review

Advances in understanding the molecular mechanisms that

maintain normal haemostasis

James S. O’Donnell, Jamie M. O’Sullivan and Roger J. S. Preston

Haemostasis Research Group, Department of Molecular and Cellular Therapeutics, Irish Centre for Vascular Biology, Royal College of
Surgeons in Ireland, Dublin, Ireland

present in the blood, and was triggered when plasma came

Summary
into contact with a diverse range of artificial surfaces (includ-
Blood clot formation to stem bleeding from an injured blood ing kaolin, micronized silica or ellagic acid) in vitro. Accord-
vessel arises from a complex series of cellular and biochemi- ing to the cascade model, once coagulation was triggered by
cal events, which, when dysregulated, predispose to an either of these independent pathways, individual clotting fac-
increased risk of thrombosis or bleeding. Similarly, haemo- tors were sequentially activated in an amplification cascade or
static regulation of clot growth and size is exquisitely con- ‘waterfall’ (Fig 1) (Davie & Ratnoff, 1964; Macfarlane, 1964).
trolled by a series of anticoagulant ‘checkpoints’, that exert In essence, circulating inactive coagulation factor proenzymes
their inhibitory activity at distinct stages in the steps leading or zymogens were specifically activated through limited prote-
to clot formation. Although the major plasma protein con- olysis by an activated upstream clotting factor, serine pro-
stituents required for haemostasis have now been largely elu- tease. This activation then proceeded in a stepwise manner
cidated and the molecular events that lead to clot formation down the cascade, ultimately leading to thrombin generation
are well understood, defining a fuller appreciation of the and formation of cross-linked fibrin. This original cascade
importance, location and regulation of each haemostatic pro- model of coagulation is still useful when it comes to clinical
cess remains a fertile area of ongoing research. In this review interpretation of abnormal prothrombin and activated partial
article, we first provide an overview of the original ‘waterfall’ thromboplastin times in clinical practise. However, accumu-
or ‘cascade’ hypothesis of blood coagulation as it was defined lating data has highlighted a number of important limitations
in the 1960s. We subsequently discuss how this original with respect to the original cascade model. In this review, we
model has been refined over time to incorporate accumulat- discuss some recent advances in our understanding of the
ing data that has enabled a more nuanced consideration of molecular mechanisms involved in maintaining normal
the role of specific proteins, receptors and lipids in dictating haemostasis. Given the complexity of blood coagulation, it is
the spatial and temporal development of a blood clot. not possible to comprehensively discuss all advances for indi-
vidual pro- and anti-coagulant proteins. Rather, we have
Keywords: coagulation, haemostasis, thrombosis, intrinsic sought to provide a primer by giving our perspective on some
pathway, extrinsic pathway. examples of the most pertinent advances. We hope that this
review will thus be useful for trainees and of interest to gen-
The original cascade model for coagulation, first described in eral haematologists interested in a brief synopsis of the state-
the 1960s, proposed that coagulation could be triggered by of-the-art in molecular haemostasis.
one of two major independent pathways termed the ‘extrinsic’
and ‘intrinsic’ pathways respectively (Fig 1) (Davie & Ratnoff,
Integrating the extrinsic and intrinsic pathways
1964; Macfarlane, 1964). Initiation of the extrinsic pathway
in physiological haemostasis
required plasma exposure to tissue factor (TF, also known as
thromboplastin, coagulation factor III or CD142), which is Although the initial cascade model proposed two separate
usually external to the blood in extravascular tissues (Wilcox pathways for coagulation initiation, it has subsequently
et al, 1989; Fleck et al, 1990). In contrast, the intrinsic path- become clear that, at least under normal conditions, this is
way for coagulation activation relied only on factors normally not actually the case in vivo. Rather, definitive evidence has
shown that TF is the key trigger for physiological haemosta-
sis. Moreover, the distinct extrinsic and intrinsic pathways
Correspondence: Prof. James O’Donnell, Irish Centre for Vascular envisaged in the cascade model have also been shown to
Biology, Royal College of Surgeons in Ireland, Ardilaun House111 St instead function as a single integrated pathway in vivo during
Stephen’s Green, Dublin 2, Ireland. normal haemostasis (Fig 2). TF is a 46 kDa transmembrane
E-mail: [email protected] glycoprotein that is abundantly expressed in adventitial

First published online 28 March 2019 ª 2019 British Society for Haematology and John Wiley & Sons Ltd
doi: 10.1111/bjh.15872 British Journal of Haematology, 2019, 186, 24–36
 13652141, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.15872 by Nat Prov Indonesia, Wiley Online Library on [05/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review

Intrinsic pathway Extrinsic pathway

HMWK
PK

FXII FXIIa

Fig 1. The cascade model of blood coagulation. FXI FXIa

Overview of the original ‘waterfall’ model of TF-FVIIa
blood coagulation cascade. This model pro-
posed that coagulation could be triggered via FIX FIXa
two distinct and independent pathways, known
as the ‘intrinsic’ and ‘extrinsic’ pathways respec-
tively. The extrinsic pathway is triggered when
tissue factor is exposed to plasma and forms a FX FVIIIa FX
complex with activated factor VII (FVIIa). In
contrast, the intrinsic pathway is initiated
in vitro when plasma comes into contact with a Common pathway FXa
range of negatively charged surfaces. These two
amplification cascade pathways ultimately
FVa
converge into a final common pathway, which Prothrombin Thrombin
results in thrombin generation and the forma-
tion of a cross-linked fibrin clot. HMWK, high
molecular weight kininogen; PK, prekallikrien;
TF, tissue factor.
Fibrinogen Fibrin

tissues, such as fibroblasts and smooth muscle cells (Wilcox requires the amplification loop that results from specific
et al, 1989; Fleck et al, 1990). Following endothelial cell (EC) intrinsic pathway factor activation.
damage, TF becomes exposed to the plasma and binds to
activated factor VII (FVIIa). The TF:VIIa complex then con-
The cellular basis of coagulation
verts zymogen factor X into its active serine protease (FXa)
via limited proteolysis (Fig 2). In turn, FXa generation com- Coagulation activation proceeds more efficiently on cell sur-
bines with its activated cofactor factor V (FVa) and substrate faces rather than in solution. In addition, it is clearly criti-
prothrombin to form a ‘prothrombinase complex’ that facili- cal that the integrated coagulation amplification cascade,
tates the conversion of prothrombin into active thrombin. once activated, remains localised to the site of injury in
However, only a limited amount of thrombin is generated order to prevent disseminated thrombotic complications.
before the extrinsic tenase complex is inhibited by tissue fac- These two observations underpin the so-called ‘cell-based’
tor pathway inhibitor (TFPI) (Maroney & Mast, 2015). Cru- model of haemostasis (Monroe & Hoffman, 2006). Accord-
cially, the thrombin ‘spark’ produced is able to initiate ing to this model, different cells play specific roles in mod-
activation of zymogen factor XI (FXI) to its active form ulating in vivo haemostasis which can be considered as
(FXIa). FXIa then activates FIX (to FIXa), which forms the consisting of three distinct but overlapping stages, termed
intrinsic tenase complex in combination with thrombin-acti- the initiation, amplification and propagation phases respec-
vated FVIII (FVIIIa) to greatly accelerate FXa generation. tively (Fig 3) (Monroe et al, 1994, 1996; Monroe & Hoff-
The net effect of this positive feedback loop amplification is man, 2006). The initiation phase is localised to TF-
that sufficient thrombin is generated to convert soluble fib- expressing surfaces (e.g. fibroblasts and smooth muscle cells
rinogen into an insoluble fibrin clot and further activate pla- exposed at sites of EC damage). The TF:FVIIa complex is
telets that have adhered at the site of vasculature injury. formed on these cells where it activates FX as previously
Thus, as illustrated in Fig 2, the extrinsic and intrinsic path- discussed. Some of the FXa produced remains localised to
ways do not function as discrete separate pathways as origi- the cell surface where it is relatively protected from plasma
nally hypothesized, but rather are intertwined in the process protease inhibition (Monroe & Hoffman, 2006). This FXa
of physiological haemostasis. Physiological coagulation is then associates with FVa to form the prothrombinase com-
therefore triggered by the extrinsic or TF pathway, but plex and results in the generation of small amounts of

