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Arterial thrombosis: a global challengeAtherothrombotic diseases are a major healthcare problem1 and are responsible for >25% of all deaths worldwide2. Long considered a disease of industrialized countries, recent World Health Organization statistics have highlighted the true global impact of this disease2, with ~80% of the world’s deaths from atherothrombosis occurring in low- and middle-income countries. The development of a clot in the coro-nary or cerebral circulation (causing acute myocardial infarction or ischemic stroke, respectively) is now the single most common cause of morbidity and mortality globally, and the prevalence of these dis-eases continues to rise, particularly in developing nations. Despite intense investigation over the last 40 years into the discovery and development of more effective antithrombotic drugs (Box 1), the effect of these therapies on mortality rates has remained disappoint-ingly small, with less than one in four individuals taking antithrom-botic therapies avoiding a fatal thrombotic event3. This situation will probably become more challenging in the future as the incidence of obesity, diabetes and the metabolic syndrome rapidly increases. These conditions are typically more resistant to the benefits of anti-thrombotic therapy4, and, thus, there is a need for the identification and development of more effective approaches to combat this global cardiovascular epidemic.

This review aims to integrate the current understanding of arterial thrombosis from insights gained from clinical and pathological studies with those obtained from experimental models. The main focus of this paper is not only that arterial thrombosis is central to vascular occlu-sion and the development of an acute, potentially life-threatening car-diovascular event, but also that it has a crucial role in accelerating the progression of atherosclerotic lesions. The formation of asymptomatic

thrombi in the arterial circulation seems to occur frequently in indi-viduals at high risk of cardiovascular disease, signaling an acute ‘flare up’ of an underlying chronic inflammatory vascular disease process. Although many of these thrombi do not cause symptoms, they are not harmless, as they promote the rapid progression of atherosclerotic lesions. The challenge remains to identify those individuals at high risk for arterial thrombosis and implement safe and effective anti-thrombotic strategies that can prevent thrombotic vascular occlusion as well as delay the accelerated progression of atherosclerotic lesions. Achieving this will require moving from a one-size-fits-all approach to the development of tailored atherothrombotic therapies.

Arterial thrombosis and atherosclerosis: evolving conceptsAtherosclerosis and arterial thrombosis have traditionally been considered separate entities with distinct pathogenic mechanisms, natural histories and therapies. Thrombosis is primarily mediated by platelets and fibrin5, whereas atherosclerosis is promoted by leukocytes, endothelial cells, smooth muscle cells and components of the adaptive immune response1. However, it is becoming increasingly clear that the cellular and biochemi-cal interactions underlying thrombosis are also directly relevant to athero-sclerosis6. Thus, dysregulated adhesive interactions between platelets, the endothelium and leukocytes are probably important throughout all stages of the atherothrombotic process7. Similarly, coagulation reactions linked to fibrin generation may also contribute to the rapid progression of athero-sclerotic lesions8,9.

As with many other chronic inflammatory diseases, which are marked by acute phases of inflammation on a background of chronic inflammation, there is a considerable body of evidence indicating that arterial thrombosis may signal an acute flare up of an underlying chronic vascular disease. That is, inflammatory changes in an unstable plaque leading to plaque rupture or fissuring stimulates a throm-botic event. These thrombi, which are often asymptomatic, have an important role in promoting the rapid progression of the underlying atherosclerotic lesions10,11. Unfortunately, this insidious cycle of acute inflammation and thrombosis is often only first recognized after the development of a catastrophic cardiovascular event. The challenge

1Australian Centre for Blood Diseases, Alfred Medical Research and Education Precinct, Monash University, Melbourne, Australia. 2Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA. Correspondence should be addressed to S.P.J. (shaun.jackson@monash.edu).

Published online 7 November 2011; doi:10.1038/nm.2515

Arterial thrombosis—insidious, unpredictable and deadlyShaun P Jackson1,2

The formation of blood clots—thrombosis—at sites of atherosclerotic plaque rupture is a major clinical problem despite ongoing improvements in antithrombotic therapy. Progress in identifying the pathogenic mechanisms regulating arterial thrombosis has led to the development of newer therapeutics, and there is general anticipation that these treatments will have greater efficacy and improved safety. However, major advances in this field require the identification of specific risk factors for arterial thrombosis in affected individuals and a rethink of the ‘one size fits all’ approach to antithrombotic therapy.

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remains to identify these high-risk phases in the disease, elucidate the specific pathogenic mechanisms in an affected individual and provide appropriate targeted therapies.

