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The comparison of protein S synthesis and processing among various cell types that produce human protein S

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Title:
The comparison of protein S synthesis and processing among various cell types that produce human protein S
Creator:
Richmond, Carla Diane
Publication Date:
Language:
English
Physical Description:
ix, 72 leaves : illustrations, photograph ; 29 cm

Thesis/Dissertation Information

Degree:
Master's ( Master of arts)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Integrative Biology, CU Denver
Degree Disciplines:
Biology

Subjects

Subjects / Keywords:
Proteins ( lcsh )
Proteins ( fast )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 63-72).
General Note:
Submitted in partial fulfillment of the requirements for the degree, Master of Arts, Biology.
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Carla Diane Richmond.

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Source Institution:
University of Colorado Denver
Holding Location:
Auraria Library
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
36392140 ( OCLC )
ocm36392140
Classification:
LD1190.L45 1996m .R53 ( lcc )

Full Text
THE COMPARISON OF PROTEIN S SYNTHESIS AND PROCESSING
AMONG VARIOUS CELL TYPES THAT PRODUCE HUMAN PROTEIN S by Carla Diane Richmond B.S., Sterling College, 1984 A thesis submitted to the University of Colorado at Denver in partial fulfillment of the requirements for the degree of
Master of Arts
Biology


This thesis for the Master of Arts
degree by
Carla Diane Richmond
has been approved
by

Date


Richmond, Carla Diane (M.A., Biology)
The Comparison of Protein S Synthesis and Processing
Between Various Cell Types that Produce Protein S
Thesis directed by Associate Professor Bradley J. Stith
ABSTRACT
In this study, the basal rate of protein S (PS)
synthesis was analyzed and compared among the cell types
that produce PS within the vascular system. The cell
types studied were the Hep G2 cell (a hepatocyte cell
line), the K562 cell (a megakaryocyte/platelet cell
line), and the human umbilical vein endothelial cell
(HUVEC). The Hep G2 cell secreted the most PS with a
basal rate of 1.85 X 10'15 gram/cell/hour. This cell was
followed by the HUVEC with a basal rate of 0.37 X 10'15
grams/cell/hour. The K562 cell secreted very little PS
(basal rate of 0.0404 X 10'15 grams/cell/hour). The HUVEC
stored 3 0.5 X 10'15 grams/cell which was 5 to 8 times more
in


PS compared to the Hep G2 cell (5.46 X 10'15 grams/cell)
and the K562 cell (3.78 X 10~15 grams/cell).
The effects of vitamin K and Warfarin on the
secretion and storage of PS were also studied. Vitamin K
had no effect on the synthesis or storage of PS in the
Hep G2 cell. In the K562 cell, vitamin K increased the
secretion of PS by 4 to 6 fold. However, vitamin K had
no effect on the storage of PS in the K562 cell. Vitamin
K induced a slight increase in the rate of secretion in
the HUVEC. The HUVEC was the only cell type in which
storage of PS was affected by vitamin K but only by 11%.
Warfarin down regulated the secretion of PS by the
Hep G2 cell by 36%, but increased the storage by 13%.
The secretion of PS by the K562 cell was not effected by
Warfarin treatment, however, paradoxically the amount
stored within the cell increased 5% to 8%. Warfarin had
no effect on the secretion or storage of PS in the HUVEC.
IV


Hepatocytes maintain the basal level of PS in the
plasma but increase the concentration at the local injury
site is supplied by the platelet (K562) and the
endothelial cell. Warfarin and vitamin K affected each
cell type differently, which explains why the plasma
levels and local site levels are different compared to
other vitamin K dependent proteins.
This abstract accurately represents the content of the
candidate's thesis. I recommend its publication.
Signe
Bradley J. Stith, Ph.D.
v


Dedicated to my parents,
Pauline and Alfred Richmond


ACKNOWLEDGMENTS
I would like to thank my advisor, Dr. Richard A.
Marlar for his guidance, patience, and support during
this project. I also want to thank my committee members,
Dr. Bradley J. Stith and Dr. Alan P. Brockway for their
guidance and support. Special thanks goes to Laurie Graf
and David Kressin for our numerous helpful discussions
and to Jo Kent and Michael Vaughan for their help in
setting up the formats of figures presented in this
thesis. Also, I would like to thank Thrombosis Research
Laboratory and the Denver VA Medical Center for providing
the financial support for this project. My greatest
appreciation goes to my family and friends for their
constant support and understanding through the difficult
times.
Vll


CONTENTS
CHAPTER
1. INTRODUCTION ................................. 1
Primary Hemostasis ........................... 1
Coagulation System ........................... 3
Regulation of the Coagulation System .... 8
Protein C System............................. 10
Protein S.................................... 13
Clinical Aspect of Protein S ................ 17
Molecular Biology of Protein S .............. 18
Cell Biology of Protein S.................... 19
Post-Translational Processing of Protein S 20
Mechanism of Vitamin K and Warfarin .... 22
Purpose of Research.......................... 24
2. MATERIALS AND METHODS........................ 26
Materials.................................... 26
Cell Types.............................. 26
Media and Media Additives............... 26
vm


Other Materials......................... 27
Methods...................................... 27
Cell Cultures........................... 27
Determination of PS Antigen......... 29
SDS-PAGE and Western Blots .......... 30
Statistical Analysis of Data....... 31
3. RESULTS...................................... 33
Production of Protein S...................... 33
Hep G2 Cells............................ 33
K562 Cells.............................. 39
HUVEC................................... 44
4. DISCUSSION................................... 48
Basal Rates of Protein S Production .... 49
Effects of Vitamin K and Warfarin on
PS Production................................ 54
Vitamin K............................... 55
Warfarin................................ 58
Conclusions.................................. 61
REFERENCES........................................ 63
IX


CHAPTER 1
INTRODUCTION
The human body requires a hemostatic balance within
the vascular system to maintain the blood in a fluid
state and prevent blood loss from the vessels. This
system is maintained by the interaction between the
procoagulant, anticoagulant, and fibrinolytic systems,
cellular components, and the vessel wall (1,2).
Primary Hemostasis
The hemostatic process is initiated when an injury
has occurred to the endothelial lining of the vessel wall
and is followed by a series of events which limit blood
loss and to halt the development of a intra-vessel clot
(thrombus) (1,3). The endothelial cells that line the
vessel wall form a barrier between the surrounding tissue
and the circulating blood. In their quiescent state,
endothelial cells provide a non-thrombogenic surface
1


(4,5). Injury to the endothelium exposes the
subendothelial layer, a highly thrombogenic surface,
which initiates platelet adhesion and aggregation, and
activates the coagulation system (4,6,7).
The role of platelets in the hemostatic process is
to adhere to the site of injury and aggregate with other
platelets to form the platelet plug (also referred to as
the primary hemostatic plug) to temporarily reduce and
stop blood loss into the surrounding tissue (8,9).
Aggregation is the accumulation of platelets with those
initially adhering at the site of injury (5). Platelets
circulate in the blood in a resting form which adheres to
the exposed collagen and undergo a series of biochemical
and morphological changes (6). In this process, numerous
intracellular proteins and small molecules are released
to accelerate the adhesion and aggregation of platelets
and the subsequent formation of the platelet plug at the
site of injury (6,8,10). Activated platelets express two
different membrane glycoprotein receptors which aid in
the adhesion and aggregation of platelets at the site of
2


