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In the face of vascular injury, a chain reaction of pro-inflammatory and wound-healing
responses is rapidly triggered. As part of the wound-healing response, a stable blood
clot, consisting of aggregated platelets and a mesh of cross-linked fibrin protein,
is formed to prevent excessive blood loss 18]. Traditionally, blood coagulation is described as occurring in two phases, primary
and secondary hemostasis, wherein platelets first aggregate to form the initial platelet
plug followed by activation of the coagulation system to form the fibrin clot. Depictions
of blood coagulation have evolved significantly and now showcase the complex interplay
between platelets, the coagulation system, and vessel wall to clot formation. This
intricate network can be described as the cell-based model of coagulation. In short,
blood coagulation is categorized into three phases: initiation, amplification, and
propagation. Initiation occurs upon vascular injury with resultant activation of the
endothelium, consisting of activated endothelial cells, and exposure to sub-endothelial
cells that include amongst others smooth muscle cells and fibroblasts. Sub-endothelial
collagen is exposed to the blood and mediates the initial adhesion of circulating
platelets to the exposed collagen surface via von Willebrand factor. The platelet
aggregate that forms is responsible for stopping blood loss. In parallel to these
events, activated endothelial cells and smooth muscle cells express the potent pro-coagulant
molecule, tissue factor (TF), which binds with circulating coagulation factor (f)
VII. TF acts as a cofactor for fVII to promote the proteolysis and activation of fVII
to become active fVII (fVIIa), wherein TF also binds with fVIIa to form the TF/fVIIa
complex. The TF/fVIIa complex then goes on to proteolytically cleave fIX and fX into
fIXa and fXa, respectively. FIXa serves to generate more fXa and fXa serves to generate
thrombin. In the amplification phase, circulating platelets that have adhered to the
site of injury become activated by thrombin and form a platelet aggregate. This provides
a surface for the activation of other procoagulant factors. Concomitantly, thrombin
proteolytically cleaves platelet-derived fV and circulating fVIII into fVa and fVIIIa,
respectively. In addition, thrombin converts fXI into fXIa, which promotes further
fIXa generation. In the final propagation phase, thrombin generation is amplified
on the surfaces of activated platelets. FVa binds with fXa to form the prothrombinase
complex, whereas fVIIIa binds with fIXa to form the intrinsic tenase complex. The
prothrombinase and intrinsic tenase complexes serve to enhance the activities of fXa
and fIXa, generating sufficient quantities of thrombin to produce large amounts of
insoluble fibrin. In this phase, thrombin also cleaves fXIII into fXIIIa, which covalently
cross-links fibrin chains to form a large fibrin mesh. Taken together, the platelet
aggregate and cross-linked fibrin forms a stable clot, which seals off the site of
injury and prevents excessive blood loss.

Since the blood coagulation system is a potent, highly effective process, tight regulation
of the blood coagulation system is essential to prevent unnecessary clot formation
18]. Any perturbations of the regulatory pathways can accordingly culminate in thrombosis.
Coagulation proteases and molecules can be inhibited by either direct inhibition of
protease activity or through degradation of coagulation factors. First, circulating
protease inhibitors, such as antithrombin, heparin cofactor II, tissue factor pathway
inhibitor (TFPI), and C1 inhibitor, bind with the active site of proteases and prohibit
the protease from cleaving its target. Table 1 indicates the respective targets for these inhibitors. Second, coagulation factors
can be degraded through activation of the protein C/protein S pathway, activation
of a disintegrin and metalloproteinase with a thrombospondin type 1 motif member 13
(ADAMTS13), or activation of tissue-type plasminogen activator (t-PA). The protein
C/protein S pathway inactivates fVa and fVIIIa and is catalyzed by the presence of
thrombomodulin and endothelial protein C receptor (EPCR). ADAMTS13 is a matrix metalloproteinase
that cleaves the multimeric strands of von Willebrand factor, disrupting platelet
adhesion. t-PA contributes to the final dissolution of the fibrin-mesh by triggering
fibrinolysis through the conversion of plasminogen to plasmin. Collectively, these
individual events come together to ensure that the blood coagulation system is highly
regulated. As a result of its location, the endothelium plays an essential role in
the development of a clot. Figure 1 illustrates the contribution of the endothelium to blood coagulation. The following
sections provide an in-depth review of some of the key components of the blood coagulation
system that originate from the endothelium.