Molecular mechanisms underlying arterial thrombosisArterial thrombosis most frequently occurs after the rupturing or erosion of an unstable atherosclerotic plaque, particularly in the coro-nary circulation, exposing potent thrombogenic elements to flowing blood12–14. At the cellular level, arterial thrombi are primarily composed of aggregated platelets because of their unique ability to adhere to the injured vessel wall (platelet adhesion) and to other activated platelets (platelet aggregation) under conditions of rapid blood flow, as it occurs in stenotic diseased arteries. The mechanisms supporting platelet adhe-sion and aggregation at sites of vascular injury have been well defined and reviewed in detail elsewhere6,15–17; therefore, here I will provide only a brief description of the role of platelets and blood coagulation in promoting thrombus growth.

Platelets. After vascular injury, a number of subendothelial matrix proteins become exposed to blood, including von Willebrand factor (vWF), fibrillar collagens, fibronectin and laminin, which all support platelet adhesion through the engagement of specific receptors; vWF binds the platelet GPIb-V-IX complex18, collagens bind the glycoprotein receptor GPVI (refs. 19,20) and integrin α2β1 (ref. 21), and fibronectin engages integrin α5β1 and laminin α6β1 (ref. 18) (Fig. 1). The relative contribution of these individual receptor-ligand interactions to the platelet adhesion process depends on the prevailing blood flow condi-tions and the extent of vascular injury. Under conditions of rapid blood flow, as occurs in arterioles and stenotic arteries, vWF is involved in recruiting platelets to the site of vascular injury22. This unique adhesive function of vWF depends on the unique biomechanical properties of vWF-GPIbα bonds, which have a very rapid on-rate23. Circulating plasma vWF has limited binding potential for GPIbα; however, once immobilized onto subendothelial collagen (type I, III and VI) and exposed to hemodynamic drag forces, the vWF macromolecule adopts an unfolded conformation that facilitates engagement of a linear array of A1 domains with multiple GPIbα receptors24,25. The bond between GPIbα and vWF has an intrinsically rapid off-rate that does not support stable adhesion independent of other ligand-receptor interactions26. As a consequence, platelets typically translocate over a vWF substrate (Fig. 1), with efficient firm adhesion requiring the engagement of col-lagen, fibronectin or laminin (Fig. 1).

This multistep adhesion mechanism, which has long been recog-nized to be important for efficient platelet adhesion to sites of vascu-lar injury, is now increasingly recognized as being crucial for platelet aggregation and thrombus growth. Thus, propagation of a thrombus

Figure 1  Adhesion and activation mechanisms supporting the hemostatic and prothrombotic function of platelets. Major ligands and receptors mediating platelet adhesion and activation at sites of vascular injury. Platelets are captured in the injured vessel wall from flowing blood through the specific interaction of the platelet GPIb-V-IX complex with collagen-bound vWF exposed on the subendothelium (top). This ligand-receptor interaction has a rapid on-off rate that supports platelet translocation at the vessel wall. Stable platelet adhesion occurs through the binding of platelet GPVI to fibrillar collagen as well as the ligation of multiple β1 integrins, including the collagen α2β1 interaction and fibronectin engagement of α5β1. Once firmly adhered, platelets undergo a series of biochemical changes that induce integrin αIIbβ3 activation, leading to the high affinity interaction with adhesion proteins including vWF, fibrinogen and fibronectin. These adhesive interactions are indispensable in the ability of platelets to form stable aggregates with other activated platelets and promote thrombus growth. Activated platelets release or locally generate soluble agonists, including ADP, TXA2 and thrombin, that can induce autocrine or paracrine platelet activation (bottom). Each agonist activates specific G protein–coupled receptors on the platelet surface, including ADP-P2Y1 or ADP-P2Y12, TXA2-TP and thrombin–human PAR1 or PAR4, stimulating intracellular signaling events that induce cytosolic calcium mobilization. This second messenger has a key role in promoting integrin αIIbβ3 activation, TXA2 generation, granule secretion and in the procoagulant function of platelets.

