separation of the ?laSi.la) To compare different adsorption-activation conditions, a reference retire used. plasma pool, prepared iz the fo!lowing i!an;lrr, was employed. Blood ‘Gas drawn froi.1 four liealthy subjects into plastic syringes and immediately mixed with li9 volui,le of 3.32 sodiun citrate dihydrate. After centrifugation, the plasmas were pooled, aliquoted, and stored at -70 “C. Deficient plasmas !ii;:h-purity Cl--inhibitor were obtained from George King, Overland “ark, KS. was kindly provided by 11. Gickerhauser, American Red Cross. Antithro[.lbin III was furnished by G. ilurano, Bureau of Biologics. B-XII.? inhibitor (AT III) isolated frol,l popcorn (11) izas provided by ‘i. tio jir:ia, YIILBI. The chr~ogenic substrate S-2302 (3-Pro-Phe-Arg p-nitroanilide) vas obtained from I:abi Soybean trypsin inhibitor (SBTI)(\:orthington,)) Diagnostica, Sweden. diisopropylphosphorofluoridate (DFP)(Sigr,la), celite (llyflo Supercel, Johns DEAT.-Sephadex A-50, SP-Sephadex C-50, Con-A Sepharose, and Sephadex :Ianville), G-150 (Pharl.lacia) i;ere purchased from the sources indicated. Other chemicals were the best available quality. Prckallikrein (PK) of >95% purity was isolated frol,l freshly frozen, titrated hur.lan plasma by sequential chromatography on DEAL-Sephadex A-50 and Con ASepharuse as described previously (9)) followed by further purification on SP-Sephadex C-50 (12) and Sephadex C-150. The product hydrolyzed S-2302 at a rate of 173 uciol~nin-l~mg-l following activation by Z-SIIa and had no detectable activity toward this substrate without prior activation. The r-XIIa used for irnmunoelectrophoretic studies ijas prepared by cilromatography of titrated human plasma on DUG-Scphadcx A-50 and C:I-Sephadex C-50 activation of Factor XII occurred during the latter procedure. Final (13); purification (~85,~:) was achieved by Seplladex G-150 chromatography. Antiserum to B-XIIa was prepared in rabbits; the 3-::IIa was inactivated with DFP, and primary i[zlmunization used 60 ug DIP-B-XIIa in complete Freund’s The rabbits were boosted after 14 and 28 days with the same adjuvant. quantity of DIP-S-XLIa in incomplete Freund’s adjuvant, and serum was Slight cross-reactivity to hur.:an albumin was noted and collected on day 35. was removed by absorption with l/10 volulle of Factor XII-deficient plasma. Antiserum (goat) to human Factor XII was furnished by J. Griffin (Scripps La Jolla, CA); this antiserum demonstrated cross-reactivity to CLinic, which was also removed by absorption with Factor XIIa27”acroglobulin, deficient plasr.la.
Protein concentrations were determined spectrophotonctrically; the Assays. absorption coefficients (a280) used were 1.21 for PK and 1.60 for 3-XILa. These values were deterl,liGed by comparison of the A280 and differential refractive index (in546) of solutions of the purified proteins. A specific refractive increl‘lcnt of 0.186 ml/g was ossuned. Similar 5280 values (1.1s for Pb, 1.55 for B-XIIa) were deterl.hined by comparing the A280 with the results of a Lowry assay in which bovine albur.lin was used as the standard. Enzyme activities of kallikrein and B-SIIa were determined with 0.5 dl S-2302 in 0.05 il Tris-HCl, 0.05 [I iiaC1 buffer (pll 8.0)(Tris-IICl buffer). The sar,ple (10 21, diluted as necessary in Tris-IICI buffer which contained 5 ng
albuninisl) , in absoroance
uas added to 4911 ~1 of substrate solution 3: 405 nn with tine was recorded. The
calculated
froi.:
the
_Aigj/K!ihute
observed
by
using
at rate
30 “C and the of hydrolysis 9,7OC
s4Qj=
change -as
for
Activities were expressed as -%oles hydrolyzed per minute. p-nitroaniline. Fixtures containing both kallikrein and 3-XIIa (i.e., celite eluates) were analyzed by measuring the rate of !:ydrolysis of S-2302 in the presence and absence of SBTI (final concentration 40 :g/:.il), which conpletely inhibits kallikrein but has only slight (
not
(concentration