Table 1. Targets of coagulation inhibitors

Fig. 1. Illustration of endothelial control over blood coagulation

Endothelial contribution to the blood coagulation cascade

Traditionally, the blood coagulation system is described as a “cascade” or “waterfall”
of Ca
²⁺
-dependent activation steps that begin with activation of the intrinsic pathway or
extrinsic pathway and culminate in the production of the penultimate enzyme, thrombin
(Fig. 2) 19], 20]. While the blood coagulation cascade model admittedly provides an over-simplified
view of blood coagulation, it does highlight all of the essential protein components
and is a useful tool for quickly understanding this otherwise rather intricate system.
Intact endothelial cells express potent inhibitors, discussed in later sections, to
prevent the synthesis and activity of thrombin. Once endothelial cells become activated,
they play an essential role to the generation of thrombin through expression of pro-coagulant
factors that contribute to both initiation and propagation of thrombin generation.

Fig. 2. Succinct schematic overview of the blood coagulation cascade. f factor, HK high-molecular-weight kininogen, TF tissue factor

In the blood coagulation cascade, thrombin generation is initiated by activation of
the extrinsic pathway, triggered by activation of fVII to form fVIIa, or by activation
of the intrinsic pathway, triggered by activation of fXII to form fXIIa. In the extrinsic
pathway, activated endothelial cells express TF on the cell surface. TF is constitutively
expressed in extravascular tissues, such as fibroblasts and smooth muscle cells 21] and the phenomenon has been described as a hemostatic envelope that limits bleeding
after vascular injury 22]. Although TF is not typically expressed in the intravascular space, activated endothelial
cells and adhered leukocytes may express active TF in response to vascular injury
or inflammatory stimuli. For example, activated endothelial cells express TF, which
has an important role in the pathogenesis of thrombosis 23], 24]. TF is also abundantly expressed in atherosclerotic plaques and is found in both
cellular (macrophages, vascular smooth muscle cells, and endothelial cells) and acellular
(foam cell-derived debris within the necrotic core) regions 25], 26]. Furthermore, under experimental conditions, cultured endothelial cells express TF
in the presence of pro-inflammatory molecules, such as lipopolysaccharide, tumor necrosis
factor-? (TNF-?), interleukin-1? (IL-1?), thromboxane A2, vascular endothelial growth
factor, and thrombin 27]–40]. In contrast, only a few studies have reported endothelial TF expression in animal
models. While some investigators have reported the presence of TF protein on the endothelial
cells of lipopolysaccharide-treated mice and rabbits 41], 42], others have been unable to reproduce the results in similar models 43], 44]. These conflicting observations may in part have arisen from the different detection
methods applied. Alternatively, TF may exist as an inactive (encrypted) form that
becomes activated (decrypted) upon vascular injury 45] and this may account for the presence of TF protein with little to no procoagulant
activity. How and why TF encryption occurs has been a matter of great debate 45], and is beyond the scope of this review. Several lines of evidence have suggested
that the effects of endothelial cell-derived TF may not be confined to the coagulation
network. The TF/fVIIa complex activates protease activated receptor (PAR)-2 24], which induces a pro-inflammatory response. TF deficiency has been associated with
decreased IL-6 expression via fXa-dependent activation of PAR-2 in a murine model
of sickle cell disease 46]. Furthermore, microvascular endothelial cells have been observed to induce angiogenesis
and collateral vessel formation through the release of TF-rich microparticles 47]. The collective data suggest that TF has a multitude of hematologic and vascular
effects although further work is required to elucidate the origin and role of endothelial
cell-derived TF.

While activated endothelial cells are typically associated with the extrinsic pathway,
they may serve additional roles with the intrinsic pathway. For example, cultured
endothelial cells induce thrombin generation in platelet-poor plasma. The presence
of a TF inhibitor has a limited effect on thrombin generation whereas low levels of
thrombin are formed in fXII-deficient plasma. Similarly, thrombin generation is enhanced
in patients undergoing coronary artery bypass grafting, suggesting that the intrinsic
pathway may be important under specific conditions. The underlying mechanisms of how
endothelial cells affect the intrinsic pathway is currently unclear although it is
plausible that endothelial cells may in part act as a barrier for intrinsic factors
from inhibition since fXIIa is protected from inhibition by C1 inhibitor in the presence
of endothelial cells 48]. Thus, endothelial cells may be critical components of both the intrinsic and extrinsic
pathways.

