Familial protein S deficiency is associated with
recurrent thrombosis.
P C Comp, … , M R Cooper, C T Esmon
J Clin Invest.
1984;74(6):2082-2088. https://doi.org/10.1172/JCI111632.
Recent studies have demonstrated that protein C deficiency is associated with recurrent
familial thrombosis. In plasma, activated protein C functions as an anticoagulant. This
anticoagulant response requires a vitamin K-dependent plasma protein cofactor, referred to
as protein S. Since the anticoagulant activity of activated protein C is dependent on protein
S, we hypothesized that patients lacking functional protein S might have associated
thrombotic disease. Two related individuals with otherwise normal coagulation tests are
described whose plasma is not effectively anticoagulated with activated protein C. Addition
of purified human protein S to their plasma restores a normal anticoagulant response to
activated protein C. We have developed a rapid one-stage clotting assay for protein S to
quantitate the level of protein S in their plasma. Plasma is depleted of protein S by
immunoadsorption with immobilized antiprotein S antibodies. The resultant plasma
responds poorly to activated protein C, but is effectively anticoagulated in a dose-dependent
fashion upon addition of purified protein S or small quantities of plasma. The affected
individuals possess less than 5% protein S activity. Using Laurell rockets, protein S antigen
was detected in the plasma but was at reduced levels of 13 and 18% in the two individuals.
When the barium eluate of the patient plasma was chromatographed on quaternary
aminoethyl Sephadex, a single peak of protein S antigen devoid […]
Research ArticleFamilial
Protein S
Deficiency
Is
Associated
with Recurrent
Thrombosis
PhilipC. Comp,RandalR. Nixon, M. RobertCooper,
and Charles T. Esmon
Section ofHematology, Department ofMedicine, andDepartment
ofBiochemistry, Universityof OklahomaHealthSciencesCenter
atOklahoma City, Oklahoma;Department ofMedicine, Wake
Forest UniversityMedicalCenter,Bowman Gray School of
Medicine, Winston-Salem, NorthCarolina; Thrombosis/
HematologyResearchProgram, OklahomaMedicalResearch
Foundation, Oklahoma City, Oklahoma 73126
Abstract. Recent studies
have
demonstrated
that
protein
C
deficiency
is associated with
recurrent
familial thrombosis.
In
plasma, activated
protein
C
functions
as an
anticoagulant.
This
anticoagulant
re-sponse
requires
a
vitamin K-dependent plasma
protein
cofactor, referred
to as
protein S. Since
the
anticoagulant
activity
of activated
protein
C
is
dependent
on
protein
S,
we
hypothesized
that
patients lacking
functional
protein S might have associated thrombotic disease.
Two
related
individuals with otherwise
normal
coagu-lation
tests are
described
whose
plasma is
not
effectively
anticoagulated with
activated
protein
C. Addition of
purified
human
protein
S
to
their
plasma
restores a
normal
anticoagulant
response to
activated protein
C.
We
have
developed
a
rapid
one-stage
clotting
assay
for
protein S
to
quantitate
the
level of
protein
S in
their
plasma. Plasma
is depleted
of
protein
S
by
immunoad-sorption
with
immobilized
antiprotein
S
antibodies.
The
resultant plasma
responds
poorly
to
activated
protein
C,
but
is
effectively
anticoagulated
in
adose-dependent
fashion
upon
addition
of
purified protein
S
orsmall
quantities
of
plasma.
The affected individuals
possess
<5%
protein
S
activity.
Using
Laurell
rockets,
protein
S
antigen
was
detected in the
plasma
but
was atreduced
levels of 13 and 18%
in the
twoindividuals.
When the
barium eluate of the
patient plasma
waschromato-graphed
on
quaternary
aminoethyl Sephadex,
a
single
Sendreprint
requests to Dr. Comp, Oklahoma Memorial Hospital, OklahomaCity,OK 73126.Received
for
publication
21February
1984andin revisedform
2July
1984.peak of protein
S
antigen
devoid of protein S
anticoag-ulant
cofactor activity
was detected early in the
chro-matogram. In contrast, the barium eluate from normal
donors separated
into
two peaks, one emerging early
and also
devoid of anticoagulant
cofactor, and the
second
peak
with anticoagulant
activity emerging later.
