Topics in Compan An Med 27 (2012) 40-45

Topical Review

New Models of Hemostasis

Maureen McMichael, DVM, DACVECC

A B S T R A C T

Keywords: Hemostasis is an essential protective mechanism that depends on a delicate balance of procoagulant and
waterfall model anticoagulant processes. The waterfall/cascade models of coagulation are useful for understanding several
cascade model
essential steps of coagulation in vitro. These have resulted in the creation of the plasma-based tests used
coagulation
thrombosis commonly and the ability to identify deficiencies in the extrinsic, intrinsic, and common pathways of
cell-based model coagulation. The model was also essential in elucidating the role of several of the inhibitors of coagulation
and is currently used to demonstrate coagulation as it occurs in plasma in a static environment that is
University of Illinois College of Veterinary devoid of endothelial interactions. The intrinsic pathway originally described by these models does not
Medicine, Champaign, IL, USA
appear to be essential for in vivo hemostasis but may play a role in pathologic thrombosis. The
Address reprint requests to: Maureen McMi- waterfall/cascade models’ lack of cellular elements sets the stage for the cell-based model of coagulation.
chael, DVM, DACVECC, University of Illinois The cell-based model of blood coagulation, which includes the varied, complicated network of factors
College of Veterinary Medicine, 1008 W. Ha- necessary for appropriate in vivo coagulation to occur, was the next step in the evolution of our
zelwood Drive, Champaign, IL 61802.
understanding of coagulation. Recently, researchers have focused on real-time, in vivo models of
E-mail: mmcm@illinois.edu. hemostasis and this research reveals unexpected phenomena. ! 2012 Elsevier Inc. All rights reserved.

In the early 1900s, research from the laboratories of W. H. Seegers
dominated the coagulation literature. It was believed that the only
enzymatic process necessary for clot formation was the conversion of
prothrombin (FII) to thrombin (FIIa).1 All other components identified
were thought to be degradation products of prothrombin and there-
fore artifacts.2 There were several important discoveries in the late
1950s that conflicted with Seegers’ theory. In 1953, Rosenthal and
coworkers discovered that a deficiency of FXI caused mild bleeding
abnormalities upon provocation (e.g., surgery, injury, etc.) and was
found mainly in Ashkenazi Jews.3 This abnormality was corrected by
the addition of normal plasma or plasma from patients with hemo-
philia A or Hageman factor (FXII) deficiency.3 After the discovery of
Stuart-Prower factor (FX) deficiency, the experimental and genetic
evidence for the presence of additional, unidentified coagulation fac-
tors continued to strengthen.4 It became clear to several researchers
that coagulation factors were present in blood in an inactive form
(zymogen) and were converted to enzymes in a step-by-step manner.
These concepts laid the groundwork for the next step in coagulation
research. In 1964, 2 independent and similar reports were published,
introducing the waterfall5 and cascade6 theories of blood coagulation.
The role of cells, particularly platelets, in coagulation occurring in
vivo emerged as an essential element for clot formation, paving the
way for the next model of hemostasis. The cell-based model of blood
coagulation, which includes the varied, complicated network of play-
ers necessary for appropriate in vivo coagulation to occur, was the
next step in the evolution of our understanding of coagulation.7 Re-
cently researchers have focused on real time, in vivo models of hemo-
stasis and this research reveals unexpected phenomena.