Once the blood coagulation cascade is triggered, thrombin is generated and amplifies
its own production through activation of cofactors fV and fVIII. While fV, as well
as most other pro-coagulant proteins, is derived from hepatocytes, the origins of
fVIII have been elusive. An early study by Webster et al. demonstrated that canines
with fVIII deficiency (Hemophilia B), that received liver transplants from normal
canines, exhibited reduced bleeding tendencies compared with non-transplanted canines
49]. Despite reduced bleeding tendencies, fVIII deficient canines possessed 50 % of the
fVIII plasma levels compared with normal canines. Similarly, normal dogs who received
liver transplants from hemophilic dogs also maintained a fVIII level of 50 %. Follow-up
investigations in in vitro settings have provided evidence suggesting that liver sinusoidal
endothelial cells, and not hepatocytes, produce fVIII in culture 50]. Extra-hepatic endothelial cells may also synthesize fVIII. Norman et al. and Veltkamp
et al. demonstrated that fVIII deficient animals who received spleen or lung transplantations
had partially restored levels of fVIII in plasma 51], 52], suggesting that extra-hepatic sources of fVIII existed. Fahs et al. demonstrated
that mice with hepatocyte-specific deletion of fVIII were indistinguishable from littermate
controls. In contrast, mice with endothelial-specific deletion of fVIII displayed
a severe hemophilic phenotype with no detectable levels of plasma fVIII activity 53]. Everett et al. also demonstrated that endothelial-specific deletion of Lman1 in mice had lower levels of plasma fVIII compared with their control group 54]. Conversely, hepatocyte-specific deletion of Lman1 in mice did not reveal any effect on plasma fVIII levels, supporting the observation
that fVIII is synthesized in multiple types of endothelial cells. Together, these
results provide solid evidence that endothelial cells are the primary source of fVIII.

Endothelial contribution to platelet adhesion and aggregation

Platelets play a fundamental role to prevent blood loss by forming the platelet hemostatic
plug, and to serve as a platform for coagulation factors. Platelet-endothelium interactions
play an integral role in the activation and regulation of platelets. While an intact
endothelium inhibits the adhesion of platelets, through the release of nitric oxide
and prostaglandin I
₂
, activated endothelial cells express a variety of molecules and receptors that increase
platelet adhesion to the site of injury. In endothelial cells, Weibel-Palade bodies
store VWF, P-selectin, angiopoietin-2, t-PA, and endothelin-1, which are active participants
of platelet adhesion, leukocyte recruitment, inflammation modulation, fibrinolysis,
and vasoconstrictor, respectively. Following vascular insult or in the presence of
vasoactive agents such as histamine, bradykinin, and thrombin, endothelial Weibel-Palade
bodies fuse with the plasma membrane and release these products into the abluminal
space wherein they perform their respective functions.

VWF, a predominant product of the endothelium, is a multimeric adhesion glycoprotein.
It is synthesized within the endoplasmic reticulum and processed by the Golgi complex
of endothelial cells, megakaryocytes, and the ?-granules of platelets 55]. In hemostasis, VWF plays a dual role. First, VWF is essential for platelet adhesion
to collagen at sites of vascular injury. Following vascular insult, VWF forms long
strings of up to several millimeters in length on the surface of endothelial cells
resulting in ultra-large VWF multimers that readily form high-strength bonds with
platelets via the platelet receptor glycoprotein Ib-V-IX, and stretch along the surface
of the endothelium or of the damaged vessel. The second function of VWF is to stabilize
circulating plasma fVIII 56]. VWF stabilizes fVIII through a non-covalent interaction, which results in a tightly
bound complex 57]. The importance of VWF in fVIII stabilization is demonstrated by the rapid clearance
of fVIII from the circulation, resulting in a moderate hemophilia-like phenotype 58], 59]. Because of its dual role, VWF is an important contribution to hemostasis by endothelial
cells.