in
incubate,
30
which
zginl)(ll),
inhibits
3-XIIa
kallikrein. centrifugation
;\ mall-scale, with celite
was
yield of S-XIIa. 600 ul of plasma (nias incubated at
procedure
used to iuvestigate Organic solvent
for
adsorption-activation
the effect (or buffer),
of
of various conditions 150 cl, was adnixed
plasma
on with
the
in a 1.5 ml polypropylene centrifuge tube and the mixture the desired tenperature. Cclite (25 ag) was added and the with frequent agitation, for a raeasured title. ni:;ture was incubated, The centrifuged (30 seconds) and the pellet was washed t:iice suspension was then with 1 nl portions of cold (5 OC) Tris-IICl buffer. After the second wash, buffer and allowed to stand, the pellet was resuspended in 1.2 121 of Tris-liC1 with occasional (“celite eluate”) of The
SBTI rate
as of
;.lixing, was
described activation
for 24 hours at 23 oC. The supernatant a-,XIIa bv using S-2302 then assayed for
in
liquid the presence
above. of
PK by
S-XIIa
was
deternined
as
described
previously
except that S-2302 was er.:ployed as the substrate for kallikrein. One (9)) hundred ul of PE: solution (10 :ig PK/nl in Tris-IlCl buffer containing 5 ng 50 ~1 of the S-XIIa solution albumin/ml) was incubated at 30 ‘C with (diluted in 5;: albunin). At intervals during the incubation, 10 LLL portions From these data, of the r.lixture were removed and analyzed for kallikrein. the first-order rate constant was computed and compared to that produced by of Plasma Protein Fraction naintained by the Bureau of PKA reference 2, a lot The prekallikrein-activating Biologics. 7OZ of that of PKA reference 1, a lot associated with hypotensive reactions
of
(PKA) activity Plasma Protein
of PKA reference 2 is Fraction that was
(2).
SDS-PAGE (14)) alkaline disc PACE (15), and imunoelectroOther methods. SDS-PAGE gels phoresis (IEP)(lG) were performed on the purified proteins. and IEP plates were stained with Coonassie Blue; alkaline disc PAGE gels, with
Amide
Black
10B.
EXPERIilEi;TAL This procedure has been Preparation of S-SIIa. with starting plasma volumes of 0.9 laboratory, ranged from 1.1 to 2.7 r.lg E-XIIa/liter of plama. outdated) plasna have been and recovered (i.e., A typical preparation is described below.
carried out four times in our Yields have to 5.04 liters. Both freshly frozen plasma used with equivalent results.
Frozen recovered plasma (5.04 liters) was thawed, warned to (1) Adsorption. each portion was processed 23 ‘C and divided into five “-1 liter portions; separately. A 1 liter portion was stirred as 250 ml of acetone was added The suspension was stirred during l-2 minutes, followed by 25 g of celite. The for 3 ninutes and then filtered through a coarse sintered-glass funnel.
.
zetite
tilter-cafke
(5
Tris-:iCl
OC)
(i)?
Activa:ion.
The
at 23 OC for periodically filtered and conbined (3) was
.Las buffer
2P t:ours. tlonitored the celite
filtrate
and
DEAE-Se?hadex applied LO a
immediately and
then
suspensions
washed
on
the
in
151: ml
suspended from
step
‘ii t h 5 120 m1 $0f
funnel
1 were
of
the
saze
combined
and
the supernatant In some experiments for GXIIa and kallikrein content. Gjas washed vith 300 ml of Iris-!lCl wash
(1,lOC
ml)
A-50 chromatography. 5 :c 12 cm column of
was
used
in
The solution DEXE-Sephadex
the
cold
buffer. stirred
s low1 v
liquid Gas The sus>ensi on was The buffer.
next
step.
from step 2 A-50 equilibrated
(1,100 at
ml)
23 ‘C buffer
with Tris-!!Ci buffer. The column was washed with 250 ml Tris-IICl and then eluted with a linear gradient from n.05 11 Tris-IICl, ‘1.05 i! to O.C!5 II Tris-hC1, 0.6 :I :;aCl, pii 8.0 (1 liter). LaCl , pi1 8.0 (1 liter) The elution profile is shown in Figure 1. Kallikrein was not bound by the column and was found in fractions were pooled and concentrated P:l-10
25-140. Fractions to a volume of
2.9
203-230, ml by
containing ultrafiltration
c-XIIa, (.Amicon
membrane).
r c
5
4
0.4
-‘
I
03
0.5
.