ª 2019 British Society for Haematology and John Wiley & Sons Ltd 25
British Journal of Haematology, 2019, 186, 24–36
 13652141, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.15872 by Nat Prov Indonesia, Wiley Online Library on [05/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review

(A)

FXI FXIa

FIX FIXa TF-FVIIa

FX FVIIIa

FXa FX
Inial thrombin spark
propagates thrombin Prothrombin FVa
burst by feedback
Thrombin

(B)

FX Thrombin Bust
FIX FV

FVIII Prothrombin
Thrombin Spark
FVIIa FVIIa FXa
Prothrombin
FIXa
FXa FX FXa FVa
FVIIIa
FVa
TF

Fig 2. An integrated model of physiological coagulation. Evidence suggests that tissue factor (TF) exposure constitutes the principal trigger
of physiological haemostasis in vivo. The TF:FVIIa complex is then able to active FX to FXa. In combination with its cofactor FVa, FXa
is then able to convert prothrombin into thrombin. Critically, the small thrombin spark generated by initial TF exposure is then able to
back activate FXI to FXIa, FVIII to FVIIIa and FV to FVa (illustrated by green lines). The net effect of this positive feedback loop ampli-
fication is that a secondary large amount of thrombin is generated. (B) Representation of sequential activation of clotting factors follow-
ing physiological activation of haemostasis triggered by exposure of subendothelial tissue factor.

thrombin on the TF-expressing cell surfaces (Versteeg et al, fibrin clot [including activating FXIII and thrombin activat-
2013). Any FXa dissociating from the cells following activa- able fibrinolysis inhibitor (TAFI) respectively].
tion is rapidly inhibited by plasma inhibitors, so that the
coagulation activation during this initiation phase remains
Redefining the biological roles of the contact
localised. During the amplification phase, the relatively
pathway
small amounts of thrombin generated on the TF-expressing
cell surface moves to nearby platelets to promote further The contact pathway comprises FXII, high molecular weight
activation, and drive FXIa, FVIIIa and FVa generation that kininogen (HMWK) and prekallikrien (PK) (Versteeg et al,
in turn can also then bind to the surface of the activated 2013; Smith et al, 2015). Although the molecular mecha-
platelets (Monroe & Hoffman, 2006; Versteeg et al, 2013). nisms involved are not clearly elucidated, when plasma
Collectively, these steps establish the key Xase and pro- comes into contact with negatively charged surfaces in vitro,
thrombinase complexes on the platelet surface that can then bound FXII and PK are able to reciprocally activate one
drive formation of much larger quantities of thrombin dur- another by limited proteolysis (Fig 4) (Muller et al, 2011).
ing the final propagation phase. The resultant thrombin The generated FXIIa subsequently activates FXI to FXIa,
‘burst’ plays critical roles in enabling formation of a stable which in turn activates FIX to FIXa, leading to the formation

26 ª 2019 British Society for Haematology and John Wiley & Sons Ltd
British Journal of Haematology, 2019, 186, 24–36
 13652141, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.15872 by Nat Prov Indonesia, Wiley Online Library on [05/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review

FIX FX Thrombin spark

FIXa
Initiation FVIIa Prothrombin
FXa
FVa Prothrombinase
Complex
TF

TF expressing fibroblasts

FXa
FXIa FIX FX
Amplification FXa
FV
FVIII FIXa
FVIIIa
FVa
Thrombin spark Xase Complex

Phospholipid membrane on activated platelets

Propagation Thrombin burst Fibrinogen

Prothrombin
FXa
FVa
Crosslinked fibrin

Activated
platelets

Fig 3. Cellular surfaces play a critical role in regulating coagulation. Cellular surfaces play a critical role in regulating coagulation
activation in vivo. The initiation phase is localised to tissue factor (TF)-expressing cell surfaces (e.g. TF-positive fibroblasts) and
leads to formation of a limited amount of FXa together with the initial thrombin spark. During the amplification stage, the rela-
tively small amounts of thrombin generated on TF-expressing cells moves to activated platelet surfaces where it can catalyse forma-
tion FXIa, FVa and FVIIIa, thereby establishing the critical tenase and prothrombinase complexes. Finally, during the propagation
phase, these complexes drive formation of much larger concentrations of thrombin, which can cleave soluble fibrinogen into insol-
uble fibrin.

ª 2019 British Society for Haematology and John Wiley & Sons Ltd 27
British Journal of Haematology, 2019, 186, 24–36
 13652141, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.15872 by Nat Prov Indonesia, Wiley Online Library on [05/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review

Contact Pathway
HMWK Plasmin Fibrinolysis
PK activation Fig 4. The contact pathway plays multiple
important biological functions. Although acti-
FXII FXIIa Angiogenesis vation of the contact pathway factors can trig-
Bradykinin ger formation of FXa, and thus coagulation,
in vitro, it does not appear to play a major role
FXI FXIa in physiological haemostasis in vivo. Interest-
Inflammation
Complement ingly, recent data suggest that activation of this
activation FIX FIXa pathway may be important in the pathogenesis
of thrombosis. In addition, accumulating evi-
Intrinsic Xase complex dence suggests that the contact pathway has a
FX FVIIIa number of other direct biological roles.
HMWK: high molecular weight kininogen; PK:
FXa prekallikrien.

of the ‘intrinsic tenase complex’ (Smith et al, 2015). Finally, haemostasis have occurred over the past decade. Some of
generation of FXa from this complex then causes thrombin these advances, and how they further redefine our current
generation and clot formation via the common pathway, as vision of an integrated cell-based model of haemostasis, will
before (Fig 2). Importantly, although in vitro triggering of now be considered. In particular, examples of specific
this intrinsic pathway leads to thrombin generation, studies advances in three key clinical aspects of physiological coagu-
over the past decade have clearly demonstrated that the lation (initiation of coagulation, the common pathway of
intrinsic pathway does not play a significant in vivo role in coagulation and regulation of blood coagulation) will be con-
physiological haemostasis. This is perhaps best exemplified sidered.
by data demonstrating that patients with FXII deficiency do
not have any significant bleeding phenotype (Renne et al,
Insights into the physiological initiation of
2012). Interestingly however, a number of in vivo activators
coagulation
for the contact pathway have recently been elucidated,
including polyphosphate and neutrophil extracellular traps
In vivo haemostasis is triggered by tissue factor
(Muller et al, 2009; Geddings & Mackman, 2014; Smith et al,
2015). Altogether, this accumulating evidence suggests that As previously discussed, TF is the principal trigger for physi-
pathological activation of the contact pathway may therefore ological haemostasis in vivo. TF is abundantly expressed in
play a critical role in the pathogenesis underlying thrombosis. adventitial extravascular cells in the blood vessel wall and
These data are reviewed in the accompanying review article consequently is not normally in contact with plasma (Wilcox
in this series (Preston et al, 2019). These findings raise the et al, 1989; Fleck et al, 1990). Rather, in simplistic terms, TF
intriguing possibility that the molecular mechanisms involved functions as a haemostatic ‘envelope’ that can trigger coagu-
in physiological haemostasis and pathological thrombosis lation activation following vascular injury (Drake et al,
exist as distinct entities that can occur in parallel but with 1989). TF differs from other coagulation cofactors in that it
multiple points of intersection. Defining the different molec- is an integral membrane protein and also does not require
ular mechanisms involved in thrombosis vis a vis haemostasis proteolytic activation. Accumulating evidence has provided
offers the exciting opportunity to develop novel anti-throm- significant new insights into how TF functions as the physio-
botic agents that may be useful in the prevention and/or logical initiator of coagulation in vivo. In particular, it has
treatment of thrombosis, but that would not necessarily be become clear that the role of TF is significantly more com-
associated with significant bleeding risks. Furthermore, a plex than simply serving as a haemostatic envelope. For
number of other important biological roles for the contact example, TF expression has been shown to vary significantly
pathway (including activation of fibrinolysis, promoting between different tissues, with high levels in the brain and
complement activation, angiogenesis and generation of kidney compared to reduced levels in skeletal muscle and the
vasoactive peptides particularly bradykinin) have also been synovium (Drake et al, 1989; Fleck et al, 1990). Importantly,
defined (Muller & Renne, 2008; Smith et al, 2015). there is also evidence that the procoagulant activity of TF on
a given cell can be regulated by allosteric structural changes.
In particular, studies have shown that TF-procoagulant activ-
Molecular mechanisms underpinning integrated
ity on extravascular cells can be increased up to 100-fold fol-
coagulation – the state of the art
lowing stimulation with calcium ionophores (e.g. ionomycin)
Given the complex nature of coagulation, it is perhaps or by freeze/thaw-induced cell disruption (Maynard et al,
unsurprising that huge insights into understanding of the 1975; Bach & Rifkin, 1990). These findings suggest that dur-
biological mechanisms involved in regulating physiological ing steady-state, the procoagulant activity of TF may exist, at

