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This is to certify that the
thesis entitled

PROTEOLYSIS OF PROTEIN C IN POOLED NORMAL
PLASMA AND PURIFIED PROTEIN C BY ACITVATED
PROTEIN C (APC)

presented by

CHRISTOPHER MICHAEL QUINN

has been accepted towards fulfillment
of the requirements for the

Master of degree in CLINICAL LABORATORY
Science SCIENCES

 

 

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2/05 p:IClRC/DaIeDue.indd-p.1

PROTEOLYSIS OF PROTEIN C IN POOLED NORMAL PLASMA AND PURIFIED
PROTEIN C BY ACTIVATED PROTEIN C (APC)

BY

CHRISTOPHER MICHAEL QUINN

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree Of

MASTER OF SCIENCE
Medical Technology Program

2004

ABSTRACT
PROTEOLYSIS OF PROTEIN C IN POOLED NORMAL PLASMA AND PURIFIED
PROTEIN C BY ACTIVATED PROTEIN C (APC)
By

Christopher Michael Quinn

Protein C is a vitamin-K dependent zymogen of the anticoagulant serine protease
activated protein C (APC). Reported are four lines of evidence that APC can activate
protein C in pooled normal plasma and purified protein C. First, the addition of APC to
protein C deficient plasma supplemented with protein C produces a prolongation of the
clotting time of plasma that is proportional to the amount of protein C. This behavior was
Observed with two different sources of APC. Second, using immunoblotting afier gel
electrophoresis, the disappearance Of epitopes for monoclonal antibodies that recognize
protein C but not APC indicates a time course for the activation by APC of protein C in
pooled normal plasma and protein C purified from plasma. Third, the same time course for
the disappearance of protein C specific epitope can be followed using ELISA. Finally,
protein C can be activated by APC as indicated by the increase in APC specific synthetic
substrate Tryp-Arg-Arg-p nitroaniline hydrolysis. Kinetic data indicate a value of 4.7 i 0.4
mM"ls'l for the activation of protein C by APC under physiological conditions and in the
presence of calcium. These observations document that APC must fimction not only in the
inactivation of activated factors V and VH1, but also in the activation of protein C. This
additional action of APC may be important to consider more broadly because of APC in the

treatment Of sepsis.

I dedicate this work to my mentor and wonderful friend Dr. Suzy Hassouna and to my

loving wife Kara, without whom none Of this work would have been possible.

iii

ACKNOWLEDGMENTS

I am gratefirl to Dr. Stephanie Watts, Associate Professor Pharmacology for her
invaluable help in teaching me the immunoblotting techniques. Also, to Dr. Ayala who
helped conduct some of the kinetic experiments in Dr Enrico DiCera's laboratory, in the
Department Of Biochemistry and Molecular Physics, at Washington University in St
Louis. I thank Dr DiCera and Dr Ayala for providing this data. This study was

supported by a grant from MSU Hematology Education and Research Foundation.

iv

TABLE OF CONTENTS

LIST OF EXPERIMENTAL PROTOCOLS ...................................... vi
LIST OF FIGURES ................................................................... vii
INTRODUCTION ..................................................................... 1
MATERIALS & METHODS ......................................................... 5
EXPERIMENTAL PROTOCOLS .................................................. 15
RESULTS ............................................................................... 17
DISCUSSION .......................................................................... 32
CONCLUSION ........................................................................ 35
BIBLIOGRAPHY ..................................................................... 36

LIST OF EXPERIIVIENTAL PROTOCOLS

Table l. Clotting Assay Materials and Methods ............................................. 15

Table 2. Time dependent activation of plasma protein C (native protein C) by
APC 121.38 enzyme: substrate weight ratio ................................................... 15

Table 3. Evaluation by Western Blotting- Activation Of plasma protein C by
Protac, Thrombin and APC/CaC12 .............................................................. 16

Table 4. Chromogenic substrate assay- Plasma protein C activation by Protac,
APC and thrombin ................................................................................. 16

vi

LIST OF FIGURES

Figure 1. Structure of protein C molecule with activation site ............................... 8

Figure 2. Plasma protein C activation by APC-110 and by APC-110
+ Argatroban measured by enzyme-linked immunosorbent assay (ELISA) ............... 18

Figure 3. Comparison of the anticoagulant responses to APC in protein C
deficient plasma versus pooled normal plasma ................................................. 20

Figure 4. Effect of plasma protein C on APC anticoagulant response
measured by APTT in seconds .................................................................. 21

Figure 5. Comparison of the reactivity Of monoclonal antibodies I-IPC-4
and H-C2 on protein C versus APC by immunoblotting ...................................... 23

Figure 6. Immunoblot analysis of time dependent APC-catalyzed
activation of plasma protein C ................................................................... 24

Figure 7. Immunoblot analysis of time dependent APC-catalyzed
activation of purified protein C .................................................................. 25

Figure 8. Immunoblot analysis Of plasma protein C activated by human
ct-thrombin by Protac and by Coatest APC/ CaClZ ............................................ 26

Figure 9. Plasma protein C activation by APC-110 and Protac measured
by enzyme-linked immunosorbent assay (ELISA) ........................................... 28

Figure 10. Time course of plasma protein C activation tO APC by
APC-145 and Protac measured as APC amidolytic activity ...... . ......................... 3O

vii

INTRODUCTION

In 1976 Stenflo published the discovery of bovine protein C, a vitamin K-
dependent factor and zymogen of a serine proteolytic enzyme [52,56]. This was followed
by several reports [17,19-21,34,36-38,57,58] on the isolation, characterization, and
mechanism Of human protein C activation by OC- thrombin and by PROTAC®, which is a
product derived from the venom of the Southern Copperhead. The anticoagulant effect of
APC on factor V and factor VIII also was recognized at that time [17,19,34,58] and in
1985 the protein C gene was sequenced permitting the identification of DNA deleterious
mutations [23]. Thus, the first biochemical cause to be identified by a persistence in
blood of both activated factor V and factor VIII was a deficiency of protein C associated
with thrombotic disease [1,4,25,26,32]. Hassouna recognized a second biochemical
cause, resistance to inactivation Of both activated factors V and factor VIII by APC
associated with thrombosis, in 1989. Afier adding PROTAC® to activate plasma protein
C of a 33-year-old patient with recurrent thrombosis since age 21, both factor Va and
factor VIIIa resisted inactivation. [28]. It was confirmed by personal communication
with Joseph P Miletich that the protein C immunoreactive levels and the amount of
protein C purified from the patient's plasma were 120% of normal [28] and genetic
testing in 1999 identified a homozygous state for the factor V Leiden mutation reported
in 1994 by Bertina et al [5]. In 1993, by an innovative approach, expected to bypass
protein C activation, Dahlback et a1 [12] added APC to plasma from a patient with
~ thrombosis and discovered that both factors Va and VIIIa resisted inactivation, which