Waterfall/Cascade Model
The waterfall/cascade models proposed that fibrinogen formation
occurs through a stepwise series of reactions in which serine pro-
teases, which normally circulate as inactive zymogens, are converted
into active enzymes.8 Originally, the essential steps of coagulation
were believed to come from a single pathway, the intrinsic pathway,
which was named because all of the components necessary were
present in blood (intrinsic to blood). The extrinsic pathway was not
mentioned in the waterfall article and only briefly mentioned in the
cascade article5,6 (Fig. 1). The models were later refined to include a
pathway dependent on a factor not believed to be in contact with
blood in health, tissue factor (TF), and was named the extrinsic path-
way. Additional subsequent modifications included linking the ex-
trinsic and intrinsic pathways and the discovery that FV and FVIII act
as cofactors rather than enzymes.8
It later became evident that the extrinsic pathway plays the major
role in in vivo blood clotting, bringing the physiological relevance of
the contact pathway into question.8 Despite the fact that high-molec-
ular-weight kininogen (HMWK) and pre-kallikrein (PK) accelerated
coagulation in vitro, individuals with deficiencies of FXII or PK did not
show any bleeding tendency in vivo. It was not clear how these factors
were relevant to normal hemostasis.9-13
The traditional waterfall/cascade model depicts the contact path-
way (intrinsic) and the tissue factor pathway (extrinsic) as indepen-
dent pathways to the generation of factor Xa (FXa). Factor Xa is essen-
tial to the formation of thrombin and ultimately fibrin. The contact
pathway (also called the kallikrein-kinin pathway) is composed of 2
zymogens (FXII and PK) and 1 cofactor (HMWK). The initial triggering
event leading to clot formation was believed to be activation of FXII,
but the in vivo activator of FXII was unknown. Autoactivation of FXII
and PK occurs whenever blood contacts an artificial surface (collec-
tion tubes, hemodialysis tubing, etc.). This is the reason that blood will
clot in a syringe or tube that does not contain anticoagulant. Blood
generally clots slowly in plastic, more rapidly in glass, and most rap-
idly when in contact with a strong negatively charged surface.14 Im-
portantly, these zymogens (FXII and PK) are not calcium dependent
and will continue to “auto-activate” despite citrate anticoagulant in
the sample. Studies have shown that the longer citrated canine blood
sits before recalcification, the greater the contact activation, presum-
ably because of continued auto-activation of FXII.15 Canine blood also
appears to undergo considerably more contact activation than human
blood.15
Factor XII is produced in the liver and circulates as a single-chain
molecule with a molecular weight of !80 kDa. It is cleaved by kal-
likrein or plasmin to the active enzyme (FXIIa) or is auto-activated
when in contact with a negatively charged or artificial (e.g., glass,

0958-3947/$ – see front matter ! 2012 Topics in Companion Animal Medicine. Published by Elsevier Inc.
http://dx.doi.org/10.1053/j.tcam.2012.07.005
 Maureen McMichael / Topics in Companion An Med 27 (2012) 40-45
Fig. 1. Waterfall/cascade models depicting the intrinsic pathway to clot formation.
diatomaceous earth, plastic) surface or with certain polymers. Factor
XIIa also activates the complement pathway, and C1 inhibitor is the
major inhibitor of FXIIa.16 Once a threshold amount of FXIIa is formed,
it activates FXI to FXIa, and this reaction requires HMWK and is accel-
erated by PK. Factor XIa then activates FIX. Factor IXa, along with
calcium, phospholipids, and FVIIIa, activate FX. Factor Xa, along with
FVa, calcium, and phospholipids, activate prothrombin (FII) to throm-
bin (FIIa). Except for the first 2 steps in the intrinsic pathway, calcium
is required for the promotion or acceleration of all reactions.
A complete absence of the FXII gene occurs in cetaceans and non-
mammalian vertebrates.17 In humans, dogs, cats, and mice a mutation
of the FXII or PK gene does not cause deficiencies of hemostasis.18 A
role for FXII in physiological hemostasis is yet to be found, but recently
a role for FXII and FXI in thrombus formation has been demon-
strated.19 The genetic mutations of FXII and FXI appear to confer some
protection against pathologic thrombosis formation in humans, sug-
gesting that the intrinsic pathway may play a role in pathologic
thrombosis formation in vivo.18,20 There is support for a theory that
FXII and FXI are important for thrombosis but not hemostasis and
that, perhaps, these are distinct entities.19 Mice deficient in FXII are
protected against thrombus formation in several models of arterial
and venous thrombosis.18,21 Recently, microparticles derived from
platelets and erythrocytes have been shown to trigger thrombin gen-
eration via FXIIa.22 Microparticles are small subcellular fragments
surrounded by a membrane bilayer and complete with membrane-
associated proteins that can arise from several different cell types.