The
first
peak
of
protein S antigen, from both the
normal
donor and the patient, chromatographed in the
region of
the complement component C4-binding
pro-tein-protein
S complex. These studies suggest that protein
S
deficiency
may
result
in
recurrent thrombotic disease.
Introduction
Protein S is
avitamin
K-dependent plasma protein that wasdiscovered in
1977(1).
Atthattime,
nofunction
wasascribed
to
protein
S.
Recently,
arole
for protein S in
theexpression
of activated
protein
C
anticoagulant activity
has been
proposed(2). Activated
protein C inhibits the blood clotting
cascade atthe levels of
Factor VandFactor
VIII. In normalplasma,
aslittle
as0.1
tg/ml
activated
protein
C
results in
significant
prolongation of the plasma clotting time.
In1980,
Walkerobserved
thatprotein
S-deficient plasma
was nolonger
effec-tively anticoagulated by activated
protein
C(2).
Subsequent
studies revealed that
protein
S
serves as acofactor for activated
protein C (3, 4). Protein S forms
a 1:1complex
with activated
protein C
onthe surface of membranes
(3).
This
complex
inactivates Factor Va more
rapidly
thanfree activated
pro-tein
C.These in
vitro
studies
suggest thatprotein
S may beessential for
expression
of
theanticoagulant
effects of
activatedprotein
C invivo. Clinical studies
have demonstrated thatpatients
whoarecongenitally
protein
C-deficientarepredisposed
to recurrent
thrombosis
(5,
6),
presumably
on the basis of abnormalregulation
of
intravascularclotting.
Sinceprotein
Sis
required
foroptimal
expression
of activated
protein
Canticoagulant
activity,
wehypothesized
thataprotein
Sdefi-ciency
couldpredispose
individualstothrombotic disease. WeJ. Clin.Invest.
©The American
Society
for ClinicalInvestigation,
Inc.0021-9738/84/12/2082/07
$ 1.00nowdescribetwobrothers withrecurrentthrombosis who lack functional protein S.
Methods
Reagents. Reagentswerepurchased fromsources previously recorded (7), and were of thehighest gradecommercially available.
Protein
purification.
Human plasma wasthe generousgift
of the Oklahoma Blood Institute. Human vitamin K-dependent factorswerepurified from plasma in the following manner. Frozen plasma was
thawed and made 10 mM in benzamidine HCI and then diluted 1:1 into 20 mM Tris HCO, pH 7.5. The vitamin K-dependent proteins from 30 liters ofstarting plasmawereadsorbedonto30 g of quaternary aminoethyl (QAE) Sephadex Q50, previously equilibrated in 0.1 M NaCI, 0.02 M Tris-HCI, pH 7.5,by gentlestirring for 1 hat 220C. Stirringwasceased, and the QAESephadexwasallowedtosettle for 30 min. The plasma was siphoned off, and the QAE Sephadexwas
packed intoa 10X60cmcolumn,andwashed with 1 liter of 0.15 M NaCI,0.02 MTris-HCl, 5mMbenzamidine HCO, pH 7.5,before the vitamin K-dependentproteins were eluted with 2 liters of 0.4 M NaCO, 0.02MTris-HCO,5mMbenzamidineHC1,pH 7.5. All of these steps were at220C.Theeluatecontainingthevitamin K-dependent factors was pooled anddiluted 1:2.7 in0.01 M Na oxalate, 5 mMbenzamidine HCO,0.02 M TrisHC1, pH 7.5. The eluatewasadsorbedonto500 g ofBaSO4by gentlemixingfor 1 h,and theBaSO4 washarvestedby centrifugationat2,500 g for 5 minat4°C.Thepelletwasresuspended in 2 liters of 0.078M
NaCO,
1 mM Nacitrate, 10 mM TrisHCO,
1 mMbenzamidineHCO, pH7.5. TheBaSO4washarvestedby centrif-ugation asdescribed above. The vitamin K-dependent factors wereelutedfrom the BaSO4 bysuspensionin 2 liters of0.1 M Nacitrate, pH 5.8. TheBaSO4washarvestedbycentrifugationasdescribedabove, and eluted a secondtime with 2 liters of the citrate buffer. Thetwo
eluateswerepooled.