28 ª 2019 British Society for Haematology and John Wiley & Sons Ltd
British Journal of Haematology, 2019, 186, 24–36
 13652141, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.15872 by Nat Prov Indonesia, Wiley Online Library on [05/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review

least partly in a quiescent or ‘encrypted’ state, which then that a number of different thiol oxidising agents can decrypt
can be upregulated when required (Versteeg et al, 2013). TF-procoagulant activity (Chen & Hogg, 2013). In contrast,
A number of different hypotheses have been advanced to antibodies inhibiting protein disulphide isomerase have also
explain how TF encryption/decryption biology may be regu- been shown to inhibit TF decryption (Ahamed et al, 2006).
lated (Fig 5) (Chen & Hogg, 2013). First, under normal con- The molecular mechanisms determining how this thiol/disul-
ditions, TF-expressing cells typically express low levels of phide switch is regulated in vivo remain to be fully eluci-
phosphatidylserine (PS) on their outer membrane leaflet. dated. Nonetheless, based upon the available data, it seems
However, activation of the cells induces redistribution of PS that exposure of PS coupled with formation of the Cys186-
from the inner to outer membrane leaflet (Fig 5A, B). This Cys209 disulphide bond may be necessary for full TF-procoa-
exposure of PS on the cell surface facilitates binding and ori- gulant activity (Chen & Hogg, 2013; Versteeg et al, 2013). In
entation of FIX and FX, thereby significantly increasing their addition, a role for lipid rafts in regulating TF encryption/de-
activation by the TF:FVIIa complex. Thus, surface exposure cryption has also been proposed. In particular, TF-procoagu-
of negatively-charged phospholipids undoubtedly plays a crit- lant activity is attenuated if it becomes sequestered in
ical role in modulating TF-procoagulant activity (Bach & cholesterol-rich microdomains or is localised to caveolae
Moldow, 1997; Kunzelmann-Marche et al, 2000). Second, TF where PS expression levels are low (Mulder et al, 1996;
contains an allosteric disulphide bond (Cys186-Cys209) located Sevinsky et al, 1996). Finally, dimerization and oligomerisa-
in its C-terminal membrane-proximal domain (Fig 5C, D) tion of TF have also been proposed as another putative
(Chen & Hogg, 2013). This bond is highly conserved, and mechanism that may contribute to its encryption (Roy et al,
reduction of the bond ablates the procoagulant activity of TF 1991; Bach & Moldow, 1997). Taken together, it is clear that
(Ahamed et al, 2006; Krudysz-Amblo et al, 2013). Further- the biological mechanisms underlying TF encryption and
more, in vitro studies using cultured EC have demonstrated how this process is regulated in vivo remain controversial.

Phospholipid exposure
(A) (B)
FVII FXa FVIIa

TF TF

v v
v

Allosteric disulphide regulation

(C) (D)

FVII
FVIIa
Reduced TF SH- Oxidised TF
S-
SH-
S-

Fig 5. Mechanisms underlying tissue factor encryption/decryption. A number of different mechanisms have been proposed to play a role in tissue
factor (TF)encryption/decryption. First, under normal conditions, TF-expressing cells express low levels of phosphatidylserine (PS) on their outer
membrane leaflet (A). Following activation, this PS is redistributed to the outer leaflet where it can facilitate FVIIa and FXa binding (B). Second,
TF contains an allosteric disulphide bond located in its C-terminal end (C). Oxidation of this disulphide bond has been proposed to induce a
conformational change and thereby enable FVIIa binding (D).

ª 2019 British Society for Haematology and John Wiley & Sons Ltd 29
British Journal of Haematology, 2019, 186, 24–36
 13652141, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.15872 by Nat Prov Indonesia, Wiley Online Library on [05/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review

Further studies will be essential to define which of the differ- B, suggesting that FIXa may play an important role in FVII
ent mechanisms is most important, or whether they may all activation in vivo (Wildgoose et al, 1992). Finally, in contrast
play some part in regulating TF-procoagulant activity. In to other activated serine proteases, the plasma half-life of
addition, it seems plausible that the mechanisms underpin- FVIIa is markedly longer (approximately 2 h) as it is rela-
ning encryption/decryption may differ between specific tis- tively resistant to protease inhibitors (Seligsohn et al, 1979;
sues and cells under differing physiological conditions. Kondo & Kisiel, 1987). Exposed TF binds to both FVII and
FVIIa with high affinity. Moreover, once FVII is bound to
TF, it is rapidly activated to FVIIa through limited proteoly-
A role for intravascular TF in haemostasis and
sis (Nemerson & Repke, 1985). Thus, the critical TF:FVIIa
thrombosis
initiator complex can be formed by TF binding to circulating
The traditional concept that TF expression was restricted to FVIIa, or by TF binding FVII with subsequent activation to
extravascular tissues and only came into contact with the FVIIa. In contrast to free FVIIa, which has limited enzymatic
plasma following vascular injury, has been challenged in activity, the combination with its TF cofactor, on a mem-
recent years. Although this is also a controversial area with brane surface expressing negatively charged phospholipids,
some conflicting data, nevertheless a number of lines of evi- markedly enhances the ability of FVIIa to cleave its substrate
dence support the hypothesis that detectable amounts of TF FX (Bom & Bertina, 1990; Komiyama et al, 1990).
are present in blood, and that this intravascular TF may play
a role in haemostasis and thrombosis particularly under
Advances in understanding the biology of the
specific conditions (Giesen et al, 1999). For example, a num-
common pathway of coagulation
ber of independent groups have reported TF expression on
monocytes, neutrophils and endothelial cells in the context
Factor V as a regulator of both pro- and anticoagulant
of hypoxia or Gram-negative sepsis (Drake et al, 1993; Yan
pathways
et al, 1999; Pawlinski et al, 2004). Similarly, there have also
been reports of TF expression on platelets, although some of Factor V (FV) circulates in plasma as a single-chain 330 kDa
these results have proved difficult to replicate (Todoroki pro-cofactor that lacks any significant procoagulant activity
et al, 1998; Giesen et al, 1999; Camera et al, 2012). In addi- (Camire & Bos, 2009). It consists of a number of discrete
tion to this blood-borne cellular expression, TF has also been domains (A1-A2-B-A3-C1-C2) (Fig 6) (Jenny et al, 1987).
described on circulating microparticles. Low levels of circu- Within the B domain, two highly charged regions have been
lating TF-positive microparticles have been reported in reported (a positive cluster between residues 963–1008 and a
healthy individuals that may be derived from stimulated negatively charged cluster between 1493–1537) (Camire,
monocytes or endothelial cells (Aras et al, 2004; Ollivier 2016). Interaction between these two charged regions has
et al, 2010). However, significantly increased levels of TF- been proposed to play a role in maintaining FV in its proco-
positive microparticles have been observed under pathologi- factor state (Dahlback, 2017). FV is activated to procoagulant
cal conditions, such as cancer, where TF expression on circu- heterodimeric FVa following limited proteolysis by thrombin
lating tumour-cell derived microparticles may play a role in at three discrete sites (Arg 709, Arg 1018 and Arg 1545)
cancer-associated thrombotic risk (Tesselaar et al, 2007). (Suzuki et al, 1982). Initial cleavage of FV occurs at Arg 709
Notwithstanding the origin of the TF-positive microparticles, and Arg 1018, which results in partially activated forms of
the pool of circulating TF has been postulated to play a role FVa that possess intermediate levels of procoagulant activity
in enabling continued thrombin generation and fibrin depo- (Camire & Bos, 2009; Camire, 2016). Subsequently, cleavage
sition at the site of vascular injury once the original throm- at Arg 1545 occurs, which results in loss of the complete B
bus layer has formed. domain and confers full FVa activation (Steen & Dahlback,
2002; Dahlback, 2017). Importantly, FV can also be cleaved
and activated by FXa, although this is less effective than
The role of FVIIa in triggering physiological haemostasis
thrombin (Thorelli et al, 1997). Once activated, FVa assem-
FVII is a 50 kDa glycoprotein that is synthesised in the liver bles into a high affinity complex with FXa on negatively
and circulates in plasma as an inactive zymogen (Kisiel & charged phospholipid surfaces. Under normal physiological
Davie, 1975; Broze & Majerus, 1980). Like other serine pro- conditions, the procoagulant properties of FVa are regulated
teases, FVII becomes activated following limited proteolysis. by activated protein C (APC) which, in combination with its
Although the molecular mechanisms through which FVII is cofactor protein S, cleaves and inactivates FVa so that further
activated remain incompletely understood, studies have esti- thrombin generation is attenuated (Norstrom et al, 2003;
mated that 1% of plasma FVII circulates as active FVIIa Dahlback & Villoutreix, 2005; Harmon et al, 2008).
(Morrissey et al, 1993). Proteases reported to be able to acti- In addition to the established procoagulant function of
vate FVII in vitro include the TF:FVa complex, as well as FVa, recent studies have demonstrated that FV(a) also plays
FIXa, FXa and thrombin. Interestingly, circulating FVIIa a number of important anticoagulant roles. First, FV has
levels are significantly reduced in patients with haemophilia been shown to function with protein S as a synergistic