These microparticles completely failed to induce thrombin genera-
tion in FXIIa-deficient plasma. In humans a deficiency of FXI is asso-
ciated with a reduced risk for ischemic stroke.23 The lack of bleeding
in patients deficient in contact factors suggests that the contact factor
pathway may be an attractive target for novel anticoagulant thera-
peutics. Artificial agents, such as glass, have been known to activate
the contact pathway in vitro, but recently in vivo activation has been
shown to occur from negatively charged compounds such as extracel-
lular RNA, collagen, and polyphosphate.24 A newly discovered inor-
ganic polyphosphate (PolyP) appears to be crucial to several phases of
coagulation.25 PolyP is secreted from platelet-dense granules upon
platelet activation and has pro-hemostatic, prothrombotic, and pro-
41
inflammatory effects.24 PolyP may also be one answer to an age old
mystery, the identity of the physiological activator of the contact
pathway in vivo.25-28
In the waterfall/cascade models, FXI is activated by FXIIa. Severe
FXIIa deficiency is not associated with a bleeding tendency in humans,
cats, or mice.29 Humans with FXI deficiency, however, may have se-
vere bleeding with surgery or trauma, suggesting another mechanism
for FXI activation (in addition to FXIIa activation) in vivo.30 Although
thrombin can activate FXI, the process is slow and inefficient but is
profoundly improved in the presence of PolyP.31-34 PolyP has been
shown to be a potent activator of both thrombin and FXI.35
Platelet-derived PolyP is much more effective in the propogation
phase than in the initiation phase of coagulation and may play an
important role in accelerating FV activation by FXa and/or throm-
bin.27 The thrombin burst occurred significantly earlier when PolyP
was added to plasma and the anticoagulant function of tissue factor
pathway inhibitor (TFPI) was severely inhibited. PolyP appears to alter
the stability of the clot and attenuates fibrinolysis.25 PolyP is also
present in bacteria and has been purified from Salmonella.27 Bacteria
appear to alter the size and amount of their PolyP based on environ-
mental cues, and bacterial PolyP is extremely potent in triggering
blood clotting via the contact pathway.36 PolyP also induces a capil-
lary leak syndrome via FXII-dependent bradykinin generation, and
the long-chain PolyP secreted from bacteria appear to be particularly
strong triggers of inflammation.27 Current and future research on
PolyP is likely to yield exciting new insights into the mechanisms of in
vivo contact activation.
In addition to coagulation, other products of the contact activation
pathway play an important role in vascular tone,37 inflammation,38
angiogenesis,39 and fibrinolysis.40 Bradykinin is primarily generated
via contact activation on negatively charged cell membranes when
FXIIa cleaves PK to kininogen (K) and then K cleaves to HMWK to
release bradykinin. Bradykinin binds endothelial cell receptors, lead-
ing to increased production of tissue plasminogen activator (TPA),
nitric oxide, prostacyclin, and endothelial-derived hyperpolarizing
factor resulting in clinically significant vasodilation.14 The deletion of
the FXII gene in mice results in defective immune responses to infec-
tion, suggesting that the contact pathway’s main physiological role in
that species may be in host response to pathogens.18,20,21 Additional
findings in support of this theory include the ability of multiple mi-
crobes to activate the contact pathway.41,42
The waterfall/cascade model was pivotal in advancing our under-
standing of several essential steps of coagulation in vitro. It has re-
sulted in the creation of the plasma-based tests used most commonly
(i.e., activated partial thromboplastin time, prothrombin time, Rus-
sell’s viper venom time, thrombin time) that are essential for the iden-
tification of deficiencies in the extrinsic, intrinsic, and common path-
ways of coagulation. The model was also essential in elucidating the
role of several of the inhibitors of coagulation and is currently used to
demonstrate coagulation as it occurs in plasma in a static environ-
ment that is devoid of endothelial interactions. The intrinsic pathway
originally described by these models does not appear to be essential
for in vivo hemostasis. The waterfall/cascade models’ lack of cellular
elements sets the stage for the cell-based model of coagulation.
Cell-based Model
There were several challenges to the relevance of the waterfall/
cascade model beginning in the late 1960s. It was noted that patients
deficient in FXII or PK did not experience abnormal hemostasis. This
conflicted with the important role the intrinsic pathway played in the
waterfall/cascade model. In 1977, Osterud and Rapaport discovered
that FVIIa-TF can activate FIX and FX, bringing the extrinsic pathway
to the forefront of coagulation research.43 In 1982, another group re-
ported that plasma deficient in either FVIII or FIX showed significantly
less activation of FX by TF compared with normal plasma, suggesting
42 Maureen McMichael / Topics in Companion An Med 27 (2012) 40-45
Fig. 2. Cell-based model of coagulation depicting the interconnection between the
intrinsic and extrinsic pathways.