Translocation

Adhesion

GPIb-V-IX

AggregationThrombusformation

GPVI

GPIb-V-IX

ITAM

DTS

Lowaffinity

Highaffinity

Solublefibrinogen

ADP

Primaryadhesion

COXAA

FibrinogenvWF FibronectinKey Collagen

Gi

TP

PAR1 or PAR4Thrombin

Gq

Gq

Flow

AbciximabEptifibatideTirofiban

SCH530348E5555

ClopidogrelPrasugrelTicagrelor

Ca2+ mobilization

IP3R

PLA2

PGH2

TXA2generation

Granulesecretion

P2Y12

αIIbβ3

α2β1

α5β1

GPVI

FcR γ -chain

β1 integrinsαIIbβ3

Integrin αIIbβ3‘activation’

δ granuleα granule

Aspirin

Box 1 Arterial thrombosis and acute coronary syndromes: historical aspects The causative link between arterial thrombosis and acute  coronary syndromes has a controversial history161,162. The initial link between coronary thrombosis and acute myocardial  infarction was recognized in the late 1800s (refs. 163–165)  and gained widespread acceptance within the medical  community throughout the early 1900s (ref. 166). But after a series of pathological studies that cast doubt on this association, alternative mechanisms were sought167–169. It was not until the use of coronary angiography in people with an acute myocardial infarction in the late 1970s (ref. 170), in combination with the success of thrombolytic therapies in the 1980s (refs. 171,172), that the causative role for thrombosis in precipitating unstable angina and acute myocardial infarction was unequivocally  established. Since then, there has been dramatic improvement  in the understanding and treatment options for the acute  coronary syndromes, with antithrombotic therapy taking center stage in the management of these diseases.

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Table 1 Antiplatelet agents in the clinic or in clinical trialsAnti-platelet strategy Drug

Type of agent; route of  administration Mechanism(s) of action Side effects and limitations or clinical trial status

Food and Drug Administration approved41,42

Blockade of prostanoid biosyntesis (TxA2)

Acetylsalicylic  acid (aspirin)

Salicylate drug; member of nonsteroidal  anti-inflammatory drugs (NSAIDs); oral

Irreversible acetylation of cyclooxygenase 1 (COX-1), inhibiting generation of TxA2

Bleeding, gastrointestinal toxicity  Weak anti-platelet agent, ~25% prevalence of  aspirin ‘resistance’

GPIIb-IIIa  inhibition

Abciximab  (ReoPro)

Mouse-human chimeric Fab of murine monoclonal antibody 7E3 (c7E3); intravenous

Reversible inhibition of integrin αIIbβ3 activation, also blocks integrin αvβ3

Bleeding, thrombocytopenia  Restrictions because of intravenous administration

Eptifibatide (Integrilin)

Synthetic disulfide-linked cyclic heptapeptide, structure based on KGD sequence of snake venom disintegrin; intravenous

Rapidly reversible, Arg-Gly-Asp (RGD) mimetic

Bleeding, small increase in profound thrombocytopenia  Restrictions because of intravenous administration

P2Y12 antagonists Tirofiban (Aggrastat)

Nonpeptide derivative of tyrosine; intravenous

RGD mimetic; rapidly reversible,  minimal effects on αvβ3

Bleeding, severe but reversible thrombocytopenia in small numbers of recipients  Restrictions because of intravenous administration

Ticlopidine (Ticlid) Thienopyridine prodrug; oral Active metabolite of parent compound irreversibly inhibits the ADP receptor P2Y12

Bleeding, gastrointestinal toxicity, rash, neutropenia, thrombotic thrombocytopenic purpura (TTP) (largely replaced by clopidogrel because of increased toxicity)

Clopidogrel (Plavix) Thienopyridine prodrug; oral Active metabolite of parent compound irreversibly inhibits P2Y12

Rash, neutropenia, TTP, major bleeding corresponding with >50% inhibition of P2Y12. Interpatient response variability

Prasugrel (Effient) Thienopyridine prodrug; oral Active metabolite of parent compound irreversibly inhibits P2Y12; improved potency and consistency over clopidoogrel. Pro-drug metabolism is more efficient.

