REVIEWS

Therapeutic strategies for thrombosis:

new targets and approaches
Nigel Mackman 1 ✉, Wolfgang Bergmeier2, George A. Stouffer 3
and Jeffrey I. Weitz 4

Abstract | Antiplatelet agents and anticoagulants are a mainstay for the prevention and treatment
of thrombosis. However, despite advances in antithrombotic therapy, a fundamental challenge
is the side effect of bleeding. Improved understanding of the mechanisms of haemostasis and
thrombosis has revealed new targets for attenuating thrombosis with the potential for less bleeding,
including glycoprotein VI on platelets and factor XIa of the coagulation system. The efficacy
and safety of new agents are currently being evaluated in phase III trials. This Review provides an
overview of haemostasis and thrombosis, details the current landscape of antithrombotic agents,
addresses challenges with preventing thromboembolic events in patients at high risk and describes
the emerging therapeutic strategies that may break the inexorable link between antithrombotic
therapy and bleeding risk.

Cardiovascular disease
Disorders of the heart and
blood vessels.
Platelets
Small enucleate cells in the
blood involved in clotting.
1
UNC Blood Research Center,
Division of Hematology and
Oncology, Department of
Medicine, University of North
Carolina at Chapel Hill,
Chapel Hill, NC, USA.
2
UNC Blood Research Center,
Department of Biochemistry
and Biophysics, University of
North Carolina at Chapel Hill,
Chapel Hill, NC, USA.
3
McAllister Heart Institute,
Division of Cardiology,
Department of Medicine,
University of North Carolina
at Chapel Hill, Chapel Hill,
NC, USA.
4
Thrombosis &
Atherosclerosis Research
Institute and Department of
Medicine, McMaster
University, Hamilton, Canada.
✉e-​mail: nmackman@
med.unc.edu
https://doi.org/10.1038/
s41573-020-0061-0
Thrombosis is the leading cause of death worldwide,
being responsible for one in four deaths1. The global
burden is likely to increase with the ageing population
because thrombosis risk increases with age. Thrombosis
can be divided into arterial thrombosis (AT) and venous
thrombosis (VT). Interestingly, patients with AT are at
increased risk of VT and vice versa2–6. In addition, var-
ious conditions, such as antiphospholipid syndrome,
malignancy, infection, and oestrogen use, are associated
with both AT and VT2.
Patients with cardiovascular disease (CVD) are at
increased risk of AT7. The main acquired risk factors
for AT are hyperlipidaemia, smoking, diabetes, hyper-
tension, chronic kidney disease and obesity8. Rupture
of atherosclerotic plaques leads to the formation of a
thrombus — a process commonly referred to as ath-
erothrombosis9 (Fig. 1). By contrast, eroded plaques are
not normally associated with the formation of occlud-
ing thrombi because they do not present a highly pro-
thrombotic surface. Arterial thrombi are platelet rich,
but are stabilized by crosslinked fibrin (Fig. 1). Clinical
manifestations of atherothrombosis primarily occur in
the heart (myocardial infarction (MI)), brain (ischae-
mic stroke) and legs (peripheral artery disease (PAD),
with its attendant risk of limb ischaemia, gangrene
and amputation).
Venous thrombi occur in deep veins most often in the
legs or arms, a process known as deep vein thrombosis10.
These clots can break off, travel to the lungs and lodge
in pulmonary arteries — a process known as pulmonary
embolism (PE). Deep vein thrombosis and PE are col-
lectively known as venous thromboembolism (VTE).
VTE is triggered by one or more factors of Virchow’s
triad — sluggish blood flow, blood hypercoagulability
and endothelial dysfunction10. Specific risk factors for
VTE include genetic factors (such as factor V (FV)
Leiden, a prothrombotic variant of FV) or deficiencies
of endogenous anticoagulants (such as antithrombin or
protein C) as well as non-​genetic factors (for example,
immobility, surgery and age)8,10. In contrast to arterial
thrombi, venous thrombi form slowly on an intact, but
likely activated, endothelium and are fibrin rich but also
contain platelets and trapped red blood cells7.
Atrial fibrillation (AF), the most common sustained
cardiac arrhythmia, is responsible for about 25% of
all ischaemic strokes. Stasis of blood in the left atrial
appendage or left atrium of patients with AF and other
factors can trigger thrombus formation11. These thrombi
can break off and travel from the left atrium to lodge
in the brain, coronary arteries, abdominal arteries or
peripheral arteries. While the left atrial appendage is
the most common site for thrombus formation, patients
with AF have haematological findings consistent with
a prothrombotic state12. Strokes in patients with AF are
more likely to be fatal or disabling than those in patients
without AF because the thrombi are larger.
Antithrombotic agents fall into three classes — anti-
platelet agents, anticoagulants and fibrinolytic agents.
Because of the preponderance of platelets in arterial
thrombi, antiplatelet agents are the mainstay for the pre-
vention and treatment of AT. There are four main types
of FDA-​approved antiplatelet agents — a cyclooxygen-
ase inhibitor (aspirin), P2Y12 antagonists (for example,
clopidogrel, prasugrel and ticagrelor), αIIbβ3 antago-
nists (for example, abciximab) and a protease-​activated
receptor 1 (PAR1) antagonist (vorapaxar) (Box 1, Fig. 2).
Antiplatelet therapy is also effective as secondary
prevention in patients with ischaemic stroke (Box 2).

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Blood vessel Endothelial

cells

Lumen

RBC Neutrophil
Platelet/fibrin thrombus

NETs
FVa–FXa Thrombin

TxA2, ADP
Fibrin

FVIIIa–FIXa Platelet

Plaque rupture
FXI

TF–FVIIa FXIIa Collagen/vWF

Nucleic acid

Fig. 1 | Formation of an occlusive arterial thrombosis. Rupture of an atherosclerotic plaque exposes collagen
and von Willebrand factor (vWF), which bind and activate platelets. Released second messengers, such as ADP and
thromboxane A2 (TxA2), further activate platelets. Tissue factor (TF) within the plaque binds factor VIIa (FVIIa), and this
complex activates the clotting cascade, resulting in fibrin formation. In addition, FXII is activated by nucleic acid and
other negatively charged surfaces and contributes to thrombin generation. Neutrophils are incorporated into the
thrombus and extrude neutrophil extracellular traps (NETs). Red blood cells (RBCs) are also incorporated into thrombi.

Anticoagulants are the cornerstone for the prevention
and treatment of VT because they attenuate the forma-
tion of fibrin, which predominates in venous thrombi.
Anticoagulants are also the main therapeutic modality
used to prevent stroke in patients with AF. There are four
classes of FDA-​approved anticoagulants — heparins,
including low-​molecular-​weight heparin (LMWH),
vitamin K antagonists (VKAs; such as warfarin), direct
thrombin inhibitors (DTIs; such as bivalirudin and dab-
igatran) and direct FXa inhibitors (such as apixaban,
edoxaban and rivaroxaban) (Box 3, Fig. 2). Oral FXa and
thrombin inhibitors are called direct oral anticoagulants
(DOACs). Finally, fibrinolytic agents are used to degrade
arterial and venous thrombi13.
All current antithrombotic agents are associated
Haemostasis
with an increased risk of bleeding because platelets and
Arrest of bleeding after crosslinked fibrin are essential components of haemo-
damage to a blood vessel. static plugs that seal leaks in the vasculature14,15. Indeed,
there is widespread underuse of anticoagulants by clin­
icians and patients because of concerns with bleeding,
particularly in the elderly. One study analysed the use of
oral anticoagulation in eligible patients with AF between
2008 and 2014 and found that there was a significant
increase in use rates from 52.4% to 60.7%16, correspond-
ing with an increase in the use of DOACs (0 to 25.8%)
during this time period. However, despite this increase,
up to 40% of patients with AF at risk for stroke fail to
receive anticoagulation therapy, which may be the result
of bleeding concerns.
There are a large number of agents targeting plate-
lets and components of the coagulation cascade that
are claimed to reduce thrombosis without affecting
haemostasis; however, most have only been studied in
preclinical models17–20. This review provides an over-
view of the current landscape of emerging antiplatelet
and anticoagulant targets and agents in development,

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particularly those that are currently being studied
in clinical trials. Ongoing challenges in the field and
potential strategies to address them are discussed.
Overview of haemostasis and thrombosis
Haemostasis
The mammalian haemostatic system has evolved to
respond rapidly to vascular injury and minimize blood
loss by forming a haemostatic plug consisting of plate-
lets and crosslinked fibrin21–27. Platelets are the major
cellular component of the haemostatic system, inter-
acting closely with components of the coagulation
cascade, namely coagulation proteins and fibrinogen/
fibrin. Platelets are small enucleate cells produced by
mega­karyocytes in the bone marrow and released into
the blood, where they circulate for 7–10 days. All com-
ponents of the coagulation cascade, except tissue factor
(TF), are present in the blood of healthy individuals21,28.
Under normal conditions, endogenous platelet inhib-
itors and anticoagulants prevent activation of the clot-
ting system29–32. For instance, the vascular endothelium
inhibits platelet activation via multiple mechanisms
that include release of prostaglandin I2 (also known as
prostacyclin) and nitric oxide as well as expression of
ectonucleotidases (which degrade the platelet activators
ATP and ADP)32,33. Activation of the coagulation cascade
is negatively regulated by various anticoagulant proteins
that act at different points in the cascade29,30. For instance,
tissue factor pathway inhibitor (TFPI) — present on
the endothelium and in the blood — inhibits FXa, FVa