30 ª 2019 British Society for Haematology and John Wiley & Sons Ltd
British Journal of Haematology, 2019, 186, 24–36
 13652141, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.15872 by Nat Prov Indonesia, Wiley Online Library on [05/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review

Thrombin cleavage sites

Arg 709 Arg 1018 Arg 1545
Heavy chain Light chain
FV A1 A2 + B - A3 C1 C2
963-1008 1493-1537

Arg 1545
Heavy chain Light chain
Parally
acvated FVa
A1 A2 B - A3 C1 C2

Heavy chain Light chain

FVa A1 A2 A3 C1 C2

Fig 6. The pro- and anti-coagulant functions of factor V are regulated by degree of proteolysis. Factor V (FV) circulates in the plasma as a
330 kDa single chain glycoprotein that consists of a number of discrete domains and has no intrinsic procoagulant cofactor activity. Two distinct
charged regions have been identified within the B domain of FV. Moreover, interaction between these regions may be important in regulating the
function of FV. FV is activated by limited proteolytic cleavage by thrombin. This cleavage occurs first at Arg 709 and Arg 1018, followed by later
cleavage at Arg 1545. Complete cleavage at all three sites results in release of the large B domain and generates heterodimeric FVa, which contains
heavy and light chains and functions as a critical procoagulant cofactor. Incomplete FV proteolysis, which can occur following FXa cleavage, can
result in the generation of intermediate FVa forms that possess intermediate pro- and anti-coagulant properties.

cofactor for APC-mediated proteolysis of FVIIIa in the tenase patients have a 10-fold increase in plasma TFPIa levels (Vin-
complex (Shen & Dahlback, 1994). Second, FV has also been cent et al, 2013) and consequently display a significant bleed-
shown to play a role in regulating the anticoagulant proper- ing phenotype (Kuang et al, 2001).
ties of TFPI (Camire, 2016; Dahlback, 2017). In particular,
studies have shown that the positively charged C terminal
Advances in understanding of the physiological
region of TFPIa can bind to FV (Duckers et al, 2008). More-
regulation of blood coagulation
over, this FV-binding may be important in protecting plasma
TFPIa against premature clearance (Dahlback, 2017). In
The multiple roles of tissue factor pathway inhibitor
addition, data suggests that FV can also significantly enhance
TFPIa-mediated FXa inhibition. Interestingly, partially acti- TFPI is a Kunitz-type serine protease inhibitor that is
vated forms of FVa (such as those released from activated released constitutively by endothelial cells and by platelets
platelets or generated by FXa) (Ayombil et al, 2013) retain upon activation (Maroney & Mast, 2015). Interestingly, TFPI
the negatively charged cluster region from the C-terminal is synthesised in two major isoforms – TFPIa and TFPIb
end of their B domain and consequently can still interact (Fig 7) (Piro & Broze, 2005). TFPIa consists of an N-term-
with TFPIa (Camire, 2016). Therefore, in the presence of inal acidic region followed by three tandem Kunitz-type pro-
TFPIa, these intermediate activated FVa forms will bind tease inhibitor domains (K1, K2 and K3) (Wun et al, 1988).
TFPIa, which will inhibit any downstream FXa generation K1 and K2 bind to the active sites of FVIIa and FXa respec-
and thereby limit assembly of the prothrombinanse complex tively (Mast, 2016). In addition, TFPIa contains a C-terminal
(Wood et al, 2013; Dahlback, 2017). region that binds tightly to some forms of FVa. As a result
Investigations into the rare East Texas bleeding disorder of alternate splicing, TFPIb contains K1 and K2 but has a
have provided novel insights into the role of FV in regulating modified C terminal region that includes a GPI-anchor
the anticoagulant effects of TFPI (Vincent et al, 2013; Dahl- sequence which allows direct association with cell surfaces
back, 2017). Studies have shown that these patients have a (Girard et al, 2012). In vivo, both TFPIa and TFPIb isoforms
FV point mutation that results in alternative splicing (Kuang are thought to inhibit the TF:FVIIa complex and thereby
et al, 2001; Vincent et al, 2013). Consequently, affected indi- shut off the extrinsic pathway after the initial stimulus for
viduals express a shorter variant of FV (FV-Short) from coagulation activation has been provided (Wood et al, 2013).
which a significant amount of the B-domain has been lost Interestingly however, TFPI is only a weak inhibitor of TF:
(Dahlback, 2017). Importantly however, FV-Short retains the FVIIa alone. Rather, data suggest that TFPI simultaneously
negatively charged cluster region of the B domain and thus binds and inhibits both FXa and TF-FVIIa (immediately after
can still interact with TFPIa. Moreover, affinity of FV-Short FX has been activated by the TF:FVIIa complex) in the form
for TFPIa is actually markedly increased compared to that of of a quaternary (Xa-TF-FVIIa-TFPI) complex (Baugh et al,
full-length FV (Vincent et al, 2013). As a result, these 1998). These observations have led to the suggestion that

ª 2019 British Society for Haematology and John Wiley & Sons Ltd 31
British Journal of Haematology, 2019, 186, 24–36
 13652141, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.15872 by Nat Prov Indonesia, Wiley Online Library on [05/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review

TFPI α
C-terminal
N-terminal K1 K2 K3
Fig 7. Tissue factor pathway inhibitor iso-
FVIIa FXa Protein S FVa forms. There are two major isoforms of tissue
Inhibition Inhibition binding binding factor pathway inhibitor (TFPI). TFPIa and
TFPIb both consist of an N-terminal acidic
region followed by two Kunitz-type protease
TFPI β
inhibitor domains (K1 and K2). K1 binds to
C-terminal the active site of FVIIa whilst K2 binds to the
N-terminal K1 K2 active sites of FXa. In addition, TFPIa also
contains a K3 domain that modulates interac-
tion with protein S, as well as a C-terminal
FVIIa FXa GPI anchor site region that binds to some forms of FVa. GPI,
Inhibition Inhibition glycophosphatidylinositol.