vascular injury (11). The receptor complex, glycoprotein
Ib/glycoprotein IX (GIb/GPIX) provides platelet adhesion
to the vascular subendothelial matrix via von Willebrand
factor (vWf) (5,11). vWf is present in plasma and
released from the a-granules (storage compartments) of
platelets upon activation of the platelets (8,11). The
second receptor complex, glycoprotein Ilb/IIIa
(GPIIb/IIIa), is involved in the aggregation process of
platelets (5,8). The GPIIb/IIIa receptor specifically
binds to fibrinogen which forms a bridge between adjacent
activated platelets (8,12). These receptor reactions set
the stage for the coagulation cascade and fibrin
formation by providing a negatively charged phospholipid
surface on activated platelets.
Coagulation System
The blood coagulation system is a cascade of
enzymatic activations leading to the formation of
thrombin, which ultimately converts fibrinogen to the
fibrin clot (8,13). One of the remarkable aspects of the
3


coagulation system is its rapid response to seal wounds
within the vascular system without the clot propagating
and occluding the blood vessel (8). The process is
regulated by a host of components with either
procoagulant or anticoagulant properties (1,10).
Imbalances in one of the components of either system can
lead to excessive bleeding (defective procoagulant
system) or to excessive fibrin formation (defective
anticoagulant system) (1,14).
The plasma proteins that are involved in the
clotting cascade are converted from a proenzyme to an
enzyme; these enzymes then initiate the formation of the
fibrin clot, which reinforces the platelet plug at the
site of injury (3,6). The blood coagulation system
consist of two pathways: Intrinsic and Extrinsic (Fig
1.1). Both pathways require proenzymes, protein
cofactors, phospholipids, and calcium ions (7). In order
for the intrinsic coagulation system to be initiated, the
plasma proteins must come in contact with a negatively
charged surface such those on collagen or
4


Extrinsic Pathway
Intrinsic Pathway
Factor VII
Tissue Factor
Factor XII
I
Factor XI
Common Pathway
Factor
Prothrombin
Fibrinogen
I
Factor IX
Thrombin
Fibrin Clot
Figure 1.1: The Coagulation System. The coagulation system is made up of two pathways,
Extrinsic and Intrinsic, that converge into the common pathway. The extrinsic pathway is initiated
when tissue factor is exposed due to an injury to the endothelium lining. Tissue factor forms a
complex with factor VII and activates factor X. The intrinsic pathway is initiated when factor XII
comes into contact with collagen-containing subendothelial layer. The activation of factor XII, in
turn, set off the sequential activation of factors XI, IX, and X. Factor X is produced by both
pathways and converts prothrombin to thrombin. Factor V (FV) and factor VIII (FVIII) are cofactors
used in the coagulation system, and are the rate limiting steps. Thrombin is the final enzyme in the
coagulation system and it converts fibrinogen to fibrin clot.
gylcosaminoglycans (contact phase). This "contact phase"
occurs when there is injury to the endothelial lining
exposing the subendothelial region of the vascular
system. Normally, the endothelial lining provides a
barrier between the circulating blood and the collagen-
5


containing subendothelial layer (4,5). However, when an
injury has occurred to the endothelial lining, it exposes
this layer and starts the coagulation system (6,7).
The intrinsic pathway is initiated when one of its
components, factor XII comes into contact with collagen-
containing subendothelial layer (3,7,8). The activation
of factor XII to factor Xlla, in turn, sets off the
sequential activation of factors XI, IX, and X (3,7).
Factor Xlla converts factor XI to its activated form,
factor XIa, with the aid of its cofactor high molecular
weight kininogen (3,6,8). Factor XIa activates factor IX
to factor IXa in the presence of calcium ions. Factor
IXa in a complex with factor Villa (a regulatory
cofactor) catalyzes the activation of factor X to factor
Xa (6,7,8).
The extrinsic pathway is initiated when tissue
factor (TF), which is also located within the
subendothelial layer, is exposed when an injury occurs to
the endothelium lining (8). TF serves as a cofactor for
factor VII activation (6). This complex of TF, factor
6


Vila and calcium ions activates factor X (3,6). The
intrinsic and extrinsic pathways converge at factor X to
form the common pathway. Factor Xa produced by both
pathways utilizes factor Va as a cofactor to convert
prothrombin to thrombin (6,7,8). The rate of fibrin
formation is determine by the concentration of the
cofactors, factors V and VIII at the rate limiting steps
of the coagulation cascade (6,15).
Thrombin is the final enzyme formed in the
coagulation cascade. It is responsible for clot
formation by converting fibrinogen to fibrin, platelet
aggregation, and the feedback activation of two
regulatory proteins factors V and VIII (15). Thrombin
also activate factors XIII which cross-links fibrin to
form an insoluble fibrin clot that intertwines with the
primary hemostatic plug (8,15). Thus, the fibrin clot
mechanically halts bleeding into the surrounding tissue
and allowing anti-inflammatory and tissue repair
responses to proceed (6).
7


Regulation of the Coagulation System
The potential for the coagulation system to stop
blood flow, if left unregulated, can be hazardous to an
individual. Therefore, it requires precise control.
Regulatory mechanisms serve to limit and localize the
clotting process within the area of tissue injury
(6,16,17). Three main regulatory processes involved in
the coagulation system are known (Fig 1.2 and 1.3).
The first is the inhibition of procoagulant serine
protease enzymes (thrombin, factors IXa, Xa, XIa and
Xlla) by antithrombin III (Fig 1.2) (14,18,19) .
Antithrombin III serves as a coagulation protease
scavenger, forming a complex with the clotting enzymes,
thus neutralizing the enzymatic activity and localizing
clot formation at the site of injury (6). These
interactions are accelerated by heparin-like
glycosaminoglycan molecules located on the surface of
endothelial cells (3,6,19). The second process involves
tissue factor pathway inhibitor (TFPI) that blocks the
8


Extrinsic Pathway
Intrinsic Pathway
Factor VII
Tissue Factor
Factor XII
1
V
\
Factor XI \
i \\
FVIII Factor IX^Q \
' \ '
\ v
Factor X/_ \ \ x
I / ----- '
I FV - -X
Prothrombin
Fibrinogen
^
Thrombin
Fibrin Clot
Figure 1.2: The Antithrombin III and the Tissue Factor Pathway Inhibitor
Anticoagulant Pathways. Antithrombin III (ATIII) forms a complex with the clotting enzymes,
neutralizing the enzymatic activity and localizing clot formation at the site of injury. Tissue factor
pathway inhibitor (TFPI) binds at the active site of factor X and induces a feedback inhibition of the
extrinsic coagulation pathway by forming a complex with the factor VII/Tissue factor complex.
activity of the factor Vlla/tissue factor/factor X
complex (Fig 1.2) (18,20) TFPI binds at the active site
of factor Xa and induces a feedback inhibition of the
extrinsic coagulation pathway by forming a complex with
the factor Vlla/tissue factor complex (21,22,23). This
interaction requires the presence of calcium ions and
occurs on the surface of membranes (24,25) .
9


The third anticoagulant regulatory process involves
the inactivation of the cofactors in the rate limiting
reaction of coagulation (factors Va and Villa) by the
protein C system (Fig 1.3)(3,16,17). The main components
of this mechanism include two vitamin K-dependent
proteins: protein C (PC) and protein S (PS), and a
membrane bound thrombin receptor, thrombomodulin
(14,15,17).
Protein C System
Protein C circulates in the blood as a proenzyme and
is converted to a serine protease by thrombin bound to
thrombomodulin, a surface protein on the endothelial cell
(16,17). Once activated, activated protein C (APC)
functions as an anticoagulant by inactivating factors Va
and Villa on the surface of negatively charged
phospholipids (3,13). PS functions as a cofactor for APC
and enhances the anticoagulant action (13).
Thrombin is the central protease of the coagulation
system but also has many other diverse activities(16,17).
10