Box 1 | A brief history of antiplatelet agents

there are currently four major types of antiplatelet agents used for the prevention and treatment of arterial thrombosis,
namely aspirin (acetylsalicylic acid), P2Y12 antagonists, αiibβ3 (also known as GPiib/iiia) antagonists and a Par1 inhibitor
(Fig. 2).
in 1956, it was proposed that the anti-​inflammatory agent aspirin could protect individuals from heart attacks, and,
several years later, two groups identified the mechanism by which aspirin inhibits platelet activation and aggregation10.
aspirin irreversibly acetylates cyclooxygenase 1, which is used by platelets to generate the second messenger thromboxane
a2 — a potent platelet agonist. the first clinical trials evaluating the use of aspirin for secondary prevention in patients
with myocardial infarction were reported in 1974 (ref.223). since then, aspirin has been successfully used both as a single
agent and in combination with other antiplatelet or anticoagulant agents.
the second class of antiplatelet agents is the P2Y12 antagonists. P2Y12 is a major aDP receptor on the platelet
surface and ADP released from activated platelets binds to P2Y12 on adjacent platelets, thereby propagating platelet
activation and aggregation.
in 1972, studies into the anti-​inflammatory effects of a thienopyridine revealed that thienopyridine derivatives exhibit
antiplatelet effects. the first-​generation thienopyridine, ticlopidine, was used globally in 1991 for the primary and secondary
prevention of stroke and to prevent stent thrombosis224. the second-​generation thienopyridine, clopidogrel, entered
clinical trials in 1987 and was approved by the FDa in 1997. all thienopyridines are oral prodrugs that are metabolized
in the liver by cytochrome P450 (CYP) enzymes, mainly 2C9 and 2C19. about 30% of patients do not metabolize clopidogrel
sufficiently to achieve a therapeutic antiplatelet effect, mainly owing to CYP2C19 polymorphisms225. the problem of
clopidogrel resistance led to the development of a third-​generation thienopyridine, prasugrel (FDa approved in 2009),
which relies less on CYP-​mediated metabolism and produces less variability in response than clopidogrel226.
ticagrelor and cangrelor belong to a new generation of reversible P2Y12 antagonists218,227,228. ticagrelor is an oral atP
analogue that competes with aDP for P2Y12 binding. ticagrelor was approved in europe in 2010 and in the usa in 2011.
Cangrelor is a rapid on and rapid off reversible P2Y12 inhibitor. it is administered intravenously and was approved by the
FDa in 2015. Oral aDP receptor antagonists are used to treat patients with coronary artery disease (CaD), particularly as
an adjunct to aspirin in dual antiplatelet treatment (DaPt) after percutaneous coronary intervention.
the third class of antiplatelet agents targets the integrin αiibβ3 (ref.229). Platelet activation leads to the conversion of
αiibβ3 into an active state that then mediates platelet aggregation by binding fibrinogen and other ligands230. the first
agent targeting αiibβ3 was a monoclonal antibody, abciximab, which was approved by the FDa in 1994 (ref.229). Other
inhibitors of αiibβ3 include eptifibatide (a cyclic peptidomimetic) and tirofiban (a non-​peptide derivative of tyrosine)229.
these agents are administered intravenously and are mainly used in patients at high risk following percutaneous coronary
intervention, such as those with st-​segment elevation myocardial infarction. attempts to develop oral agents in this class
failed due to prothrombotic effects231,232.
the last class of FDa-​approved agents is a protease-​activated receptor 1 (Par1) antagonist. thrombin activates platelets
by cleavage of both Par1 and Par4 (ref.233). vorapaxar is an oral, slowly reversible Par1 inhibitor derived from a natural
product, himbacine234. addition of the Par1 antagonist vorapaxar to standard of care (single antiplatelet therapy or DaPt)
for secondary prevention in patients with CaD and peripheral artery disease decreased the risk of cardiovascular death or
ischaemic events but increased intracranial haemorrhage (NCt00526474)95. a second trial in patients with acute coronary
syndrome (traCer) was terminated early because of an increase in intracranial haemorrhage in patients with a history
of stroke (NCt00527943)96. together, these two studies demonstrate that triple antiplatelet therapy is associated with a
significant increase in bleeding. Despite the increase in intracranial haemorrhage, the FDa concluded that the benefits
of vorapaxar outweighed the risks and approved vorapaxar in 2014 for secondary prevention in patients with CaD or
peripheral artery disease combined with single antiplatelet therapy or DaPt (excluding patients with a history of stroke
or transient ischaemic attack). the efficacy of vorapaxar as sole antiplatelet therapy is unknown. Despite its approval,
vorapaxar is not used, presumably because of concerns with bleeding. atopaxar is a reversible, orally administered Par1
inhibitor that was tested in several phase ii clinical trials. However, its development was halted, in part owing to liver
toxicity (NCt01000727; NCt00312052)235–237.
Despite targeting different receptors or a second messenger, the major side effect of all currently available antiplatelet
agents is bleeding.

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Platelet cascade
Platelet agonists
Aspirin
TxA2 ADP Thrombin
• Clopidogrel
• Prasugrel
• Ticagrelor
• Cangrelor P2Y12
• Vorapaxar PAR1
• Abciximab
αIIbβ3
• Eptifibatide
• Tirofiban
Platelet aggregation
Haemostatic plug/thrombus
Fig. 2 | Targets of current antithrombotic drugs. Antiplatelet agents act either by preventing the formation of second
messengers (thromboxane A2 (TxA2)), blocking receptors (P2Y12 and PAR1) involved in platelet activation or inhibiting
platelet aggregation by binding to the integrin αIIbβ3. Anticoagulant agents act by either inhibiting post-​translational
modification of coagulation proteins, such as factor Xa (FXa) and prothrombin, inhibiting FXa and thrombin by increasing
the activity of antithrombin (AT), by directly inhibiting FXa or by directly inhibiting thrombin. VKA, vitamin K antagonist.
and the TF–FVIIa initiating complex34. The endothelial
protein C receptor–thrombomodulin–thrombin path-
way generates activated protein C (APC), which cleaves
and inactivates the cofactors FVa and FVIIIa28. Finally,
antithrombin inhibits most of the coagulation proteases,
particularly FXa and thrombin35.
Both platelets and the coagulation system are acti-
vated by vascular injury. Adhesion of platelets to the
injury site is mediated by transient bridging of the gly-
coprotein (GP) Ib–V–IX complex on the platelet sur-
face to subendothelial collagen via von Willebrand
factor (vWF)36 (Fig. 3). Two platelet receptors — α2β1
and GPVI — are critical for platelet activation and
accumulation on collagen37 (Fig. 3). Adherent platelets
secrete ADP and thromboxane A2, which act as soluble
agonists that recruit and activate circulating platelets27
(Fig. 3). Thrombin is also a potent activator of platelets
and mediates crosstalk between the platelet and coagu-
lation pathways. Activated platelets express an activated
form of the integrin αIIbβ3 on their surface, which binds
fibrinogen and other ligands to link adjacent platelets
together, a process known as platelet aggregation38,39
(Fig. 3). Finally, αIIbβ is also critical for platelet-​mediated
clot retraction, a process that helps seal the injury site
and initiate wound healing40.
Vascular injury exposes TF, a transmembrane protein
that serves as a receptor for FVII/FVIIa21. The TF–FVIIa
complex is the physiological trigger of blood coagula-
tion. TF is expressed by adventitial cells that surround
blood vessels and forms a haemostatic envelope to
limit blood loss41. TF surrounding blood vessels, except
in the brain, has bound FVII that allows rapid initiation
of coagulation after vessel injury42,43. Components of
the intrinsic pathway (FVIII, FIX and FXI) are required
for amplification of thrombin generation because the
TF–FVIIa complex is rapidly inactivated by TFPI44.
Indeed, individuals with deficiencies of these proteins
Coagulation cascade
Extrinsic/intrinsic pathways
Plasma clotting cascade
• Apixaban
FXa
• Betrixaban
• Edoxaban
AT • Rivaroxaban
VKA
Heparins
AT
Thrombin • Bivalirudin
• Argatroban
• Dabigatran
Fibrin
exhibit different degrees of bleeding45–48. During haemo-
stasis, FIX is activated by the TF–FVIIa complex and, to
a lesser extent, by the thrombin–FXIa pathway21 (Fig. 4).
Thrombin is the central protease in the coagulation
cascade49. It activates the cofactors FV and FVIII, con-
verts soluble fibrinogen to fibrin monomers, which then
polymerize to form fibrin strands that tie the platelet
aggregates together, and activates FXIII, which is a trans-
glutaminase that crosslinks and stabilizes fibrin. Finally,
thrombin and other agonists activate platelets that pro-
vide a negatively charged surface for the assembly of
the intrinsic tenase (FVIIIa–FIXa) and prothrombinase
(FVa–FXa) protease/cofactor complexes that enhance
thrombin generation by several orders of magnitude.
The fibrinolytic system removes haemostatic plugs
from the vasculature after they have sealed the wound50.
The plasminogen activators tissue plasminogen activator
(tPA) and urokinase plasminogen activator convert plas-
minogen to plasmin, which degrades fibrin. Fibrinolysis
is inhibited by plasminogen activator inhibitor 1 (PAI1),
which inhibits the plasminogen activators α2 antiplas-
min, (a plasmin inhibitor) and thrombin activatable
fibrinolysis inhibitor51.
Thrombosis
Excessive activation of platelets and coagulation leads to
the formation of thrombi in blood vessels that partially
or completely block blood flow23,26. For instance, rupture
of an atherosclerotic plaque exposes blood to collagen,
vWF and TF and leads to atherothrombosis9 (Fig. 1). TF is
also induced in peripheral blood mononuclear cells after
total knee arthroplasty and likely contributes to VTE52.
Indeed, TF appears to play a role in all forms of throm-
bosis, with the exception of those triggered by artificial
surfaces such as mechanical heart valves21. FXII contrib-
utes to thrombosis but not haemostasis and undergoes
autoactivation on negatively charged surfaces, such as
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silica or kaolin (the FXII activators used in the activated
partial thromboplastin time (aPTT) assay), and bioma-
terials such as catheters, mechanical heart valves and
extracorporeal circuits. More recently, natural negatively
charged polyphosphates — RNA and DNA released from
damaged or activated cells, inorganic phosphate released
from activated platelets and the DNA portion of neutro-
phil extracellular traps (NETs) extruded from activated
neutrophils — have been shown to activate FXII53,54.
In addition, NETs, which are present in both AT and
VT in humans54,55, have been shown to contribute to VT
in a mouse model56. Studies with mice also showed that
FXII and FXI contribute to AT, VT and atherothrombo-
sis57–60. Likewise, FXII or FXI knockdown or inhibition
attenuates catheter thrombosis in rabbits and clotting on
arteriovenous shunts in baboons53,61. Furthermore, FXIIa
inhibition prevented clotting to the same extent as hep-
arin in a rabbit extracorporeal membrane oxygenation
(ECMO) model but was associated with less bleeding62.
Because the formation of haemostatic plugs and throm-
botic clots are regulated by many of the same pathways,
it is difficult to separate protective haemostasis from
pathologic thrombosis.
Dysregulation of the fibrinolytic system can con-
tribute to thrombosis. For instance, elevated levels of
PAI1 and thrombin activatable fibrinolysis inhibitor in
disorders such as obesity are associated with thrombo-
sis63,64. In addition, a polymorphism in the PAI1 gene is
associated with VTE65.
It is important to remember that bleeding and
thrombosis do not usually occur in the same vascular
territories. Haemostatic plugs repair the vascular wall
and have little impact on blood flow, whereas patho-
logical thrombi grow without stopping, leading to vas-
cular occlusion and clinical manifestations. Increased
amounts of soluble accessible matrix components and
soluble activators of platelets and procoagulants, as well
as loss of endothelial inhibitory mechanisms in diseased
vessels, could be responsible for the deregulated growth
of pathogenic thrombi. Antithrombotics need to be pow-
erful enough to prevent thrombi in such prothrombotic
environments, yet of course they can cause bleeding in
damaged healthy vessels.
Clinical experience with antithrombotics
Antiplatelet agents
AT is prevented and treated with antiplatelet agents20,66
(Box 1, Fig. 2). These agents can be divided into paren-
teral and oral agents. Parenteral agents, such as αIIbβ3
antagonists or cangrelor, are generally used to prevent
or treat acute thrombosis. Single antiplatelet treatment
(SAPT) with oral agents (aspirin or a P2Y12 inhibitor
such as clopidogrel) is generally used for secondary
prevention in patients with stable coronary artery dis-
ease (CAD), PAD, non-​cardioembolic stroke or transient
ischaemic attack (TIA). By contrast, patients with acute
coronary syndrome (ACS) and/or undergoing percuta-
neous coronary intervention (PCI) receive short-​term
treatment with an anticoagulant (heparin or a parenteral
DTI such as bivalirudin) and then dual antiplatelet
treatment (DAPT) — the combination of aspirin and
a P2Y12 inhibitor — for secondary prevention (Table 1).
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Clopidogrel used to be the most commonly used
P2Y12 antagonist in DAPT, but this is being replaced
with the more potent agents prasugrel and ticagre-
lor. Occasionally, αIIbβ3 antagonists are used for ACS
and/or PCI, but these agents are used less than they
were in the past.
Approved antiplatelet agents either prevent the
generation of second messengers (thromboxane A2),
inhibit receptors activated by soluble agonists (P2Y12,
PAR1) or block the binding of fibrinogen and other
ligands to αIIbβ3 (Box 1, Fig. 2). However, because all
these pathways are required for platelets to maintain
haemostasis, the major side effect of existing antiplatelet
agents is bleeding. The risk of major bleeding (generally
defined as bleeding that requires medical attention) is
approximately 1% per year with aspirin and all the P2Y12
inhibitors15,67. In general, the use of antiplatelet agents
that are more potent than aspirin or the combination
of antiplatelet agents, are associated with a concomitant
increase in bleeding67. There are no guidelines on the
reversal of antiplatelet therapy68. In general, it is thought
that platelet transfusion is ineffective at reversing the
antiplatelet effects of P2Y12 inhibitors69,70. One study
found that platelet transfusions restored platelet function
in patients treated with aspirin but not in those treated
with clopidogrel71. However, the Platelet Transfusion
versus Standard of Care after Acute Stroke due to
Spontaneous Cerebral Haemorrhage Associated with
Antiplatelet Therapy (PATCH) trial found that platelet
transfusion increased the risk of death compared with
a non-​transfused group72. Ticagrelor is the only plate-
let inhibitor that has a reversal agent — a monoclonal
antibody fragment called PB2453 that binds to ticagre­
lor and its metabolite with high affinity70. Aspirin and
P2Y12 inhibitors, such as clopidogrel and prasugrel, have
short half-​lives in the circulation but bind to their tar-
gets in an irreversible manner such that their inhibitory
effects last the lifetime of the platelets. Transfusion of
Box 2 | Antiplatelet therapy for ischaemic stroke
antiplatelet therapy is effective as secondary prevention
in patients with non-​cardioembolic transient ischaemic
attack or non-​cardioembolic ischaemic stroke, but the
most effective agents and optimal duration remain
controversial. the 2014 Guidelines for the Prevention
of stroke in Patients with stroke and transient ischaemic
attack238 recommended an aspirin dose of 325 mg per day
(with the option of aspirin plus clopidogrel for 90 days)
in patients with a stroke or transient ischaemic attack
caused by 50% to 99% stenosis of a major intracranial
artery. these recommendations are supported by a
2002 meta-​analysis from the antithrombotic trialists
Collaboration, which analysed 195 randomized controlled
trials comparing antiplatelet therapy, primarily aspirin,
with placebo in the prevention of stroke, myocardial
infarction and vascular death239. in the subset of patients
with prior cerebrovascular disease, antiplatelet therapy
reduced the risk of secondary stroke, myocardial infarction
or vascular death by 22%. For most patients with ischaemic
stroke, the long-​term use of aspirin plus clopidogrel does
not have a greater benefit for stroke prevention than
either agent alone but does substantially increase the risk
of bleeding complications.
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Box 3 | A brief history of anticoagulants