The pooled eluates were diluted 1:3.3 into 0.02 M Tris HCO, 5
mMbenzamidine
HCO,
pH7.5, and the vitamin K-dependentfactors wereadsorbed ontoQAE SephadexQ50 (150 ml settled volume), and equilibrated in 0.18 MNaCO,
5 mM24N-morpholino)ethanesulfonic
acid(MES)', 5mMbenzamidine HCO, pH 6.0, for 1 hat22°C.The QAESephadex wasallowedtosettlefor 30 min, the supernatant was removed by siphoning, and the QAE Sephadex was collected in a Buchnerfunnel overlayed with nylon cloth, and washed with 200 ml of 0.18 M NaCO, 5 mM MES, 5 mM benzamidine HCI, pH 6.0. A thick slurry of this QAE Sephadex was then packed on top of a prepacked 2.5 X90 cm column of QAE Sephadex Q50 equilibrated in this same buffer to give a total column height of 120 cm. From melting the plasma to this step requires 10 h. The column was developedat4°Cwithalineargradient(1 liter/reservoir) from 0.18 to 0.5M
NaCO
in 5 mM MES, 5 mM benzamidineHCO,
pH 6.0. The columnrequires'30
h to develop. Protein C and protein S emerged infront of the prothrombin peak, and were almost completely resolved from prothrombin. The protein S emerged slightly ahead of the protein C,andthetwoproteins were only partially resolved.Protein S was further purified by a modification of the method of Dahlback and Stenflo (8). Routinely, the protein S peak was identified by the functional protein S assay described below. Protein C was 1. Abbreviations used in this paper: C4bp, complement component C4-bindingprotein; MES,2-(N-morpholino)ethanesulfonic acid;QAE, quaternaryaminoethyl.
removed from the protein S by adsorption on amurine monoclonal IgG antibody to human protein C coupled to Affigel 10 (Bio-Rad Laboratories, Richmond, CA). The protein S peak was partially purified by (NH4.SO4 fractionation. Solid(NH4)2SO4 was added at
4VCtobring the sample to 25%saturation, and stirred for 30 min. Theprecipitatewasremoved bycentrifigation at 15,000 g for 30min
at4VC.The supernatant containing the protein Swasbroughtto70% saturation by adding(NH4.SO4, and stirred for 30 min. The pellet was collected by centrifugation as described above. The pellet was
resuspended in 5-10 ml of 50 mM Tris HCI, pH 7.5, and 2 mM diisopropyl fluorophosphate. The precipitate (10 ml, 5 mg/ml) was
dialyzed against 0.05 M NaCl, 1 mM EDTA, 1 mM benzamidine HCO, 0.02% Na azide, 50 mM TrisHCO, pH 7.5, and chromatographedon ablue Sepharose column (1.5 X 52 cm) at 220C. The columnwas
developed with alineargradient from 0.05 M NaCI to0.2 MNaCl (150ml/reservoir) in 1 mM EDTA, 1 mM benzamidine, 50 mM Tris HCO, pH 7.5. Protein Sactivitywasdetected in the firstproteinpeak
to emerge from the column. Most of the trace contaminants were separated from the protein S by chromatographyat22°C onacolumn (0.6X25 cm) of heparin-agarose. The heparin was linked to Biogel A 15 M(20040 mesh; Bio-Rad Laboratories) asdescribed previously (9) forlinkageto Bio-GelA 5 M. The protein S peak from the blue Sepharosewasdialyzedinto 0.05 M imidazoleHCO,1 mM benzamidine HCI, 1 mM CaCl2, pH 6.0 before applicationtothe heparin-agarose columnpreviouslyequilibrated in the above buffer. The columnwas
developed with a linear gradient (30 ml/reservoir) containing the equilibrationbuffer in the leadreservoir,and 0.05 M imidazoleHCO,
ImMEDTA, 1 mM benzamidine HC1, pH 6.0, in the trailing buffer. Protein Sactivitywasassociated with the lastprotein peaktoemerge from the column. This peak constituted >80% of the applied protein. TheproteinSpreparedin thismanneris shown inFig. 1.
Treatmentof thepurified protein Swith thrombin destroyed the ability to serve as a cofactor for activated protein C as previously reported by others (4) (Fig. 2). The loss of the activated protein C cofactor activity correlated with the disappearance of the protein S as monitoredonpolyacrylamidegels (Fig. 3).