they named APC resistance. APC resistance is a laboratory clotting value measured in an

assay wherein standardized amounts of APC added to plasma have no influence on
plasma levels of protein C [12,16]. APC resistance also became the clinical name applied
to a familial thrombotic disorder, and the first notable exception Of a clinical entity
ascribed to a laboratory clotting value [15,35]. The genetic basis for most of the
hereditary APC resistance cases (about 90%) was identified by Bertina et al in 1994 as a
point mutation in the gene for coagulation factor V (f V R506Q) [5] while the underlying
biochemical cause for increased risk of venous thrombosis associated with a reduced
sensitivity for APC in the absence of factor V Leiden remained unrecognized [16].
Protein C, a vitamin-K dependent zymogen of the anticoagulant serine proteolytic
enzyme, activated protein C (APC) has a major role in regulating intravascular clotting
[17,19,34,36,56-58] and its deficiency is associated with the thrombosis phenotype
[6,8,44,46,47]. Plasma protein C has: Mr 63,000, plasma concentration 60 nM (4 ug/mL),
and half-life 0.25 day [41]. It is rapidly activated to APC by thrombin-thrombomodulin
on endothelial cell surfaces with the 12 amino acid residue activation peptide being
rapidly eliminated from the circulation [17,19]. Thrombin activation Of human protein C
by cleavage of the peptide bond between Arg 169 and Leu 170 is very slow [36]. In vitro,
protein C is activated to APC by Ot-thrombin [36] and by a protease [37,38] and an
activator protein derived from the Southern Copperhead Agkistrodon contorlrix
contortrix venom [21]. Conversion of protein C to APC was described by Kiesel to occur
in 120 minutes when human protein C (2.3mg) was incubated with human oc-thrombin
(40 ug) at an enzyme to substrate weight ratio Of 1:50 [36]. In 1983 Esmon et a1, [20]
reported a dose dependent inhibition by calcium ions of the activation Of bovine protein C

by bovine thrombin, however, this has not been verified for human protein C. In 1987,

PROTAC® a novel protein C activator, (PROTAC® is a component purified from crude
Southern Copperhead venom and other venoms) provided the first means to determine
the biological activity of plasma protein C [43]. Protac®, an Mr 37,000 protein C
activator, has no significant detectable enzyme activity, is not inactivated by DFP or
antithrombin-heparin, and the molecular mechanism Of protein C activation by
PROTAC® is not fully elucidated [21]. Specific protein C activators isolated from the
same venom activate human and bovine protein C at 121000 enzyme: substrate weight
ratio [38].

APC is an anticoagulant which reduces the velocity Of thrombin formation from
prothrombin by the inactivation of activated coagulation cofactors V and VIII [31,60,61]
and APC is slowly neutralized in plasma with a half life in the circulation estimated at 15
to 20 minutes. Thus, the powerful feedback loop by which thrombin generates more
thrombin from its zymogen prothrombin is held in check. In vitro, bovine and human
APC exhibit anticoagulant activity in the presence of phospholipids and calcium ions
[36].

It is well documented that two biochemical causes result in persistence Of
activated factors V and VIII. One is the protein C deficiency and the other is resistance
to inactivation by APC. Both of these biochemical causes enhance the risk Of thrombosis.
[24,39,48]. The special coagulation laboratory at Michigan State University has been
involved for several years in the study of these disorders [28,30]. Despite the widespread
use of the APC resistance assay, APC effect on native plasma protein C and on purified
protein C has never been studied. In 1997, the anticoagulant APC effects in pooled

normal plasma versus protein C deficient plasma were compared using a commercial kit,

Coatest® APCTM (Chromogenix, Mohndal, Sweden, purchased from Dia Pharm,
Cincinnati OH, USA) it was observed that APC activates its zymogen protein C. The
following research was undertaken to verify this observation by a number of

experimental approaches. Results confirm that APC functions in the activation of protein

C.

MATERIALS AND METHODS

Protein C and plasma reagents

In all experiments, two sources Of protein C were used. One source, pooled
normal plasma (plasma protein C) stored at -80°C was prepared in our laboratory from
the blood of 40 healthy medical students (IRB # 99-200, category 2-B approval by the
University Committee on Research Involving Human Subjects) drawn in 0.105 M sodium
citrate (9 parts blood to 1 part anticoagulant). The second source was protein C isolated
from human plasma, (purified protein C) Lot I-IPC 1120 (1.25 mg/lml in 20mM Tris-
HCL /0.1 M NaCl/ pH 7.4) from Enzyme Research Laboratories Inc., South Bend IN
USA. Pooled normal plasma heat defibrinated at 56°C for 5 minutes was used as a
source for protein C in the amidolytic assays. Heat treatment of pooled normal plasma at
56°C for 5 minutes does not inhibit plasma protein C activation by thrombin or Protac
[29]. FACT (normal plasma control) and plasma immunO-depleted of protein C (protein
C deficient plasma) were purchased from George King Bio-Medical Inc., Overland Park,
KS. Protein C deficient plasma which contains zero unit protein C provides an exquisitely
selective tool to study APC influence on plasma protein C [Craig M. Jackson, personal
communication]. To vary protein C concentrations, pooled normal plasma was diluted in

protein C deficient plasma and purified protein C was diluted in sample buffer for all

assays.