there are four major classes of anticoagulants used for the prevention and treatment of venous thromboembolism (vte)
and the prevention of stroke in patients with atrial fibrillation (aF), namely heparins (parenteral), vitamin K antagonists
(vKas; oral), direct thrombin inhibitors (Dtis; parenteral and oral) and direct Factor Xa (FXa) inhibitors (oral)196 (Fig. 2).
the first anticoagulant, heparin (also referred to as unfractionated heparin), was originally isolated from liver in 1918
(ref.240). a protocol for its purification was described 15 years later241. Heparin is a naturally occurring glycosaminoglycan
with a negative ionic charge synthesized by basophils and mast cells. Heparin was first used as an anticoagulant
in humans in 1937 (refs242,243). the heparin family of anticoagulants now includes heparin, low-​molecular-​weight
heparin (LMwH), which is generated by chemical or enzymatic degradation of heparin, and fondaparinux (a synthetic
pentasaccharide)244, all of which are administered parenterally245. enoxaparin was the first LMwH to be approved by the
FDa in 1993 (with a generic version gaining approval in 2010)246, while fondaparinux was approved in 2001 (ref.247).
several additional LMwH preparations have been approved by the FDa and have antithrombotic activities like that of
enoxaparin yet with distinct biochemical and pharmacological profiles.
Heparin has several limitations, including heparin-​induced thrombocytopenia (Hit), a limb-​threatening and
life-​threatening side effect in which antibodies form against complexes of heparin and platelet factor 4 and induce
platelet activation and a prothrombotic state. Fortunately, the risk of Hit is lower with LMwH than with heparin and
almost non-​existent with fondaparinux248. a challenge with the use of heparin is the significant patient-​to-​patient
variation in its anticoagulant effect. By contrast, LMwH and fondaparinux have more predictable effects on coagulation
and thus can be administered in weight-​based doses without the need for coagulation monitoring249.
all heparins exert their anticoagulant effects by enhancing the activity of antithrombin, a naturally occurring inhibitor
of coagulation250. Heparin enhances antithrombin inhibition of both FXa and thrombin, whereas LMwHs are more
selective for FXa, and fondaparinux only inhibits FXa. therefore, heparins are indirect FXa and thrombin inhibitors.
Notably, positively charged protamine sulfate completely reverses the anticoagulant activity of heparin but only partly
reverses the anticoagulant activity of LMwH and has no effect on fondaparinux activity251.
vKas were the first oral anticoagulants. the anticoagulant activity of dicoumarol (3,3’-methylene-​bis[4-​hyroxycoumarin])
was discovered in 1940 (ref.252), and warfarin was approved by the FDa in 1954 (ref.253). Like all 4-​hyroxycoumarin drugs,
dicoumarol and warfarin are competitive inhibitors of vitamin K epoxide reductase; this leads to a reduction in vitamin K,
which is required for post-​translational modification of glutamic acid residues to carboxyglutamic acid254. several
proteins in the coagulation cascade require this modification to create a positively charged Gla domain, which enables
their binding to negatively charged phospholipids exposed on damaged or activated cells255. vKas induce a transient
prothrombotic state owing to inhibition of anticoagulant proteins, followed by an anticoagulant state due to inhibition
of procoagulants256. vitamin K reverses the effect of warfarin257. vKas interact with multiple drugs and their activity
is affected by dietary vitamin K intake. Because of these interactions, vKas require frequent monitoring and dose
adjustments. in addition, polymorphisms in CYP2C9 and vKOrC1 account for up to 50% of the interindividual variability
of warfarin dosing, which led to a label change in 2007 (refs258,259).
Dtis can be divided into parenteral (bivalirudin (FDa approved in 2000), lepirudin and argatroban) and oral (dabigatran)
agents. Bivalirudin, lepirudin and argatroban are used to treat patients with Hit260. Dabigatran etexilate, a prodrug of
dabigatran, was the first direct oral anticoagulant to be developed and was approved by the FDa in 2008 (ref.261).
idarucizumab is an inhibitory monoclonal antibody fragment that binds dabigatran with an affinity 350-​fold greater than
the affinity of dabigatran for thrombin262–265. idarucizumab was approved by the FDa in 2015 for dabigatran reversal.
there are four direct oral FXa inhibitors (rivaroxaban, apixaban, edoxaban and betrixaban) that were approved by the
FDa between 2008 and 2017 for the prevention and treatment of vte and for the prevention of stroke in patients with
aF196,266. andexanet alfa is a catalytically inactive FXa lacking the membrane-​binding γ-​carboxyglutamic acid domain that
acts as a decoy to bind FXa inhibitors267. andexanet alfa restored haemostasis in patients treated with a FXa inhibitor who
presented with acute major bleeding268. andexanet alfa was FDa-​approved in 2018 for the reversal of all FXa inhibitors269.
as a class, the direct oral anticoagulants are at least as effective as warfarin for stroke prevention in patients with aF or
for the treatment of vte, but they are associated with a 50% reduction in intracranial bleeding and are more convenient
to administer270. Nonetheless, the major side effect of all currently approved anticoagulants is bleeding.

sufficient numbers of normal platelets should replace
the inhibited platelets and enable haemostasis. By con-
trast, ticagrelor and its active metabolite bind reversibly
to P2Y12. Despite the reversible binding, it takes 4 or
5 days for recovery of platelet function after ticagrelor
is stopped. Platelet transfusion is ineffective for patients
treated with ticagrelor because free ticagrelor is available
to bind to the transfused platelets. Therefore, there is a
need for a reversal agent for ticagrelor but not for other
agents. Other concerns with the use of current antiplate-
let agents are as follows: up to 30% of patients are not
responsive to clopidogrel; prasugrel is contraindicated
in patients ≥75 years of age, with a weight <60 kg or with
a prior history of stroke; and ticagrelor can cause dysp-
noea and is contraindicated in patients with a history of
intracranial haemorrhage.
Another key challenge in the use of current anti-
platelet therapies is related to the variability in patient
response and side effects. Indeed, numerous studies
have demonstrated substantial interpatient variability
in platelet inhibition in those treated with clopidogrel,
depending on the method of testing and the defini-
tion of response73. In addition, patients with type 2
diabetes have increased platelet reactivity and are less
responsive to antiplatelet therapy74. While prasugrel
and ticagrelor exhibit less variability than clopidogrel,
prasugrel has been associated with higher rates of
major bleeding and ticagrelor has been associated
with higher rates of non-​coronary artery bypass graft
surgery-​related bleeding in head-​to-​head comparisons
with clopidogrel. In addition, both agents are more
expensive than clopidogrel and are associated with