VWF secretion has been described as constitutive, constitutive-like, and regulated,
whereby an agonist acts as the trigger 60]–62]. Constitutive secretion occurs when pro-VWF is released from resting endothelial
cells via a canonical constitutive secretory pathway. Constitutive-like, or basal,
secretion of VWF occurs when VWF is released from storage granules without stimulation.
Despite extensive research, the exact mechanism by which VWF is secreted remains poorly
defined. Several studies have suggested that VWF secretion may be dependent on different
molecular pathways. In mice with endothelial cell specific knockout of G protein subunits
(G?12 and G?11), thrombin-induced secretion of VWF was reduced whereas basal secretion
of VWF was decreased in G?12 knockout mice 63], suggesting that VWF secretion is G protein dependent. Another study implicated autophagy,
an intracellular process, as a regulator of VWF secretion 64]. In mice with endothelial cell specific knockout of ATG7, a critical component of autophagy, VWF secretion is decreased and maturation of
VWF is hindered. In addition, these mice have prolonged bleeding times compared with
control. Interestingly, secretion of VWF is also influenced by circulating sodium
levels 65]. In mice subjected to water restriction, protein levels of VWF in the endothelium
rose along with an increased number of microthrombi inside capillaries 65].

Endothelial regulation of the blood coagulation system

Under physiological conditions, the endothelium prevents thrombosis by providing a
surface that discourages the attachment of cells and clotting proteins 66]. The endothelium regulates clot formation in part via its activation of the intravascular
PARs. PARs exist as four isoforms, PAR-1, PAR-2, PAR-3, and PAR-4, and are expressed
in arterial and venous endothelial cells 67]–69]. Acute release of endothelial products in coagulation is largely mediated via PAR-1
70]. Endothelial PARs serve as sensors for proteases and initiate a cascade of cell signals
upon activation by thrombin, APC, fXa, the TF/fVIIa/fXa complex, high concentrations
of plasmin, and matrix metalloproteases 69]–71]. First, thrombin-mediated activation of PAR-1 is responsible for the production of
nitric oxide and prostacyclin, which limits platelet activation. Second, thrombin-mediated
activation of PAR-1 induces the activation of Weibel-Palade bodies, releasing VWF
and t-PA. Lastly, thrombin-mediated activation of PAR-1 mediates the surface exposure
of TF. Taken together, PAR-1 plays an important role in the pro-coagulant response
upon stimulation.

An intact and healthy endothelium expresses various anticoagulants, such as TFPI,
thrombomodulin, EPCR, and heparin-like proteoglycans 72]. Endothelial cells also secrete ectonucleotidase CD39/NTPDase1, which metabolizes
the platelet agonist ADP, and platelet inhibitors, such as nitric oxide and prostacyclin
66]. As such, the endothelium actively regulates the powerful coagulation response through
equally potent inhibitory processes.

One of the most important endothelium-derived inhibitors of the blood coagulation
cascade is TFPI. A detailed overview of the structure and function of TFPI is extensively
reviewed elsewhere 73]. Briefly, TFPI is a Kunitz-type protease inhibitor that inhibits the coagulation
cascade by direct inhibition of free fXa and the TF/fVIIa/fXa complex. TFPI is present
in endothelial cells but can also be found in megakaryocytes and platelets 73], and can be released upon treatment with heparin 74]–76]. While TFPI exists as three isoforms (?, ? and ?), TFPI? and TFPI? are the predominant
isoforms. TFPI also possesses a cofactor, Protein S. Protein S aids in the TFPI-induced
inhibition of fXa and stabilizes the TFPI/fXa inhibitory complex thereby delaying
thrombin generation by prothrombinase 77]. However, the catalytic activity of protein S is limited to platelet- and endothelial
cell-derived TFPI? whereas protein S has no effect on cell surface-associated TFPI
77]. The importance of TFPI in hemostasis has been made very apparent in in vitro and
animal models. Early studies demonstrated that inhibition of TFPI decreases the time
to clot in normal plasma, suggesting that TFPI is an important regulator of coagulation.
Mice with systemic homozygous deletion of the first Kunitz domain of TFPI (TFPI
^tm1Gjb
; tfpi
^?/?
) suffer from intrauterine lethality due to coagulopathy 78]. Those with hematopoietic-specific deletion of TFPI experience increased clot volume
following vascular injury, whereas mice with endothelial-specific deletion of TFPI
have decreased time to vascular occlusion following ferric chloride-induced injury
79]. Complete TFPI deficiency is a condition that has not been reported in humans, likely
due to embryonic lethality. Patients with TFPI levels at or less than the 10th percentile
of the normal reference range for TFPI are at slightly increased risk for venous thrombosis
and coronary heart disease 80], 81]. This corroborates the notion that endothelium-derived TFPI is important for prevention
of thrombosis.