TFPI is, in fact, a FXa-dependent inhibitor of the TF:FVIIa of VTE in individuals with quantitative antithrombin defi-
complex (Mast, 2016). Protein S has recently been demon- ciency (Croles et al, 2018). Qualitative antithrombin defi-
strated to play a pivotal role as a cofactor for TFPI inhibition ciency is often correlated with defects in antithrombin-
of FXa activity by binding to the K3 domain of TFPIa and heparin binding, substrate recognition and post-translational
thereby promoting binding to FXa (Hackeng et al, 2006). In glycosylation, which appears to significantly contribute to
particular, the high-affinity of protein S for anionic phospho- antithrombin anticoagulant activity (Lane et al, 1996; Bays-
lipids enables it to bind TFPI and localise on the cell surface, ton & Lane, 1997; Preston et al, 2013). Recent evidence sug-
making inhibition of phospholipid-bound FXa approximately gests that antithrombin anticoagulant activity is also tightly
10-fold more efficient (Wood et al, 2014). regulated by other post-translational modifications, such as
In addition to this function as the key regulator of the citrullination, where arginine residues are modified by the
extrinsic tenase complex, more recent studies have demon- action of protein arginine deiminases (Chang et al, 2005;
strated that TFPIa but not TFPIb can also inhibit forms of Ordonez et al, 2009; Tilvawala et al, 2018). Specifically,
the prothrombinase complex assembled during the initial citrullination of the P1 arginine in the antithrombin reactive
stages of blood coagulation (Wood et al, 2013). Specifically, centre loop was found to completely ablate antithrombin
as discussed above, FVa isoforms that retain the FVa B ability to recognise and inhibit its protease substrates. Sur-
domain acidic region, such as FXa-activated FVa and platelet prisingly, heparin was found to further enhance the rate of
FV, can tightly bind TFPI and thereby inhibit prothrombi- antithrombin citrullination (Ordonez et al, 2009; Tilvawala
nase activity (Camire, 2016; Mast, 2016; Dahlback, 2017). et al, 2018). Further studies to identify the prevalence of
This novel anticoagulant mechanism implicates a key role for antithrombin citrullination in VTE disease cohorts will be
TFPI in dampening thrombin generation initiated by FV(a) useful in ascertaining the role of this phenomenon in VTE
isoforms generated early in response to vascular injury and pathogenesis.
before substantive thrombin generation has occurred (Wood
et al, 2013).
The anticoagulant protein C pathway
Protein C (PC) is a vitamin K-dependent plasma zymogen
Antithrombin-heparin anticoagulant activity
that becomes activated in response to thrombin generation
Antithrombin is a member of the coagulation serine protease (Griffin et al, 2007). When thrombin is bound to its antico-
inhibitor (serpin) family and represents a crucial stoichio- agulant endothelial cofactor receptor thrombomodulin (TM),
metric inhibitor of coagulation protease activity, particularly the function of thrombin is skewed towards conversion of
thrombin and FXa (Huntington, 2011). Antithrombin binds zymogen PC to APC (Esmon & Owen, 1981). APC genera-
to thrombin and FXa, eventually forming a covalent complex tion is further enhanced by the presence of PC binding to
devoid of enzymatic procoagulant activity that is subse- the endothelial cell PC receptor (EPCR), which further accel-
quently cleared from the circulation. Antithrombin anticoag- erates PC activation by the thrombin-thrombomodulin com-
ulant activity is significantly enhanced by the presence of plex (Fig 8) (Gleeson et al, 2012). The molecular basis of
endothelial heparan sulfates or mast cell-derived heparin, how EPCR enhances PC activation is currently unknown but
which dramatically increases antithrombin recognition of ser- is likely to arise from the co-localisation of PC on the
ine protease targets by both structural modification of endothelial cell surface. Notably, EPCR also exists in a sol-
antithrombin and provision of a scaffold for protease- uble form (sEPCR) that circulates in blood at ~100 ng/ml
antithrombin co-localisation. The importance of antithrom- and has the capacity to inhibit APC anticoagulant activity
bin in coagulation regulation is exemplified by the high risk (Zheng et al, 2007). Interestingly, three EPCR haplotypes

32 ª 2019 British Society for Haematology and John Wiley & Sons Ltd
British Journal of Haematology, 2019, 186, 24–36
 13652141, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.15872 by Nat Prov Indonesia, Wiley Online Library on [05/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review

Protein
Thrombin:Thrombomodulin FVa
C APC APC
Protein
TM S FVa

Protein Inacvated FVa

Thrombin C

EPCR

Phospholipid surface

Endothelium

Fig 8. Activated protein C anticoagulant pathway. The protein C pathway is initiated when protein C bound to its endothelial cell receptor
(EPCR) is activated by the thrombin-thrombomodulin complex. Once activated, activated protein C (APC) detaches from EPCR, where it forms
an anticoagulant complex with its cofactor protein S and its substrate activated factor V (FVa). FVa proteolysis at Arg 506 and Arg 306 by APC
results in loss of FVa procoagulant cofactor activity and reduced thrombin generation.

exist, one of which (present in ~20% of individuals) is asso- associated with a high risk of mortality (Griffin et al, 1981).
ciated with 10-fold increased sEPCR levels and has been pos- Heterozygous PC deficiency is more common and results in
tulated as a candidate risk factor for VTE (Saposnik et al, a moderately increased thrombotic risk. The physiological
2004). importance of FVa proteolysis by APC is underscored by the
Once generated, newly generated APC must detach from elevated prothrombotic risk for individuals that carry the FV
EPCR and re-bind to the anionic phospholipid-containing Leiden mutation (Arg506Gln), which prevents proteolysis at
cell surface, where it forms an anticoagulant complex with its one of the APC cleavage sites (Bertina et al, 1994; Greengard
cofactor protein S to enzymatically degrade FVa. Protein S is et al, 1994). FV Leiden mutation increases VTE risk 7-fold in
a prerequisite for APC anticoagulant activity in plasma, how- heterozygous carriers and 80-fold in homozygous carriers
ever, what protein S does to facilitate APC proteolysis of FVa (Van Cott et al, 2016).
is not fully understood (Fernandez et al, 2017). Previous
studies have indicated that protein S interaction with APC is
Conclusions
entirely dependent upon co-localisation of both proteins on
a phospholipid cell surface and results in higher retention of In conclusion, landmark studies performed back in the 1960s
APC on the phospholipid surface and re-location of the APC provided critical insights into how circulating inactive coagu-
serine protease domain to enable more efficient substrate lation factor zymogens and cofactors could be specifically
recognition (Yegneswaran et al, 1997). Recently, novel bind- activated through limited proteolysis in a stepwise manner,
ing sites on the APC light chain were shown to be crucial for ultimately resulting in thrombin generation and cross-linked
protein S interaction and support a model where protein S fibrin formation. Subsequent data has enabled refinement of
makes multiple contacts with the APC light chain when both this original model of coagulation, and lead to a more
are bound to the anionic phospholipid surface of the platelet nuanced understanding of the roles played by specific pro-
cell membrane (Preston et al, 2006; Fernandez et al, 2017). teins, receptors and cellular surfaces in regulating clot devel-
FVa proteolysis by APC and protein S occurs via proteolysis opment. In view of the complex biology underpinning
at two sites within the FVa A2 domain (Arg 506 and Arg in vivo haemostasis, it is not surprising that a whole series of
306), which leads to the destruction of FVa procoagulant novel insights into the molecular mechanisms involved have
cofactor activity (Nicolaes et al, 1995). The PC pathway rep- emerged over recent years. Moreover, it seems highly likely
resents a dynamic negative feedback loop, whereby the that further discoveries will continue to come to light in the
amount of thrombin generated in response to vascular dam- medium term. Given the huge global morbidity and mortal-
age is both controlled (by APC proteolysis of FVa) and ity associated with cardiovascular disease, elucidating the
restricted to the locale of the developing clot (by APC gener- molecular mechanisms underlying the complex processes
ation on intact endothelial cells). Homozygous PC deficiency involved in both haemostasis and thrombosis are clearly not
is associated with perinatal purpura fulminans and is only of basic scientific interest, but also of direct clinical