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 Reviews

higher discontinuation rates75. As a result, clopidogrel a minority of patients of European ancestry carry the
remains the most commonly prescribed P2Y12 inhibi- cytochrome P450 (CYP; CYP2C19) loss-​of-​function
tor in clinical practice, accounting for over 50% of filled allele that is associated with decreased platelet respon-
prescriptions after PCI in 2016 (ref.75). siveness to clopidogrel78. This gene–clopidogrel response
Individuals exhibit a broad range of platelet reactiv- relationship was first reported in 2007, but it took more
ity, which makes it difficult for clinicians to know the than a decade to perform a clinical trial. Importantly, the
best agent and dose to use. Attempts have been made POPular Genetics trial found that the use of a CYP2C19
to develop point-​of-​care ADP-​specific platelet reactiv- genotype-​guided strategy (carriers received ticagrelor
ity tests such as PFA-100, VerifyNow, and Multiplate or prasugrel and non-​carriers received clopidogrel) to
Electrode Aggregometry76. A meta-​analysis of 10 clini- guide the selection of oral P2Y12 inhibitor therapy in
cal trials comparing a standard dose of clopidogrel with patients undergoing PCI was non-​inferior to treatment
an intensified dose based on platelet reactivity testing with ticagrelor or prasugrel at 12 months in the primary
showed a reduction in cardiovascular mortality, stent outcomes but with a lower incidence of bleeding79. This
thrombosis and MI; however, this difference was driven suggests a benefit of using a CYP2C19 genotype-​guided
by a decrease in stent thrombosis in the intensified dose strategy for antiplatelet therapy. The success of this trial
group77. These results suggest that more work is needed should lead to avoidance of clopidogrel use in carriers
to improve point-​of-​care platelet reactivity testing, which of the CYP2C19 loss-​of-​function allele, and a CYP219
would allow better use of current antiplatelet agents. genotype-​guided strategy is gaining wider acceptance for
Another approach to improving the use of antiplate- use in clinical practice.
let drugs is genetic testing. For instance, it is known that Another challenge in antiplatelet therapy is defining
the optimal duration of DAPT in patients with ACS,
Soluble agonists particularly those receiving an intracoronary stent.
With first-​generation drug-​eluting stents, long-​term or
Thrombin lifelong DAPT was recommended because of reports
of late stent thrombosis80. With the newer generations of
ADP TxA2 drug-​eluting stents, vascular healing is improved and
BMS-986141 PAR1 PZ-128 P2Y1 stent thrombosis is less common, enabling the duration
PAR4 P2Y12 of DAPT to be reduced, especially in patients at high
BMS-986120
risk of bleeding. The 2016 ACC/AHA Guideline Focused
TP Update on Duration of Dual Antiplatelet Therapy in
Patients with Coronary Artery Disease recommended
6 months of DAPT in patients with stable CAD who
received an intracoronary stent and 12 months for
patients with ACS treated medically or who received
an intracoronary stent81. The 2017 European guidelines
PI3Kβ were similar and recommended 1–6 months (depending
AZD6482 on bleeding risk) of DAPT in patients with stable CAD
and 12 months for patients with ACS82. Several recent
Activation
studies have provided more evidence that 1 month of
BTK DAPT is sufficient in patients with stable CAD at high
bleeding risk 83. Whether the duration of DAPT in
GPVI
patients with ACS can be reduced to less than 1 year is
Fcγ CLEC2 αIIbβ3
ACT017 unknown and will require further study.
These problems associated with existing antiplatelet
agents have led to a search for new platelet targets.
Revacept Collagen
GPIb–V–IX
Anticoagulants
Fibrinogen
Anticoagulants are divided into those that require par-
PDPN
• AJW200 enteral administration and those that are given orally
• ALX-0081 vWF Platelet aggregation (Box 3, Fig. 2). The commonly used parenteral antico-
• ARC1779
agulants include heparin, LMWH, fondaparinux and
Collagen bivalirudin. The commonly used oral anticoagulants
are VKAs and DOACs. Heparins are used as a first line
Adhesion receptors therapy to treat acute thrombotic events. Both parenteral
(such as heparins) and oral agents (such as VKAs and
Fig. 3 | Targets of antiplatelet agents in development. Platelet receptors can be DOACs) are used to prevent and treat VT. Because of
divided into three groups: adhesion receptors (glycoprotein Ib (GPIb) and integrins),
the preponderance of fibrin in venous thrombi and in the
insoluble ligands (GPVI), soluble agonist receptors (thromboxane receptor (TP), P2Y1,
P2Y12, PAR1 and PAR4) and an aggregation receptor (αIIbβ3). In addition, intracellular thrombi that form in the left atrial appendage in patients
kinases (Bruton’s tyrosine kinase (BTK) and phosphoinositide 3-​kinase-​β (PI3Kβ)) are with AF, anticoagulants are the cornerstone for the pre-
required for platelet activation. The names and targets of antiplatelet agents in vention and treatment of VTE and for stroke preven-
development are shown. CLEC2, C-​type lectin-​like 2; PDPN, Podoplanin; TxA2, tion in patients with AF (Box 3, Table 1). Anticoagulants
thromboxane A2; vWF, von Willebrand factor. are also used to prevent clotting on mechanical heart

Nature Reviews | Drug Discovery

Reviews
PolyP, nucleic acid
FXIIa CSL312
• IONIS-416858
• Osocimab Intrinsic
Extrinsic TF FVIIa FXIa • AB023
pathway pathway
• BMS-986177
FIXa
FXa
Common
pathway
Platelet
activation Thrombin FXIIIa
Fibrin monomers
Crosslinked fibrin
Fig. 4 | Targets of anticoagulant agents in development. The coagulation cascade can
be divided into three parts: extrinsic (tissue factor (TF), factor VIIa (FVIIa)), intrinsic (FXIIa,
FXIa, FIXa and FVIIIa) and the common pathway (FXa, FVa and thrombin). Activation of
the coagulation cascade leads to the generation of thrombin. Thrombin plays a central
role in the coagulation cascade by cleaving fibrinogen to fibrin monomers, activating
FXIII, which then crosslinks fibrin monomers, and by activating the cofactors FV and FVIII.
In addition, thrombin is a potent activator of platelets. All coagulation factors except
FXII are required for haemostasis. Components of the extrinsic and common pathways
are essential for haemostasis, whereas components of the intrinsic pathway are required
to amplify thrombin generation. Anticoagulant agents in development target either FXII,
FXI, FIX or activation of FXI by FXIIa.
valves or on extracorporeal circuits such as those used
for haemodialysis, cardiopulmonary bypass surgery
and ECMO.
Although VKAs are effective, they are difficult to
manage because of within-​patient and between-​patient
differences in dosing. These differences reflect a combi-
nation of factors, including common genetic polymor-
phisms in CYP2C9 and vitamin K epoxide reductase
complex subunit 1 (VKORC1) (which account for ~30%
of the interindividual variation) that affect the metabo-
lism of VKAs, variations in dietary intake of vitamin K
and multiple drug interactions84,85. Because of this vari-
ability, frequent monitoring of the international normal-
ized ratio and dose adjustments to maintain it within the
therapeutic range are necessary. The many limitations
of VKAs prompted a long search for oral anticoagulants
that were at least as effective as VKAs, but could be
administered in fixed doses without the need for routine
coagulation monitoring.
DOACs overcome many of the limitations of VKAs86.
DOACs are direct anticoagulants because they bind
to the active site of their target enzyme and block its
activity. Thus, dabigatran inhibits thrombin, whereas
apixaban, betrixaban, edoxaban and rivaroxaban
inhibit FXa. In contrast to VKAs, DOACs are given in
fixed doses without monitoring. Such dosing is possible
because DOACs produce a more predictable anticoag-
ulant response than VKAs because their metabolism is
unaffected by genetic polymorphisms or variations in
dietary vitamin K intake and there are fewer drug–drug
interactions. Therefore, DOACs are more convenient to
administer than VKAs. In randomized clinical trials that
included over 100,000 patients, DOACs were shown to
be at least as effective as VKAs for the prevention of
stroke in patients with AF and for the treatment of VTE
but were associated with less bleeding, and in particular
less intracranial bleeding. Given their similar efficacy,
greater safety and convenience compared with VKAs,
DOACs are now given preference over VKAs for AF and
VTE indications in recent guidelines87.
Nevertheless, although DOACs are a major advance
over VKAs, there are several issues with DOACs, including
increased bleeding in some patient populations, their ina-
bility to be used in certain patients and a lack of efficacy
in others. For instance, rates of gastrointestinal bleeding
are higher with DOACs than with VKAs. Studies compar-
ing either edoxaban or rivaroxaban versus dalteparin for
the prevention of recurrent VTE in patients with cancer
observed an increase in bleeding in patients with gas-
trointestinal cancers treated with DOACs compared to
dalteparin88. Furthermore, even though the risk of major
bleeding is 50% lower with DOACs than with VKAs, the
annual rates of major bleeding with DOACs can be as
high as 2–3% in elderly patients with AF89. All DOACs
are cleared through the kidneys and, therefore, there is a
risk of drug accumulation and bleeding in patients with
renal dysfunction. All DOACs have different recommen-
dations for use in patients with impaired renal function,
with a dose reduction for renal impairment. However,
apixaban has recently been licenced for patients with
end-​stage renal disease (ESRD)90. Like VKAs, DOACs
pass through the placenta, which could pose a risk to
the foetus; consequently, DOACs are contraindicated
in pregnancy. In addition, unlike VKAs, DOACs may
pass into breast milk and are therefore also contraindi-
cated in nursing mothers. There is a black box warning
against the use of DOACs in patients with mechanical
heart valves because a study comparing dabigatran with
warfarin for this indication showed a trend for more
thromboembolic and bleeding events with dabigatran91.
Consequently, VKAs, such as warfarin, remain the only
approved anticoagulants for this patient population.
In addition, VKAs remain the anticoagulant of choice for
patients with triple-​positive antiphospholipid syndrome
(positive for lupus anticoagulant, anticardiolipin and
anti-​β-​glycoprotein I antibodies) following the early ter-
mination of a study comparing rivaroxaban and warfarin
in such patients due to excess thromboembolic events in
the rivaroxaban arm (NCT02157272)92. Finally, DOACs
also have more drug–drug interactions than LMWHs93.
Therefore, there is still a need for safer anticoagu-
lants, particularly those with minimal renal clearance
and the capacity to attenuate clotting induced by medi-
cal devices such as catheters, heart valves, left ventricle
assist devices and extracorporeal circuits.
Combination antiplatelet and anticoagulant therapy
ACS, CAD and PAD. More potent antiplatelet agents
and/or the use of multiple antiplatelet therapies sig-
nificantly increase the risk of bleeding, which suggests
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 Reviews

that we have reached the limit for targeting platelets
alone in patients with CVD94–96. Fibrin is also present
in AT. The concept of combining antiplatelet and anti-
coagulant therapy to prevent thromboembolic events in
patients with CVD was examined more than 10 years
ago. A meta-​analysis of 10 trials concluded that addition
of a VKA to aspirin decreased MI and ischaemic stroke
in patients with ACS compared with aspirin alone, but
increased major bleeding97. However, these studies do
not reflect current practice because they were performed
before the widespread use of PCI and DAPT.
Because DOACs are associated with a lower risk of
intracranial bleeding compared with VKAs, the concept
of dual pathway inhibition (DPI) — combined anti-
platelet and anticoagulant therapy — was revisited. Two
trials determined the effect of adding a FXa inhibitor to
standard antiplatelet therapy in patients with ACS. The
first trial, APPRAISE-2, which administered apixaban
at the same dose used to prevent stroke in patients with
AF or to treat patients with VTE98, resulted in signifi-
cantly increased major bleeding without reducing the
primary outcome of cardiovascular death. The second
trial, ATLAS ACS 2–TIMI 51 (ref.99), which used two
doses of rivaroxaban that were lower than those used
to treat or prevent VTE or for stroke in patients with
AF, the primary end point of cardiovascular death, MI
and ischaemic stroke was reduced with both doses of
rivaroxaban compared with standard antiplatelet ther-
apy alone. However, as expected, rivaroxaban increased
rates of major bleeding and intracranial bleeding. The
European Commission approved the use of rivaroxaban
for the secondary prevention after an acute coronary
event in 2013. However, it has not been widely adopted
owing to concerns with bleeding.
DPI has also been investigated in patients with stable
CAD and PAD. The standard therapy for these patients
is a single antiplatelet agent, either aspirin or clopi-
dogrel. The Cardiovascular Outcomes for People Using
Anticoagulation Strategies (COMPASS) trial compared
the effect of three treatments: aspirin alone, rivaroxaban
alone and the combination of the two agents in patients
with stable CAD or PAD100–102. The trial was stopped early
owing to the overwhelming benefit of DPI compared
with aspirin alone on the primary outcome (the com-
posite of cardiovascular death, MI or stroke) being signif-
icantly reduced100,102. Although major bleeding (mostly
gastrointestinal bleeding) was significantly increased in
the aspirin plus rivaroxaban group compared with the
aspirin alone group, there was no significant increase in
fatal bleeding or intracranial bleeding. Notably, the net
clinical benefit of DPI was greater in patients with PAD
compared with patients with CAD, as well as in patients
with PAD with the highest risk of atherothrombotic
events102. In 2018, the FDA approved the combination
of aspirin and rivaroxaban for patients with CAD or
PAD. The results of the COMPASS trial are exciting and
support the notion that a combination of an antiplate-
let and anticoagulant agent may be more efficacious in
reducing cardiovascular death, MI or stroke than a single
agent for some indications. The VOYAGER trial is cur-
rently evaluating the effect of rivaroxaban on acute limb
ischaemia or major amputations as well as cardiovascular
death, MI and stroke in patients with PAD undergoing
revascularization procedures (NCT02504216).