some form of dependency between the 2 pathways.44 In the 1990s
Broze and Gailani presented evidence that clotting, in vivo, is initiated
by TF-VIIa and that the contact factors (FXII, PK) are not essential for
physiological hemostasis.32 Several areas of interaction between the
“intrinsic” and “extrinsic” pathways became apparent. These ad-
vances led to a revised theory of coagulation, which includes the vital
contribution of cellular elements to hemostasis. Coagulation complex
formation requires a phospholipid membrane surface and the addi-
tion of calcium. In vivo, the procoagulant membrane is currently
thought to consist mainly of activated platelets, although this is being
questioned.45 In addition to undergoing a shape change, activated
platelets expose anionic phospholipids, mostly in the form of phos-
phatidylserine, on their surface. Other cells (monocytes, smooth mus-
cle cells, endothelial cells) can also function as the procoagulant sur-
face. In an atherosclerotic plaque, smooth muscle and endothelial
cells express phosphatidylserine after exposure to low-density lipo-
protein.46
In 1992, Mann proposed what has become known as the “cell-
based model” of coagulation7 (Fig. 2). The model has 2 phases: an
initiation phase and a propagation phase. An amplification phase oc-
curs in some of the literature. The initiation phase begins when TF
comes in contact with circulating FVIIa, forming a complex that re-
sults in the generation of a relatively small amount of thrombin. The
propagation phase is where the bulk of thrombin is generated and this
phase is necessary for appropriate hemostasis. In this model, contact
factors are not necessary for coagulation and there are multiple inter-
actions between the intrinsic and extrinsic pathways. The cell-based
model of coagulation answers several important questions left unre-
solved by the waterfall/cascade models. Questions such as why pa-
tients with hemophilia bleed if there are 2 independent pathways to
clot formation, why deficiencies in FXII or PK do not cause coagulation
abnormalities if the contact pathway is essential for hemostasis, and
how TFPI plays an important role in normal hemostasis are all ad-
dressed by the cell-based model.
Initiation—Tissue Factor
Tissue factor, also called thromboplastin or FIII, is synthesized by a
number of different cells and is expressed on the surface of the cell.47
Clotting occurs in vivo when blood comes in contact with TF, an inte-
gral membrane protein. TF is expressed in a variety of cells that do not
normally come in contact with blood and has been likened to a hemo-
static envelope initiating clot formation immediately upon a breach of
the endothelial barrier. Adventitial cells (the layer of cells that sur-
round blood vessels), smooth muscle cells, and keratinocytes express
TF on their surface (Fig. 3). TF appears to be essential for survival
because there are no reports of its absence.48
Although the majority of TF is not in contact with blood, a small
amount is detectable in plasma. Exactly what role this blood-borne TF
plays is unclear. Some researchers believe that it is encrypted and can
only become activated when cleaved by an enzyme.48 TF begins to
bind the plasma serine protease FVIIa as soon as it comes in contact
with blood, which begins the initiation phase of blood coagulation.
Initiation—FVII/FVIIa
Factor VII, a vitamin K– dependent protein produced in the liver,
has the shortest half-life of the procoagulation factors and is the only
coagulation factor that circulates in both the active and inactive
forms. In humans, the active form (FVIIa) constitutes approximately
1% of total FVII circulating in plasma.49 Factor VII can be activated by
FIXa, FXa, FXIIa, thrombin, plasmin, or FVII-activating protease. The
most significant physiological activator of FVII is still a mystery, with
some suggesting that it undergoes autoactivation.48 Factor IX likely
plays a significant role in the activation of FVII because humans with
hemophilia B have very low levels of FVIIa.50 The FVII-TF complex can
also be autoactivated by the FVIIa-TF complex.48 Free FVIIa is not
inactivated by any plasma protease inhibitor, and therefore has a long
half-life (2 hours).14 Factor VIIa is protected from inactivation unless it
is bound to TF. The TF-VIIa complex appears to be the only physiolog-
ical activator of coagulation in vivo.51
The TF-VIIa complex then activates FIX to FIXa and FX to FXa. FXa
consequently generates a trace amount of thrombin.47 However, the
TF-VIIa-FXa complex is rapidly inhibited by TFPI. Antithrombin (AT), a
serine protease inhibitor, neutralizes FXa and thrombin. The initiation
phase, therefore, only results in a trace amount of thrombin before
rapid neutralization of FXa occurs, and further thrombin generation is
dependent on the success of the propagation phase.