Enzmes and enzyme inhibitor
APC commercial reagents: APC1120 (APC-110) 0.1mg / 91 pl (20mM Tris-HCL /0.1 M
NaCI/ pH 7 .4) and APC 1370P(APC-145) 0.1mg / 91ul (20mM Tris-HCL /0.1 M NaCl/
pH 7.4) were prepared by thrombin activation of the zymogen protein C, Enzyme
Research Laboratories Inc, South Bend, IN, USA. Concentrations Of APC: 92.5ng
(1.65uM) in 4.92 mM CaCl/ 10ul, prepared in our laboratory and frozen at -80°C were
varied in sample buffer according to assay requirement. Human (Jr-thrombin with activity
1660.13 U/ml and concentration 0.45mg/ml [provided by John Fenton II] was diluted to
3ng/ul (0.8 uM) in 100111 distilled water immediately prior to use in the assays.
PROTAC® (Protac) a direct protein C activator (3 U/ml) from American Diagnostica,
Greenwich, CT, USA, was reconstituted to lunit/lml distilled water. Coatest APC/CaClz
(Dia Pharm, Cincinnati OH, USA) was prepared as specified by the manufacturer to
activate plasma protein C in immunoblot experiments. Argatroban,
(2K4R)-4-methyl- l -[N2-[(RS)-3-methyl-1,2,3 ,4-tetrahydrO-8-quinolinosulfonyl-L-
argynyl]-2-pyperindinecarboxylic acid hydrate] Mr 526.66 , binds rapidly to thrombin at
the catalytic site and apOIar region at a diffusion controlled rate [9,10].

Argatroban, [a gift (500mg) Of Mitsubishi Pharma Corporation, Tokyo, Japan]
stock solutions (4mg/ml in distilled water) frozen at -80° C were appropriately diluted in
distilled water to detect contaminant thrombin in commercial APC reagents. Because

Argatroban is a competitive inhibitor it was added to APC at a higher stochiometry.

Buffers and gels for SDS-PAGE and ELISA: were prepared in our laboratory from
chemicals available from Sigma, St. Louis, and MO. Acrylamide/Bis (30%); 1.5M Tris-
HCl (pH 8.8); 0.5M Tris-HCI (pH 6.8); 10% (w/v) SDS; 10% ammonium persulfate
(w/v) TBS-Ca (10m M Tris, 15m M NaCl ,lm M Ca C12 pH 7.5); TBST-Ca solution
(10m M Tris, 15m M NaCl, lmM CaClz, 0.05% Tween 20 pH 7.5); 10% SDS-PAGE
gel; 5% stacking gel (30% acrylamide mix, 0.5M Tris pH 6.8, 10% SDS, 10% APS,
TEMED).

SDS-buffer: (62.5mM Tris-HCl, 20% Glycerol, 20% SDS, 0.5% w/v pH 6.8) and
bromophenol blue in water 0.4ml; Sample buffer: prepared by adding SDS-buffer
(1.33ml) to 90 mM calcium chloride (50 ul) and distilled water (3.07 ml). M
M (Tris base 0.03g, Glycinel4.4g, methanol 200ml to IL in distilled water);
Coating buffer: (15mM Na2C03 , 34.9 mM NaHCOg, 3.08 mM NaN3 pH 9.6). Ming
m (1% PBS- Tween-20, 0.15 M NaCl, 0.01M Na2I-IPO4 pH 7.4)

Antibodies: Monoclonal antibody (MOAb) HPC4 to protein C from human plasma

(200ug lyophilized) purchased from Boehringer Mannheim, Indianapolis, IN, USA; was

prepared lug/ml in PBS for immunoblotting experiments and lug/50 ml PBS for the
ELISA. Monoclonal anti-human protein C purified mouse IgG] clone HC-2, Sigma
P5305 (4mg/ml) was diluted 1.22ug/ml in PBS for immunoblotting, and lug/50 ml PBS
for the ELISA. Polyclonal anti-human protein C purified rabbit IgG] Sigma P-4680 was
diluted 1/ 1000 in coating buffer. Polyclonal anti-mouse IgG (h-l) peroxidase conjugate,
affinity purified from goat, Boehringer Mannheim Co. (1414168) and anti-rabbit IgG

purified in goat, horseradish peroxidase conjugated Sigma A0545 were dilutedl/ 16000 in

PBS.

MOAb HPC4 and HC-2 have identical specificity. Both clones bind specifically
to an epitope on the heavy chain Of protein C Spanning the thrombin cleavage site.
Neither binds to activated protein C or to the released activated peptide. Figure 1 shows

the structure Of protein C and the area Of the activation peptide where the MOAb

binds.

 

II

 

 

 

 

    

 

1w vx‘fl'wy-

 

Activation Peptide

 

 

A;
our—

 

 

 

 

Figure 1. Structure of protein C with activation site. Adapted from The Molecular Basis of Blood Diseases

2"d Edition pg. 603

Clotting Assays

Refer to table 1 under experimental protocols. The anticoagulant effect Of APC
prepared by activation Of purified protein C with thrombin and that Of APC/CaCl2 were
compared in plasma and in plasma selectively immunodepleted Of protein C. The
activated partial thrombOplaStin time (APTT) assay was performed with activated
ThrombcfaxTM reagent Optimized, or Thrombosil*1 activated PTT, and OrthOTM calcium

chloride solution purchased from OrthO Diagnostics Inc, Raritan, NJ, USA. In the

clotting assays 100ul plasma was used which equates to approximately 64.5nM/L protein
C. The APTT clotting times measured in pooled normal plasma, in normal plasma
control, and in protein C deficient plasma (0 unit protein C) respectively were 26.7 i
1.53.; 27.25 i 1.2 and 28.5 d: 1.5 s which is within the normal range previously reported
by the Special Coagulation laboratory [29]. In both pooled normal plasma and protein C
deficient plasma, factor V and factor VIII activities measured were 100% and 98% within
the established normal range (80 to 100%) [29].
Evaluation of contaminant thrombin in commercjrl APC preparations

Complete protein C activation by thrombin was confirmed on 10% SDS-PAGE
gels and thrombin removal from the commercial APC preparations was verified in
clotting assays and in enzyme-linked immunosorbent assays for marker of coagulation
activation (thrombin-antithrombin complexes) and for thrombin inhibition by argatroban.

In clotting assays, thrombin removal was tested by adding APC-110 (10ng) and
APC-145 (10ng) to citrate pooled normal plasma (100uL) and to an 18mg/mL fibrinogen
solution (lOOuL). The mixtures with added APC and controls consisting of citrate
pooled normal plasma and fibrinogen solutions without APC were incubated at 37°C for
2 hours after which a wooden stick was gently spun by hand in the mixtures and in the
controls, then removed and examined for macroscopic evidence of fibrin. Verification of
minute thrombin contamination was accomplished by comparing ELISA measurements
of thrombin-antithrombin complexes (TAT) in plasma mixtures and in controls before
and after the 2 hours incubation at 37°C. Enzygnost TAT Micro kits were purchased

from Behringwerke AG, Marburg, Germany.