Extrinsic Pathway
Intrinsic Pathway
Prothrombin
Fibrinogen
Thrombin
.X
Thrombomodulin
Fibrin Clot
Figure 1.3: Protein C Anticoagulation System. The Protein c (PC)
anticoagulation system inactivates factors V and VIII, the rate limiting reaction of the
coagulation system. PC is activated when thrombin binds to thrombomodulin on the surface
of endothelial cells. Once activated, activated protein C (APC), inactivates factors V and VIII
with the aid of protein S (PS). PS enhances the anticoagulant activity of APC by increasing
the binding affinity of APC to the membrane surface of platelets and endothelial cells.
When bound to thrombomodulin, thrombin acts as a feedback
inhibitor of the coagulation system by generating, APC
(17,26). Thrombomodulin is a single-chain integral
membrane glycoprotein, located on the surface of
endothelial cells (3,8,17). Structurally, thrombomodulin
consists of five domains. These include, an amino-
11


terminal domain, a region containing six consecutive
epidermal growth factor (EGF)-like domains, an O-linked
carbohydrate side chain region, a transmembrane domain,
and a carboxyl-terminal intracellular region (27,28,29).
Thrombomodulin forms a one-to-one stoichiometric complex
with thrombin. This complex induces a modification in
thrombin's substrate specificity (7,8,16). The binding
site for thrombin on thrombomodulin is located in the
fifth and sixth EGF-like domain (27,30). Thrombomodulin
controls coagulation in two ways: 1) thrombomodulin
binding to circulating thrombin causes thrombin to lose
its procoagulant activity; and 2) generating the
anticoagulant, APC (16,17,31).
The activation of PC by the thrombin-thrombomodulin
complex requires calcium ions and a negatively charged
cell surface (17,26). The role of the surface is to
expose the third and fourth EGF-like domains on the
thrombomodulin molecule allowing interaction with PC
through a calcium bridge (3,28,32).
12


APC is a highly specific enzyme, inactivating
factors Va and Villa by limited proteolysis of peptide
bonds on the heavy chain of the activated molecules
(33,34) Both factor V and factor VIII circulate in the
blood as pro-cofactors and are converted to their active
forms by thrombin cleavage(7). Only the activated forms
of factors V and VIII are sensitive to APC inactivation
(7,26).
In addition to its anticoagulant activity, APC also
exerts a profibrinolytic activity thought to occur by
neutralizing plasminogen activator inhibitor type 1
(35,36,37). Plasminogen activator inhibitor type 1
inhibits the activity of plasminogen activator (36).
Plasminogen activators in turn convert plasminogen to
plasmin, the enzyme responsible for lysing the fibrin
clot (36).
Protein S
Protein S serves as a cofactor in the anticoagulant
and fibrinolytic activities of APC (7,37,38). Its main
13


function appears to be to enhance the binding of APC to
the membrane surface of platelets and endothelial cells
(37,38). It also facilitates APC to catalyze the
inactivation of factors Va and Villa (26,39). The
concentration of total PS in human plasma is
approximately 20-24 mg/L, and, approximately, 60% is
bound to C4b-binding protein (C4b-BP, a component of the
complement system) (14,40). The remaining 40% is unbound
or free (13,14,40). Unbound PS is the functional
fraction (13,14,41). PS and C4b-BP form a one-to-one
stoichiometric complex (13,14). When PS is bound to C4b-
BP, it loses its cofactor activity for APC. However, the
complement regulatory function of C4b-BP is not affected
(13,41). The biological significance of PS in the
complement system is yet to be confirmed.
PS is a single-chain molecule with a molecular
weight of 70,000 Daltons (14,42,43). Its structure is
similar to the other vitamin K-dependent proteins (Fig
1.4). The amino-terminal of PS (residues 1-37) contains
11 Y~carboxyglutamic acid (Gla) residues and is referred
14


to as the Gla domain (44,45) The function of the Gla
domain is to facilitate membrane binding via a calcium
bridge (7,13,46). Following the Gla domain is an unique
region that is sensitive to thrombin cleavage (residues
47-75) (13,42,43,44). Thrombin cleavage in this region
appears to induce a structural change causing the loss of
affinity for negatively charged phospholipid surfaces and
its cofactor activity (13,47). PS contains four EGF-like
domains (residues 76 to 242), but the functions of these
domains are unknown (47,48). Each of these domains of PS
contain one of two modified amino acids: 3-hydroxylated
aspartic acid (Hya) and 3-hydroxylated asparagine (Hyn)
residues. The mechanisms by which these amino acids are
modified is also unknown. The Hya residue is present in
the first EGF-like domain at residue #95, whereas the
other three EGF-like domains contain a Hyn at residue
#138 (second EGF-like domain), #178 (third EGF-like
domain), and #217 (fourth EGF-like domain) (49). The
function of Hya in other vitamin K-dependent proteins is
15


Gla TSD EGF1 EGF2 EGF3 EGF4 SHBP
Figure 1.4: Schematic Diagram of Human Protein S. Gla-Gamma Carboxyl Glutamic
Acid Residue; TSD-Thrombin Sensitive Domain; EGF-Epidermal Growth Factor; SHBP-Sex
Hormone Binding Protein. See text for more detail.
thought to be involved in Gla-independent calcium binding
(13). In PS, the Hyn containing EGF-like domains (#2-4)
contains a high affinity calcium binding site, which
stabilizes the EGF structures (13). The EGF-like domains
and the thrombin-sensitive region contain the site(s) for
APC interactions (13). The remaining carboxyl-terminus
region (residues 243-635) has no homology to the other
16


vitamin K-dependent proteins serine protease domains
(13) . This region does not have enzymatic activity and
its function is unknown. It is similar to the rat
androgen-binding protein and the human sex hormone-
binding globulin (14,43).
Clinical Aspect of Protein S
A hereditary deficiency of PS represents a risk
factor for thrombosis in young adults. This genetic
disorder is inherited as an autosomal dominant trait
(14) . The major clinical problems associated with a
hereditary deficiency of PS are deep vein thrombosis,
pulmonary embolism, and superficial thrombophlebitis
(1,50). These clinical manifestations resemble
deficiencies of PC and antithrombin III (1,14). The
first thrombotic event usually occurs between the age of
15 and 45 (43). The chances of PS-deficient individuals
remaining free of thrombosis up to the age of 35 years is
only 32% (1,50). The standard treatment for venous
thrombosis in PS-deficient individuals is the initial
17


heparin therapy followed by long term administration of
the oral anticoagulant, Warfarin (1,50).
Molecular Biology of Protein S
Two PS genes, PSa and PS3, have been identified in
the human genome and are located on chromosome 3 near the
centromere (14,44,48). However, only the PSa gene is
transcriptionally active, and PS3 gene is a
pseudogene(44). The PSa gene is approximately 8752
nucleotides long and the PS3 gene is 5414 nucleotides
long (51). These two genes share a high degree of
homology (approximately 97%) in the 3' untranslationed
region (44,48). The PSa gene is organized with 14
introns and 15 exons which code for the PS mRNA. PS mRNA
is approximately 3.5 to 4 kilobases long (41,44).
The PS3 gene is a pseudogene because it: 1) lacks a
5' exon containing the initiation methionine, 2) contains
a splice site mutation, 3) has several stop codons, and
4) has a frame shift mutation (31). Pseudogenes are DNA
sequences that have a significant homology to a
18