FVIIa appears to provide surveillance of the circulation to assess
for sites of endothelial damage (e.g., TF exposure). Trace quantities of
FXa and thrombin provide feedback that barrier damage has occurred.
Activity is tightly monitored (TFPI, AT) to prevent an abundance of
thrombin generation for a false alarm. Procoagulant triggering only
proceeds if TF is exposed at high enough levels (e.g., massive tissue
injury) to overcome inhibition by TFPI and AT.
Fig. 3. Layers of the endothelium showing tissue factor located in the adventitial
layer and vWF and collagen in the intima.
 Maureen McMichael / Topics in Companion An Med 27 (2012) 40-45
Amplification
The small amount of thrombin generated in the initiation phase
can diffuse away and have widespread effects on multiple areas of
coagulation. This prothrombotic ramping-up period is referred to as
the amplification phase and results in activation of platelets with ex-
posure of membrane phospholipids and the creation of a procoagu-
lant membrane and release of granule contents. Thrombin cleaves FXI
to FXIa and FV to FVa on the platelet surface. Thrombin also cleaves
von Willebrand factor (vWF) off of FVIII to release FVIII and then ac-
tivates FVIII to FVIIIa.52 Platelets, recruited to the site of damage dur-
ing this phase, provide the procoagulant phospholipid surface for the
assembly of the factors necessary for the propagation phase.
Propagation
The initiation phase succeeds in activating FX, FIX, and cofactors
FV and FVIII (these are activated by the trace amount of thrombin that
is produced). Then, FIXa, along with FVIIIa, binds to an appropriate
membrane, often the platelet, forming the tenase complex. The “te-
n”ase complex activates factor “ten,” resulting in a rapid generation of
FXa and is composed of FIXa, FVIIIa, FX, and calcium. Most of the Xa
formed physiologically is produced through the action of the tenase
complex, not through TF-VIIa complex activation. In fact, the tenase
complex is thought to be !50-fold more efficient at activation of FX
than is the TF-FVIIa complex.53 Activated FX initiates assembly of the
prothrombinase complex, which is composed of FVa, FXa, and cal-
cium. The prothrombinase complex activates prothrombin to throm-
bin, and activation of the prothrombinase complex results in an ex-
plosive generation of thrombin and subsequent fibrin clot
formation.52 In the absence of FVIII (hemophilia A) and FIX (hemo-
philia B), the initiation of coagulation is normal (dependent on the
TF-VIIa complex), but the propagation steps are severely diminished,
leading to inadequate clot formation and the inability to maintain
appropriate hemostasis when challenged.
Coagulation, in vivo, is initiated by FVIIa coming into contact with
TF. The complex TF-FVIIa activates FX and FIX. Once FXa is generated,
the TF-VIIa-FXa complex is rapidly inhibited by TFPI.53,54 The amount
of FXa generated is insufficient to sustain hemostasis, but the trace
amounts of thrombin generated enhance its own generation via acti-
vation of the cofactors FV and FVIII, by activating FXI, and by stimu-
lating platelet aggregation. Further generation of FXa is dependent on
assembly of the tenase complex (FVIIIa and IXa). Once a stable clot is
formed at the site of damaged endothelium, coagulation must be
dampened. Healthy quiescent endothelium, located downstream
from the clot, does not support coagulation, partly because of lack of a
procoagulant membrane. Any FXa that diffuses away from the clot
will not be able to generate thrombin. FXa and thrombin that diffuse
away are inhibited by endothelial cell surface anticoagulants, AT and
TFPI. Thrombin can also bind to thrombomodulin, which results in
loss of thrombin’s procoagulant activity.
The contact factors do not appear necessary for the initiation phase
of coagulation, but components of the intrinsic pathway (FVIII and
FIX) are critical to sustained thrombin generation and the formation
of a clot. In the absence of FVIII and FIX, thrombin generation during
the propagation phase is significantly suppressed. This explains, in
part, the bleeding manifestations of people with hemophilia A (FVIII)
and B (FIX), which appears to be more profound in areas that lack TF
(e.g., joints).55 Therapeutic use of high-dose recombinant FVIIa leads
to increased concentrations of FXa, which can overcome the rapid
inhibition of TFPI and AT. Patients with hemophilia often have anti-
bodies to FVIII, so treatment with FVII is more effective (Table 1).55
Fibrinolysis
Fibrinolysis is essential for clot dissolution but also appears to be
important in tissue repair.14 During health, in flowing blood, fibrino-
43
Table 1
Summary of in vivo model findings
1. An interconnection between platelet thrombus formation, platelet activation,
and fibrin generation appears both temporally and spatially.