Table 1 | use of current antithrombotics

indication subtype Agent comments
Arterial thrombosis Coronary stable CAD ASA or ASA + riva Need for new agents
ACS DAPT
PCI high risk AP + AC (heparin or DTI)
Cerebral ASA
Peripheral ASA or ASA + riva
VTE – AC (DOAC, VKA, heparins) Guidelines give preference to
DOAC vs VKA treatment
AF – AC (DOAC, VKA) Guidelines give preference to
DOAC vs VKA treatment
APS – AP + AC –
Medical devices — mechanical – VKA Black box warning against the
heart valves and LVADs use of DOACs for use with
mechanical vent valve
Extracorporeal circuits — – Heparin FXIIa and FXIa inhibitors may be
cardiopulmonary bypass, associated with less bleeding
haemodialysis and ECMO
Prevention of recurrent VTE in – AC (DOAC, LMWH, VKA) DOAC > LMWH > VKA
patients with cancer
Prevention of recurrent stroke – ASA Heparin, LMWH and heparinoids
have failed; need for new agents
AC, anticoagulant; ACS, acute coronary syndrome; AF, arterial fibrillation; AP, antiplatelet; APS, antiphospholipid syndrome;
ASA, acetylsalicyclic acid (aspirin); CAD, coronary artery disease; DAPT, dual antiplatelet treatment; DOAC, direct oral anticoagulant;
DTI, direct thrombin inhibitor; ECMO, extracorporeal membrane oxygenation; F, factor; LMWH, low-​molecular-​weight heparin;
LVAD, left ventricular assist device; PCI, percutaneous coronary intervention; riva, rivaroxaban; VKA, vitamin K antagonist;
VTE, venous thromboembolism.

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Reviews
Interestingly, addition of the PAR1 inhibitor vora-
paxar to SAPT or DAPT for patients with CAD or PAD
also reduced major adverse limb events and revascu-
larization in patients with PAD, but it did not reduce
mortality95. Further studies are needed to better under-
stand the mechanism by which combined antiplatelet
and anticoagulant therapy as well as PAR1 inhibition is
protective in patients with CAD or PAD. It is possible
that DPI reduces both thrombosis and PAR-​dependent
vascular inflammation.
Patients with CVD and AF. Patients with CVD and
AF require both antiplatelet agents and anticoagulants
as they are at risk for both AT and VT. In the past,
patients with AF who experience an ACS or underwent
PCI were treated with DAPT plus an anticoagulant
(labelled as triple therapy). Five recent studies (WOEST,
PIONEER AF-​PCI, RE-​DUAL PCI, AUGUSTUS and
ENTRUST-​AF-​PCI) examined the effects of using SAPT
or DAPT plus an anticoagulant (VKA or DOAC). A
meta-​analysis of four of these studies103 compared the
efficacy and safety of the different groups. There were no
statistically significant differences in the rates of major
adverse cardiovascular events (MACE) amongst the dif-
ferent groups, but there was more bleeding in the VKA
plus DAPT group. In addition, strategies omitting aspi-
rin caused less bleeding, without a significant difference
in MACE, compared with strategies including aspirin.
This indicates that DOACs are safer than VKAs when
used in combination with antiplatelet agents and that
aspirin might not be necessary in patients treated with a
DOAC or VKA and a P2Y12 inhibitor.
The AFFIRE study of 2,236 patients in Japan found
that rivaroxaban monotherapy was non-​inferior to
dual therapy (rivaroxaban plus an antiplatelet agent) in
patients with AF and CAD, with neither requiring inter-
vention for more than 1 year after revascularization104.
The primary efficacy end point was stroke, systemic
embolism, MI, unstable angina requiring revasculari-
zation and all-​cause mortality, while the primary safety
end point was major bleeding. The study was stopped
early because of higher rates of all-​cause mortality in
the dual therapy arm. Therefore, a single anticoagulant,
rivaroxaban, was superior to dual therapy in this patient
population.
Fibrinolytic therapy
Acute ischaemic stroke is treated with fibrinolytic ther-
apy and/or mechanical thrombectomy. tPA is approved
for use within 3 h of onset and there are data that out-
comes are improved when administered within 4.5 h of
the onset of symptoms105. As with antithrombotics, the
major side effect of fibrinolytics is bleeding. Studies have
shown that treatment of MI and PE with systemic throm-
bolysis is associated with a 1–1.5% risk of intracerebral
bleeding106,107. tPA also affects blood–brain barrier per-
meability13. The ongoing MOST trial (NCT03735979)
is determining whether combining tPA with either
the direct thrombin inhibitor argatroban or with the
αIIbβ3 antagonist eptifibatide is superior to tPA alone
in improving the 90-​day Rankin score for patients with
acute ischaemic stroke.
APC has both anticoagulant and cytoprotective
activity via activation of PAR1 on the endothelium108,109.
3K3A-​APC is a variant of APC with full cytoprotective
activity but significantly reduced anticoagulant activity.
This variant was protective in a mouse stroke model110.
A phase II study showed that addition of 3K3A-​APC to
tPA for the treatment of acute stroke was associated with
a trend towards less bleeding, possibly owing to protec-
tion of the endothelium111. An ongoing phase II trial
is comparing the effect of 3K3A-​APC with placebo in
tPA treatment, medical thrombectomy or both in acute
ischaemic stroke (NCT02222714).
Current guidelines recommend systemic throm-
bolytics for the treatment of massive PE but not for
sub-​massive PE unless the patient deteriorates after
beginning anti-​coagulation and the bleeding risk is low87.
Interestingly, the MOPETT trial showed that low-​dose
tPA significantly reduced the combined end point of
death plus recurrent PE in patients with moderate PE112.
Another study found that both half-​dose systemic throm-
bolysis and ultrasound-​facilitated catheter-​directed
fibrinolysis were beneficial in the treatment of patients
with PE but systemic thrombolysis was more cost effec-
tive113. More recent trials have suggested that low-​dose
tPA delivered directly into the pulmonary arteries by a
catheter that provides high-​frequency, low-​power ultra-
sound (termed ultrasound-​enhanced thrombolysis)
can decrease right ventricle dilation, reduce pulmonary
arterial pressures and decrease thrombus burden114.
Thrombolytic agents are also a beneficial treatment
for acute ST-​segment elevation myocardial infarction,
although their use has largely been supplanted by PCI.
Antiplatelet drugs in development
A number of antiplatelet targets and associated agents
are currently being evaluated in clinical trials (Table 2).
Inhibitors of thrombin-​mediated platelet activation
Thrombin activates human platelets by proteolytic
cleavage of two G protein-​coupled receptors, PAR1 and
PAR4115,116 (Fig. 3). PAR1 is activated by low concentra-
tions of thrombin (sub-​nanomolar) and produces a rapid
but transient signal, whereas PAR4 requires ~10-​fold
more thrombin for its activation and mediates pro-
longed signalling that is required for stable thrombus
formation. Early studies focused on developing PAR1
antagonists and culminated in the FDA approval of vora-
paxar in 2014 (Box 1). However, vorapaxar is a slowly
reversible inhibitor that may interfere with thrombin
activation of platelets during haemostasis. Therefore,
recent efforts have been made to develop rapidly revers-
ible PAR1 antagonists and PAR4 inhibitors that may
have less effect on haemostasis. Importantly, PAR1 and
PAR4 are expressed on numerous cell types in the vas-
culature, including endothelial cells117. Therefore, inhi-
bition of PAR1 and PAR4 may have effects on cells other
than platelets.
PAR1 antagonists. PZ-128 is a pepducin and there-
fore represents a second class of PAR1 inhibitors
with a distinct mechanism of action. Pepducins are
membrane-​ tethered, cell-​ p enetrating lipopeptides
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 Reviews

Table 2 | Antiplatelet agents in development

Name/company Agent/route of Target Disease clinical trial Trial iD comments
administration
PZ-128/Tufts Medical Pepducin/IV PAR1 NA Phase I NCT01806077 Completed; safety study
Center
BMS-986120/ Small molecule/oral PAR4 NA Phase I NCT02439190 Completed; safety study
Bristol-​Myers Squibb
BMS-986141/ Small molecule/oral PAR4 NA Phase I NCT02341638 Completed; safety study
Bristol-​Myers Squibb
Recurrent stroke Phase II NCT02671416 Study terminated early
Revacet/Advance Cor Fusion protein/IV GPVI ligand Stable CAD Phase I NCT01042964 Completed; safety study
undergoing PCI
Phase II NCT03312855 Completed; no data
Symptomatic Phase II NCT01645306 Ongoing
carotid stenosis
ACT017/Acticor Biotech Antibody/IV GPVI NA Phase I NCT03803007 Completed; safety study
ARC1779/Archemix DNA aptamer/IV vWF NA Phase I NCT00432770 Completed; safety study
Cerebral Phase II NCT00742612 Completed; reduced cerebral
embolization embolization
AZD6482/AstraZeneca Small molecule/IV PI3Kβ NA Phase I NCT00688714 Completed; safety study
Phase I NCT00853450 Completed; safety study
Isoquercetin/Beth Israel Small molecule/oral ND NA Phase I NCT01722669 Completed; safety study
Isoquercetin/NHLBI Small molecule/oral ND VTE in advanced Phase II/III NCT02195232 Completed; reduced D-​dimer
cancer and P-​selectin
CAD, coronary artery disease; GPVI, glycoprotein VI; IV, intravenous; NA, not available; ND, not determined; NHLBI, National Heart, Lung, and Blood Institute; PAR,
protease-​activated receptor; PCI, percutaneous coronary intervention; PI3Kβ, phosphoinositide 3-​kinase β; vWF, von Willebrand factor; VTE, venous thromboembolism.