functional gene but contain mutations which are incapable
of being expressed or does not produce a functional
protein (52,53).
Cell Biology of Protein S
While much is known about the anticoagulant
mechanism of PS, little is known about its synthetic and
cellular regulation. PS is synthesized and post-
translationally modified to produce a functional PS
molecule in three cell types: hepatocytes (54),
megakarycytes (55), and endothelial cells (10). The
processing, secretion and/or storage of PS appears to
vary depending on the cell type. The hepatocytes are the
major producer of plasma vitamin K-dependent coagulation
proteins, including PS (54). The megakarycytes and
endothelial cells appear to synthesis and/or store PS,
releasing it upon specific stimulation. PS is stored in
the a-granules of platelets (end cell product of
megakarycytes) and is released by thrombin activation of
the platelets (55). The a-granules of platelets contain
approximately 2.5% of the PS in whole blood (1,55). The
19


manner in which PS is stored and released by the
endothelial cell is unknown. Stern and colleagues (39)
have suggested that the mechanism for release of PS is
similar to that of vWf release from the endothelial cell,
although the storage mechanism is different. The release
of PS from the endothelial cell occurs in response to
stimulation by ionophores, such as ionomycin and A23187,
which induce cytosolic calcium influx (39). The
regulation of PS synthesis by the platelets and
endothelial cells may reflect the role these cells play
in localizing the PC anticoagulant system.
Post-Translational Processing of Protein S
Before PS is secreted or stored in the cell, it
undergoes several post-translational modifications:
Signal peptide and propeptide are removed, specific
glutamic acids are ycarboxylated, specific aspartic
acids and asparagine are [3-hydroxylated, disulfide bonds
are formed, and carbohydrate moieties are added to the PS
polypeptide (14,46).
20


PS and the other vitamin K-dependent proteins are
synthesized in a pre-protein form with a signal peptide,
a propeptide, and the precursor form of the protein
(56.57) . After the protein is translocated to the rough
ER, the signal peptide is cleaved off by a signal
peptidase (18,46,56). The propeptide contains the y-
carboxylation recognition site which designates the
residues in the precursor form of PS for carboxylation
(56.58) . Significant homology exists between the
propeptides of the vitamin K-dependent proteins(46,59).
The conserved amino acids among these propeptides are:
phenylalanine -16, alanine -10, hydrophobic amino acids
at residues -17, -7, and -6. and basic amino acids at
residues -4, -3, -2, and -1 (58). Residues -18, -17, -
16, -15, and -10 characterize the carboxylation
recognition site (46). Residues -1 to -4 are important
for the cleavage of the propeptide from the mature
protein before secretion into the circulation (59).
21


Mechanism of Vitamin K and Warfarin
The propeptide is involved in the interaction of
these proteins with the enzyme, vitamin K-dependent
carboxylase enzyme, that catalyzes the y-carboxylation of
specific glutamic acid residues in the Gla domain
adjacent to the propeptide (59). The propeptide has two
main functions: 1) To regulate the activity of vitamin K-
dependent carboxylase, and 2) To orientate the enzyme
with its substrate (59,60). The vitamin K-dependent
carboxylase is an integral membrane enzyme of the ER
(7,46,57). The carboxylation reaction requires vitamin
K, molecular oxygen, carbon dioxide, and a precursor form
of the vitamin K-dependent protein as the substrate
(7,58). Vitamin K is an essential vitamin and is
obtained from green leafy vegetables and natural
intestinal floral (58,61). Vitamin K is reduced to
vitamin hydroquinone (vitamin KH2) by vitamin K reductase
and vitamin KH2 serves as a cofactor to vitamin K-
dependent carboxylase (9). During the carboxylation
reaction, Glu side chains are modified at the appropriate
22


residues on the polypeptide chain and vitamin KH2 is
converted to vitamin K epoxide. This is a two step
reaction: first, the proton at Y_Psition is removed from
the glutamyl residue by a carbanion mediated mechanism
(61,62,63) Next, carbon dioxide is added to the y-
position of the glutamyl residues and forms the y-
carboxyl glutamic residue (61,62,63). The energy
required for the y-carboxylation reaction is provided
when vitamin KH2 is oxidized to vitamin K epoxide (62) .
The vitamin K epoxide is recycled to vitamin KH2 by the
action of vitamin K epoxide reductase and vitamin K
reductase (7,46). The vitamin K antagonist, Warfarin,
inhibits vitamin K epoxide reductase and causes the
accumulation of the epoxide metabolite (61). The
accumulation of vitamin K epoxide causes uncarboxylated
and partially carboxylated forms of vitamin K-dependent
proteins. That have low activity, to be secreted into
the plasma (59).
23


Purpose of Research
Since the mechanism and regulation of PS synthesis
in the various cell types have not been elucidated, the
effects of PS synthesis, secretion, and storage on the
thrombotic process is still unknown. Examination of the
mechanism of PS synthesis and regulation by the different
cell types will provide a more complete understanding of
the role of PS in normal hemostasis and thrombotic
process.
The purpose of this research is to compare the basal
rate of and synthesis among the different cell types
which produce PS. The cell types studied will be: 1)
human hepatocellular carcinoma (Hep G2) cells, a
hepatocyte cell line; 2) human chronic myelogenous
leukemia (K562) cells, a megakaryocyte cell line; and 3)
human umbilical endothelial cells (HUVEC). To determine
the basal rate of synthesis, the amount of PS secreted
and stored by the various cell types will be measured in
media samples and cell lysates respectively by running
enzyme-linked immunosorbent assay on the sample. The
24


different molecular weight forms of PS secreted and/or
stored by the various cells will be qualitatively
analyzed by SDS gel electrophoresis and Western blot
technique. The effects of vitamin K and Warfarin, a
vitamin K antagonist, on the process, secretion, and
storage of PS by each cell type will be examined. The
results obtained will be analyzed and compared among the
Hep G2 cells, K562 cells, and HUVEC.
25


CHAPTER 2
MATERIALS AND METHODS
Materials
Cell Types
The cell types used in this project were: 1) human
hepatocellular carcinoma (Hep G2) cells; 2) human chronic
myelogenous leukemia (K562); and 3) human umbilical
endothelial cells (HUVEC). The K562, and Hep G2 cells
were obtained from American Type Culture Collection
(Rockville, MD) and the endothelial cells were kindly
provided by Rubin Tuder, MD of Department of Pathology,
University of Colorado Health Sciences Center, Denver,
CO.
Media and Media Additives
Eagle's minimal essential medium (MEM), Dulecco's
modification of Eagle's medium IX (DMEM), RPMI-1640
medium were purchased from Fisher (Denver, CO). Fetal
26


calf serum (FCS), horse serum, heparin, gentamicin, and
gelatin were purchased from Sigma (St. Louis, MO). Media
additives included: nonessential amino acids, sodium
pyruvate, penicillin-streptomycin, and L-glutamine
(Fisher, Denver, CO). Fibroblast growth factor was
purchased from Promega (Madison, WI).
Other Materials
Anti-PS antibodies were made as previously described
(64). Horseradish Peroxidase (HRP)-conjugate rabbit
anti-human Protein S was purchased from DAKO
(Carpinteria, CA). Vitamin K (AquaMEPHYTON) was obtained
from Merck Sharp and Dohme, (West Point, PA). Warfarin
(3-(a-acetonylbenzyl)-4 Hydroxycoumarin) was purchased
from Sigma.
Methods
Cell Cultures
The following media additives were used in all media
for each cell type: 1% non-essential amino acids, 1 mM
27