2. Platelet adhesion, originally believed to be exclusively dependent on vWF inter-
actions, now appears to involve multiple proteins, including vWF, fibrinogen,
and possibly fibronectin.
3. Platelet-platelet binding, originally thought to be dependent on glycoprotein
IIb/IIIa interaction with fibrinogen, appears to involve numerous adhesion mol-
ecules.
4. Blood-borne TF is brought to a developing thrombus and this process is depen-
dent on P-selectin and P-selectin glycoprotein ligand 1.
5. The critical membrane surface, in vivo, is still a mystery and the belief that
activated platelets provide this surface is being questioned.
lysis is kept quiescent because of an abundance of fibrinolysis inhibi-
tors. Fibrinolysis also requires fibrin as a cofactor for optimal function,
which serves to inhibit fibrinolysis in the absence of clot formation.
Plasminogen, synthesized in the liver, is transformed by plasmin-
ogen activators to plasmin, and plasminogen activation is enhanced
by fibrin. Plasmin then degrades polymerized fibrin to form fragments
referred to collectively as fibrinogen degradation products. Cross-
linked fibrin molecules are created by FXIIIa, and the D-dimer frag-
ment is produced by degradation of these molecules. The presence of
D-dimers is indicative of the breakdown of a crosslinked fibrin clot,
whereas the presence of fibrinogen degradation products can occur
from the breakdown of fibrinogen, fibrin monomers, or crosslinked
fibrin.
TPA and urokinase plasminogen activator (UPA), both named after
the source from where they were originally isolated, are the 2 physi-
ological activators of plasminogen in mammals.56 TPA is the major
plasminogen activator in the vasculature and UPA is the major activa-
tor in extravascular tissue. TPA is secreted by endothelial cells in re-
sponse to a variety of triggers including bradykinin, histamine, ace-
tylcholine, alpha adrenergic agents, and platelet-activating factor.
TPA enzymatic activity is very weak in the absence of fibrin. UPA is
synthesized by fibroblast-like cells, epithelial cells, monocytes, and
endothelial cells. Unlike TPA, UPA can activate plasminogen in the
absence of fibrin.56
Alternative Method of Thrombus Removal
The concept of site-specific hemostasis is gaining momentum with
the discovery of significant variation in the endothelial milieu of ar-
teries, veins, arterioles, venules, capillaries, and different organ
beds.57,58 Fibrinolysis is dependent on plasminogen being converted
to plasmin. In the central nervous system, where thromboemboli can
arise from the arterial or venous system or the heart, plasminogen
expression varies according to region in mice.59
Injecting fluorescence-labeled microemboli into the capillaries of
the central nervous system of mice, Lam et al showed that approxi-
mately half were cleared within 2 hours, presumably via fibrinolysis
and hemodynamic forces.60 The other half were cleared over the
course of several days by extravasation. Fibrin clots were detected by
electron microscopy outside the lumen, and time-lapse imaging cap-
tured endothelial membrane projections that formed a vesicle-like
structure around the thrombus, which opened to the perivascular
space to release the clot.60
Interestingly, the older mice had fewer extravasated emboli, sug-
gesting that aging may predispose to microvascular occlusion.60 What
happens to the clot after extravasation and what triggers the extrav-
asation are still mysteries.