Protein C and activatedproteinCwereprepared eitherby
conven-tional chromatographic methods (7) or by chromatography on a
murine IgGmonoclonal antihuman proteinCantibody. Briefly,this antibodyisconformationally specificandrequires Ca2+for interaction withprotein C. Removal ofCa2'resultsin elution of human protein C(unpublisheddata). Protein C andactivated protein C prepared by either method functionedidenticallyin theprotein S functional assay. Laurell rocket electrophoresis of protein S. Protein S in human plasma circulates both as a free protein and as a complex with complementcomponentC4-bindingprotein (C4bp) (10). The complex does not dissociate during conventional agarose gel electrophoresis (1 1). Toestimate immunologic levels of protein S, conventional Laurell rocketelectrophoresis was employed with 7 mM EDTA, and with 2% polyethyleneglycol 6,000 in the agar plates. Agarose used in the plates (1% Seakem ME agarose, FMC Corp., Marine Colloids Div., Rockland, ME) was made up with Paragon B-2 buffer (Beckman Instruments Inc., Fullerton, CA). The same buffer with 7 mM EDTA added was employedfor electrophoresis. Goat IgG directed against human proteins S and C wasproducedas previously described (7). Samples of 15 Ml were electrophoresed at 2 mA/cm at 80C for 18 h. Under these conditions,free plasma protein S can easily be detected, and at least
Figure 1. Purified hu-man protein S. Samples ofproteinS were elec-trophoresed on 10% polyacrylamidegels containingsodium do-decyl sulfate. The sam-ple on theleftwas de-natured withsodium dodecylsulfate before electrophoresis,andthe sample on therightwas reducedwith beta-mer-captoethanoland dena-tured withsodium do-decylsulfate before elec-trophoresis.
antibodydid not cross-react with human protein S on Ouchterlony doublediffusionoverawiderange ofantibodyandantigen concentra-tions.
Protein S-deficient plasma. Goat IgG directed against human proteinS was bound toAffigel10(Bio-Rad Laboratories)afterdialysing
Figure 2. Loss of func-1OOS - - *-/. tional protein S activity
afterthrombin
treat->
75%
ment. 50ug proteinS/0
F | ml of Tris buffer, pH
cL> 1 7.4,containing0.1 M
-' I s0
NaCl
wastreated witha < \ human thrombin ata
25% final concentration of 5
LU
|g/ml (-)
orwithbuffercc * (o). Thesampleswere
5 10 15 30 incubated at37°C and INCUBATION TIME at times indicated,
5S-IA
(MINUTES) aliquots were re-moved and addedto50
slI
antithrombin III (1.6 mg/ml concentra-tion inTris/saline buffer).After 30 minincubation, thesampleswereassayed for residual protein S activityandexpressedas apercentage of initialactivity.
Figure3.Time course of protein S degradation by thrombin. Protein S wasincubated withthrombin under conditions outlined in Fig. 2. Attimes indicated, aliquots were removed and denatured by addition ofsodium dodecyl sulfate and beta-mercaptoethanol, and immediate immersionof the tubes containing the samples in a boiling water-bath. Samples were electrophoresed on 10% polyacrylamide gels. Gel
1, unreduced starting proteinS; gel 2, reduced starting proteinS;gels 3,4, 5, and 6, reduced samples at 15 s, 2 min, 5 min, and 30min. The band with relative mobility of 58% is thrombin. Protein S incubatedwithout thrombin showed no degradation.
theantibody against0.1 M3-(N-morpholino)propanesulfonic acid, pH 7.5.The antibody was bound at -10 mg/ml gel. A 1.5 X 10 cm goat-antihumanprotein S column was prepared and washed extensively in 20 mMTris buffer, pH 7.4, containing0.1 M NaCl. Over this column 35 mlcitrated normal human plasma was passed at 7 ml/h. Fractions containing the plasma were then pooled. No protein S could be detected in thepooled deficient plasma by rocket immunoelectrophoresis
orby the functional assay. Theprotein S-deficient plasma contained normal levels of FactorsXand V,assayed as previously described (12, 13), and normal levels of protein C, measured functionally and immunologically (7). The activated partial thromboplastin and pro-thrombin timeswereunchanged afterimmunoadsorption.