sodium pyruvate, 50 mg/ml penicillin-streptomycin, 2 mM
L-glutamine, and 0.5 mg/ml gentamicin. The Hep G2 cells
were grown to confluence in 100 mm2 tissue culture plates
in MEM containing 10% heat-inactivated horse serum. The
endothelial cells were grown to confluence in 100 mm2
tissue culture plates precoated with 2 mg/ml gelatin in
media containing 50% DMEM and 50% RPMI-1640 medium, 20%
heat-inactivated FCS, 50 mg/1 sodium heparin, and 10 /ig/1
fibroblast growth factor. The K562 cells were maintained
in 75 cm2 tissue culture flasks in RPMI-1640 medium with
10% heat-inactivated FCS by passage of at least 107 cells
per flask.
Once the cells reached confluency (107 cells per
flask), culture media was removed and fresh media with or
without vitamin K or Warfarin was added to the cell
cultures. The amount of vitamin K and Warfarin added to
the media varied from 5 fig/ml to 15 /xg/ml and 10 ng/ml to
20 ng/ml, respectively. Media samples and cell lysates
were obtained at 0, 6, 12, 24, 48, and 72 hours and the
amount of PS secreted/stored within the cell by each cell
28


type was analyzed by enzyme-linked immunosorbent assay
(ELISA). An aliquot of each media sample and cell lysate
was analyzed for the molecular forms of PS by SDS-PAGE
and Westerns blots.
Determination of PS Antigen
The amount of PS antigen was determine by ELISA.
Nunc-Immuno 96 well plates (Nunc Inter Med) were used in
these experiments. The plates were coated with a
solution containing 1:250 dilution of rabbit anti-human
PS IgG in 0.05 M sodium carbonate buffer pH 9.6 (100
/il/well) and incubated overnight at 4C. After
incubation, the plates were washed 3 times with ELISA
wash buffer (0.02 M Tris-base, 0.14 M NaCl, pH 7.4,
0.025% Tween-80). Then, 100 jxl/well standards, controls,
media and cell lysate were added to wells and incubated
for 2 hours at room temperature. The standard curve was
made from normal plasma at a 1:100 dilution, and then
serially diluted. The cell lysates and media samples
were diluted at a 1:2 in dilution buffer (50 mM Tris,
29


0.14 M NaCl, 3 mM KCL, pH 7.4, 0.2% Tween-80, 0.4% PEG-
8000) After incubation, the plates were washed 6 times
with ELISA wash buffer. Then, 100 fil of a 1:2000
dilution of HRP-conjugated rabbit anti-human PS in
dilution buffer was added to each well and incubated for
one hour at room temperature. The plates were then
washed 6 times with ELISA wash buffer. 100 /il of o-
Phenylenediamine Dihydrochloride/Urea peroxide solution
(Sigma, St Louis, MO) was added to each well and
incubated for 15 minutes at room temperature. The
reaction was then stopped with 50 /il/well of 3 M H2S04,
and read at an absorbance of 490 nm with a Dynateck
MR5000 ELISA reader.
SDS-PAGE and Western Blots
To determine the different molecular weight forms of
PS produced by the various cell types, SDS-PAGE and
Western blot analysis were performed. Fifty /zl of
samples were electrophoresed on a 10% SDS-polyacrylamide
slab gel containing prestained molecular weight standard
30


from BioRad (Hercules, CA). Slab gels were washed in
Western blotting buffer (2.5 mM Tris-base, 192 mM
glycine, pH 8.3, and 20% methanol) for one hour and then
transferred onto a nitrocellulose membrane (BioRad,
Hercules, CA) for four hours at 50 volts. The
nitrocellulose membrane was blocked overnight in 10% non-
fat dry milk solution and washed three times with wash
buffer (0.02 M Tris-base, 0.14 M NaCl, pH 7.4, 0.025%
Tween-80) for 30 minutes each. Then the nitrocellulose
membrane was incubated for one hour in a 1:2,000 dilution
of HRP-conjugated rabbit anti-human PS. The blot was
again washed three time with wash buffer for 30 minutes
each. The membrane was then incubated for one minute in
Enhanced Chemiluminescence (ECL) detection reagents
(Amersham Arlington Heights, IL), and exposed for 2 to 30
minutes on film, Kodak X-OMAT RP (Sigma St Louis, MO).
Statistical Analysis of Data
In this study, the mean of the measurements of three
experiments were calculated one standard deviation for
31


each set of experiments. The p values were calculated by
Student t-test using a computer program called Sigma Plot
(Jandel Scientific Software).
32


CHAPTER 3
RESULTS
Production of Protein S
Media samples and cell lysates from Hep G2, K562,
and HUVEC were collected over a 72 hour period and
analyzed by ELISA for PS antigen and determining the
molecular forms produced by each cell type by SDS-PAGE
and Western blot. The information gathered from these
experiments paved the foundation for determining the
effects of vitamin K and Warfarin on the synthesis of PS
by each cell type. In order to determine the effects of
vitamin K and Warfarin on the rate of PS synthesis and/or
storage in Hep G2 cells, K562 cells, and HUVEC, the cells
were grown in serum free media treated with multiple
concentrations of vitamin K or Warfarin.
Hep G2 Cells
The consistent production of PS, demonstrating the
linear increase of PS secretion into the media with
33


respect to time by the Hep G2 cells, was shown by ELISA
determination (Fig 3.1A) and Western blot (Fig 3.2). The
amount of PS secreted into the media was 13 0 X 10'15
grams/cell within 72 hours. The basal rate of PS
secretion of Hep G2 cells was 1.85 X 10'15
grams/cell/hour. The amount of PS stored in the cells
stayed constant over time at 5.46 X 10'15 grams/cell (Fig
3.IB). This is minimal compared to the amount secreted
from the cell.
Western blot analysis confirms a progressive
increase in PS synthesis over time(Fig 3.2). Lanes 1
through 6 represent the increase of PS secretion into the
media by Hep G2 cells over time. In the media samples,
no band was observed at time point 0 (Lane 1), however, a
faint 70 kilodalton bands started to appear at 6 hours
and the signal increased over 72 hours (Lanes 2-6).
Lanes 8 through 13 represents the processing of PS that
occurred in the Hep G2 cells over time. In the cell
34


Figure 3.1: Protein S Production in Hep G2 Cells: A) Time-course of PS antigen
secreted by Hep G2 cultures. B) The amount of PS stored in Hep G2 cells over time. All values
represent the average of three experiments + 1 SD. Hep G2 cells were grown to confluence in MEM
+ 10% HS. At each time point, cell lysates and media samples were collected. (See method section
for more details.)
lysates, a 70 kilodaton band appeared at time 0 and the
same intensity signal also persisted over 72 hours (Lanes
8-13). Lanes 7 and 14 are controls of a 1:400 dilution
of normal plasma and show a 70 kilodaton band.
PS antigen levels are depicted to show the effects
of vitamin K on the Hep G2 cells (Fig 3.3). Vitamin K
had no significant effect on the amount of PS secreted,
35


1 2 3 4 5 6
#
7 8 9 10 11 12 13
'-133 i,?
§|i f '
41.6
30.6
1*1
'** 17.8
> *
HRS. 0 6 12 24 48 72 NP
O 6 12 24 48 72
Figure 3.2: Autoradiogram of Protein S Secreted and Stored in the Hep G2
Cells. Fifty pi of media samples or cell lysates were analyzed in SDS-10% polyacrylamide slab gels
in the present of reducing buffer. Lanes 1 through 6 shows the migration of PS molecule secreted into
the media by Hep G2 cells. Lanes 8 through 13 shows the processing of PS molecule in the Hep G2
cells over time. Lanes 7 and 14 shows the migration of PS in normal plasma (Control). Standard
(Lane 15) were: myosin (202,000), P-galactosidase (133,000), bovine serum albumin (71,000),
carbonic anhydrase (41,800), soybean trypsin inhibitor (30,600), lysozyme (17,800), and aprotinin
(6,900).
the amount of PS stored within the cell, or the basal
rate of PS antigen synthesis (p=0.22).
Warfarin did not significantly (p=0.11) down
regulated the secretion of PS antigen into the media
compared to the control (Fig 3.4A). The amount of PS
36