0.3 E
2 2
0.2
1
0.1
T Y ZA
FRACTION NO. (10 ml) FIG. Chromatography in polypropylene S-2302 activity
of
1
celite eluate on DEAE-Sephadex. Fractions tubes and assayed for protein (A2SO; (-a----O-). i:aCl concentration (-).
were collected ) and
(4) Sephadex G-15D chromatography. The concentrate from step 3 was applied to a 1.5 x 90 cm column of Seohadex C-150 equilibrated at 4 OC with 0.05 II sodium citrate, 0.20 ;I XaCl buffer (ptl 5.0): The elution profile is shown in Figure 2. Fractions 29-33 (constant specific activity) were pooled and exhaustively dialyzed at 23 OC against Tris-tIC1 buffer, then concentrated to a final volume of 6.0 nl. The concent rate was stored at -70 ‘C in 0.25 ml aliquots.
A summary of the preparation is given in Table of the (anticoagulated) plasma was assumed to
XII content The Factor 1. be 23 q/ml (17), :-Jhich upon
complete
zg
conversion
to
S-XILa
would
yield
8.6
?-XIIa/nl.
30, .L
E
-4
!
I 30 NO. (4.2 ml)
20 FRACTION FIG. Sephadex flow rate
C-150 was
chromatography 15 ml/hour.
of A280
the
2 6-XIIa );
( TABLE
Purification
of Total
Step
B-XIIa Protein
(A280.ml)
PlaSW
302,000
Celite
Eluate
from
Human Plasrla
).
678(c)
(5.04
liters)
Purification Factor
Yield (X)
0.0055
( .)
(100)
0.329
60
Specific Activity(b)
1,663
2,060
The
I
Recovered
Total Activity(a)
concentrate from step 3. S-2302 activity (I) ---a
41
DEAE-Sephadex
45.5
612
13.45
2,44
37
Sephadex
18.6
452
24*3(d)
4,420
27
(a) (b) (c) SBTI (d) (a?80
C-150
Total activity as mol.min-1. Specific activity as umol~min-1.A2~~-1.m1-1. The activity at this step was assayed with to inhibit kallikrein. See .&says. This corresponds to a specific activity of = 1.6).
S-2302 38.9
in
the
presence
uc~ol*ain-l.og-l
of
-./ Lilr rnte (5 tep 2)
01_ +XIIa (ah.! was deter:.iineJ
ka tlikrein) release in sonr e:cperil.lents
substantially complete in 18 hours, ‘were occasionally used. ::aXj, 0.?5 z..~hrn tii.ics 1,2-t ilours ::ere ei.iployed.
of
Activation
Activation Solvent AC e t ohr Ethanol ;ietttanol l-Proparlol(h) ?-Prupanol(b) Tris-!ICL buffer
23 23 23
Acetone Acetonc‘ ;\cetone
2 11, 37
Acetone Acetone Acetone
23 23 23 23
Acetone (a) the (b)
Assessed presence Sorle
after 24 of SbTI.
precipitation
Conditions
hours
tines in the
upon
the
Recovery
of
Tir.ie
(-!i n)
Eluate
of (“),
occurred
23
‘C;
with
i:as
<-KITa 3-XIIa
Celite
-i.101 *nib-l*nl-1
0.080 0.070 Q.004 0.034 0.032
3 3 3 1 10 20
0.001 0 *0 6 4 0.1132
45
0.063
O.OSl o.v3a 0.076
S-2302 both
in
0. 080
3 3 3 3 3 3
at
step release
(up to 75 hours) suspension buffer
Activitv
(0:) 73 23 23
acrivatlon :-XII3
II
Conditions Temp.
(20,:)
tile 3).
although longer :, :Kas included
Th3LE Effect
durihg (Figure
l-
hydrolysis and
was
determined
in
2-propanol.
FIG.
3
Release of kallikrein and B-XIIa from celite during the activation step. The celite eluate was assayed at variolus tines for 5-XIIa and for c-> kallikrein ( CD- - - - - -0 by the procedures described in Abssavs.
INCUBATION
TIME
ih)
)
roi.2S,
7.
2
313
f0.i
To deline the role, if any, of tieneracion of 8-XIIa in deficient plasmas. other contact factors (PI:, Factor XI, and high ?lr kininogen) in the generation of &XIIa, i:e examined various deficient plasmas by the smallAcetone (202) and a 3-minute contact time at scale centrifugation technique. 23 'C were employed in these experiments, and the celite eluates were assayed for 8-XIIa and for kallikrein aEter 1, 6, and 18 hours of incubation. PK is required for rapid (i.e., 1 hour) release of 3-XIIa (Table III). High lir kininogen also accelerates 3-XIIa generation, perhaps by increasing the amount of PK initially adsorbed to the celite (1).