Argatroban was used in the ELISA to detect inactivation of contaminant thrombin
in commercial APC preparations. Argatroban (Sug/SOul saline) was added in molar
excess to APC-110 (92.5 ng/ 10p.l) or to APC-145 (92.5 ng/ 10LIL) in TBS /CaC12 buffer
(300ul total volume) and to plasma protein C (100111 plasma). Aliquots added to the
capture antibody were processed as described later and OD readings were performed at 5
minutes intervals for up to 40 minutes.

APC resistme assays

Commercial kits Coatest® APCTM Resistance V (Chromogenix, Mohndal,
Sweden) were purchased from Dia Pharm, Cincinnati OH, USA. Anticoagulant APC
effects in pooled normal plasma, in protein C deficient plasma, and in protein C deficient
plasma augmented with plasma protein C were tested as described in the first commercial
kit Coatest® APCTM [15,35]. Commercial reagent APC-110 (10ng/100uL) purchased
from Enzyme Research, West Bend IN was reconstituted in 3.2% saline without adding
calcium. In the Coatest ® APCTM Chromogenix kit, APC is provided as APC/CaClz. Clot
end points were detected on a CoA Screener (American LaBor, Raleigh, NC, USA) or on
a KoaguLab 6OS (Ortho Diagnostics Inc).

Imrflnoblotting analysis of plasma protein C and purified protein C activation.

Refer to experimental protocols tables 2 and 3. Activation reactions of plasma
protein C or purified protein C by APC in immunoblot experiments were conducted as
follows: varying concentrations of APC in 80ul sample buffer, or 3ng Ot-thrombin (0.1U/
80ul distilled water) or Protac (0.1U/80ul distilled water) were added to pooled normal
plasma (120ul) or to purified protein C (120ul in sample buffer). Incubation times at

37°C were varied from 10 minutes to 180 minutes after which 20 ul activation mixtures

10

were removed, added to sample buffer (60ul) and slowly mixed, then 27ul of activation
mixtures were added to 8 III sample buffer and 1.6 LII PMSF (200Mm diluted 1/ 10) gently
mixed and incubated at 37°C for 1 hour. After centrifugation for 4 minutes, 36111 aliquots
of non-reduced activation mixtures containing purified protein C or plasma protein C
reacted with APC-110, with ct-thrombin, or Protac were separated on 10% SDS-PAGE in
1 mm thick slab gels according to the method of Laemmli [40]. Protein transfer to a
PROTAN pure nitrocellulose transfer and immobilization membrane with a pore size of
0.45 pm was accomplished at 100 V for 2 hours at 4°C or 20V overnight at 4°C. Purified
protein C and plasma protein C were probed by two commercial anti-human protein C
MoAb’s, characterized by Miletich [45,55]. Purified mouse IgGl MOAb HC-2 (MOAb
HC-2) and anti-protein C mouse MOAb HPC4 to protein C from human plasma (MOAb
HPC4). Neither MOAb reacts with APC, but the formation of the MOAb HPC4 /protein C
epitope tag complex is dependent on the presence of calcium while MOAb HC-2 is a
divalent cation independent antibody. Molar concentration of protein C activator to
substrate was varied, and in one experiment, Coatest APC/CaClz was used to activate
pooled normal plasma protein C. Enhanced chemoluminescence density of protein C
immunoprecipitated with MOAb HC-2 or MOAb HPC-4 was measured in pixels by public
domain Scion Image software, Science Corporation, Science Technology Division
(scioncorpcom). The intensities of the 62kD bands in the lanes were quantitated and the
value at zero time before protein C activation was set at 100%. In the experiments,
pooled normal plasma protein C and purified protein C were activated by APC-110,

APC-145, by Coatest APC/CaClz, by Ot-thrombin, or Protac.

Plasma rotein C activation measured by enzyme-linked immunosorbent assay (ELISA)

ll

MOAb HC-2 and two antigen affinity purified rabbit antihuman protein C
polyclonal antibodies were used to develop a three site ELISA system based on the
change in antigen structure resulting from protein C activation. The capture antibody for
fixation of protein C and products of protein C activation (protein C and APC) is a
commercial polyclonal rabbit anti-human protein C IgG diluted 1/ 1000 in coating buffer
(15mM Na2C03, 34.9mM NaHCO3, and 3.08mM NaNg) was adsorbed onto wells in
plates manufactured by Nunc-ImmunoTM (MaxiSorpTMSurface, Nalge Nunc International,
Denmark). MOAb HC-2 (1/50 in 100 ILL PBS) added to each well blocked the
unoccupied epitope on the plasma protein C heavy chain that is lost when the activation
peptide is released. MOAb HC-2 does not react with APC. The detection antibody, a
commercial horseradish peroxidase conjugated-polyclonal anti-human protein C rabbit
IgG measured the captured APC.

Plasma protein C activation assay conditions were as follows. Aliquots of pooled
normal plasma (300 ILL) in 1% TBS-Tween, 0.2 M CaClz to a total volume 500 ILL were
separately reacted with 14.8nM APC-110, or with Protac (0.1U/ 80ul in distilled water)
for 20, 30, 40 and 50 minutes at 37°C. Reaction mixtures (100uL) were left for 1 hour at
37°C to react with the capture antibody. After repeated washing, MOAb HC-2 (1/50 in
IOOILL PBS) added for 1 hour at 37°C to each well blocked the unoccupied epitope on the
plasma protein C heavy chain. Afier more washing, an enzyme conjugate of the same
polyclonal anti-protein C rabbit IgG was added to detect the captured APC. Color
formed by horseradish peroxidase reaction with O-phenylene diamine (OPD) substrate
was read at 492 nm on Spectra Max Plus microplate reader. APC from plasma protein C

activation by APC-110 and Protac was estimated from changes in absorbance using the

12

standard curves to find the corresponding points. Standard curves were derived by linear
regression analysis of the changes in absorbance plotted versus 1.8 nM, 0.9 nM, and 0.45
nM of the reactive APC bound to the horse radish peroxidase conjugated-polyclonal anti-
human protein C rabbit IgG in 0.2 M CaClz TBS buffer. Quantity of APC from protein C
activation was derived from the absorbance of color readings against the corresponding
points on the standard curves.
Substgte-Based Functional Assay