ª 2019 British Society for Haematology and John Wiley & Sons Ltd 33
British Journal of Haematology, 2019, 186, 24–36
 13652141, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.15872 by Nat Prov Indonesia, Wiley Online Library on [05/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review

significance. Consequently, there is significant promise that

Authorship
improved understanding of coagulation biology and, in par-
ticular, how it differs between physiological haemostasis and Contribution: All authors drafted the first version of different
pathological thrombosis, may provide exciting opportunities sections of the manuscript and all critically reviewed the final
to develop new therapeutic approaches. manuscript.

References Camire, R.M. & Bos, M.H. (2009) The molecular cofactor, protein S. Blood Advances, 1, 1423–
basis of factor V and VIII procofactor activation. 1426.
Ahamed, J., Versteeg, H.H., Kerver, M., Chen, Journal of Thrombosis and Haemostasis, 7, 1951– Fleck, R.A., Rao, L.V., Rapaport, S.I. & Varki, N.
V.M., Mueller, B.M., Hogg, P.J. & Ruf, W. 1961. (1990) Localization of human tissue factor anti-
(2006) Disulfide isomerization switches tissue Chang, X., Yamada, R., Sawada, T., Suzuki, A., gen by immunostaining with monospecific,
factor from coagulation to cell signaling. Pro- Kochi, Y. & Yamamoto, K. (2005) The inhibi- polyclonal anti-human tissue factor antibody.
ceedings of the National Academy of Sciences of tion of antithrombin by peptidylarginine deimi- Thrombosis Research, 59, 421–437.
the United States of America, 103, 13932–13937. nase 4 may contribute to pathogenesis of Geddings, J.E. & Mackman, N. (2014) New players
Aras, O., Shet, A., Bach, R.R., Hysjulien, J.L., Slun- rheumatoid arthritis. Rheumatology (Oxford), 44, in haemostasis and thrombosis. Thrombosis and
gaard, A., Hebbel, R.P., Escolar, G., Jilma, B. & 293–298. Haemostasis, 111, 570–574.
Key, N.S. (2004) Induction of microparticle- Chen, V.M. & Hogg, P.J. (2013) Encryption and Giesen, P.L., Rauch, U., Bohrmann, B., Kling, D.,
and cell-associated intravascular tissue factor in decryption of tissue factor. Journal of Thrombosis Roque, M., Fallon, J.T., Badimon, J.J., Himber,
human endotoxemia. Blood, 103, 4545–4553. and Haemostasis, 11 (Suppl 1), 277–284. J., Riederer, M.A. & Nemerson, Y. (1999)
Ayombil, F., Abdalla, S., Tracy, P.B. & Bouchard, Croles, F.N., Borjas-Howard, J., Nasserinejad, K., Blood-borne tissue factor: another view of
B.A. (2013) Proteolysis of plasma-derived factor Leebeek, F.W.G. & Meijer, K. (2018) Risk of thrombosis. Proceedings of the National Academy
V following its endocytosis by megakaryocytes venous thrombosis in antithrombin deficiency: a of Sciences of the United States of America, 96,
forms the platelet-derived factor V/Va pool. systematic review and bayesian meta-analysis. 2311–2315.
Journal of Thrombosis and Haemostasis, 11, Seminars in Thrombosis and Hemostasis, 44, Girard, T.J., Tuley, E. & Broze, G.J. Jr (2012)
1532–1539. 315–326. TFPIbeta is the GPI-anchored TFPI isoform on
Bach, R.R. & Moldow, C.F. (1997) Mechanism of Dahlback, B. (2017) Novel insights into the regula- human endothelial cells and placental micro-
tissue factor activation on HL-60 cells. Blood, tion of coagulation by factor V isoforms, tissue somes. Blood, 119, 1256–1262.
89, 3270–3276. factor pathway inhibitoralpha, and protein S. Gleeson, E.M., O’Donnell, J.S. & Preston, R.J.
Bach, R. & Rifkin, D.B. (1990) Expression of tissue Journal of Thrombosis and Haemostasis, 15, (2012) The endothelial cell protein C receptor:
factor procoagulant activity: regulation by 1241–1250. cell surface conductor of cytoprotective coagula-
cytosolic calcium. Proceedings of the National Dahlback, B. & Villoutreix, B.O. (2005) Regulation tion factor signaling. Cellular and Molecular Life
Academy of Sciences of the United States of Amer- of blood coagulation by the protein C anticoag- Sciences, 69, 717–726.
ica, 87, 6995–6999. ulant pathway: novel insights into structure- Greengard, J.S., Eichinger, S., Griffin, J.H. & Bauer,
Baugh, R.J., Broze, G.J. Jr & Krishnaswamy, S. function relationships and molecular recogni- K.A. (1994) Brief report: variability of thrombo-
(1998) Regulation of extrinsic pathway factor Xa tion. Arteriosclerosis, Thrombosis, and Vascular sis among homozygous siblings with resistance
formation by tissue factor pathway inhibitor. Biology, 25, 1311–1320. to activated protein C due to an Arg–>Gln
Journal of Biological Chemistry, 273, 4378–4386. Davie, E.W. & Ratnoff, O.D. (1964) Waterfall mutation in the gene for factor V. New England
Bayston, T.A. & Lane, D.A. (1997) Antithrombin: sequence for intrinsic blood clotting. Science, Journal of Medicine, 331, 1559–1562.
molecular basis of deficiency. Thrombosis and 145, 1310–1312. Griffin, J.H., Evatt, B., Zimmerman, T.S., Kleiss,
Haemostasis, 78, 339–343. Drake, T.A., Morrissey, J.H. & Edgington, T.S. A.J. & Wideman, C. (1981) Deficiency of pro-
Bertina, R.M., Koeleman, B.P., Koster, T., Rosen- (1989) Selective cellular expression of tissue fac- tein C in congenital thrombotic disease. The
daal, F.R., Dirven, R.J., de Ronde, H., van der tor in human tissues. Implications for disorders Journal of Clinical Investigation, 68, 1370–1373.
Velden, P.A. & Reitsma, P.H. (1994) Mutation of hemostasis and thrombosis. American Journal Griffin, J.H., Fernandez, J.A., Gale, A.J. & Mosnier,
in blood coagulation factor V associated with of Pathology, 134, 1087–1097. L.O. (2007) Activated protein C. Journal of
resistance to activated protein C. Nature, 369, Drake, T.A., Cheng, J., Chang, A. & Taylor, F.B. Jr Thrombosis and Haemostasis, 5 (Suppl 1), 73–80.
64–67. (1993) Expression of tissue factor, thrombo- Hackeng, T.M., Sere, K.M., Tans, G. & Rosing, J.
Bom, V.J. & Bertina, R.M. (1990) The contribu- modulin, and E-selectin in baboons with lethal (2006) Protein S stimulates inhibition of the tis-
tions of Ca2+, phospholipids and tissue-factor Escherichia coli sepsis. American Journal of sue factor pathway by tissue factor pathway
apoprotein to the activation of human blood- Pathology, 142, 1458–1470. inhibitor. Proceedings of the National Academy of
coagulation factor X by activated factor VII. The Duckers, C., Simioni, P., Spiezia, L., Radu, C., Sciences of the United States of America, 103,
Biochemical Journal, 265, 327–336. Gavasso, S., Rosing, J. & Castoldi, E. (2008) 3106–3111.
Broze, G.J. Jr & Majerus, P.W. (1980) Purification Low plasma levels of tissue factor pathway inhi- Harmon, S., Preston, R.J., Ni Ainle, F., Johnson,
and properties of human coagulation factor VII. bitor in patients with congenital factor V defi- J.A., Cunningham, M.S., Smith, O.P., White, B.
Journal of Biological Chemistry, 255, 1242–1247. ciency. Blood, 112, 3615–3623. & O’Donnell, J.S. (2008) Dissociation of acti-
Camera, M., Brambilla, M., Facchinetti, L., Can- Esmon, C.T. & Owen, W.G. (1981) Identification vated protein C functions by elimination of pro-
zano, P., Spirito, R., Rossetti, L., Saccu, C., Di of an endothelial cell cofactor for thrombin-cat- tein S cofactor enhancement. Journal of
Minno, M.N. & Tremoli, E. (2012) Tissue factor alyzed activation of protein C. Proceedings of the Biological Chemistry, 283, 30531–30539.
and atherosclerosis: not only vessel wall-derived National Academy of Sciences of the United States Huntington, J.A. (2011) Serpin structure, function
TF, but also platelet-associated TF. Thrombosis of America, 78, 2249–2252. and dysfunction. Journal of Thrombosis and Hae-
Research, 129, 279–284. Fernandez, J.A., Xu, X., Sinha, R.K., Mosnier, mostasis, 9 (Suppl 1), 26–34.
Camire, R.M. (2016) Rethinking events in the L.O., Sanner, M.F. & Griffin, J.H. (2017) Acti- Jenny, R.J., Pittman, D.D., Toole, J.J., Kriz, R.W.,
haemostatic process: role of factor V and TFPI. vated protein C light chain provides an Aldape, R.A., Hewick, R.M., Kaufman, R.J. &
Haemophilia, 22 (Suppl 5), 3–8. extended binding surface for its anticoagulant Mann, K.G. (1987) Complete cDNA and derived