Once the cascade is initiated, thrombin generation is amplified in the presence of
activated cofactors. Since fVa and fVIIIa are essential cofactors for the amplification
of thrombin production, their inactivation provides an avenue for regulating the blood
coagulation cascade. This can be achieved through thrombin-mediated activation of
the protein C pathway, which is catalyzed by endothelial cells via thrombomodulin
and EPCR. Thrombomodulin is a 60-kDa transmembrane protein that is predominately synthesized
by endothelial cells and expressed in all tissues, except for the microvasculature
of the brain. Its transcription is regulated by shear stress and involves the transcription
factor KLF-2 82]. Thrombomodulin has been implicated in coagulation, inflammation, cancer development,
and embryogenesis. Evidence to date suggests that thrombomodulin possesses three independent
anticoagulant activities: catalyzing thrombin-induced activation of protein C to activated
protein C, binding with thrombin to prevent conversion of fibrinogen to fibrin and
activation of platelets, fV, fVIII, fXI, and fXIII, and catalyzing the inhibition
of thrombin by antithrombin 83]. As the concentration of thrombin rises during coagulation, thrombomodulin forms
a complex with thrombin to create the thrombin-thrombomodulin complex that proteolytically
activates protein C and generates activated protein C, which confers anticoagulant
activities 84]. Activated protein C degrades fVa and fVIIIa, and thus, attenuates thrombin generation.
The thrombin-thrombomodulin complex enhances the conversion of protein C to activated
protein C by 1000-fold compared with thrombin alone 85]. Although thrombin-thrombomodulin-mediated protein C activation is relatively efficient
in the microvasculature, protein C cleavage in larger vessels requires the presence
of EPCR, which is primarily localized on the endothelial layer of larger vessels 16]. EPCR is a type 1 transmembrane glycoprotein that possesses a similar homology to
the major histocompatibility complex-class 1/CD1 family of molecules 86]. EPCR recruits protein C at the endothelial cell surface thereby facilitating the
interaction between protein C and the thrombin-thrombomodulin complex. In the presence
of EPCR, the conversion of protein C to activated protein C is increased by 20-fold
compared with the thrombin-thrombomodulin complex alone 87]. EPCR has also been shown to bind with activated protein C 88]. The importance of EPCR is highlighted in studies involving mice. Mice with total
deficiency in EPCR die in utero 89], suggesting that EPCR is not only important for suppression of thrombosis but is
essential for normal embryonic development. Taken together, thrombin-mediated activation
of protein C requires the presence of thrombomodulin and EPCR to efficiently generate
activated protein C.

Endothelial contribution to clot resolution

Once the wound is repaired, endothelial cells release pro-fibrinolytic molecules to
degrade the clot and metalloproteases to cleave platelet aggregates. Dissolution of
the clot is induced by fibrinolytic agents, such as t-PA and urokinase-type PA (u-PA).
Although t-PA and u-PA perform the same function, t-PA is predominantly found in endothelial
cells while u-PA is expressed in endothelial cells, macrophages, renal epithelial
cells and some tumor cells. For simplicity, we primarily focus on t-PA since endothelial
cells are the primary source of t-PA. t-PA is a 70-kDa protein that catalyzes the
degradation of fibrin within the clot 90]. t-PA performs this function by activating the fibrinolytic system through conversion
of plasminogen to plasmin in blood and body cavities 91]. In cultured endothelial cells, there is evidence that t-PA is stored in two types
of storage granules: Weibel-Palade bodies 92] and small storage granules 93]. After wound repair is complete, the high local increase in t-PA at the site of thrombin
generation allows for efficient removal of fibrin deposits at the luminal side of
an intact vascular endothelium and is important for reducing tissue damage. The importance
of t-PA has been demonstrated in t-PA deficient mice wherein clot lysis was impaired,
especially when combined with u-PA deficiency 94], 95]. In experimental primate models of acute disseminated intravascular coagulation,
plasma concentrations of t-PA was acutely increased by more than two orders of magnitude,
due to thrombin-mediated release from storage granules in endothelial cells 96]. Thus, t-PA provides an essential method for removal of blood clots.