that target the cytoplasmic domain of receptors118,119.
PZ-128 was developed as a rapidly acting, reversible
antiplatelet agent to prevent ischaemic events associated
with PCI. The safety, efficacy and pharmacokinetics of
PZ-128 were evaluated in a phase I study in patients
with vascular disease or who had two or more CAD
risk factors (NCT01806077)120. Intravenous adminis-
tration of PZ-128 inhibited PAR1-​dependent platelet
activation ex vivo but not activation by other agonists,
such as ADP, and did not cause bleeding. Further tri-
als are planned with PZ-128 in high-​risk patients with
CAD or ACS undergoing PCI (A. Kuliopulos, personal
communication).
Another class of PAR1 inhibitors is the non-​peptidic
small molecules named parmodulins, which also target
intracellular domains121. Mechanistic studies demon-
strated that parmodulins selectively interfere with
Gαq but not Gα12/13 signalling downstream of PAR1.
Consequently, parmodulins inhibit PAR1-​mediated
platelet integrin activation and aggregation. In addition,
parmodulins have also been shown to inhibit proin-
flammatory signalling in endothelial cells while pre-
serving cytoprotective signalling in endothelial cells122.
No clinical trials have yet been conducted with these
new agents.
PAR4 antagonists. Inhibition of PAR4 blocks the sus-
tained activation of platelets by high concentrations
of thrombin116 — conditions that are more likely to be
present during atherothrombosis — while leaving tran-
sient PAR1 signalling intact. The rationale for targeting
PAR4 is that this receptor may be critical for platelet–
platelet cohesion during AT while PAR1 signalling alone
mediates thrombin-​induced platelet adhesion during
haemostatic plug formation. A variety of agents, such as
inhibitory antibodies, pepducins and small molecules,
have been developed against PAR4.
BMS-986120 is a potent and reversible PAR4-​specific
small molecule antagonist123 (Fig. 3, Table 2) that blocks
platelet activation by γ-​thrombin or a PAR4 activa-
tion peptide. In a non-​human primate thrombosis
model, BMS-986120 and clopidogrel decreased throm-
bus weight to a similar extent but, unlike clopidogrel,
which prolonged the bleeding time, BMS-986120
had little effect123. In a phase I study, orally adminis-
tered BMS-986120 had a half-​life of 4 h and selectively
inhibited PAR4-​induced platelet activation ex vivo
(NCT02439190) 124. Importantly, administration of
BMS-986120 to healthy volunteers did not cause spon-
taneous bleeding but reduced thrombus growth ex vivo
to a similar extent as aspirin plus clopidogrel. BMS-
986141, which is more potent than BMS-986120, was
also evaluated in healthy volunteers (NCT02341638).
In addition, a phase II trial was initiated to determine if
BMS-986141 reduced recurrent stroke compared with
placebo in patients with a recent stroke or TIA, all of
whom received aspirin (NCT02671461). However, the
study was terminated early for undisclosed reasons.
A pepducin against PAR4 and an inhibitory PAR4
monoclonal antibody have been generated but neither
has been evaluated in humans118,119,125.
Antagonists of GPVI collagen-​mediated platelet
activation
GPVI is a promising new target for antiplatelet therapy126
(Fig. 3). It is the main platelet receptor for collagen127,
and recent studies suggest that it also binds fibrin128,129.
However, the interaction between GPVI and fibrin is

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controversial, and one study concluded that dimeric
GPVI is not a receptor for fibrin128,130–133.
Ruptured atherosclerotic plaques are rich in collagen
and thus provide a robust surface for GPVI-​mediated
platelet adhesion. Indeed, several preclinical studies
suggest that GPVI is critical for platelet adhesion to
plaque material under flow conditions134,135 and to rup-
tured plaques in vivo134,136. However, the role of type I
and type III fibrillar collagen in thrombosis triggered
by plaque erosion is less clear. Patients lacking func-
tional GPVI generally exhibit only a mild bleeding
diathesis137 except when they have moderate to severe
thrombocytopenia138. Taken together, these results sug-
gest that targeting GPVI may reduce thrombosis caused
by ruptured or possibly eroded atherosclerotic plaques
with less bleeding than that produced by conventional
antiplatelet agents. However, it remains to be seen how
important GPVI is for embolic stroke.
Inhibitors of GPVI–collagen/fibrin interaction. Two
agents have been developed to inhibit the function
of GPVI. Revacept targets the GPVI ligand collagen,
whereas ACT017 targets GPVI itself (Fig. 3).
Revacept is a fusion protein that contains the extra-
cellular domain of GPVI fused to a human Fc immuno­
globulin region139. Revacept binds to subendothelial
collagen to prevent platelet adhesion and activation.
A recent study found that revacept did not bind to
fibrin132. In a mouse stroke model, revacept improved
the efficacy of tPA without increasing bleeding140.
In addition, revacept dose-​d ependently inhibited
collagen-​induced platelet activation without impairing
haemostasis in a phase I trial (NCT01042964)141. A com-
pleted phase II trial evaluated the efficacy of revacept in
patients with symptomatic carotid artery stenosis, ama­
urosis fugax, TIA or stroke (NCT01645306). However, no
results have been reported. A second ongoing phase II
trial is comparing the effect of two doses of revacept
versus placebo on top of DAPT in patients with stable
CAD undergoing PCI (NCT03312855)142.
ACT017 is a humanized Fab fragment that has
a high antigen-​binding specificity and affinity for
GPVI143. Injection of ACT017 into macaque monkeys
dose-​dependently inhibited collagen-​induced plate-
let activation ex vivo without inducing thrombocyto­
penia or bleeding144. A phase I study in healthy subjects
evaluating the pharmacokinetics, pharmacodynam-
ics, safety and tolerability of ACT017 demonstrated
favourable safety and tolerability profiles and inhibi-
tion of collagen-​induced platelet aggregation without
effects on platelet count or platelet GPVI expression
(NCT03803007)144. A phase II study in patients with
stroke is planned (NCT03803007). Therefore, revacept
and ACT017 represent novel antiplatelet agents that
may reduce thrombosis with less bleeding than current
antiplatelet agents.
Inhibitors of vWF–GP1bα-mediated platelet activa-
tion. Platelets are captured at sites of vascular injury by
interaction of GPIbα with vWF exposed on the extra-
cellular matrix (Fig. 3). This interaction is particularly
important under high shear stress, such as is found in
atherosclerotic arteries and arterioles. Studies in pre-
clinical animal models suggested that the vWF-​GPIbα
interaction is more important in arterial thrombosis and
ischaemic stroke than during haemostatic plug forma-
tion145,146. However, one must be cautious when extra­
polating results from preclinical models because they do
not necessarily equate to results in the clinical setting.
Increased vWF levels and/or activity can lead to
thrombotic microangiopathy, which is thrombosis
in capillaries and arterioles due to endothelial injury.
For instance, in thrombotic thrombocytopenia pur-
pura (TTP) there is a reduction in the level of the
protease ADAMTS13 (inherited or acquired), which
cleaves ultra-​large vWF multimers into smaller forms.
Accumulation of these large vWF multimers leads to
life-​threatening microvascular thrombosis and thrombo­
cytopenia147. A similar phenotype is observed in patients
with type 2B von Willebrand’s disease, who express
a mutant form of vWF that binds spontaneously to
GPIbα148.
vWF inhibitors that are being investigated include
ARC1779, which is a DNA-​based aptamer, two mono­
clonal antibodies (AJW200 and 82D6A3) and two
bivalent nanobodies (caplacizumab (ALX-0081) and
ALX-0681) 149–153. Caplacizumab was demonstrated
to be well tolerated with no signs of bleeding in two
phase I clinical trials (NCT0289733 and NCT03172208).
Moreover, caplacizumab was associated with a lower
incidence of recurrence of TTP compared with pla-
cebo in a phase III trial154, leading to its FDA approval
in 2019 for adult patients with acquired TTP in com-
bination with plasma exchange and immunosuppres-
sive therapy. ARC1779 has a high affinity for the A1
domain of vWF 150; a phase I trial showed that the
aptamer dose-​dependently inhibited platelet function
ex vivo (NCT00432770) 150 whereas a phase II trial
(NCT00742612) showed that it also reduced cerebral
embolization after carotid endarterectomy155.
The clinical development of GPIbα inhibitors has
lagged behind drugs targeting the vWF A1 domain.
Various agents, including two anti-​GPIbα antibodies
(SZ2 and h6B4-​Fab), a snake toxin (anfibatide) and
a fusion protein that contains the amino terminus of
GPIbα fused to human IgG1 Fc, showed promise in
preclinical testing149,156–158. It should be noted that loss
of GPIbα is associated with increased platelet clearance,
and this must be avoided when targeting GPIbα159.
Despite the development of several agents that inhibit
the interaction between vWF and GPIbα, these agents
have not been tested as antiplatelet agents in patients
with CVD (except for a small study in patients under-
going carotid endarterectomy). It will be interesting to
see if there will be future trials of these agents in patients
with CVD.
Inhibiting PI3Kβ
The phosphoinositide 3-​kinase-​β (PI3Kβ) member
of the PI3K family of lipid kinases plays an impor-
tant role in signalling downstream of various platelet
receptors and is required for stable platelet adhesion
under shear stress160 (Fig. 3). Indeed, p110β-​deficient
mice have reduced arterial thrombosis161. Two PI3Kβ
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inhibitors, AZD6482 and MIPS-9922, were shown to
reduce thrombosis in dog and mouse models with min-
imal effects on bleeding162,163. AZD6482 was well toler-
ated in healthy volunteers (NCT00688714)162 and, in
combination with aspirin, exhibited greater antiplatelet
activity and less bleeding than clopidogrel plus aspirin
(NCT00688714)164. Further studies are needed to deter-
mine if targeting PI3Kβ will be an effective strategy to
inhibit thrombosis without increasing bleeding.
Inhibiting PDI
Thiol isomerases are oxidoreductases best known for
their role in protein folding. Protein disulfide isomerase
(PDI), a member of the thiol isomerase family, is secreted
by vascular cells and modifies targets involved in throm-
bus formation165. Mechanistic studies demonstrated an
important role for PDI in platelet activation. The proposed
mechanism of action includes reshuffling of disulfide
bonds within key surface receptors such as αIIbβ3 inte-
grin. The flavonol quercetin-3-​rutinoside (isoquercetin)
was the first identified PDI inhibitor with antithrombotic
activity. Administration of isoquercetin protected mice in
several models of thrombosis166. As it is commonly con-
sumed in fruits, vegetables and food supplements, iso-
quercetin quickly moved into phase I (NCT01722669)167.
A subsequent phase II trial demonstrated that isoquerce-
tin decreased levels of D-​dimer and soluble P-​selectin
in patients with advanced cancer and no patients devel-
oped VTE (NCT02195232)168. Further phase III trials
are planned with isoquercetin in patients with cancer
(J. Zwicker, personal communication).
Other antiplatelet strategies
Several additional antiplatelet strategies are emerging.
For example, targeting the active conformation of αIIbβ3
with a single-​chain variable fragment has been shown to
inhibit thrombosis without an increase in bleeding time169.
This has led to the concept that antithrombotic agents
can be delivered to developing thrombi, which in theory
should require a lower concentration of the antithrom-
botic and therefore be associated with less bleeding.
Indeed, single-​chain variable fragments have been
coupled to a variety of antithrombotic agents, including
the FXa inhibitor tick anticoagulant peptide170, and all
conjugates have exhibited antithrombotic activity in pre-
clinical studies without affecting haemostasis20. Another
approach is to target intracellular interactions that are
required for αIIbβ3 activation. One study found that dis-
rupting the binding of the intracellular domain of αIIbβ3
to Gα13 with a myristoylated peptide did not inhibit initial
platelet adhesion or primary aggregation, but inhibited
thrombosis in a mouse model171.
Platelets contain an oxygenase called 12(S)-
lipoxygenase (12-​LOX) that converts arachidonic acid
released from phospholipids into bioactive metabolites
(12(S)-hydroperoxyeicosatetraenoic acid and hydro­
eicosatetraenoic acid)172. 12-​LOX was found to regulate
various platelet responses, including integrin activation
and secretion, in response to stimulation with collagen
or thrombin173. In addition, 12-​LOX is required for
FcγRIIa-mediated platelet activation during immune-
mediated thrombosis in patients with heparin-​induced
Nature Reviews | Drug Discovery
Reviews
thrombocytopenia and sepsis174. ML355 is a 12-​LOX
inhibitor that shows >50-​fold selectivity compared with
other LOX paralogs175. In mice, ML355 was protective
against microvascular arterial thrombosis with no effect
on tail bleeding175. In vitro, ML355 shows comparable
inhibitory activity towards aggregation of human and
murine platelets, suggesting that studies in mice can be
translated to humans.
Bruton’s tyrosine kinase (BTK) is expressed in
platelets and is a critical component of the signalling
downstream to GPVI. The small molecule inhibitor,
ibrutinib, is an oral irreversible BTK inhibitor used to
treat patients with B cell malignancies176. One of the side
effects of ibrutinib is low-​grade bleeding events, which
occur in ~35% of treated patients177. Ex vivo studies on
collagen-​induced and plaque material-​induced platelet
aggregation under static and flow conditions support
the notion that ibrutinib inhibits platelet activation178. The
effects of BTK inhibitors on platelet function and their
usefulness as antithrombotic agents are currently under
investigation.
Development of new anticoagulants
All current anticoagulant agents either directly or indi-
rectly target components of the common pathway —
FXa and thrombin (Box 3, Figs 2,4). The goal of this
strategy is to partially inhibit these proteases to reduce
thrombosis. However, these proteases are essential for
haemostasis and therefore bleeding is the primary side
effect of current anticoagulants. Table 3 shows a list of
anticoagulant agents and their targets that have been
evaluated in clinical trials.
Inhibiting the TF–FVIIa complex
The TF–FVIIa complex plays an essential role in haemo-
stasis but also contributes to most forms of thrombosis21.
This complex can be inhibited by the endogenous inhib-
itor TFPI or by antibodies, pharmacological agents (such
as active site-​inhibited FVIIa) and proteins expressed
by blood-​sucking ticks (ixolaris) and worms (nematode
anticoagulant protein C2 (NAPc2))21. The antithrom-
botic effect of these inhibitors has been tested in preclin-
ical and clinical studies. NAPc2 suppressed prothrombin
F1.2 and reduced ischaemia without increasing bleed-
ing in patients with non-​ST-​segment elevation ACS
(NCT00116012)179. However, no further studies have
been performed. Active-​site inhibited FVIIa was eval-
uated in patients with acute lung injury/acute respira-
tory distress syndrome but was stopped early because of
increased bleeding and mortality180.
Targeting the intrinsic coagulation pathway
Components of the intrinsic pathway (FXII, FXI, FIX
and FVIII) are attractive targets for the development of
anticoagulants because their inhibition is less likely to
cause bleeding compared with anticoagulants that tar-
get the TF–FVIIa complex or common pathway181–184.
However, there is a trade-​off between efficacy and safety.
Earlier trials focused on targeting FVIII or FIX but these
agents were halted owing to a lack of efficacy and an
allergic reaction to the conjugate as well as for addi-
tional unknown reasons. We have included a discussion
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Table 3 | clinical trials of anticoagulant agents