In Vivo Models
The cell-based model has provided a major step forward in de-
scribing in vivo hemostasis and it continues to evolve as the contribu-
44 Maureen McMichael / Topics in Companion An Med 27 (2012) 40-45
tion of additional cellular and subcellular elements becomes more
apparent. Cells not normally associated with coagulation, such as
erythrocytes and white blood cells, are now known to be intimately
involved. Microparticles are rapidly moving toward the forefront of
thrombosis research and have been found to be increased in several
pathologic states. Microparticles can arise from a variety of cell types
and have been shown to increase with storage time in canine eryth-
rocyte concentrates.61
The most recent attempts to understand the complexities of he-
mostasis focus on thrombus formation in living animals. Utilizing in-
travital microscopy, originally introduced to study leukocyte and en-
dothelium interactions,62,63 these models shed new light on the
complexities involved in thrombus formation.45 Thrombus formation,
in vivo, is a dynamic process dependent on shear flow, turbulence, the
concentration of cells (especially platelets) in the flowing blood, the
state and location of the endothelium (e.g., veins, arteries, venules,
arterioles, capillaries), and a host of other factors. Together, these
influence the final architecture of the clot. Some of the more recent
discoveries refute longstanding beliefs regarding thrombus forma-
tion.
In experiments using a laser to initiate injury to the endothelium,
researchers have observed the overlap of both platelet thrombus for-
mation and fibrin clot formation almost simultaneously.45 This is in
contrast to the classical models of thrombus formation, which are
based on the belief that platelets are activated first and the forming
platelet clot is subsequently stabilized by fibrin.
This simultaneous activation of both platelets and thrombin may
be partially explained by additional studies involving collagen. Colla-
gen is present in the subendothelial matrix and TF is present in the
medial (smooth muscle) and adventitial layers of the endothelium.
Exposed collagen triggers accumulation and activation of platelets.64
TF exposure triggers thrombin generation and this converts fibrino-
gen to fibrin and also activates platelets. Collagen and TF are essential
for the maintenance of the closed circulatory system as neither of
these are exposed to flowing blood under normal conditions. It ap-
pears that collagen is the first line of defense in a breach of the endo-
thelium and TF is the second line of defense. This constitutes 2 inde-
pendent pathways to platelet activation and, depending on the injury
or the disease, one or the other may predominate.65,66
vWF is currently thought to be essential in the facilitation of plate-
let binding to local endothelium. In one experiment, platelets accu-
mulated on the endothelium of arterial vessels, although at a signifi-
cantly diminished rate, in mice with a complete absence of vWF
(fibrinogen-null mice).67 The fact that platelets can bind at all in the
absence of vWF suggests the presence of a mechanism for arterial
thrombus formation that is independent of vWF.
In addition to vWF, fibrinogen is also thought to be absolutely
essential for platelet-platelet bridging during the formation of a fibrin
clot. In an experiment evaluating the contribution of fibrinogen to
arterial thrombus formation, Ni et al studied clot formation in fibrin-
ogen-null mice.67 Thrombus formation was the same for wild-type
mice and fibrinogen-null mice, which argues for a platelet-platelet
receptor that is independent of fibrinogen. The thrombi in the fibrin-
ogen-null mice were unstable and tended to embolize as they became
larger.67 Thrombi even formed in both fibrinogen-null and vWF-null
mice, but again the thrombi were unstable.67 The fact that thrombi
formed in the absence of both fibrinogen and vWF suggests the pres-
ence of additional undiscovered adhesive molecules that participate
in the platelet-vessel wall synapse.67
TF was previously thought to be located exclusively outside of
blood vessels and was thought to be activated only when the blood-
tissue barrier was breached. However, several cell types, including
circulating microparticles, have been shown to express TF and these
cells accumulate in the developing thrombus via P-selectin.68 The ac-
cumulation of these TF-bearing cells allows delivery of TF, followed by
accumulation of TF in the thrombus, which may lead to the critical
concentration of TF needed to initiate coagulation.68 TF concentration
in the thrombus, which is significantly higher than circulating TF, may
be a critical component of physiological hemostasis. Cho et al pro-
posed that the activation of encrypted TF by protein disulfide isomer-
ase initiates coagulation and that this isomerase is secreted upon ac-
tivation of platelets and endothelial cells.69 The isomerase converts
inactive TF on cells or microparticles to its active form. With direct
tissue damage, the TF in the endothelium may already be in the active
form and would not require the isomerase.64
Conclusions
The waterfall/cascade models are helpful to understanding in vitro
clot formation, but the lack of cellular elements limits their utility to in
vitro diagnostics. The cell-based models will continue to evolve as
new information is gained from the real time, in vivo, studies being
carried out. Site-specific clot dissolution research is still in its infancy
and will likely expand beyond the central nervous system to shed
light on other areas of interest. Lastly, PolyP may provide an answer to
the longstanding mystery of the in vivo activator of the contact path-
way.
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