Refer to experimental protocols table 4. Chromogenic substrate H-D-Trp-Arg-
Arg-p-nitroaniline specific for APC [13] was generously provided by Dr Enrico DiCera.
The source of plasma protein C was pooled normal plasma heat defibrinated at 56°C for 5
minutes [29]. Time-dependent activation Of plasma protein C to APC by APC-110
(5.1nM), and Protac (0.1U), was monitored at 405 nm on a Spectra Max Plus microplate
reader (Bio-Tek Instruments, Inc, Winooski, VT, USA). APC from plasma protein C
activation by APC-110 and Protac was estimated from changes in absorbance using the
standard curves for the corresponding points. Standard curves were derived by linear
regression analysis of the changes in absorbance plotted versus 1.27nM, 2.55nM, and
5.1nM APC-110 in 0.2M CaClz TBS buffer. Changes in absorbance from chromogenic
substrate p-nitroaniline release by APC-110 (5.1nM) added to activate plasma protein C,
were subtracted from data points.
Kinetic Experiments

Kinetic experiments were performed as follows. Human protein C and activated
protein C were purchased from Enzyme Research, South Bend, IN. The chromogenic

substrate (D)-DRR was Obtained from Midwest Bio-Tech and S2266 from Chromogenix.

13

Progress curves following the release Ofp-NA after DRR hydrolysis were measured at
405nm. Assay conditions were 145mM NaCl, SmM Tris pH 7.4 at 37 °C, 0.1% PEG,
lOrnM CaClz. Typical concentrations of activated protein C and protein C were 10-50nM
and 10-100nM, respectively. The progress curves were analyzed with KINSIM and
FITSIM to obtain kw/Km for chromogenic substrate hydrolysis and protein C cleavage [2].

The progress curves were analyzed assuming the following mechanism for PC activation:

aPC + PC :3 aPC-PC :3 ZaPC (1)

aPC+S .—_s aPC-S : aPC-P .—_e aPC+P (2)

where the rate constants for equation 2 were independently determined in the absence of

PC, and these values were used to obtain the value of law/Km for PC activation [2,14].

14

EXPERIMENTAL PROTOCOLS

CLOTTING AS SAYS:

Table 1. Clotting assay materials and methods

 

Pooled Normal Plasma (PNP)

Source for native protein C. PNP is
prepared from citrated blood of 40 healthy
individuals.

 

Immunodepleted protein C deficient
plasma (George King Biomedical Inc.)

Standard curve dilutions are performed in
protein C deficient plasma with PNP to a
total of 100u1

 

APC/CaC12 (Chromogenix AB Molndal,
Sweden

APC/CaC12 used according to
manufacturer instructions is added to PNP
(100ul) and to protein C deficient plasma

@0110

 

 

APC (Enzyme Research Ind.) APC reconstituted in deionized water
(100ng/10u1) is added to PNP (100ul) and
to protein C deficient plasma (100ul)

APTT assay Reagent: Thrombofax (ODSI) ellagic acid

& purified phospholipids incubation time 4
min. Clot forms with 0.02M CaC12

 

 

APTT assay (Chromogenix AB Molndal,
Sweden

 

Reagent: Colloidalsilica & purified
phospholipids incubation time 5 min. Clot
forms with 0.025M CaC12

 

INIMUNOBLOTTING ANALYSIS OF PLASMA PROTEIN C AND PURIFIED

PROTEIN C ACTIVATION:

Table 2. Time dependent activation Of plasma protein C (native protein C) by

APC 1:1.38 enzyme: substrate weight ratio

 

Pooled Normal Plasma (PNP)

200ul (800ng Native protein C or 64.5nM)

 

Purified APC (APC)

10.8ul (1000ngL

 

Incubation mixture

200u1 PNP (800ng native protein C) +
10.8ul APC (1000ng) (61.19nM PC +

 

 

84.5nM APCL
Incubation Times 5, 10, 20 mins
Incubation Temp. 37 C

 

Samples from incubation mixture

20u1 added to 60u1 sample buffer = 80ul
(15.3nM PC + 21.1nM APC)

 

Sample size loaded into gel

27ul from the 80ul above + 9ul buffers
(11.3nM PC +15.6nM APC)

 

Control sample

PNP (12nM native plasma protein C)

 

SDS-PAGE

10%

 

MOAb clones

HPC4

 

 

Chemiluminescence density in pixels

 

Software: Scion Image

 

15

 

 

Table 3. Evaluation by Western Blotting- Activation of plasma protein C by

Protac, Thrombin and APC/CaC12

 

Pooled Normal Plasma (PNP)

Serial diluted to give 16.1nM PC (80ng
native plasma protein C/100u1 (0.02U))

 

 

 

 

APC/CaC12 100ul
APTT Reagent 100ul
Protac 0. lU/ 100ul
Thrombin 0. 1U/100ul

 

Incubation mixtures:

100ul 0.02U PNP + 100ul APTT reagent +
100ul APC/CaC12

100ul 0.02U PNP + 100ul buffer + 100ul
Protac

100ul 0.02U PNP + 100ul buffer + 100u1
Thrombin

100ul 0.02U PNP + 200ul buffer (Control)

 

Incubation Times

30 mins

 

Incubation Temp.

37C

 

Samples from incubation mixture

27ul

 

Sample size loaded into gel

27ul from incubation + 9u1 buffer = 36ul
(4nM native protein C in each well)

 

Control sample

PNP (4nM native plasma protein C)

 

SDS-PAGE

10%

 

MOAb clones

HC-2

 

 

Chemiluminescence density in pixels

 

Sofiware: Scion Image

 

SUBSTRATE-BASED FUNCTIONAL ASSAY:

Table 4. Chromogenic substrate assay- Plasma protein C activation by Protac,

APC and thrombin

 

Reagents & Methods

Activation & Detection Methods

 

Test Plasma

Heat defibrinated PNP (3 00ul)

 

Protein C Activators

50ul Protac (100ng), APC (100ng) and
Thrombin (100ng)

 

Incubation mixture

Defibrinated PNP (3 00ul) + Activator
(50ul) + tris buffer (200ul)

 

Sample size

50ul

 

D-tryp-arg-ppnitroanalide

50ul

 

Time dependent measurements

2,5,10,15,20,30,40,50 min

 

 

Equipment: Spectra MaxPlus

 

Reaction read at 405nm

 

16

 

 

RESULTS

Evaluation Of contaminant thrombin in commercial APC prepmtions

 

The commercial APC reagents APC-110 and APC-145 did not induce fibrin to
form in citrate pooled normal plasma or in fibrinogen solutions and the TAT complexes
measured in pooled normal plasma augmented with APC and in the control pooled
normal plasma were within the same range. APC-110 and APC-145 did not increase
TAT complexes above baseline.