Name/company Agent/route of Target Disease clinical trial Trial iD comments
administration
TB-402/ThrombGenics Antibody/IV FVIII NA Phase I NCT00612196 Completed; safety study
TKA Phase II NCT00793234 Completed; reduced VTE
THA Phase II NCT01344954 Completed; no data
REG1/NHLBI/Regado RNA Aptamer/IV FIX NA Phase I NCT00113997 Completed; safety study
Biosciences
CAD Phase II NCT01848106 Terminated early; allergic reaction
ACS Phase III NCT00932100 Terminated early; allergic reaction
TTP889/vTv Therapeutics Small molecule/oral FIX Hip repair Phase II NCT00119457 Completed; no effect
LVAD Phase II NCT00909298 Terminated
Ionis-416858/Ionis ASO/SC FXI TKA Phase II NCT02553889 Ongoing
Pharmaceuticals
ESRD on Phase II NCT03358030 Ongoing
haemodialysis
BAY-1213790/Bayer Antibody/IV FXI TKA Phase II NCT03276143 Ongoing
ESRD on Phase II NCT03787368 Ongoing
haemodialysis
MAA868/Novartis Antibody/IV FXI AF Phase II NCT04213807 Ongoing
BMS-986177/BMS Small molecule/oral FXI Recurrent stroke Phase I NCT02608970 Completed; safety study
Phase II NCT03766581 Ongoing
TKA Phase II NCT03891524 Ongoing
AB023/Aronora Antibody/IV FXI ESRD on Phase I NCT03097341 Completed; safety study
haemodialysis
Phase II NCT03612856 Ongoing
CSL312/CSL Behring Antibody/IV FXII Hereditary Phase II NCT03712228 Ongoing
angioedema
ACS, acute coronary syndrome; AF, atrial fibrillation; ASO, antisense oligonucleotide; CAD, coronary artery disease; ESRD, end-​stage renal disease; F, factor;
IV, intravenous; LVAD, left ventricular assist device; NA, not available; NHLBI, National Heart, Lung, and Blood Institute; SC, subcutaneous; THA, total hip arthroplasty;
TKA, total knee arthroplasty; VTE, venous thromboembolism.

of FVIII and FIX inhibitors herein because this infor-
mation may provide insights into the future success or
failure of targeting FXI or FXII.
Because individuals with congenital deficiency of
FXII do not bleed44, targeting FXII should be safe, but
efficacy may be reduced because TF–FVIIa-​dependent
and thrombin-​FXIa prothrombotic pathways will not be
inhibited. By contrast, targeting FXI will inhibit throm-
bin generation by blocking both FXIIa-​dependent and
thrombin-​dependent activation of FXI.
FVIII inhibitors. TB‐402 is a synthetic monoclonal
antibody against FVIII that inhibits FVIII activity by
80–90%. In a phase I study, TB-402 prolonged the aPTT
in a dose-​dependent manner, and, with higher doses,
the effect lasted up to 56 days185. Furthermore, in a
phase II study in patients undergoing knee replacement
surgery186, a single postoperative intravenous TB‐402
infusion resulted in a lower rate of VTE than with the
approved LMWH enoxaparin. The rates of major and
clinically relevant non-​major bleeding with the two low-
est doses of TB-402 were the same as with enoxaparin.
However, despite these promising results, development
of TB-402 was halted for unknown reasons.
FIX inhibitors. TTP889 is a small molecule oral inhib-
itor of FIXa. In a study comparing a once daily dose of
TTP889 with placebo for extended thromboprophylaxis
in 261 patients who had undergone surgery for hip frac-
ture187, there was little difference in the primary efficacy
outcome of VTE between patient groups. There were
no major bleeding events and two clinically relevant
non-​major bleeding events with TTP889. Because of
the lack of efficacy compared with placebo, develop-
ment of TTP889 was halted. TTP889 was also admin-
istered to patients with left ventricular assist devices to
determine if it would reduce levels of thrombin activa-
tion markers in the plasma, but no results have been
reported (Table 3).
SB249417 is a monoclonal antibody against FIX
that binds to the membrane-​binding carboxylation-​
carboxyglutamic (Gla) domain of human FIX. SB249417
attenuated thrombosis and infarct volume in rat mod-
els of arterial thrombosis and stroke, respectively188,189.
In addition, SB249417 demonstrated rapid and dose-
dependent prolongation of the aPTT in cynomolgus
monkeys and humans190,191 but was not carried forward
into a phase I trial for unknown reasons.
Pegnivacogin (REG1) is a FIXa-​directed inhibitory
RNA aptamer. A strength of using an RNA aptamer
is that it can be rapidly inhibited by a complementary
aptamer anivamersen (RB007). In phase I studies, intra-
venous REG1 prolonged the aPTT and activated clot-
ting time in a dose-​dependent manner, and these effects
were rapidly reversed with RB007 (ref.192). In the phase II
RADAR study, 640 patients with non-​ST-​s egment