Figure 3.3: The Effects of Vitamin K on Protein S Production in Hep G2 Cells.
A) Time-course of PS antigen secreted by Hep G2 cultures after vitamin K treatment. B) The effects
of vitamin K on the storage of PS in Hep G2 cells over time. PS levels were determine in media
samples and cell lysates from cultures of Hep G2 cells treated with either 5 pg/ml vitamin K (--), 10
pg/ml vitamin K (-A-), or without vitamin K (Control All values represents the average of three
experiments + 1 SD.
secreted slighly decreased over 72 hours when Hep G2
cells were treated with 10 ng/ml Warfarin. When Hep G2
cells were treated with 15 ng/ml Warfarin, secreted PS
levels also slightly decreased over 72 hours (Fig 3.4A).
The rate of secretion for cells treated with 10 ng/ml
Warfarin was 0.90 X 10'15 grams/cell/hour, and with 20
37


Figure 3.4: The Effects of Warfarin on Protein S Production in Hep G2 Cells.
A) Time-course of PS antigen secreted by Hep G2 cultures after Warfarin treatment. B) The effects of
Warfarin on the storage of PS in Hep G2 cells over time. PS antigen levels were determined in media
samples and cell lysates from cultures of Hep G2 treated with either 10 ng/ml Warfarin (--). 20
ng/ml Warfarin (-A-), or without Warfarin (Control All values represent the average of three
experiments + 1 SD.
ng/ml Warfarin was 0.92 X 10'15 grams/cell/hour. The rate
of secretion decreased slightly for cells treated with
both 10 ng/ml Warfarin, and 20 ng/ml Warfarin compared to
the control.
Figure 3.4B shows the effects of Warfarin on the
storage of PS in the Hep G2 cells. Storage of PS
38


increased slightly over 72 hours with 20 ng/ml Warfarin
treatment compared to the control (p = 0.025) however, no
significant change in storage was found in Hep G2 cells
treated with 10 ng/ml Warfarin (p = 0.25).
K5S2 Cells
Figure 3.5A represents the basal rate of PS
secretion in K562 cells (0.0404 X 10'15 grams/cell/hour)
and PS production in K562 cells with respect to time.
The amount of PS stored in the K562 cells over time was
3.78 X 10-15 grams/hour (Fig 3.5B) .
Western blot analysis showed that K562 cells did not
increase PS synthesis but was constant over time (Fig
3.6), which confirms the ELISA results. Lanes 1 through
6 show a molecular weight of 69 kilodalton for PS
secreted from the K562 cells. Lanes 9 through 14 show
intacellular PS(MW=69 kilodaltons). Western blot
analysis confirmed that the K562 cells store PS instead
39


Figure 3.5: Protein S Production in K562 Cells. A) Time-course of PS antigen secreted
by K562 cultures. B) The amount of PS stored in K562 cells over time. All values represents the
average of three experiments + 1 SD. See text for details.
of secreting it. Lane 8 is the PS control of a 1:400
dilution of normal plasma and with a 70 kilodalton band.
Figure 3.7A shows the average of three time course
experiments demonstrating the effects of vitamin K on PS
secretion in K562 cells. With Vitamin K, the quantity of
PS secreted by the K562 cells significantly increased
(p=0.044). The amount of PS secreted increased with 10
40


HRS. 0 6 12 24 48 72 NP
0 6 12 24 48 72
Figure 3.6: Autoradiogram of Protein S Secreted and Stored in the K562 Cells.
Fifty pi of media samples or cell lysates were analyzed in SDS-10% PAGE gels in the present of
reducing buffer. Lanes 1 through 6 shows the of PS molecule secreted into the media by K562 cells.
Lanes 8 through 13 shows the processing of PS molecule in the K562 cells over time. Lanes 7 and 14
shows the migration of PS in normal plasma (Control). Standard (Lane 15) were: myosin (202,000),
(i-galactosidase (133,000), bovine serum albumin (71,000), carbonic anhydrase (41,800), soybean
trypsin inhibitor (30,600), lysozyme (17,800), and aprotinin (6,900).
lig/ml vitamin K, and 15 fig/ml of vitamin K. The rate of
secretion in the presence of vitamin K increased 4 to 6
fold compared to the control. The rate of PS secretion
was 0.25 X 10'15 grams/cell/hour when treated with 10
/tg/ml vitamin K and 0.16 X 10~15 grams/cell/hour vitamin K
compared to the control of 0.04 X 10'15 grams/cell/hour.
41


Figure 3.7: The effects of Vitamin K on Protein S Production in K562 Cells. A)
Time-course of PS antigen secreted by K562 cultures after vitamin K treatment. B) The effects of
vitamin K on the storage of PS in K562 cells over time. PS levels were determine in media samples
and cell lysates from cultures of K562 cells treated with either 10 pg/ml vitamin K (--), 15 pg/ml
vitamin K (-A-), or without vitamin K (Control All values represents the average of three
experiments + 1 SD.
Vitamin K had no significant effect on the quantity
of PS stored within the K562 cell over time (p = 0.57 for
10 /xg/ml vitamin K and p = 0.47 for 15 /ig/ml vitamin K)
(Fig 3.7B).
Warfarin had no significant (p=0.21) effect on the
secretion of PS from the K562 cells (Fig 3.8A). However,
42


Figure 3.8: The Effects of Warfarin on Protein S Production in K562 Cells. A)
Time-course of PS antigen secreted by K562 cultures after Warfarin treatment. B) The effects of
Warfarin on the storage of PS in K562 cells over time. PS antigen levels were determined in media
samples and cell lysates from cultures of K562 cells treated with either 10 ng/ml Warfarin (--), 20
ng/ml Warfarin (-A-), or without Warfarin ( Control All values represent the average of three
experiments + 1 SD.
the quantity of PS stored within the K562 cells slightly
increased over time when incubated with Warfarin
(p=0.01).
43


HUVEC
The HUVEC showed a slight increase in the rate of PS
secretion with respect to time from 0 X 10'15 grams/cell
(time 0) to 26 X 10'15 grams/cell (72 hours) (Fig 3.9A) .
The basal rate of synthesis was 0.37 X 10'15
grams/cell/hour. The average amount of PS stored in the
HUVEC was constant over time (30.5 X 10'15 grams/cell)
(Fig 3.9B). Compared to the other cell types, HUVEC
stored 5 to 8 times more PS per cell.
During the incubation of 5 jxg/ml vitamin K with
HUVEC, PS secretion increased over time (p = 0.032) (Fig
3.10A) When HUVEC were treated with 10 fig/ml vitamin K,
PS secretion slightly increased (p=0.03). The rate of PS
secretion slightly increased. The rates of PS secretion
was 0.3 X 10'15 grams/cell/hour for 5 ^ig/ml vitamin K and
0.33 X 10'15 grams/cell/hour for 10 /xg/ml vitamin K
compared to 0.27 X 10~15 grams/cell/hour for the
control.
Figure 3.10B shows the effects of vitamin K on the
quantity of PS stored within the HUVEC. The amount of PS
44