In Figure 2, progress curves indicate a time dependent increase in protein C
activation following incubation of plasma protein C with APC-110 or APC-110 and
argatroban. The percentage of plasma protein C activation detected in the ELISA by a
horse radish peroxidase-conjugated polyclonal anti-human protein C rabbit IgG is
calculated from the absorbance of color at zero incubation set at 0%. Standard curve for
data points is depicted in the top inset. On that basis, protein C activation rate by APC-

110 with or without argatroban is comparable reaching 68% at 40 minutes.

17

Standard Curve

 

 

 

 

 

 

 

 

 

 

14 .3
fig 12 3
° 10 i
E a
e 8 '5
ca 6 i
g 1
o 4 ‘3
2 «3
O T'— W Y I I
0 0.1 (12 (L3 (L4 (15
Mean Value
y==rA-+13x-+(3x I\ 13 C3_m Iiz
1.418 -22.552 92.209 0.998
° Std (Standards: Mean Value vs Concentration)
80
70<
:: 60<
.2
iii
.2: 50 . .
8 —O—- protern C+APC
43:
U 40 ~ _
,5 + protern C+APC
o
E 30 ‘ +argatroban
.:\° 20 .
10~
0 ‘ T u I v f

Figure 2. Plasma protein C activation by APC-110 and by APC-110 + Argatroban measured by enzyme-
linked immunosorbent assay (ELISA). Progress curves indicate % plasma protein C activation to APC by
APC-110 and APC 110 + Argatroban after pooled normal plasma (100 uL) was reacted with APC-110
(92.5 ng) and APC-110 (92.5ng) + argatroban (Sag/501d for 40 minutes). OD readings were taken at 5

minute intervals. Plasma protein C activation to APC was detected by blocking non-activated protein C

with a MOAb

standard curves for the corresponding points, quantity of APC bound to horseradish-conjugated polyclonal

antihuman protein C rabbit IgG was estimated from changes in absorbance afier reaction with OPD

substrate.

 

 

 

 

0 5 10 15 20 25 30 35 40

Time (min)

HC-2 specifically reactive with the plasma protein C and does not react with APC. Using the

18

Effect of glam gotein C on anticoagu_lant response to APC in protein C deficient
claim

Comparison of the anticoagulant responses to APC in protein C deficient plasma
versus pooled normal plasma indicates plasma levels of protein C provide an added
anticoagulant response. When APC-110 and Coatest APC/CaC12 were added to protein C
deficient plasma containing 0 unit plasma protein C the APTT clotting times increased
from baseline (28.5 i 1.5 s) tO 56.6 i 2.2 s (n=16) and 73.6 i 3.2 s (n=16) respectively.
When APC-110 and Coatest APC/CaC12 were added to pooled normal plasma containing
0.08unit protein C the clotting times increased from baseline 26.7 d: 1.55 to 106.7 i 2.63
(n=16) and 96.2 i 2.95 (n=16) respectively [Fig 3]. Furthermore, when plasma protein C
concentrations (0 unit to 0.008 unit) were varied by adding serial dilutions of pooled
normal plasma to protein C deficient plasma, a steady linear increase in the anticoagulant

response to added APC proportionate to increases in plasma protein C concentrations

was obtained [Figure 4].

19

 

 

  

 

 

 

Hn(wu

 

110 -

90~

70~

APTT (sec)

 

 

30 T f I l T I
0 0.008 0.016 0.024 0.04 0.08

Pooled normal plasma (protein C unit)

 

Figure 4. Effect of plasma protein C on APC anticoagulant response measured by APTT in seconds To
vary protein C concentration, pooled normal plasma was serially diluted in protein C deficient plasma The

APC used in the experiments were APC-110 (A ) and Coatest APC/Ca C12 ( C1 ).

Immunoblot analysis of plasma protein grid purified protein C actifivation

Indication that plasma levels Of protein C provide an added anticoagulant
response was probed fiirther by means of two commercial MOAb described by Miletich
[35] raised against peptides containing the thrombin cleavage site on protein C. Human
protein C is activated to APC by thrombin cleavage of the peptide bond between Arg 169
and Leu 170, releasing an activation peptide of lZ-amino acid residues [36]. The mouse
monoclonal antibody (MOAb) HC-2 recognizes an epitope on human protein C and
inhibits its activation to APC. This monoclonal antibody is specifically reactive with the
zymogen @rotein C). In contrast, it does not recognize either of the activation products:

neither the activation peptide nor the APC [45]. Similarly, the mouse MOAb I-IPC-4

21

recognizes the peptide EDQVDPRLIDGK of protein C; (this peptide is lost during
activation from protein C to APC). Thus, MOAb HPC4 binds with high affinity to this
sequence in protein C or in proteins tagged with this epitope . It does not react with APC
[45,55]. The specificity of these two monoclonal antibodies was confirmed. Panel A of
Figure 5 shows that MOAb HPC4 immunoblotted a 62 kD band in samples of purified
protein C (lanes 1 and 2) but not APC (lane 3). This antibody also immunoblotted a
62kD band in pooled normal plasma (lane 4) but not in protein C deficient plasma (lane
5). Similarly, panel B of Figure 4 shows that MOAb HC-2 yielded a 62kD band only
with samples containing protein C (lanes land 2). The 62kD band was not observed with
either APC (lane 3) or with protein C deficient plasma (lane 5).

Pooled normal plasma was incubated with APC and the level of protein C was
monitored as a function of time by immunoblotting with MOAb HPC4. The initial
concentration of protein C in pooled normal plasma is about 12nM. The data obtained
using 15.6nM of APC (protein C to APC ratio of 1:1) are Shown in Figure 6. There was
a time-dependent decrease in the intensity of the 62kD band corresponding to protein C
(Figure 5, lanes 3-7). The intensities of the 62kD bands in the lanes were quantitated and
the value at zero time was set as 100%. On this basis, 58% of the protein C was
converted in 20 minutes (Figure 6 inset on the right). Since MOAb HPC4 recognizes only
the zymogen protein C, but not the activated product APC, it is inferred from these data
that the disappearance Of the immunoblotted 62kD band corresponds to activation Of
protein C.