34 ª 2019 British Society for Haematology and John Wiley & Sons Ltd
British Journal of Haematology, 2019, 186, 24–36
 13652141, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.15872 by Nat Prov Indonesia, Wiley Online Library on [05/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review

amino acid sequence of human factor V. Pro- factor mutant selectively deficient in promoting Preston, R.J.S., O’Sullivan, J.M. & O’Donnell, J.S.
ceedings of the National Academy of Sciences of factor VII activation. Blood, 81, 734–744. (2019) Advances in understanding the molecular
the United States of America, 84, 4846–4850. Mulder, A.B., Smit, J.W., Bom, V.J., Blom, mechanisms of venous thrombosis. British Jour-
Kisiel, W. & Davie, E.W. (1975) Isolation and N.R., Ruiters, M.H., Halie, M.R. & van der nal of Haematology, 186, 13–23.
characterization of bovine factor VII. Biochem- Meer, J. (1996) Association of smooth muscle Renne, T., Schmaier, A.H., Nickel, K.F., Blomback,
istry, 14, 4928–4934. cell tissue factor with caveolae. Blood, 88, M. & Maas, C. (2012) In vivo roles of factor
Komiyama, Y., Pedersen, A.H. & Kisiel, W. (1990) 1306–1313. XII. Blood, 120, 4296–4303.
Proteolytic activation of human factors IX and Muller, F. & Renne, T. (2008) Novel roles for fac- Roy, S., Paborsky, L.R. & Vehar, G.A. (1991) Self-
X by recombinant human factor VIIa: effects of tor XII-driven plasma contact activation system. association of tissue factor as revealed by chemi-
calcium, phospholipids, and tissue factor. Bio- Current Opinion in Hematology, 15, 516–521. cal crosslinking. Journal of Biological Chemistry,
chemistry, 29, 9418–9425. Muller, F., Mutch, N.J., Schenk, W.A., Smith, S.A., 266, 4665–4668.
Kondo, S. & Kisiel, W. (1987) Regulation of factor Esterl, L., Spronk, H.M., Schmidbauer, S., Gahl, Saposnik, B., Reny, J.L., Gaussem, P., Emmerich,
VIIa activity in plasma: evidence that antithrom- W.A., Morrissey, J.H. & Renne, T. (2009) Plate- J., Aiach, M. & Gandrille, S. (2004) A haplo-
bin III is the sole plasma protease inhibitor of let polyphosphates are proinflammatory and type of the EPCR gene is associated with
human factor VIIa. Thrombosis Research, 46, procoagulant mediators in vivo. Cell, 139, 1143– increased plasma levels of sEPCR and is a can-
325–335. 1156. didate risk factor for thrombosis. Blood, 103,
Krudysz-Amblo, J., Jennings, M.E. 2nd, Knight, T., Muller, F., Gailani, D. & Renne, T. (2011) Factor 1311–1318.
Matthews, D.E., Mann, K.G. & Butenas, S. XI and XII as antithrombotic targets. Current Seligsohn, U., Kasper, C.K., Osterud, B. & Rapa-
(2013) Disulfide reduction abolishes tissue factor Opinion in Hematology, 18, 349–355. port, S.I. (1979) Activated factor VII: presence
cofactor function. Biochimica et Biophysica Acta, Nemerson, Y. & Repke, D. (1985) Tissue factor in factor IX concentrates and persistence in the
1830, 3489–3496. accelerates the activation of coagulation factor circulation after infusion. Blood, 53, 828–837.
Kuang, S.Q., Hasham, S., Phillips, M.D., Wolf, D., VII: the role of a bifunctional coagulation cofac- Sevinsky, J.R., Rao, L.V. & Ruf, W. (1996) Ligand-
Wan, Y., Thiagarajan, P. & Milewicz, D.M. tor. Thrombosis Research, 40, 351–358. induced protease receptor translocation into
(2001) Characterization of a novel autosomal Nicolaes, G.A., Tans, G., Thomassen, M.C., Hem- caveolae: a mechanism for regulating cell surface
dominant bleeding disorder in a large kindred ker, H.C., Pabinger, I., Varadi, K., Schwarz, H.P. proteolysis of the tissue factor-dependent coagu-
from east Texas. Blood, 97, 1549–1554. & Rosing, J. (1995) Peptide bond cleavages and lation pathway. Journal of Cell Biology, 133,
Kunzelmann-Marche, C., Satta, N., Toti, F., Zhang, loss of functional activity during inactivation of 293–304.
Y., Nawroth, P.P., Morrissey, J.H. & Freyssinet, factor Va and factor VaR506Q by activated pro- Shen, L. & Dahlback, B. (1994) Factor V and pro-
J.M. (2000) The influence exerted by a restricted tein C. Journal of Biological Chemistry, 270, tein S as synergistic cofactors to activated pro-
phospholipid microenvironment on the expres- 21158–21166. tein C in degradation of factor VIIIa. Journal of
sion of tissue factor activity at the cell plasma Norstrom, E.A., Steen, M., Tran, S. & Dahlback, B. Biological Chemistry, 269, 18735–18738.
membrane surface. Thrombosis and Haemostasis, (2003) Importance of protein S and phospho- Smith, S.A., Travers, R.J. & Morrissey, J.H. (2015)
83, 282–289. lipid for activated protein C-mediated cleavages How it all starts: initiation of the clotting cas-
Lane, D.A., Kunz, G., Olds, R.J. & Thein, S.L. in factor Va. Journal of Biological Chemistry, cade. Critical Reviews in Biochemistry and Molec-
(1996) Molecular genetics of antithrombin defi- 278, 24904–24911. ular Biology, 50, 326–336.
ciency. Blood Reviews, 10, 59–74. Ollivier, V., Wang, J., Manly, D., Machlus, K.R., Steen, M. & Dahlback, B. (2002) Thrombin-
Macfarlane, R.G. (1964) An enzyme cascade in the Wolberg, A.S., Jandrot-Perrus, M. & Mackman, mediated proteolysis of factor V resulting in
blood clotting mechanism, and its function as a N. (2010) Detection of endogenous tissue factor gradual B-domain release and exposure of the
biochemical amplifier. Nature, 202, 498–499. levels in plasma using the calibrated automated factor Xa-binding site. Journal of Biological
Maroney, S.A. & Mast, A.E. (2015) New insights thrombogram assay. Thrombosis Research, 125, Chemistry, 277, 38424–38430.
into the biology of tissue factor pathway inhibi- 90–96. Suzuki, K., Dahlback, B. & Stenflo, J. (1982)
tor. Journal of Thrombosis and Haemostasis, 13 Ordonez, A., Martinez-Martinez, I., Corrales, F.J., Thrombin-catalyzed activation of human coagu-
(Suppl 1), S200–S207. Miqueo, C., Minano, A., Vicente, V. & Corral, J. lation factor V. Journal of Biological Chemistry,
Mast, A.E. (2016) Tissue factor pathway inhibitor: (2009) Effect of citrullination on the function 257, 6556–6564.
multiple anticoagulant activities for a single pro- and conformation of antithrombin. FEBS Jour- Tesselaar, M.E., Romijn, F.P., Van Der Linden,
tein. Arteriosclerosis, Thrombosis, and Vascular nal, 276, 6763–6772. I.K., Prins, F.A., Bertina, R.M. & Osanto, S.
Biology, 36, 9–14. Pawlinski, R., Pedersen, B., Schabbauer, G., Ten- (2007) Microparticle-associated tissue factor
Maynard, J.R., Heckman, C.A., Pitlick, F.A. & cati, M., Holscher, T., Boisvert, W., Andrade- activity: a link between cancer and thrombosis?
Nemerson, Y. (1975) Association of tissue factor Gordon, P., Frank, R.D. & Mackman, N. (2004) Journal of Thrombosis and Haemostasis, 5, 520–
activity with the surface of cultured cells. The Role of tissue factor and protease-activated 527.
Journal of Clinical Investigation, 55, 814–824. receptors in a mouse model of endotoxemia. Thorelli, E., Kaufman, R.J. & Dahlback, B. (1997)
Monroe, D.M. & Hoffman, M. (2006) What does Blood, 103, 1342–1347. Cleavage requirements for activation of factor V
it take to make the perfect clot? Arteriosclerosis, Piro, O. & Broze, G.J. Jr (2005) Comparison of by factor Xa. European Journal of Biochemistry,
Thrombosis, and Vascular Biology, 26, 41–48. cell-surface TFPIalpha and beta. Journal of 247, 12–20.
Monroe, D.M., Roberts, H.R. & Hoffman, M. Thrombosis and Haemostasis, 3, 2677–2683. Tilvawala, R., Nguyen, S.H., Maurais, A.J., Nem-
(1994) Platelet procoagulant complex assembly Preston, R.J., Ajzner, E., Razzari, C., Karageorgi, mara, V.V., Nagar, M., Salinger, A.J., Nagpal, S.,
in a tissue factor-initiated system. British Journal S., Dua, S., Dahlback, B. & Lane, D.A. (2006) Weerapana, E. & Thompson, P.R. (2018) The
of Haematology, 88, 364–371. Multifunctional specificity of the protein C/acti- rheumatoid arthritis-associated citrullinome. Cell
Monroe, D.M., Hoffman, M. & Roberts, H.R. vated protein C Gla domain. Journal of Biologi- Chemical Biology, 25, e696.
(1996) Transmission of a procoagulant signal cal Chemistry, 281, 28850–28857. Todoroki, H., Higure, A., Okamoto, K., Okazaki,
from tissue factor-bearing cell to platelets. Blood Preston, R.J., Rawley, O., Gleeson, E.M. & K., Nagafuchi, Y., Takeda, S., Katoh, H., Itoh,
Coagulation & Fibrinolysis, 7, 459–464. O’Donnell, J.S. (2013) Elucidating the role of H., Ohsato, K. & Nakamura, S. (1998) Possible
Morrissey, J.H., Macik, B.G., Neuenschwander, carbohydrate determinants in regulating role of platelet-activating factor in the in vivo
P.F. & Comp, P.C. (1993) Quantitation of acti- hemostasis: insights and opportunities. Blood, expression of tissue factor in neutrophils. Jour-
vated factor VII levels in plasma using a tissue 121, 3801–3810. nal of Surgical Research, 80, 149–155.