Functionalprotein S assay. The assay is based onthe ability of protein S to serve as a cofactor of the anticoagulant effects of activated proteinC. The prolongation of the Factor Xa one-stageclottingtime by the activatedproteinC isdependent ontheprotein Scontentof thetestplasma.Incontrast, proteinS doesnotinfluence this assayin
theabsenceof activated
protein
C.Comparedwith one-stageclotting
assays for othercoagulation factors, relatively
high
concentrations of normalplasmaarerequiredtoobserve theprotein
Seffects. To avoid significant changes in the levels ofclotting
factors in this assay, the standardcurve wasprepared by dilutingnormalplasmaintoprotein
S-deficient plasma to give 0,
12.5,
25, and 50% normalprotein
Slevels. Theassaywasperformedin the
following
manner. to 100 Ml of protein S-deficient plasmaat 370Cwasadded 100;d cephalin (Sigma
I
I
mmll::l:
t
.-44
i
z.
'k
i.--ki
N
ChemicalCo., St.Louis, MO),100y1A of 25 mMCaCl2,and 100
,l
of bovine Factor Xa. ThepurifiedFactor Xa(14)wasdiluted into0.1M NaCl, 0.02 M Tris HCI, 1 mg/mlBSA, pH7.5 togive
a30-sclotting time in the above assay. Thesameclottingtime(±1 s)wasobtained whenthe normal plasma dilutions were substituted for the protein S-deficientplasma. Thus, in the absence of activatedprotein
C, this coagulationassay isunaffected by proteinS. To prepare the standard curve, 10 jl of activatedproteinC(5.6pg/ml)
is addedtoeither the proteinS-deficient plasma or the normalplasma dilutionsjustbefore addingthe other components of theclottingmixture. In the presence of activatedprotein C,alinearrelationshipwasobtained between the percent normalplasma and theprolongation
of theclotting
time(Fig.
4).Thelower limitfor detection ofproteinSis -5%. Purified protein S andplasma dilutionsexhibitedthesamedilutioncurve
slope.
Basedonthis assay the functionalproteinScontentof normalplasmaseems
tobe -8
pg/ml,
in good agreement with the 10 ug/mlof free protein S estimatedimmunologically byDahlback(15).Thestandard deviation of the assay was 5.2%, and standarderrorwas 1.2%.Preliminary
data suggest that Russell's viper venom may be employed in the assay instead of Factor Xa.Plasmasfrom patients onwarfarin therapyorfrompatientswith liver failure have increased sensitivityto theanticoagulant effects of activated protein C. The standard dilutional curves generated using theseplasmasare notparalleltothat madebymixingnormalplasma withproteinS-deficient plasma. This assay is,therefore,notappropriate for thesepatients.
Barium adsorption precipitation,
elution,
andchromatography of patientplasma. 8mlof 1 M bariumchloride wasslowlyadded with constant stirring to 100 ml ofcitrated patient plasma containing 1 mMbenzamidine at 4VC. The bariumcitrate precipitatewascollected bycentrifugationat2,000 rpmat4VCfor 10 min. NoproteinS could be detected immunologically in the supernatant. Thesurface of the precipitatewaswashedwith 5 mlof cold 20mMTrisbuffer,pH 7.4, containing0.1 M NaCl, and theprecipitatewasresuspended in 10ml0.2M Tris buffer, pH 7.4, containing 0.2 M EDTA and 1 mM
benzamidine. Whenthe precipitatehaddissolved with gentle stirring, thebarium eluate was desalted on a G-75 Sephadexcolumn(2.5X50 cm), equilibrated, and developed with 5 mM MES buffer, pH 6.0, containing0.2 M NaCl.Fractionscontainingelutedproteins from this columnwereloadedon a0.6X40cmQAE-Sephadexcolumn equili-bratedwith 5 mM MESbuffer,pH6.0,containing 0.2 M NaCl. The
I
o
0I~~~~
12'.5
25 37.5 50 PERCENT NORMAL PLASMAFigure 4. Standardcurve
forfunctional proteinS as-say.Increasing
concentra-tionsofpoolednormal
plasmawereempooledto
constructthestandard
curve.
columnwasdeveloped withalinear0.2-0.5 M NaCl gradient with 50 ml in each gradient chamber. Normal plasmawas processed in an
identicalmanner as acontrol. Functional andimmunologic proteinS levels were assayed as described above. Neither eluate contained fractions that inhibited the functional assay.