Purified protein C was incubated with APC and the level of protein C monitored

as a fiinction of time by immunoblotting with MOAb HPC4. The data obtained using

22

purified protein C (60nM), and APC (6nM) (protein C to APC ratio of 10: 1) are shown in
Figure 7. There was a time-dependent decrease in the intensity of the 62kD band
corresponding to protein C (F igure7, lanes 3-7). The intensities of the 62kD bands in the
lanes were quantitated and the value at zero time was set as 100%. On this basis, 77% of

the protein C was converted in 180 minutes (Figure 7 inset on the right).

 

1 2 3 4 5 A

 

.62kD

 

“62kD

 

 

 

Figure 5. Comparison of the reactivity of monoclonal antibodies HPC-4 and H-C2 on protein C versus APC
by immunoblotting. (A) HPC-4; (B) HC-2. For both panels A and B, the following samples were
electrophoresed in: Lane 1 purified protein C (22nM), Lane 2 purified protein C (44nM), Lane 3 APC
(48.7nM), Lane 4 pooled normal plasma (4.3 uL). Lane 5 protein C deficient plasma (4.3uL). Enhanwd

chemoluminescence was measured by public domain Scion Image software.

23

 

   

 

 

 

% decrease from initial plasma
protein C reactrvrty
A N u 8 0| 0) N on
o o o o e o e o
l L l 1 1 l 1 41

 

 

0
GI

10 20

Time (min)

Figure 6. Immunoblot analysis of time dependent APC-catalyzed activation of plasma protein C ratio of
protein C to APC-110, 1:1 (APC at 15nM); The following samples were electrophoresed in: Lane 1
purified protein C marker (44nM), Lane 2 APC-110, Lane 3 pooled normal plasma (4.3uL), Lane 4 pooled
normal plasma (4.3pL) afier incubation with APC for 2 min , Lane 5 pooled normal plasma (4.3uL) after
incubation with APC for 5 min, Lane 6 pooled normal plasma (4.3 pL) afier incubation with APC for 10
min, Lane 7 pooled normal plasma (4.3pL) after incubation with APC for 20 min. Lane 8 purified protein
C marker (44nM). Chemoluminescence density was measured in pixels by public domain Scion Image

software. The graph depicts the percentage of APC-catalyzed activation of plasma protein C.

24

 

 

 

80 a
U
.5 70 ‘
a
E- 60 —
'U
2
*5 50 -
a o-
:5 °§ 4o —
.E a
a 2’.
g 30 -
8
a 20 -
E
'U
°\e 10 ‘

o 1 l fir I

0 90 120 180

Time (min)

Figure 7. Immunoblot analysis of time dependent APC-catalyzed activation of purified protein C Ratio of
protein C (protein C at 60nM) to APC-110 (APC at 6nM), 10:1. The following samples were
electrophoresed in: Lane 1 APC marker (44nM), Lane 2 purified protein C marker (60nM), Lane 3 purified
protein C after incubation with APC for 30 min, Lane 4 purified protein C after incubation with APC for 60
min, Lane 5 purified protein C afier incubation with APC for 90 min, Lane 6 purified protein C after
incubation with APC for 120 min, Lane 7 purified protein C after incubation with APC for 180 min.
Chemoluminescence density was measured in pixels by public domain Scion Image software. The graph

depicts the percentage of APC-catalyzed activation of purified protein C.

25

Im_munoblot mlysis of plam protein C activation by thrombin, Protac and Coatest
APC/CaCl_2.

The data in Figure 8 was obtained using plasma protein C (4nM) incubated for 30
minutes with thrombin (0.1U/80uL) and Protac (0.1U/80pL). There is total
disappearance of the 62kD band corresponding to protein C (Figure 7, lanes 2-5)
following incubation for 30 minutes of plasma protein C with ct-thrombin and Protac. In
Figure 8, intensities of the 62 kD bands in the lanes 6 and 7 corresponding to protein C
incubated with Coatest APC/CaC12 show 54% and 63% (mean 60%) of the protein C was

converted by Coatest APC/CaC12 (quantitated from the value at zero time set as 100% ).

 

h62kD

   

 

 

 

Figure 8. Immunoblot analysis of plasma protein C activated by human (at-thrombin by Protac and by
Coatest APC/CaC12. The following samples were electrophoresed in: Lane 1 pooled normal plasma
(4.3 pL), Lanes 2 and 3 plasma protein C (4nM)after incubation with thrombin (0.1U/80uL) for 30 min,
Lanes 4 and 5 plasma protein C after incubation with Protac (0.1U/80 pl) for 30 min, Lanes 6 and 7 plasma
protein C after incubation with Coatest APC/CaC12 (lOOuL). Imrnunoprecipitate chemoluminescence

density was measured in pixels by public domain Scion Image software.

26

Llaml protein C activation measured by enzyme-linked immunosorbent assay (ELISA)
The APC from plasma protein C activation by APC-110 and Protac was detected
by a horseradish peroxidase-conjugated polyclonal anti-human protein C rabbit IgG. The
data in figure 9 show the percentage of protein C activation following incubation of
plasma protein C with APC-110 or Protac. The bar graphs depict a time dependent
increase in protein C activation following incubation of plasma protein C with APC-110
or Protac. The absorbance of color at zero incubation was set at 0%. On that basis,
protein C activation by APC-110 was 8% at 20 minutes; 31% at 30 minutes, 65% at 40
minutes and 73% at 50 minutes. Activation of protein C by Protac was 5% at 20 minutes;

28% at 30 minutes; 62% at 40 minutes and 68% at 50 minutes.

27

I APC-110
I Protac

% Plasma protein C activation

 

 

Time (min)

Figure 9. Plasma protein C activation by APC-110 and Protac measured by enzyme-linked immunosorbent
assay (ELISA). Bar graphs represent % plasma protein C activation to APC by APC-110 and Protac afier
pooled normal plasma (300uL) was reacted separately with APC-110 (14.8nM) and Protac (0.1U/100uL)
for 20, 30. 40, and 50 minutes. Plasma protein C activation to APC was detected by blocking non-activated
protein C with a MoAb HC-2 specifically reactive with the plasma protein C and does not react with APC.
Using the standard curves for the corresponding points, quantity of APC bound to horseradish-conjugated
polyclonal antihuman protein C rabbit IgG was estimated from changes in absorbance after reaction with

OPD substrate.