Bleeding (more haemorrhagic side-effects than clopidogrel)

Ticagrelor (Brilinta) Cyclo-pentyl-triazolo-pyrimidine;  oral

Direct and reversible P2Y12 antagonist Recently approved (Box 3). Approved indication is for use with aspirin. Higher rate of major bleeding (compared to clopidogrel); increased dyspnoea and ventricular pauses

Agents that elevate camp and/or cGMP

Dipyridamole (Persantine)

Pyrimidopyrimidine derivative;  oral

Inhibitor of cyclic nucleotide phosphodiesterase (PDE), thromboxane synthase and adenosine deaminase.  Also inhibits reuptake of adenosine;  anti-platelet activity and vasodilatory properties

Headache, dizziness, hypotension, flushing, gastrointestinal toxicity, rash

Cilostazol (Pletal) Oral Selective type 3 phosphodiesterase  (PDE3) inhibitor, elevates cAMP to inhibit platelet aggregation; causes arterial vasodilation

Bleeding; headache; diarrhea; palpitations; dizziness; rash; pancytopaenia (~15% of individuals discontinue use because of side effects)

Investigational drugs currently in clinical trials41

P2Y12 antagonists Cangrelor ATP analog; intravenous administration

Direct acting, reversible P2Y12  antagonist

No significant increase in major bleeding events compared to clopidogrel. Two phase 3 trials (CHAMPION-PLATFORM and CHAMPION-PCI) halted due to lack of efficacy; phase 3 (BRIDGE) program aimed at demonstrating safe ‘bridging’ of subjects during the pre- and post-surgical period of risk is continuing (Box 3)

Elinogrel For the chemical structure,  see Box 3; oral (long term) or intravenous (acute)

Reversible P2Y12 antagonist. Phase 2 trials (INNOVATE-PCI) comparing elinogrel with clopidogrel showed promise; phase 3 trials suspended, August 2011 (Box 3)

Thrombin receptor antagonists

SCH530348 (Vorapaxar)

Synthetic tricyclic 3-phenylpyridine analog of himbacine; oral

High affinity, reversible PAR1  antagonist

Phase 2 trials, no major bleeding, generally well tolerated139; phase 3 trial (TRACER) recently halted (January 2011), potentially because of unacceptable bleeding; second phase 3 trial (TRA 2P TIMI 50) scaled back for 25% of enrolled participants with a history of stroke (Box 3). Further drug development is unclear

E5555 (Atopaxar) Low–molecular-weight molecule;  the chemical structure is  available159; oral

PAR1 antagonist Currently undergoing phase 2 trials; results show a trend to increase ‘nuisance’ bleeding, safety issues regarding liver dysfunction and QT interval prolongation require addressing159

Thromboxane antagonism

S-18886 (Terutroban)

2-aminotetralin derivative; terutroban sodium; oral

Selective prostaglandin endoperoxide  (TP) receptor antagonist: shows  anti-thrombotic, anti-vasoconstrictive  and anti-atherosclerotic properties

TAIPAD study, well tolerated, with a safety profile similar to aspirin (Box 3); well tolerated when taken in combination with aspirin160. Currently under evaluation in a phase 3 trial (PERFORM) for secondary prevention of acute thrombotic complications

Isoform selective inhibitors of  PI 3-kinase

AZD6482 Reversible; intravenous Isoform selective inhibitor of  PI 3-kinase p110β

Phase 1 trials, 2009, acceptable safety and tolerability profile (Box 3)

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into the lumen of an injured artery prompts blood flow to accelerate at the surface of the growing thrombus27, subjecting platelets and immobilized ligands, such as vWF, to extensional drag forces. As a consequence, platelet recruitment onto the thrombus surface becomes increasingly dependent on vWF and GPIbα. The formation of stable platelet-platelet adhesion contacts relies on a second adhesion step mediated by integrin αIIbβ3 binding to vWF28,29, fibrinogen30, fibrin and/or fibronectin31 (Fig. 1). Thus, irrespective of the type of vascular injury, thrombotic occlusion of blood vessels is dependent on the vWF-GPIb interaction as well as on integrin αIIbβ3. These findings help explain the nonredundant roles of these molecules in platelet thrombus formation and why qualitative or quantitative defects in these proteins lead to a bleeding disorder32; defects in vWF, GPIb-V-IX and integrin αIIbβ3 cause von Willebrand’s disease, Bernard-Soulier syndrome and Glanzmann’s thrombasthenia, respectively. These findings also explain the attractiveness of these molecules as therapeutic targets.

Stable platelet adhesion and activation. Once platelets firmly adhere to the vessel wall or to the surface of a growing thrombus, they undergo a complex series of morphological and biochemical changes that lead to the release of the contents of the platelet granules and upregulation of the adhesive function of integrin αIIbβ3 (refs. 33–35) (Fig. 1). Central to irreversible platelet activation is the generation and release of soluble agonists at sites of vascular injury. These include thromboxane A2 (TxA2) and dense-granule ADP, which act in an autocrine or a paracrine manner to potentiate platelet activation through specific G protein–coupled receptors, including the ADP purinergic receptors P2Y1 (ref. 36) and P2Y12 (ref. 37) and the thromboxane receptors TPα and TPβ (ref. 38). Both ADP and TxA2 stimulate platelets in a cooperative manner to enhance thrombus formation; thus, pharmacological targeting of TxA2 generation39 and/or the P2Y12 receptor40 are effective strategies to reduce thrombus propagation at sites of vascular injury41,42 (Table 1).