Patients. The protein S-deficient proband (E.G.) is a 17-yr-old white male. At age 14 hewas hospitalized for venographically docu-mented leftlower extremity deep vein thrombosis with clots in both calf and thigh. At age 15 the patient had an episode ofmultiple pulmonaryemboli resultingfrom documented lower extremity deep vein thrombosis. At age 16 he again had documented deep vein thrombosis. All coagulation studies, including prothrombin time,
partial
thromboplastin time, antithrombin III (104%), and protein C (110% functional, 100% immunologic), were normal. The patient was studied when he had been offanticoagulants for 7 wk.
The proband's brother (W.G.) is 27 yr old. He developed throm-bophlebitis of the left leg following a motorcycle injury at age 15. He again developed deep vein thrombosis of the right calf at 26 yr of age. Both thrombosis episodes were documented venographically. The patient's coagulation profile, including partial thromboplastin time, antithrombin III (90%), and protein C (87% functional, 95% immu-nologic), is normal. The patient had been off warfarin and heparin for 45 d when hewasstudied.
The parents andtwoothersiblings, ages 16 and 23 yr, have no history of thrombosis. The maternal grandmother had deep vein thrombosis at an advanced age. There is no consanguinity in the family.
Normal plasma samples for determining normal ranges and for makinganormalpool were obtained from20healthy individuals,not onanticoagulant therapy, and with no personal or family history of thrombosis, ranging from 21to62 yr of age.
Results
Normal plasma is
anticoagulated
by activated protein C.The
addition of activated protein Ctothe plasma oftwobrothers with severe recurrent venous
thrombosis
did not anticoagulate their plasma, as evidenced by no significant change in the clotting time after additional activated protein C (TableI).
TableI.
Effects
of
Activated ProteinC onFactor XaOne-stage ClottingTimeClottingtime
Plasma
Plasma +proteinS Plasma +activated + activated
only proteinC proteinC
(s) (s)
Normal 30.1±0.3 55.0±2.0 57.0±2.2
Patient(E.G.) 29.2±0.3 29.4±0.3 47.7±1.7 Patient(W.G.) 30.2±0.4 30.1±0.4 56.9±2.1
Plasma was supplemented with activated protein C (0.56 sg/ml con-centration in plasma) immediately before determination of the clot-tingtime,aswasprotein S (final concentration, 20 sg/mlinplasma). Threedeterminations were made on each sample. Addition of pro-tein S alone did not significantly change the clotting times deter-mined for the plasma samples without added activated protein C.
Lu
a 24
z~-022 I- 02 00 Ow , cn16 0
°1 i:-C12
O
a 00
0 XL
When the
plasma
of these twoindividuals
wassupplemented
with purified protein S,
theaddition
ofactivated
protein Cresulted
inanticoagulation.
Theaddition
ofprotein
S alonedid not
affect
theclotting
time.To
quantitate
thefunctional protein
S activity present inthe
patients' plasma,
wedeveloped
thefunctional
protein Sassay
described
inMethods.
Using this assay, wedetermined
that normal
individuals
haveprotein
Sactivity between
160 and68%
with
a meanof 100%.
We alsodetermined
that the brothers withrecurrent thrombosis
had nodetectable
protein S activity (Fig. 5),whereas
two othersiblings
and one grand-parenthad normal levels of protein S.
Themother
andfather
had
reduced levels
of 30 and 15%,respectively. Neither parent
has had any
thrombotic disease.
To
determine if
anyimmunologically functional
levels ofdetectable protein S
werepresent
in the plasma of
thedeficient
brothers,
Laurellrocket
electrophoresis
wascarried
out.Analysis
of the plasma of the brothers with thrombosis (Fig. 6) revealed
levels
of 18
and 13%for W.G.
and E.G.,respectively. Certain
other
family
members
had
less
immunoprecipitable
protein
Sthan normal plasma. These included the mother with 49%,
the
father with 71%, and
anunaffected
youngerbrother
with
50%.
Protein S is known
toexist in plasma both
free
andcomplexed
tocomplement
componentC4bp
(8). Note that
the
immunoelectrophotic
technique employed does
notpermit
quantitation of the relative
amountof free protein S
versusthe amount
of protein S in the protein S-C4bp complex.