28

Time dependerflctivation of plasml protein C by APC-145 and Protac msured in a
subflate—based fiinctionaLfisay

Plasma protein C activation by APC-145 and Protac was measured by hydrolysis
of chromogenic substrate H-D-Trp-Arg-Arg-p-nitroaniline specific for APC. Progress
curves in figure 10 indicate plasma protein C activation to APC monitored by p-
nitroanilide release at 405nm. Quantity of APC released from protein C activation was
derived from the absorbance of color readings against the corresponding points on
standard curves depicted in the inset. The absorbance of color at zero incubation was set
at 0%. On that basis, after 120 minutes, 62% of protein C is activated to APC by APC-

145 and 73% of protein C is activated to APC by Protac.

29

 

0.8

0.7 .
0.6 - —B—APC-145 (0.89 pmol/L)

—0—APC-145 (0.446 pmol/L)

      

0-5 ‘ —A—APC-145 (1.78 pmol/L)

0.11 (405 nm)

 

 

 

 

 

 

0.4 4
0.3 ‘i
0.2 -
1.2 T
1.0 .
.. 0.8 -
E
C
§ 0.6 «
C!
o on -
0.2 ~
0.0 I I I if T I r I fir I T I
1 10 20 30 40 50 60 70 80 00 100 110 120
Time (mln)

Figure 10. Time course of plasma protein C activation to APC by APC-145 ( — ) and Protac ( — — —)

measured as APC amidolytic activity. The amidolytic response is measured at 405 nm as released p-
nitroanilide from chromogenic substrate H-D-Tryp-Arg-Arg—p-nitroanilide specific for APC. Pooled normal
plasma contained 0.24 unit of plasma protein C. Enzyme and protein C activator used in these experiments
were SOuL APC-145 (5.1nM), and 50uL Protac (7.14nM) . Standard curves are depicted in the insert The
APC concentrations used to produce the standard curves were APC-145 (0.46pmol/L) APC-145 (0.89pmol/L)

and APC-145 (1.78pmol/L).

30

Kinetic Data

The hydrolysis of small chromogenic substrates by activated protein C (APC) was
significantly enhanced in the presence of the zymogen form of protein C. The hydrolysis of
chromogenic substrate DRR by protein C alone was performed to detect any trace of
contamination in the protein C sample. There was practically no hydrolysis of substrate in
the absence of APC consistent with a mechanism of activation of protein C by APC. The
values obtained for substrate hydrolysis agreed with previous studies under the same
conditions where Kan/Km = 1.0 mM'ls'l [14]. A value of4.7 i 0.4 mM‘ls'l for the activation

of protein C by APC was obtained under physiological conditions and in the presence of

calcium.

31

DISCUSSION

The above results demonstrate [figures 2-9] that APC must fiinction not only in
the inactivation of activated factors V and VIII, but also in the activation of protein C.
This additional APC function widens the scope for both the laboratory APC resistance
assay and the APC resistance clinical disorder to include protein C and protein S
phenotypic and genotypic variations. Given that APC has the same responsiveness on

plasma protein C as ct-thrombin and Protac, the diagnostic specificity for the APC

resistance assay is that of a global test for the protein C anticoagulant pathway [50, 51].
In the absence of factor V Leiden, variations in plasma protein C levels as a cause for
reduced sensitivity for APC have been reported. The effect of activated protein C
resistance was reported by Simoni et al [53,54] who Observed a high incidence of
apparent type H protein C deficiency in homozygous and heterozygous members of two
out of three kindred’s with factor V Leiden when using a Behring PC clotting assay. In a
study of 6 heterozygotes and 2 homozygotes for the factor V Leiden mutation, Ireland et
al [33] reported similar finding with the Behring method, but found no influence of
activated protein C resistance on the Instrumentation Laboratory (IL) PC Proclot kit. In
contrast, Faioni et a1 [22] reported low levels of protein C with the IL Proclot assay in 25
patients all with APC resistance. Moreover Cooper et al [11] provide evidence that
activated protein C resistance can be diagnosed as inherited functional protein S
deficiency. Similar findings in a study of 301 healthy individuals by de Ronde et al [15]
identify a profound effect on the APC resistance sensitivity ratio by protein S an

Obligatory cofactor for optimal protein C function.

32

This data [ figures 5-9 ] closely follow the time course for the activation of human
protein C by human oc-thrombin, itself a poor activator of protein C. As reported by
Kiesel, human protein C (3.6nM) activation by human oc-thrombin (12an) at enzyme to
substrate molar ratio 3 :1 was completed afier 120 minutes incubation [3 6]. In this report
of kinetic studies using isolated protein C in vitro, a value Of 4.7 i 0.4 mM'ls'l for the
activation Of protein C by APC was Obtained under physiological conditions and in the
presence of calcium, denoting poor interactions. It is well documented that optimal
expression of the anticoagulant activity of protein C requires the expression of cofactor
aetivity. This concept of a protein C anticoagulant system emerged in 1981, when Esmon
and Owen [18] described the isolation of a protein from the membrane of rabbit
endothelial cells: thrombomodulin, which could accelerate the activation Of protein C by
thrombin about 1,000-fold. Esmon also determined that formation of the thrombin-
thrombomodulin complex results in more than 20,000-fold increase in the activation rate
of protein C by thrombin [17]. Furthermore it was shown by Salem et al [50,51] that
factor Va enhances the rate of protein C activation by thrombin by 50 fold and protein S

accelerates activated factor V inactivation by selectively promoting the slow cleavage at
Arg 506 (20 fold) [49]. Additionally, optimal expression of anticoagulant activity of
APC requires the presence of negatively charged phospholipids [59] and human protein C
undergoes Ca2+ induced conformational changes required for activation by the thrombin-
thrombomodulin complex [55]. It is therefore possible to speculate that the expression of
a cofactor activity in viva, perhaps factor Va or VIIIa might enhance the activation of

protein C by activated protein C. Until the identity of this cofactor activity is elucidated,

33

the physiological relevance for APC-mediated protein C activation remains open to

speculation.

34

CONCLUSION

This data represents four lines of evidence that APC can activate plasma protein C
and purified protein C. These results indicate comparable activation of plasma protein C
and purified protein C by APC by ant-thrombin and Protac. On the basis of these
Observations, APC must function not only in the inactivation of activated factors V and
VIII, but also in the activation of protein C. This additional APC function defines the
laboratory APC resistance assay as a global test for the protein C anticoagulant pathway,
and classifies the clinical APC resistance disorder to include variations in plasma protein
C levels. Furthermore activation of plasma protein C by APC may be important to

consider more broadly because of APC in the treatment of sepsis [3].

35

10.

11.

12.

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