Figure 3.9: Protein S Production in HUVEC. A) Time-course of PS antigen secreted by
HUVEC culture. B) The amount of PS stored in HUVEC over time. All values represents the
average of three experiments + 1 SD. See text for details.
stored by the HUVEC slightly increased when incubated
with 5 /ig/ml vitamin K compared to the control (p=0.7) .
However, when the HUVEC are treated with 10 /xg/ml vitamin
K, no significant change in PS storage was observed
(p=1.0).
Warfarin (at all concentrations) had no significant
effect on the secretion (p=0.18), the amount stored
45


40
Figure 3.10: The Effects of Vitamin K on Protein S Production in HUVEC. A)
Time-course of PS antigen secreted by HUVEC cultures after vitamin K treatment. B) The effects of
vitamin K on the storage of PS in HUVEC cells over time. PS levels were determine in media
samples and cell lysates from cultures of HUVEC treated with either 5 pg/ml vitamin K (--), 10
pg/ml vitamin K (-A-), or without vitamin K (Control All values represents the average of three
experiments + 1 SD.
within the cell
(p=0.76)
or the basal rate of
PS
synthesis (Fig 3.11).
46


40
8
a
E
M
a
35
30
25
20
15
10
5
0
TIME (Hours) TIME (Hours)
Figure 3.11: The Effects of Warfarin on Protein S Production in HUVEC. A)
Time-course of PS antigen secreted by HUVEC cultures after Warfarin treatment. B) The effects of
Warfarin on the storage of PS in HUVEC overtime. PS antigen levels were determined in media
samples and cell lysates from cultures of HUVEC treated with either 10 ng/ml Warfarin (--), 20
ng/ml Warfarin (-A-), or without Warfarin ( Control All values represent the average of three
experiments + 1 SD.
47


CHAPTER 4
DISCUSSION
Cardiovascular diseases and thromboembolic processes
play a major role in a broad spectrum of diseases and
American deaths each year. Among the antithrombotic
regulatory processes, the Protein C (PC) system is an
important control path that regulates thrombus formation
by providing both anticoagulant and profibrinolytic
control. One of the central components of this system is
the cofactor protein S (PS). PS is a vitamin K-dependent
glycoprotein with similar homology to other vitamin In-
dependent coagulation proteins. PS serves as a cofactor
to activated protein C in the inactivation of factors Va
and Villa on the cell surface.
The physiological importance of PS is evident by the
fact that even a heterozygous hereditary deficiency of PS
predisposes individuals to an increased risk of
thrombosis. Much is known about the mechanism of PS
48


action, but little is known about its synthesis and
cellular regulation. PS is synthesized and post-
translationally modified in a variety of cell types. In
this study, the basal rate of PS synthesis was analyzed
in the three major cell types that produce PS: Hep G2
cells (hepatocyte linage), K562 cells
(megakaryotic/platelet cell linage), and HUVEC
(endothelial cell). The effects of vitamin K and
Warfarin on the synthetic process of each cell type were
also studied.
Basal Rates of Protein S Production
Platelets and endothelial cells have been shown to
be major players in the initiation, propagation and
regulation of hemostasis and thrombosis within the
vascular system (8,9,10,55) One mechanism used by these
cells is the synthesis and storage of specific proteins
involved in the amplification and regulation of the
coagulation system (8,10).
49


Platelets store and release high molecular weight
kininogen, von Willebrand factor (vWf), and factor V, all
of which are involved in clot formation via coagulation
and platelet aggregation (10,55). Also platelets store
and release PS which is responsible for limiting clot
formation by the PS-enhanced inactivation of factors Va
and Villa by activated PC on the cell surface (10).
Endothelial cells have been shown to provide the
surface features necessary for the initiation and
propagation of coagulation (10,64). In addition, these
cells store and express most of the cofactor activities
associated with the activation and regulation of clot
formation and clot breakdown (10). Among these
components are tissue factor, vWf, factor VIII, factor V,
high molecular weight kininogen, tissue plasminogen
activator, plasminogen activator inhibitor-1,
thrombomodulin, and PS (10).
The endothelial and platelet cell types have been
shown to produce a small amount of these coagulation and
regulatory proteins. However, the primary source of the
50


proteins are synthesized and produced by the liver
(54,65). The Hep G2 cells, an immortalized liver cell,
have been shown to have morphological characteristics of
liver parenchymal cells and are capable of synthesizing
many of the plasma proteins that are derived from the
liver, and therefore is a very good model for assessing
the regulation of synthesis and secretion of the
coagulation plasma proteins (54,65). These plasma
proteins produced by the Hep G2 cell line share the same
immunochemical, structural, and functional characteristic
as their plasma counterparts (54,65). The Hep G2 cell
responds to modulation by known pharmacologic agents,
such as, vitamin K (54,65), Warfarin (54,65), and IL-6
(66) .
In this study, the Hep G2 cell produced the majority
of PS with a basal rate of 1.85 X 10'15 grams/cell/hour
(Table 4.1). Once PS is synthesized in the Hep G2 cells,
the molecule is rapidly secreted into the media with a
constant rate, storing very little in the cell (Fig 3.1).
51


Table A :.l: Basal Rates of Protein S Production
Cell Type Media Samples (Grams/cell/hour) Cell Lysates (Grams/cell)
Hep G2 1.85 X 10'15 5.46 X 1015
HUVEC 0.37 X 10-15 30.5 X 10-15
K562 0.0404 X IQ'15 3.78 X lO'15
The small amount of PS stored within the Hep G2 cells
remained constant at 5.46 X 10~15 grams/cell (Fig 3.1).
The apparent molecular weight (MW) of PS secreted
and stored by the Hep G2 cell was 70 kilodaltons under
reducing conditions and was similar to the MW of the
plasma form (Fig 3.2). In this study, the PS molecule
produced by the Hep G2 cell was slightly smaller than the
75 to 77 kilodatons published by Fair and Marlar (54).
This is attributed to the different experimental methods
used. The MW of PS secreted and stored by the K562 cell
was 69 kilodaltons (Fig 3.6), similar to the Hep G2 cell.
The basal rate of PS secretion by the HUVEC was 0.37
X 10'15 grams/cell/hour. The synthesis and secretion of
PS in the HUVEC was constant and similar to the Hep G2
52


cell (Fig 3.9). However, the basal rate of secretion was
5 times less than the rate for Hep G2 cell. But the
HUVEC stored 5 times more PS compared to the K562 cell
and 8 times more than the Hep G2 cell. Although, the
amount of PS antigen within the cell lysate may be
attributed to PS being stored within the cell, it could
be also attributed to PS being secreted, and subsequently
tightly bound to the surface of the endothelial cell.
The storage of PS by the HUVEC may be biologically
significant, since the endothelium serves as a functional
surface for the assembly of the activated PC-PS complex
promoting factor Va and Villa inactivation (10,67,68).
PS levels increase upon addition of thrombin to
endothelial cells, providing both for the activation of
PC and assembly of APC-PS complex (10,67,68). The
increase level of PS may be necessary to obtain maximum
activated PC function in the inactivation of factors Va
and Villa (10). Thrombin's role in the release of PS
must be further studied to help clarify the link between
53


the activation of PC and the release and subsequent
assembly of APC-PS complex.
The K562 cell secreted very little PS compared to
the other two cell types (Table 4.1). The K562 cell
basal rate of secretion was 45 times less than the Hep G2
cells and 9 times less than the HUVEC. One-fourth of the
PS synthesized by the K562 cell is stored within the
cell. The storage of PS by the K562 cells is
significant, since the megakaryocyte cells form the a-
granules of platelets. Also, platelets serve as a
functional surface for the assembly of the activated PC-
PS complex similar to the endothelium.
Effects of Vitamin K and Warfarin on PS Production
The main characteristic of vitamin K-dependent
proteins is the post-translational modification of
specific glutamic acid residues to ycarboxyglutamic acid
residues near the amino terminus by a microsomal vitamin
K carboxylase (69). During the carboxylation reaction,
vitamin K is oxidized to vitamin K epoxide which provides
54