TABLE III Ceneration
of 8-XIIa and Kallikrein in Celite Eluates Prepared from Deficient Plasmas
Kallikrein
B-XIIa Activity(a)
Activity(a)
Plasma
lh
Rormal
0.069
0.079
0.113
0.134
0.161
0.173
Factor XII-def.
u. 006
0.031
o.oio
0
0
0
Factor XI-def.
0.027
0.048
0.089
0.112
0.124
0.126
Iligh ii, kininogen-def.
0.005
0.011
0.031
0.030
0.101
0.143
PK-def.
0
0.001
0.007
0.001
0.058
0.083
6h
lh
18 h
6h
18 h
(a) As nnol~uin-l~(ml plasma)-1. 8-XIIa activity was measured in the presence of SBTI to inhibit kallikrein; kallikrein activity was computed as total S-2302 activity (in the absence of SBTI) minus 8-XIIa activity.
Assessment of purity. The specific activities of four preparations of f3-XIIa ranged from 37.0 to 38.9 unolwmin -1 *mg -1 . Disc PAGE revealed three closely-spaced prealbumin bands characteristic of this enzyme (1X) (Figure 4, left). SDS-PACE (61 gels) of 8-XIIa gave a single band corresponding to an apparent Ill:of 30,000. Preincubation of the enzyme (3 Vg) with Cl-inhibitor (15 ng) or AT III (8 ug) before application to SDS gels resulted in the disappearance of the 30,000 I& band and the appearance of new bands corresponding to the 6-XIIa-inhibitor complexes (Figure 4, right). Inuunoelectrophoresis of o-XIIa and B-XIIa was performed with anti-(DIPB-XIIa) serum (rabbit) and anti-(Factor XII) serum (goat). B-XIIa produced anodal arcs against both antisera, with a mote intense precipitin arc against anti-(DIP-8-XIIa) than against anti-(Factor XII). By contrast, o-XIIa produced arcs near the well and reacted more strongly with anti-(Factor XII) serum (Figure 5). Both the relative electrophoretic mobilities and the pattern of inmunoreactivity are consistent with the respective properties of a-XIIa and 5-XIIa reported by other investigators (17).
FIG. PAGE of
r, Gel
3-XIIa.
1 (left):
alkaline
SDS-PAGE gels of S-PIIa, Ci-inhibitor, AT III, and 3-XIIa + 5-XIIa + CT-inhibitor, AT III are shown as gels 2-6 (left to right). disc
PAGE of
15
s
SXLIa.
ACtivation of PK. In the foregoing experiments, all measurements of 5-SIIa To establish that activity were made with the synthetic substrate S-2302. 3-XIIa prepared by the present method retains enzyr.lic activity toward its (presumed) natural substrate and to quantitate this activity, the isolated enzyme was incubated with purified PK. For this study B-XIIa was diluted in 59 albumin (Na+ concentration, 150 meq/liter; PKA activity, 0) to a concentration of 45 ng/ml and the PK-activating ability of this solution was determined as described in Assays. The progress curve for the activation is the first-order rate constant for this reaction was ?~!~5ir~i~t~u;Pn~ht, Figure 6). In a nur.tber of recent investigations (3,5,11,23) concentrations of PKA have been reported in terms of the activity of a reference preparation (2). The availability of highly purified PKA and the knowledge of its activity toward PK provided the opportunity to determine the actual a-XIIa content of this reference. an analysis similar to that described To make this determination, in the preceding paragraph and Figure 6 was performed with a sample of PKA reference 2 that had been adjusted (with 5 !l I;aCl) to a !{a+ concentration of 150 meq/liter.** The first-order rate constant obtained was 0.0039 Din-l. By comparison of this value with that determined for purified PKA, the PKactivating activity of PKA reference 2 was computed to be equivalent to a concentration of 26 ng B-XIIa/r.Il.
**The
i;a+
without for_:he
min
concentration
adjustment activation
.
first-order previously
The effect rate (9,lO).
of of
of the
PKA reference NaCl
concentration,
2 is
116 meq/liter. the
firsr-order
I/hen rate
assayed constant
PK by Pl:A reEerence 2 was determined to be 0.0408 ionic strength upon the Km (and hence upon the constant, V/?&I) of this reaction has been reported of
Innunoelectrophoresis (top
well)
well). contained and the :i-XIIa)
of
3-XIIa
and r-XIIa (botton The top and bottom troltghs
anti-(Factor center trough, Anode seruc.