We
wished
todetermine if
anyfree, but functionally
inactive, protein
Scould
beidentified in the plasma of the
functionally
deficient individuals.
Todetermine
this,
plasma
from
thebrother with 18%
immunologically
detectable protein
S
wassubjected
tobarium citrate
adsorption
and
elution
to concentratethe
vitamin K-dependent plasma proteins. The
barium eluate
wasthen
subjected
toion
exchange
chromatog-raphy
(Fig. 7).
Ashas been
seenby others
(10),
the
bariumeluate from normal plasma contained
twodistinct
peaks
of
immunologically
detectable
protein
S:the
first peak in the
region of the protein
S-C4bp complex,
and the
secondpeak
in the
region
where
free
proteinS
is eluted. Functional
protein
S
activity
is associated with the
second(free) protein
S
peak.
The
barium eluate from
thepatient plasma
contained
only
asingle
protein
S
peak,
eluting
from the column
inthe
region
of the
protein
S-C4bp complex.
Noprotein
S
activity
waspresent.
The Laurell rockets of these
two columnsareshown12
3
4
5
6
7
8
9
10
Figure
6. Laurellrocketelectrophoretic analysis of a protein S.deficientfamily.Wells 1-3, 100, 50, and 25% poolednormalplasma; well4, grandmother (104%); well 5,father (71%); well 6,mother (49%); well 7, proband (13%);well 8, proteinS-deficient brother (19%);wells 9 and 10, unaffected brother andsister.
3.0 NORMAL
2.0
2O(
E 30N
C I 0
-0
~~~~~~~~0
0 1o0 20 30 40 50 60 70
z
13. T N
~PATIENT
101% 15% 3%
<5% S5% <5~ 80%
Figure 5. Familywithrecurrent
thrombosis. Theproband is indicated by thearrow. Functional
protein
S levelsareshown for eachfamily
member. NoproteinS
activity
could be detected in thetwoaffectedbroth-ers atthe lowerlimitof
sensitivity
of the assay.Figure 7. Ionexchange
chromatography
of bariumeluateof protein S-deficient individuals and of normal plasma. Barium citrate adsorp-tionandelution withsubsequent desalting
andchromatography
over QAE-Sephadexaredescribed in Methods. A 0.2to0.5MNaCl
gradientwasinitiated atfraction 20. The absorptionat280 nM (-)is shown asisprotein S detected
immunologically (*) by
Laurell rocket electrophoresis and by the functional protein Sassay(C).
C4bp (detectedbyOuchterlony doublediffusion)
waspresentin the major peakfollowingthebreakthrough(fractions
30-45)
asobserved by others.A!
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-
Aj
~AAAA A A A A
A
AAA
AA
A
A
w
Figure 8. Rocket immu-noelectrophoretic analysis ofeluates of QAE-Se-phadex columns shown in Fig. 7. Wells 1, 2, and 3 contained30jsg/ml,20
Ag/ml,
and 10Ag/ml
pu-rifiedprotein S. Even-numbered column frac-tions were analysed as in-In dicatedacrosscolumn
eluates.
in
Fig. 8. The rockets from
theprotein S-C4bp peak
haveimmunoprecipitated material
insidethem, suggesting
contin-uous
separation
of theC4bp complex during
electrophoresis.The
rockets
in theregion of
thefree
protein
S
give
asharp
outline
closely resembling
that seen withpurified protein
S.The
previous
demonstration
that both brothers
responded
to
addition of
purified
protein
S
suggested
thattheirdeficiency
was not due to the presence
of
aninhibitor.
Toexclude
thepossible
presenceof
aslow-acting inhibitor,
normalplasma
was
combined
in equal partswith
plasmafrom each of
thetwo brothers. The plasma
mixtures
wereincubated
at370C,
and
remaining protein S activity
wasdetermined
over a 2-hperiod (Fig. 9).