ª 2019 British Society for Haematology and John Wiley & Sons Ltd 35
British Journal of Haematology, 2019, 186, 24–36
 13652141, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.15872 by Nat Prov Indonesia, Wiley Online Library on [05/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review

Van Cott, E.M., Khor, B. & Zehnder, J.L. (2016) hemophilia A and B patients. Blood, 80, 25– domains. Journal of Biological Chemistry, 263,
Factor V Leiden. American Journal of Hematol- 28. 6001–6004.
ogy, 91, 46–49. Wood, J.P., Bunce, M.W., Maroney, S.A., Tracy, Yan, S.F., Mackman, N., Kisiel, W., Stern, D.M. &
Versteeg, H.H., Heemskerk, J.W., Levi, M. & P.B., Camire, R.M. & Mast, A.E. (2013) Tissue Pinsky, D.J. (1999) Hypoxia/Hypoxemia-
Reitsma, P.H. (2013) New fundamentals in factor pathway inhibitor-alpha inhibits pro- Induced activation of the procoagulant pathways
hemostasis. Physiological Reviews, 93, 327–358. thrombinase during the initiation of blood coag- and the pathogenesis of ischemia-associated
Vincent, L.M., Tran, S., Livaja, R., Bensend, T.A., ulation. Proceedings of the National Academy of thrombosis. Arteriosclerosis, Thrombosis, and
Milewicz, D.M. & Dahlback, B. (2013) Coagula- Sciences of the United States of America, 110, Vascular Biology, 19, 2029–2035.
tion factor V(A2440G) causes east Texas bleed- 17838–17843. Yegneswaran, S., Wood, G.M., Esmon, C.T. &
ing disorder via TFPIalpha. The Journal of Wood, J.P., Ellery, P.E., Maroney, S.A. & Mast, Johnson, A.E. (1997) Protein S alters the
Clinical Investigation, 123, 3777–3787. A.E. (2014) Protein S is a cofactor for platelet active site location of activated protein C
Wilcox, J.N., Smith, K.M., Schwartz, S.M. & Gor- and endothelial tissue factor pathway inhibitor- above the membrane surface. A fluorescence
don, D. (1989) Localization of tissue factor in alpha but not for cell surface-associated tissue resonance energy transfer study of topography.
the normal vessel wall and in the atherosclerotic factor pathway inhibitor. Arteriosclerosis, Throm- Journal of Biological Chemistry, 272, 25013–
plaque. Proceedings of the National Academy of bosis, and Vascular Biology, 34, 169–176. 25021.
Sciences of the United States of America, 86, Wun, T.C., Kretzmer, K.K., Girard, T.J., Miletich, Zheng, X., Li, W., Gu, J.M., Qu, D., Ferrell, G.L.,
2839–2843. J.P. & Broze, G.J. Jr (1988) Cloning and charac- Esmon, N.L. & Esmon, C.T. (2007) Effects of
Wildgoose, P., Nemerson, Y., Hansen, L.L., Niel- terization of a cDNA coding for the lipoprotein- membrane and soluble EPCR on the hemostatic
sen, F.E., Glazer, S. & Hedner, U. (1992) associated coagulation inhibitor shows that it balance and endotoxemia in mice. Blood, 109,
Measurement of basal levels of factor VIIa in consists of three tandem Kunitz-type inhibitory 1003–1009.

36 ª 2019 British Society for Haematology and John Wiley & Sons Ltd
British Journal of Haematology, 2019, 186, 24–36