XII) serun, an:i-(DIPis at right.
1.0
0.8 0.6 0.4 0.2 0.2
1
INCUBATION TIME (mid I
1
I
I
I
20
40
60
80
100
INCUBATION TIME (mid FIG. Activation
of
PK by S-XLIa.
PK (final
6 concentration,
15 ng/n?) in incubated at 30 OC with B-SIIa (final, containing albuxin (final, 22). At neasured tines, was assayed for kallikrein as described in U Assavs. of the early phase of the reaction.
6.67
pg/nl)
was
Tris-HCl buffer 10 ~1 of the incubate Inset: first-order plot
DISCUSSIOG The preparative binding properties
procedure for 6-XIIa of Factor XII in
described a manner
above utilizes the surfacethat significantly increases the Numerous investigators have yield and the simplicity of the operation. employed kaolin or celite as an activator of Factor XII in plasi.>a, particu0 ‘C) which prevent or retard inhibition larly at low temperatures (i.e., This inhibition requires the prior forrlation of by plasr.la inhibitors (19). which occurs upon cleavage of the zynogen nolecule. a serine active center, Thus by removing the celite (with adsorbed contact-activation factors) from the plasr.la milieu before substantial cleavage of the Factor XII has occurred, Al though it is possible to separate the activation and inhibitory processes. of the total plasma ‘kallikrein (or Pt:) is only a small proportion (<5X)
:;01.25,
so.4
adsorbed under the conditions we employ, this amount is sufficient to cleave the adsorbed Factor XII (and probably o-XIIa as well ) with the format ion of 3-SIIa. The latter lacks the surface binding regions of Factor XII or o-XIIa Once it is present in (20) and is :herefore released into the fluid phase. this phase, ;-XIIa can be separated from other plasma proteins by conventional chromatographic methods. B-XIIa prepared by this method has been used for pharmacologic (3) and kinetic (cf. 9,lO) investigations, and for the development of an assay for plasma prekallikrein (21). The convenience of the procedure and the continuing clinical importance of PK4 (5,6,,o2,23) suggest that it could be used to provide reference preparations for standardizing laboratory analyses, as well as naterial for sequence and other structural studies. REFEREICCES 1.
GRIFFI:;, J.H. and COCHRAKE, C.G. Recent advances in the understanding of Sen. Thromb. Ilemostas., 2, 254-273, 1979. contact activation reactions.
2.
ALVI?;G, 3.:1., KiOJItlA,Y., PISANO, J.J., :lASO?Z,B.L., BUCKIKGI1Aff,R.E., H.11. and FI!:LAYSO?<,J. S. ftypotension associated with preJK., ilOZEl:, kallikrein activator (Hagenan factor fragments) in plasma protein fraction. S. Engl. J. fled., 299, 66-70, 1978.
3.
B-L., ROSSI, F., ARONSON, D.L. and ALVING, B.?l., Tx(KERSLEY, D.L., ilASON, FIKLAYSON, J.S. Contact activated factors: contaminants of immunoglobulin preparations with coagulant and vasoactive properties. J. Lab. Clin. iled., -96, 334-346, 1980.
4.
KUWAILARA, S.S. Prekallikrein activator (Hagenan factor fragment) Transfusion, _?_(I, 433-439, 1980. plasma fractions.
5.
HEIROKEi:, J., PELTOLA, K., HIllBERG, J.J. and SUOMELA, H. Ilypotensive effect of prekallikrein activator (Pf:A) in plasma protein fraction (PPF). Develop. Biol. Standard., 4J, 129-130, 1981.
6.
VAN ROSEVELT, R.F., BLEEKER, W.K., BAKKER, J.C., UFKES, J.G.R., SINCLAIR, D., VAN IlOURIG, J.A. and VAI; AKEN, W.C. Bradykinin mediated hypotension after infusion of prekallikrein activator containing plasma 259, 1981. Thrombos. tiaemostas., %, protein fraction.
7.
A prealbunin KAPLAR, A.P. and AIJSTEN, K.F. J. Imnunol., 105, 802-811, 1970.
8.
VEidKEKOD, A.11. and LAME, K. Isolation and characterization of a prekallikrein activator from acetone-activated hur!an plasma. Thromb. Res., 6, 103-118, 1974.
9.
TAM
activator
in human
of prekallikrein.