Nosignificant
change in
protein S level
wasobserved in normal
plasma incubated with
theplasma from
either
brother.Discussion
The in
vitro demonstration
by Walker(2)
thatexpression of
bovine-activated protein C anticoagulant activity requires
pro-tein S
suggested to us thatfunctional protein
Sdeficiency,
likeprotein
Cdeficiency,
might be manifested as an increasedthrombotic
tendency. Theseobservations
also suggested thatprotein
Sdeficiency
could be monitored in plasma as aninability
to respond to activated protein C. A simple FactorXaone-stage clotting assay done in the presence of activated
Figure 9. Effectonprotein
, 8 _ ^ S level in normal
plasma
ofincubation
with plasma5> from deficient patients.
Fem75 Plasmafromtheproband
(A), proband'sbrother (o),
LU z 50 and a normalindividual
(.)
wereeachmixed in
equal
I° 25 partswith
pooled
normal plasma andincubated at 37°C. Atthetimes indi-3o 60 9oNWo
cated, functional protein SINCUBATION TIME(MIN) levels weredetermined.
protein
C allows forrapid screening
ofpatients
with potentialprotein
Sdeficiency. Presumably,
this assay would detectpatients
with functionalprotein
Sdeficiency, patients lacking
the
protein
Santigen,
orpatients
with circulating protein Sinhibitors.
The two brothers described here lack detectable functional
protein
S,
andhave
evidence
of recurrent thrombotic disease.The
biochemical basis
of the functional protein S deficiencyin these
patients is
not clear.Although the
brothers do haveprotein S antigen based
onthe
chromatographic
studiespre-sented
here,
all of theprotein
S appears to becomplexed
toC4bp.
Suchbinding
has been described in normal plasma byDahlback
andStenflo (8). This
complex from
either thepatient
plasma or
from normal plasma
does not support activatedprotein
Canticoagulant
activity. Why only
thecomplex
isobserved
inpatient plasma
is uncertain. Either thepatients'
protein S could
have anincreased
affinity for C4bp
or theirC4bp could have increased affinity
forprotein
S. The latterdoes not seem
likely
because the addition of patient plasma to normalplasma does
notinhibit the functional
protein
Sactivity of normal plasma.
Whatever
thepathophysiologic abnormality,
theinheritance
pattern suggests
that
the total lack
of functional
protein
S
activity
may be ahomozygouscondition.
The levelsof
15 and30% in the
parents areconsistent with heterozygous
individuals,
who would produce totally deficient children. Neither parent has had
thromboembolic disease, suggesting
that evensignifi-cantly
reducedprotein S levels
may besufficient
to preventthrombosis. This finding
may besimilar
toprotein
C deficiencyin
which
certain individuals with significantly reduced levelsof protein
C (presumably heterozygous for protein C deficiency)do not
have
ahistory of thrombosis
(16).The
availability of
a rapid, sensitive one-stage functionalassay for protein S limits the necessity for immunological
detection. This is fortunate since
protein S seems to circulatein at least two forms; free, and complexed to C4bp (8). The presence
of
the complex complicatesinterpretation
ofelec-troimmunoassays.
Under our immunoelectrophoresiscondi-tions,
atleast some of the protein S in the complex is detectedby
immunoprecipitation,
since clearly visible precipitation4?A
i
1:
A
lw
rockets develop in that portion of the QAE Sepharose elution
profile
where the protein S-C4bp complex is known to run.More exact measurement of the total immunologically
detect-able
protein
S will require methods for separating the complexordetecting
protein
Swhile it is bound.Because the only known clotting abnormality in these
patients
is afunctional
protein Sdeficiency,
it is likely thatthis
deficiency
causes the observedthrombotic
tendency. Thesefindings suggest that, like
proteinC
(5, 6, 17),antithrombin
III
(18), and plasminogen (19) deficiencies, protein S deficiency
should
be examined
asapossible cause of recurrent thrombosis. AcknowledamentsWe thank Deborah Doray for her excellent technical assistance, as well as GaryFerrell and Eric Wassilakfortheirs. The help of Germaine Bohlman inobtainingpatientsamples is also greatly appreciated, as istheassistance ofDr.Jim Atkins. We would also liketothank Miss DeVonna Hill and Mrs.BarbaraIrish forassistancein themanuscript preparation.
This research was supported by National InstitutesofHealth grants
1-R01-HL-30443,
1-RO-HL30340, and 5-RO-HL29807, and wasperformed
during
Dr.Edmund'stenure as anEstablishedInvestigator of theAmericanHeartAssociation,withfunds contributedin part by theOklahoma Affiliate.References
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