the energy needed for this reaction. In order to
regenerate the metabolically active vitamin, the epoxide
is reduced by the action of vitamin K epoxide reductase
and vitamin K reductase (7,46,65,70). Coumarin-type
anticoagulants, such as Warfarin, inhibit vitamin K
epoxide reductase and causes the accumulation of the
epoxide metabolite (60,70). Consequently, the
carboxylation reaction is impaired and partially
carboxylated (and non-functional) forms of vitamin K
dependent proteins are secreted into the plasma (54,70).
Vitamin K
Table 4.2 shows the effect of vitamin K on the
secretion and storage of PS in the Hep G2 cells, K562
cells and HUVEC. Vitamin K had no significant effect on
synthesis of PS antigen in Hep G2 cells which corresponds
with the work of Fair and Marlar (54). However, with
other vitamin K-dependent proteins, such as prothrombin
and factor X, vitamin K increased their synthesis
(20,54,69,70). In Fair and Bahnak study, the rate of
55


Table 4.2: Effects of Vitamin K on PS Production
Cell Type Vitamin K Cone en t ra t i on (/zl/ml) PS Secretion (Grams/cell/hou r) PS Storage (Grams/cell)
Hep G2 0 1.46 X 1015 5.46 X 10'15
5 No change No change
10 No change No change
K562 0 0.0404 X 10-15 3.78 X 10'15
10 Significant increase No change
15 significant increase No change
HUVEC 0 0.37 X 10-15 30.5 X lO'15
5 Slight increase trend Slight increase trend
10 Slight increase trend No change
prothrombin secretion was the same as in untreated
cultures, but the amount secreted was twice the control
(20). Also, the intracellular levels of prothrombin was
reduced by 30% to 40%, but did not account for the two
fold increase in secretion (20) The other vitamin In-
dependent proteins may be more sensitive to vitamin K
than PS.
56


In the K562 cells, vitamin K caused an up-regulation
of PS secretion (Table 4.2) Ten fig/ml of vitamin K had
a greater effect on the rate of PS secretion in K562
cells than 15 /ig/ml of vitamin K, suggesting an optimal
concentration for vitamin K of 10 ^ig. Vitamin K had the
greatest impact on the secretion of PS in the K562 cells,
compared to the other two cell types. Vitamin K caused a
4 to 6 fold increase in secretion, whereas in the HUVEC
only a slight increase and in the Hep G2 no change was
observed. This suggests that the K562 cells are more
sensitive and capable of responding to vitamin K than the
other cell types. Vitamin K had no effect on the storage
of PS in the K562 cells.
Vitamin K induced a slight increase in both the rate
of secretion and storage of PS in the endothelial cells.
This corresponded with work done by Fair et. al. (10).
Out of the three cell types, the HUVEC was the only cell
type where vitamin K effected the amount stored within
the cell. Low levels of vitamin K had an effect on the
57


amount of PS stored within the cell suggesting an optimal
concentration of 5 ^g.
Warfarin
Table 4.3 shows the effects of Warfarin on the rates
of secretion and storage of PS in the three cell types.
Warfarin significantly decreased the secretion rate of PS
antigen in the Hep G2 cells. Higher levels of Warfarin
also significantly increased storage consistent with the
toxic effect of Warfarin. This is consistent with other
vitamin K-dependent proteins (20,70). Fair and Bahnak
demonstrated that when Hep G2 cells were treated with
Warfarin, the rate and amount of prothrombin secreted by
the cell decreased 3 to 4 fold (20). However, the
intracellular levels of prothrombin were not effected by
Warfarin (20). They suggest that the rate of
transcription or translation is decreased by Warfarin.
Jamison et. al. also demonstrated that prothrombin
secretion decreased by 81% to 87% (70). In addition,
they showed that the quantity of prothrombin mRNA was not
58


Table 4.3: Effects of Warfarin on PS Production
Cell Type Warfarin Levels (ng/ml) PS Secretion (Grams/c el1/hour) PS Storage (Grams/cell)
Hep G2 0 1.46 X 1015 6.3 X 10-15
10 Decreased No Change
20 Decreased Increased trend
K562 0 0.0404 X 10-15 4.65 X 10'15
10 No Change Increased trend
20 No Change Increased trend
HUVEC 0 0.37 X 10'15 30.5 X 10-15
10 No Change No Change
20 No Change No Change
effected by Warfarin treatment suggesting the effects of
Warfarin are post-transcriptional. In this study, the
intracellular levels of PS increased by 13% when Hep G2
cells were treated with 20 ng/ml Warfarin indicating that
the effects of Warfarin on PS synthesis are post-
transcriptional .
In the K562 cell, Warfarin treatment had no
significant effect on the secretion of PS, but caused a
slight increase in the cellular storage of PS. The
effects of Warfarin on the storage of PS was similar to
59


the Hep G2 cell, suggesting that Warfarin effects are
post-transcriptional. Under-y-carboxylated forms of PS
were produced by both cell types and they may have been
targeting these forms to an intracellular PS pool for
degradation by interaction with other proteins, possibly
a chaperone protein. Further investigation is required
to determine if a chaperone protein or another protein is
involved in this process. A study conducted by Zhang and
Suttie (65) showed that the fate of intracellular
prothrombin in Warfarin-treated H-35 cell line was
degraded rather than secreted. They suggested that the
under-y-carboxylated prothrombin pool is targeted for
degradation by another protein and that this association
is decreased after carboxylation (65).
Warfarin did not significantly effect the secretion
or storage of PS in the HUVEC. However, in the Fair et.
al. study, Warfarin caused a slight decrease in the
secretion of PS (10). The amount of Warfarin used in
their study was 1 /xg/ml of Warfarin, whereas in this
study 10 to 20 ng/ml was used.
60


Conclusions
In the vascular system, three main cell types have
been shown to produce and secrete PS and these are the
hepatocyte, endothelial cell, and the platelet. This
study was initiated to help further our understanding of
how these cells might contribute to the concentration of
PS in the vascular system and what these cellular roles
might be in the pathology of thrombosis.
The basal level of PS in plasma stays constant until
an injury occurs within vascular system causing PS levels
to increase locally. The hepatocyte maintains the basal
plasma level of PS and very little is contributed by the
endothelial cell. However, individuals with liver
disease show only a 25% reduction in PS levels (10).
This may suggest that the basal level of PS also is
partially maintained by another cell type or can be taken
over by another cell. In this study, the endothelial
cell may be the likely candidate. HUVEC secreted a low
level of PS at the initial time point and once a higher
PS concentration was reached the HUVEC rate of secretion
61


slowed down. In contrast,Whereas, the Hep G2 cell
secreted moderate level at the initial time point and the
rate of secretion continued to increase over time. This
might suggest that when the basal level of PS is reached
in the media or bloodstream, PS causes an auto-down-
regulation of PS synthesis within the endothelial cell.
However, when an injury occurs, a stimulator(s) of the
endothelial cell causes a local increase in the PS
concentration via release by the endothelial and
platelets. In this study, the mechanisms and stimuli by
which the endothelial cells and megakaryotes release PS
were not studied. Further studies needs to be undertaken
to determine these mechanisms and the molecules which
releases PS at the site of injury.
Vitamin K and Warfarin affected each cell type
differently, thus explaining why the plasma levels and
local site levels are increased (or changed) compared to
other vitamin K dependent proteins.
62


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