ILILLER-ANDERSSOK, Il., PIERCE, J.V. and D *L \ 10. HOJI:LA, Y., TANI'ERSLEY PISABO J .J . Enzymatii prop:rties of human liagenan factor fragment with Thromb. Res., 18, 417-430, plasma'prekallikrein and synthetic substrates. 1980.
i:.
iiO_iI::A,
Y.,
ifihibitor
2, 1.z.
?I,::CZ, in
ii9-162,
I;IGCI”S*I , r~o?rcular
j.!“‘.
corn
.X:3
?ISXX,
ib$e;;;an
J.;.
purification
seeds:
and
factor
BOi;l.-\,
R.C., wei:qht
a.:;.
,
‘Aininoden
COCt!RX;E, in
C.i;.
a;ld
surface-binding
GRIFFI:;,
prekallikrein.
Cil.i:X, J. Y. C. h;;D :!O?‘hT, 11.2. Purification 0E Factor Thror.!b. Res., 5, 337-349, 1976. fr0i.i I\ur.lan plasrla.
14.
WEBER,
‘K.
The reliability OSBOR:;, :!. J. b] dodecyl-sulfate-polyacr:.*la?ide
and
deter_iinations
15.
serum 16.
Chen.,
D,\VIS,
B.J.
4406-G&12,
244,
r)isc
Ann.
;;.Y.
P . iii;D !:ILLI:GIS
propriit& protiines. 193-194,
;!etllod
.icad. C. :i.
Glcctrophori;iques Application au 1953.
Sci.,
131,
:lGthods
et s&urn
18.
c:OCH&\;;t,
20.
Ai;D \:UEPPER,
R.D.
in hul.lan and XII (ilagernan
rabbit factur).
I;.
plaslia r.lethod.
witi arginine proesterase Thrornb. Res., 2, 409-421,
KEVX,
S. If.
Invest., ALVI:;C,
4i;D
23.
COCIlRA;;E,
in inman activities j7, R.LL. ,
I’. ,
of
Clesterase
inhibitor. J.T.,
the
LSh,
factor)
application
to
hur.an
conjugge
des
1964. I’Gtudc
de hcta,
2,
A. R. and !II:GLI, T.E. Structural of human liagenan factor.
first
conponent
plasm. J.
of
Its relationship 134, Exp. Lied., of
Factor
(prekallikrein). 1973.
II.
The
relationship The association regions of the
T.,
ILASON, B.L.,
SI;;CLAIR,
hypotensive J.
of
plasma Clin. D.,
the
kinin-
to 986-1004,
1971.
SII in huctan Studies on the
of
structure of enzynatic J. molecule.
Thorac.
TAXKEKSLEY,
prekallikrein Res., 2, 323h,
Drl;lEK,
effect
of
J. plasm
Cardiovasc.
STEIN, L.D., REYNOLDS, five-year irmune globulin: reactions in imunodeficiency. and Uses of Intravenous DllilS Publication J. S. Finlayson (Lds.) U.S. Governnent Printing Office, 1980,
Intravenous and adverse Characteristics
higil
and and Clin.
1976.
I:lA:;ARI,
Inhibition
CASSIDY,
C.G.
Deternination factor fragl.lent.
VA:; DEI: STARIX,
and 404-427,
Determination
I!agman factor. with separate
852-860,
PISAKO, J.J. with Hager.~an 22.
VEi
The
LhXE,
function binding
21.
C.G. systel.1 Factor and
of
nolecular weight gel electrophoresis.
pet%ettant
REV%, S.D. , COCllICAi;I:, C.G. , JOIIKSTO:;, changes accor.qanying enzymatic activation J. Clin. Invest., 2, 619-627, 1974.
19.
(!1a7e.an ~ :?
il.i~.;unvclricliques d’un clglange sa2guin. Biochin. Biophys.
17.
forming clotting
of
:
of Sci.
1969.
electropiioresis-II.
proteins.
C i&\B‘ii’\
Zes.,
?,ole
J. il.
ani! activation. Proc. :;atl. Acad.
13.
Viol.
Thronb.
1980.
coagulation Factor X1 ani! i636-GG&G, 1977. 74 _’
J.
fragzent
characterization.
and
D.L.
and
by direct 1381.
BI:L’:EIELHUIS, protein
Surg.,
activation
ii.
solutions
2,
738-740,
by 1980.
R.T. and :lAGILAVY, D.B. clinical experience with efficacy In: Inuunoglobulins : Preparations. B. 11. .Uving and i;o. (F&X)-80-9005, Washington: pp. 161-166.