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Elevating the Process of Clot Regulation
GRAFTON
HIGH SCHOOL
The Role of Endothelial Protein C Receptor in Blood Clotting and the Immune System
Grafton SMART Team: Lisa Borden, Mariah Fox, Sean Gasiorowski, Grace George, Lucas Mullens, Emily Volkmann, Jacob Zirbel
Advisor: Dan Goetz, Fran Grant
Mentor: Andrea Ferrante, M.D.
Abstract
The coagulation of blood is essential to normal bodily function, but is especially important upon blood vessel injury. An excess of coagulation can lead to complications as well. One regulatory mechanism between pro- and anti-coagulant mechanisms involves Protein C. Protein C assists
in the regulation of blood clotting by acting in a negative feedback response to the production of thrombin, a protein with procoagulant activity in the serum. Thrombin loses its procoagulant functions when it interacts with a membrane-bound protein expressed on endothelial cells
called thrombomodulin. The thrombin/thrombomodulin complex promotes conversion of Protein C into Activated Protein C (APC). If protein C binds to the thrombin/thrombomodulin complex in the presence of endothelial protein C receptor (EPCR), the rate of this activation increases
twentyfold. As a soluble molecule in the bloodstream, APC functions as an anti-coagulant factor. When APC remains bound to EPCR, the APC/EPCR complex can interact with the receptor protein, Protease-activated Receptor 1 (PAR1), resulting in decreased tissue damage during
inflammation. Because EPCR is similar in structure to the Major Histocompatibility Complex (MHC), an important receptor in activating an immune response by presenting antigens to T-cells, it may have a similar function. While the MHC binds to peptides to perform its immune
activation function, EPCR is thought to bind to lipids and may potentially be involved in cytoprotective mechanisms during sepsis. Because of its importance in blood clotting and its possible immune function, the Grafton High School SMART (Students Modeling A Research Topic) Team
chose to model EPCR using 3D printing technology.
The Importance of Clot Regulation
Within the United States, 140,000 people die from strokes each year1. This
statistic, coupled with the common occurrence of heart attacks, demonstrates
the importance of blood clot regulation. While these disorders are caused by
an excess of clotting, hemophilia and other similar disorders are prime
examples of the dangers of too little clotting. As such, it is important for the
body to have a finely controlled and properly functioning mechanism for
maintaining homeostasis in the bloodstream. The functioning of a few specific
molecules is crucial to this homeostatic condition, as well as to the prevention
of widespread development of clotting disorders and the preservation of
human life.
Protein C Activation
Possible Immune Function
The interaction of Protein C with the thrombin/thrombomodulin complex is weak.
Without EPCR, it interacts only when the chance of “bumping” into the
thrombin/thrombomodulin complex is higher, such as in small blood vessels. EPCR allows
this close contact to occur anywhere in the bloodstream, specifically in larger vessels, and
greatly facilitates the molecular interaction.
• The immune system operates through the activation of various processes in
response to the physical-chemical features of “trigger” particles known as
antigens.
• The Major Histocompatibility Complex (MHC), is a molecule responsible for
presenting antigens to T-cells (a subset of white blood cells). If T cells exist that
can recognize that particular antigen/MHC complex, an antigen-specific
immune response is initiated.
• EPCR and MHC have similar shapes, which would suggest a similar function.
• However, there is no conclusive evidence for such a potential immune
function.
Structural Comparison of MHC and EPCR
1 www.strokecenter.org/patients/about-stroke/stroke-statistics
Influence of EPCR on Protein C
The Protein C Anticoagulant Pathway
• This graph shows the effect of EPCR on the rate
A
of Protein C activation (APC).
• The data was gathered by measuring the rate
of activation at various concentrations of Protein
B
C using phosphatidylcholine vesicles (structures
similar to cells) that contained thrombomodulin.
Activation rate was measured both with and
without EPCR.
• It can be seen that the rate of production of
Activated Protein C with EPCR (line A = ▵) is
faster than the rate without EPCR (line B = ○).
Key:
The third line (line ●) does not pertain to the
In the presence of EPCR = Line A (▵) focus of this poster.
In the absence of EPCR = Line B (○)
Steps preceding the anticoagulation response of Protein C:
1. Vascular injury or endotoxin/inflammatory cytokines
2. Initiation of coagulation cascade
3. Generation of thrombin, formation of blood clot
4. Thrombin binds to thrombomodulin on endothelial cells
Activated protein C (APC), a physiological anticoagulant, is generated from the
inactive precursor protein C "on demand" in response to thrombin formation.
Briefly, vascular injury or endotoxin/inflammatory cytokines initiates the
coagulation cascade, ultimately resulting in thrombin generation and blood
clot formation. Excess thrombin then triggers the protein C pathway which
provides feedback inhibition of coagulation. The protein C pathway is initiated
when thrombin (T) binds to thrombomodulin (TM) on the endothelial cell
surface. The thrombin-thrombomodulin complex rapidly converts zymogen
protein C (PC) to its active form APC. Protein C activation is augmented by the
endothelial cell protein C receptor (EPCR) which binds circulating protein C and
presents it to the thrombin-thrombomodulin complex. APC then dissociates
from EPCR and, in combination with its cofactor protein S (S), acts as an
anticoagulant by degrading factors Va and VIIIa, key cofactors in coagulation.
Adapted from diagram in Lisa J. Toltl, Lucy Y.Y. Shin, Patricia C. Y. Liaw. (2007) Activated protein C in sepsis and beyond: Update 2006. Frontiers in Bioscience 12, 1963-1972
3HLJ.pdb
1L8J.pdb
MHC
EPCR
As visible in the above figure, MHC proteins and EPCR feature a striking
structural similarity. MHC molecules have a membrane-proximal domain,
which allows for the “anchoring” of the protein to the membrane, and a
membrane-distal domain, which is the known peptide binding site. Here, the
binding site is shown from the top, as a T cell would “see” it with its receptor.
This binding site is a groove formed by two parallel alpha-helices and a floor of
beta-sheets.
©1999 by American Society for Biochemistry and Molecular Biology
The Structural Basis of EPCR Activity
• Glu86 (Dark Green) binds to Ca2+ ions on
Protein C to facilitate activation
• Thr157, Leu82 (Green) and Tyr154 (Light
Blue) create a network of hydrophillic
interactions with Protein C
• Gln150 and Arg87 (Blue) create hydrogen
bonds with Protein C
1L8J..pdb
Conclusion
EPCR is a vital part of the blood clotting process. Without EPCR, people would
be more susceptible to thrombosis and cardiovascular events. Thus, EPCR
constitutes a crucial molecule for the maintenance of blood homeostasis. In
addition, the structural similarities between Endothelial Protein C Receptor
and the Major Histocompatibility Complex may be an indication of a potential
immune function for EPCR. Addressing this hypothesis would further enhance
our understanding of the immune system and lead to an increased ability to
treat diseases.
Oganesyan, Vaheh. "The Crystal Structure of the Endothelial Protein C Receptor and a Bound Phospholipid."Journal of
Biological Chemistry. 277.28 (2002): 24851-24854. Print.
A SMART Team project supported by the National Institutes of Health Science Education Partnership Award (NIH-SEPA 1R25RR022749) and an NIH CTSA Award (UL1RR031973).