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Antisense oligonucleotides
(ASOs). Antisense
oligonucleotides reduce
protein expression by binding
to mRNA and increasing its
degradation.
Nature Reviews | Drug Discovery
elevation ACS with planned early cardiac catheteriza-
tion via femoral access were randomized to REG1 with
RB007 reversal or to heparin193. At least 50% reversal
of REG1 was needed to prevent bleeding after sheath
removal, indicating that targeting FIX is associated with
bleeding. Allergic reactions were noted in a small num-
ber of patients. In the phase III REGULATE PCI trial,
patients undergoing PCI for non-​ST-​segment elevation
ACS were randomized to REG1 with 80% RB007 rever-
sal or to bivalirudin194. The study was stopped early after
partial enrolment owing to severe allergic reactions194,195.
The rate of the primary efficacy end point, a composite of
all-​cause death, MI, stroke and unplanned target lesion
re-​vascularization, was 7% and 6% with REG1 and biv-
alirudin, respectively. Major bleeding occurred in <1% of
patients in both groups. The allergic reactions with REG1
have been attributed to the polyethylene glycol conjugate
used to prolong the half-​life of the aptamer. However,
this adverse reaction halted the development of REG1.
FXI inhibitors. The most exciting new target for anticoag-
ulant therapy is FXI. There are several different develop-
ment programmes that involve antisense oligonucleotides
(ASOs), monoclonal antibodies, aptamers and small
molecules196,197 (Table  3). Small molecule inhibitors
can be delivered orally or parenterally, whereas ASOs,
antibodies and aptamers require parenteral adminis-
tration. ASOs reduce protein expression indirectly by
binding to target mRNA and causing its degradation. A
disadvantage of this approach is that it takes 3 to 4 weeks
of ASO treatment to lower FXI levels into the therapeu-
tic range. By contrast, FXI can be inhibited rapidly by
antibodies, aptamers and small molecules.
A particularly exciting agent in development is IONIS-
416858, an ASO directed against FXI198. A phase II trial
evaluated two subcutaneous doses of IONIS-416858 for
postoperative thromboprophylaxis in patients undergo-
ing elective knee replacement surgery199, with the higher
dose demonstrating superiority to enoxaparin in terms
of VTE with no increase in bleeding. Two phase II stud-
ies are currently assessing the safety and tolerability of
IONIS-416858 in patients with ESRD on haemodialysis
(NCT02553889 and NCT03358030).
Osocimab (BAY-1213790) is the most advanced anti-
​FXIa antibody200. In a phase II trial called FOXTROT,
BAY-1213790 was compared with enoxaparin for
throm­b oprophylaxis in patients undergoing knee
arthroplasty (NCT03276143). The study showed that
preoperative BAY-1213790 was superior to enoxaparin
for reducing asymptomatic DVT201. In addition, a small
phase II study is assessing the safety and tolerability of
BAY-1213790 in patients with ESRD on haemodialysis
(NCT0378368).
MAA868 is an antibody that binds to both FXI and
FXIa202. It reduced thrombosis in a mouse ferric chloride
carotid artery model and prolonged the aPTT in cyno-
molgus monkeys. In addition, a phase I trial reported that
a single subcutaneous dose of MAA868 prolonged the
aPTT for 4 weeks202. MAA868 is currently being evaluated
in a phase II trial in patients with AF (NCT04213807).
BMS-986177 is a small molecule oral inhibitor
of FXIa200 that was well tolerated in a phase I study
Reviews
(NCT02608970). Currently, BMS-986177 is being
compared with enoxaparin for thromboprophylaxis
after elective knee replacement surgery in a phase II
clinical trial (NCT03891524). In addition, it is being
compared with placebo in a phase II trial for second-
ary prevention in patients with stroke or high-​risk TIA
(NCT03766581). In the latter study, all patients receive
aspirin plus clopidogrel for 21 days followed by aspirin
alone thereafter. Evidence of new stroke or intracra-
nial bleeding on repeat brain imaging are the primary
efficacy and safety outcomes, respectively.
AB023 (xisomab) is a humanized version of 14E11,
an antibody that binds to the apple 2 domain of FXI
and inhibits FXIIa-​mediated FXI activation but not
thrombin-​mediated FXI activation203. AB023 has no
effect on FXIa activity. Therefore, even though it binds
FXI and not FXII, AB023 serves as an inhibitor of
FXIIa-​dependent activation of coagulation. In a phase I
study, AB023 prolonged the aPTT in a dose-​dependent
manner204. AB023 exhibits a short half-​life with low doses,
which likely reflects rapid binding of AB023 to plasma
FXI. However, once FXI is saturated with high doses of
AB023, the half-​life of the free antibody increases, likely
reflecting its slower clearance (NCT03097341)204. A
phase II trial is currently evaluating the safety and effi-
cacy of AB023 compared with heparin in patients with
ESRD while on haemodialysis (NCT03612856). The level
of clotting in the circuit and plasma will be assessed.
It is notable that three trials are analysing the effect
of FXIa inhibitors on thrombosis in patients with ESRD
on haemodialysis. However, TF has been shown to
contribute to haemodialysis-​induced thrombosis, rais-
ing concerns about the efficacy of inhibiting FXI205.
Nevertheless, we are eagerly awaiting results from these
trials to see the efficacy and safety of targeting FXIa.
FXII inhibitors. The most advanced inhibitor of FXII
is 3F7, a fully humanized monoclonal antibody. 3F7
attenuated thrombosis to the same extent as heparin in
a rabbit ECMO model62. CSL312 is an affinity matured
variant of 3F7. Notably, no thrombosis trials have been
reported for CSL312. Instead, CSL312 is being evaluated
in a phase II trial in patients with hereditary angioedema
(NCT03712228) because FXIIa also leads to the genera-
tion of bradykinin and increased vascular permeability in
these patients. Importantly, patients with a point mutation
in FXII that leads to increased autoactivation of FXII pres-
ent with hereditary angioedema type III owing to exces-
sive activation of the bradykinin-​forming kallikrein–kinin
pathway but not with thrombosis206. Indeed, the role of
FXII in thrombosis in humans is still unclear44.
Other anticoagulant strategies
An emerging approach to inhibit the activation of FXII
is to block its activators. Nucleic acid scavengers and
polyphosphate inhibitors have been shown to reduce
thrombosis in mouse models207,208. Studies have shown
that administration of DNase I, which degrades NETs
and cell-​f ree DNA, reduces venous thrombosis in
tumour-​bearing and non-​tumour-​bearing mice209–212.
Pulmozyme® is a commercial DNase used as an inhaled
therapy to remove extracellular DNA from the lungs
Reviews
of patients with cystic fibrosis. One study evaluated
the effects of intravenous followed by subcutaneous
dosing of recombinant DNase I in patients with lupus
nephritis213. DNase I was well tolerated but did not
reduce serum markers of disease. To date, these agents
have not been evaluated in thrombosis trials.
Challenges developing antithrombotic agents
One of the major challenges in the development of
new antithrombotic drugs is that we already have a
rich armamen­tarium of antiplatelet and anticoagulant
agents (Fig. 2, Boxes 1,3). Therefore, new agents must
demonstrate either superior efficacy and safety or
equivalent efficacy and improved safety compared with
existing agents.
Another challenge is the expense of the large clini-
cal trials required to assess new agents, particularly with
antiplatelet agents. For instance, the PAR1 antagonist
vorapaxar was assessed in the TRACER trial, which
involved 12,944 patients with ACS, and in the TRA
2P–TIMI 50 trial, which involved 26,449 patients with a
history of MI, ischaemic stroke or PAD95,96. However, the
TRA 2P–TIMI 50 trial included a rather diverse group of
patients who may have had different underlying diseases.
There are similar challenges with VTE trials. Guidelines
recommending VKAs for stroke prevention in AF were
based on randomized trials comparing them with aspirin
or no treatment in less than 3,000 patients. By contrast,
guidelines giving preference to DOACs over VKAs are
based on data in over 70,000 patients, and the recom-
mendation to use the combination of low-​dose rivarox-
aban plus aspirin over aspirin alone in selected patients
with CAD or PAD is based on a trial that enrolled over
27,000 patients100. Therefore, head-​to-​head studies com-
paring new anticoagulants with DOACs will require large
numbers of patients. To avoid this, niche indications need
to be identified such that the new agents can be compared
with placebo or standard-​of-​care agents that are known
to be problematic. Examples of such indications include
the use of FXII inhibitors to prevent central venous cath-
eter thrombosis in patients with cancer, an indication
where LMWH and VKAs have failed; the prevention of
recurrent stroke in patients with acute ischaemic stroke,
an indication where heparin, LMWH and heparinoids
have failed; the prevention of stroke in patients with AF
undergoing haemodialysis, an indication where there
is equipoise as to whether the benefits of VKAs out-
weigh their harms214; and the prevention or treatment of
VTE in patients with active cancer, an indication where
DOACs are superior to placebo or LMWH, respectively,
but cause more bleeding. Therefore, thoughtful selec-
tion of indications and creative trial designs are needed
to gain approval for the next generation of anticoagu-
lants. However, the selected patient population needs
to be large enough for the pharmaceutical companies to
make a profit on their investment. Of note, it would be
interesting to determine if a FXIIa antagonist can reduce
thrombosis associated with a mechanical heart valve.
Another consideration is determining in which spe-
cific indication the new agent may be most effective.
Thrombosis has been divided into AT and VT. However,
there are numerous different triggers of thrombosis in
different patients, leading to thrombosis at a variety of
sites. Thrombosis may be triggered by the activation
of platelets, of the coagulation system and/or of the
endothelium. For instance, patients with myeloprolifer-
ative neoplasia often have splanchnic vein thrombosis215.
In patients with CVD, the primary trigger is rupture or
erosion of atherosclerotic plaques, whereas the triggers
in patients with PAD are not known. In terms of VTE,
triggers range from artificial surfaces to inflammation
induced by surgery, which likely induces TF expression
on monocytes10,52. This means that, in contrast to VKAs
and inhibitors of the common pathway, a new anticoag-
ulant, such as a FXII antagonist, may be effective for one
indication but not for others.
Outlook
Thrombosis is the leading cause of death in the world.
Although major progress has been made in developing
effective antiplatelet agents (such as P2Y12 antagonists
for AT) and anticoagulants (such as the DOACs for
VT), incomplete protection and bleeding complications
associated with the use of currently available drugs call
for the development of novel therapeutic approaches
and the identification of new targets.
Notably, the PROSPECT study showed that the
cumulative rate of MACE in patients with ACS was
20.4% at 3 years216. In addition, three major trials (CURE,
TRITON and PLATO) have demonstrated that, irrespec-
tive of the combination of drugs, about 10% of patients
with ACS treated with DAPT are not protected94,217,218.
Furthermore, the risk of recurrence after stopping anti-
coagulants in patients with unprovoked VTE is about
10% at 1 year. Recent studies have shown that, compared
with placebo, aspirin significantly reduces recurrent
VTE219–221, which suggested a role for platelets in VTE.
However, a subsequent study showed that the FXa inhib-
itor rivaroxaban exhibited superior efficacy to aspirin in
preventing recurrent VTE for extended thromboprophy-
laxis and reduced the rate of recurrent VTE222. Similar
to DAPT, the optimal duration of anticoagulation after
VTE is unclear.
Improved strategies to prevent recurrent ischaemic
stroke (Box 2), where the current treatment is an anti-
platelet agent, are also needed. Ongoing trials are eval-
uating the effect of other available antithrombotics as
well as a variant of APC together with tPA on the treat-
ment of acute ischaemic stroke. Trials are also being
conducted to determine if PAR4 and FXI inhibitors —
novel antithrombotic agents — can reduce recurrent
stroke. FXI antagonists are further being evaluated in
preventing thrombosis in patients with ESRD on hae-
modialysis, a complication where the intrinsic pathway
of coagulation has a well-​documented role.
A more personalized therapeutic approach to throm-
bosis prevention would be beneficial. Indeed, one trial
found that genotyping patients for CYP2C19 genetic
variation improved antiplatelet therapy with P2Y12
antagonists. The development of similar genetic and
functional tests for predicting or measuring a patient’s
response to antiplatelet or anticoagulant therapy will be
critical for providing the best possible treatment to indi-
viduals. New challenges includes the global epidemics of
www.nature.com/nrd

obesity, metabolic syndrome and diabetes. For instance,
patients with type 2 diabetes exhibit increased platelet
reactivity and are less responsive to antiplatelet therapy74.
Major challenges in the establishment of novel thera-
pies include the demonstration of non-​inferiority of new
agents compared with approved agents and the high cost
associated with such large trials. However, while these
new agents are expected to cause less bleeding, the
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Acknowledgements
The authors would like to thank Cihan Ay, Jonathan Erlich,
Steven Grover, Raj Kasthuri, Carolyn Mackman and Dougald
Monroe III for helpful comments and Yohei Hisada for help
with preparing the manuscript.
Competing interests
G.A.S. and W.B. have no competing interests. N.M. has a grant
from Bayer and is a consultant for Bayer. J.I.W. is a consultant
for Bristol-Myers Squibb, Pfizer, Daiichi Sankyo, Bayer,
Janssen, Boehringer Ingelheim, IONIS Pharmaceuticals,
Merck, Portola, Perosphere, Servier, Anthos and Tetherex.
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