In a similar fashion, dissolution of platelet aggregates is mediated by ADAMTS13 97]. Endothelial cells produce and release functionally active ADAMTS13, a zinc/calcium
VWF-specific metalloprotease, and are an important source of plasma ADAMTS13 98]. The importance of ADAMTS13 is highlighted by the observation that patients with
thrombotic thrombocytopenia purpura, the levels of ADAMTS13 is severely reduced or
absent 99]. This lack of ADAMTS13 may be due to inadequate production/release of ADAMTS13 or
the activity ADAMTS13 is inhibited by auto-antibodies.

Endothelial contribution to tissue repair and angiogenesis

While the blood coagulation system forms the stable clot that stems the loss of blood,
it also contributes toward subsequent healing processes such as tissue repair and
angiogenesis. Since endothelial cells are important component of blood vessels, they
can be triggered to induce angiogenesis upon stimulation. For example, in addition
to its anti-coagulant abilities, activated protein C has been shown to stimulate angiogenesis
in brain endothelium 100]. Second, cross-linked fibrin serves as a scaffold for endothelial cells to synthesize
new blood vessels 101], 102]. Third, platelets contain a rich source of vasoactive agents and chemokines, such
as serotonin, thromboxane A2, platelet activating factor and RANTES, and pro-angiogenic
growth factors, such as VEGF, all of which are contained in platelet ?-granules 103], 104]. These compounds stimulate endothelial cell proliferation and promote the growth
of new blood vessels. Lastly, the expression of TF has been shown to induce tumor
angiogenesis in colorectal cancer and breast cancer models through TF-fVIIa-dependent
PAR-2 activation 105]–107], which induces the expression of VEGF, IL-8, MMP-7, and CXCL-1. In the past 10 years,
studies have highlighted the role of a TF isoform, alternatively spliced TF 108]–110], which displays no pro-coagulant activity but may play a prominant role in promoting
angiogenesis. Taken together, the role of the coagulation system plays a major role
in the development of angiogenesis.

Endothelial contribution to aberrant clot formation

The endothelium is an essential component of the blood coagulation system and necessary
to maintain hemostasis. However, endothelial dysfunction can occur in response to
a variety of conditions, including elevated cholesterol, diabetes or smoking, which
can induce vascular inflammation. Endothelial dysfunction is responsible for inflammation
and blood coagulation, and is associated with cardiovascular disease, such as hypertension,
coronary artery disease, chronic heart failure, peripheral artery disease, diabetes,
chronic kidney failure, and viral infections. During endothelial dysfunction, endothelial
cells become activated and contribute to the pathogenesis of thrombosis. For example,
hypoxic conditions often lead to endothelial dysfunction. As such, hypoxia has been
shown to promote the release of VWF from Weibel-Palade bodies in endothelial cells.
Inflammation has also been associated with endothelial dysfunction and can be accompanied
by thrombosis. For example, in vitro and in vivo studies have demonstrated that pro-inflammatory
cytokines, such as TNF-? and IL-1, up-regulate the production of TF and VWF, while
attenuating the expression of thrombomodulin and nitric oxide and prostaglandin I
₂
. In rabbits, immunodepletion of TFPI leads to increased susceptibility to disseminated
intravascular coagulation (DIC) 111], and generalized Schwartzman reaction, characterized by fibrin deposition and hemorrhagic
necrosis in kidneys following infusion of TF 112]. The Protein C pathway has also been implicated in the development of disseminated
intravascular coagulation with associated organ dysfunction. Patients with systemic
inflammation show an impaired protein C system due to impaired protein C synthesis
and impaired protein C activation. While protein C is synthesized by hepatocytes,
endothelial cells can regulate protein C activation through the expression of thrombomodulin.
As such, thrombomodulin levels are significantly down-regulated by the presence of
pro-inflammatory cytokines, such as TNF-? and IL-1, resulting in diminished protein
C activation. These events result in a shift from anti-thrombotic to pro-thrombotic
conditions.