Brief Overview
of Regulation of Hemostasis by Serpins and Two On-going Vascular Biology
Projects in the Laboratory
Introduction: Hemostasis is described
as the balance between procoagulant and anticoagulant forces that act in
concert to maintain the fluidity of blood under physiologic conditions.
This system responds rapidly to vascular injury via the coagulation cascade,
whose primary role is to prevent the flow of blood from the vasculature.
When a vessel is injured, hemostasis is achieved by several mechanisms, including
vessel spasm, formation of a platelet plug, blood coagulation, and eventual
healing of the injured area. Platelets adhere to extracellular matrix
proteins that are exposed from damaged blood vessel endothelial cells, and
then become activated and secrete factors that attract and activate nearby
platelets. The coagulation “cascade” is activated both by factors exposed
on and secreted from the injured tissues and platelets. These events
ultimately result in the formation of fibrin threads that enmesh platelets,
blood cells, and plasma proteins to form the hemostatic plug or thrombus.
If left unchecked, the clotting cascade would result in widespread thrombosis
and vessel blockage; thus “anticoagulant” pathways are also immediately activated
that regulate the activities of procoagulant factors.
Regulation of Blood Coagulation by Antithrombin
(AT), Heparin Cofactor II (HCII), Protein C Inhibitor (PCI) and Plasminogen
Activator Inhibitor-1 (PAI-1): The majority of coagulation
proteases are inhibited by two serpins, antithrombin (AT; systematic name
SERPINC1) and heparin cofactor II (HCII; systematic name SERPIND1).
The serpin-protease complexes are then cleared from the plasma by the non-specific
low-density lipoprotein receptor related protein found on hepatocytes.
AT inhibits thrombin, factors IXa, Xa, XIa, XIIa, kallikrein, and plasmin.
However, a comparison of the inhibition rates indicates that only the reactions
with thrombin, factor Xa, and factor IXa are physiologically important.
Thrombin is the only coagulation protease that HCII inhibits. Target
protease inhibition by AT and HCII is greatly accelerated by glycosaminoglycans,
such as heparin and heparan sulfate. The anticoagulant effect of commercial
heparin is based upon its ability to accelerate the inhibition of thrombin,
factor Xa, and factor IXa by AT. In vivo, heparan sulfate-containing
proteoglycans that are localized to the endothelial cell membrane serve to
accelerate this reaction. HCII inhibition of thrombin is also accelerated
by dermatan sulfate and dermatan sulfate-containing proteoglycans, which
are localized on the plasma membranes of extravascular cells. Thus,
it is postulated that HCII plays an important role in the regulation of thrombin
activity in the extravasculature. Additionally, both protein C inhibitor
and plasminogen activator inhibitor-1/vitronectin can also inhibit thrombin,
although the physiological relevance of these serpin-thrombin inhibition
reactions is not clear.
Protein C System and the Role of APC in
Cell Migration/Invasion: Once activated in the coagulation pathway,
thrombin can proceed down one of several pathways. It can continue
to cleave fibrinogen and activate Factor V and Factor VIII, thus stimulating
clot formation, or it can bind to thrombomodulin (TM). TM is a membrane
bound protein on the surface of endothelial cells. Thrombin now recognizes
another zymogen of a serine protease, protein C, as a substrate. Zymogen
protein C binds to endothelial cell protein C receptor (EPCR). Thrombin-TM
can then activate protein C bound to EPCR. Activated protein C (APC)
requires protein S as a cofactor for full activity. APC specifically
cleaves the two cofactors involved in thrombin generation, Factor Va and
Factor VIIIa. The destruction of these essential cofactors results
in cessation of thrombin generation, and thus helps stop thrombus formation.
The importance of this pathway is apparent in the discovery of familial thrombophilia
due to the Factor V Leiden mutation. We are trying to better understand
the process by which PCI and PAI-1/VN regulate the protein C system proteases,
and to refine their inhibition mechanism by directly looking at assay systems
with phospholipids and with endothelial cells that generate both thrombin
and APC. Interestingly, APC has other activities beyond its role as
an anticoagulant protease. APC is currently being used to treat sepsis
to replace decreased APC levels, e.g., in children with severe meningococcal
sepsis. Yet the mechanism of how APC achieves this anti-inflammatory
role is just beginning to be understood. Three groups have recently
analyzed various gene chip arrays and have identified several anti-inflammatory
genes that are up or down regulated on vascular endothelium following incubation
of APC with cells. Joyce et al found that APC directly down-regulated
major gene in a pattern of anti-inflammatory and cell survival pathways.
Riewald et al. reported that the major anti-inflammatory effect of APC was
due to activation of the classic thrombin receptor PAR-1 (protease activated
receptor-1), dependent on APC being bound to EPCR. We are currently
studying the effect of the protein C system in cellular migration and invasion
using various cell types, and we are working on the molecular mechanism of
this interaction of APC with cell surface receptors. Furthermore, we
are characterizing the role of serpins to down-regulate the migration/invasion
response, and the signaling pathways promoted by APC-cell interactions.
Localization of Heparin Cofactor II in Atherosclerotic
Lesions: The role of HCII in atherosclerosis is poorly understood,
we and others have explored the occurrence of HCII and other serpins in normal
and diseased vessels, we and others have characterized DSPG/HSPG from these
diseased lesions compared to normal artery wall and found as the disease
progressed the character of the GAG changed to be less effective at catalyzing
thrombin inhibition by HCII. Thus, we initiated this project to further
explore the occurrence of HCII compared to AT. Multiple microscopic
sections from 33 individual cases of human left anterior descending (LAD)
coronary artery with some degree of atherosclerosis were obtained from the
Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Study.
Liver sections obtained from Surgical Pathology at the University of North
Carolina were used as positive controls because both HCII and AT are synthesized
in the liver and therefore should be detectable using immunohistochemistry
(these results are not shown). All subjects in this study were persons
from the ages of 15-34 who died of external causes (accidents, homicides,
suicides) from fifteen cooperating centers managed by the Department of Pathology
at Louisiana State University Health Science Center. Data from each
case was recorded in a database for analysis including, age, gender, race,
total cholesterol and high-density cholesterol (HDLC) and this long-term
study was termed PDAY.
Each case was probed for the presence of heparin cofactor II (HCII) or antithrombin
(AT). Liver slides for each run were subjected to the exact same conditions
as the LAD slides being probed. HCII antigen was detected by first
incubating with goat anti-human HCII affinity purified polyclonal IgG.
AT was probed for using goat anti-human AT polyclonal IgG. Mouse anti-human
maspin monoclonal IgG was used as one negative control because it is a serpin
that is not produced in the liver and not thought to be in the blood vessels
or atheromas. Goat IgG was used as another negative control to ensure
that any antigen binding was not nonspecific binding due to IgG interactions.
Using the American Heart Association (AHA) classification system, each atherosclerotic
lesion from each case was blindly graded by three individuals. The intensity
of staining was also independently ranked on a scale of 0 to 3 with 0 indicating
no staining, 1 indicating weak staining, 2 indicating intermediate staining
and 3 indicating strong staining. As evidenced by the Spearman
correlation coefficient of 0.76 (two-sided p-value <0.0001) and the Nonzero
correlation statistic of 16.9039 (exact p-value <0.0001), there is a significant
positive correlation between lesion severity and HCII staining intensity.