Blood coagulation and a-thrombin generation. An important func-tion of activated platelets is their ability to support the assembly of coagulation complexes on their plasma membrane, which is necessary for localized α thrombin generation and fibrin formation. Thrombin is among the most potent activators of platelets through proteolytic cleavage of surface protease-activated receptors, including PAR1 and PAR4, which act on human platelets43. Both thrombin stimulation of platelets and fibrin generation are important for thrombus growth and stability, and as a consequence, drugs that target thrombin generation (vitamin K antagonists and FXa inhibitors), thrombin catalytic func-tion (direct thrombin inhibitors) or thrombin activation of platelets (PAR1 antagonists) inhibit arterial thrombus growth41.

Although both platelet activation and blood coagulation are important for the formation and stability of arterial thrombi, the spatial and temporal regulation of these events varies considerably. For example, platelet recruit-ment preferentially occurs at regions of high shear and disturbed flow, leading to the formation of predominantly ‘white thrombi’ over the site of vascular injury, whereas maximal fibrin generation occurs in regions of low flow, often leading to the development of a ‘fibrin-rich thrombus tail’ (Fig. 2, top; Box 2). Thus, although antiplatelet agents are the cor-nerstone therapies of atherothrombotic disease, therapies that reduce thrombin generation (anticoagulants) or lyse the fibrin component of thrombi (fibrinolytic therapies) also have important roles in the manage-ment of this disease.

Role of platelets in the progression of atherosclerosisPlatelet proinflammatory function. Although the central importance of platelets in arterial thrombosis is well defined, an emerging field of research is focused on how platelets promote the initiation and propa-gation of atherosclerosis7. Adhesive interactions between platelets and endothelial cells at atherosclerotic-prone sites can enhance the recruitment and activation of proatherogenic monocytes. Through the release of cytokines (interleukin 1β (IL-1β)), chemokines (platelet factor 4 (PF4) and chemokine ligand 5 (CCL5, also called RANTES)), proinflammatory molecules (platelet activating factor (PAF) and leukotrienes) and other biological response modulators (CD40L, also called CD154), the interaction between platelets, endothelial cells and leukocytes establishes a localized inflammatory response that can accelerate the early formation of atherosclerotic lesions (Fig. 3). Platelets also release growth factors, such as platelet-derived growth factor (PDGF), which can stimulate smooth muscle proliferation and

Fibrin

Red thrombus:fibrin and red cells

Plaque

White thrombus:platelets

a

b

Vessel wall breach

Plaque rupture

Hemostaticplug

Thrombosis

Figure 2  Differential composition and localization of arterial thrombi relative to hemostatic plugs. (a) Occlusive arterial thrombi at sites of atherosclerosis plaque rupture. Angioscopy studies of the coronary arteries of people with an acute myocardial infarction have shown the presence of a white thrombus (platelet-rich mural thrombus) developing at the site of atherosclerotic plaque rupture. A ‘red thrombus’, composed of red blood cells and fibrin, preferentially forms in the low flow recirculation zones on the downstream margin of the developing thrombus. The distinct spatial localization of platelets and fibrin is directly caused by local blood flow alterations at the site of vascular injury. (b) A primary hemostatic plug forms rapidly at the site of vascular injury, extending into the extravascular space created by the wound. Notably, there is minimal extension of the clot into the lumen of the healthy artery. In contrast, a platelet-rich thrombus builds up on the surface of a disrupted atherosclerotic plaque, beginning in the intima and propagating into the intraluminal space, where it can induce major hemodynamic changes at the site of injury.

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angiogenesis in the plaque44. Thus, the concept has emerged that platelets may have an important role in the atherosclerotic process and that future therapeutic strategies to combat this disease may need to take into consideration both the prothrombotic and proinflamma-tory functions of platelets. It should be emphasized that most of the information regarding a role for platelets in atherogenesis has been derived from experimental animal models and that the importance of platelet-endothelial and platelet-leukocyte interactions in promoting atherogenesis in humans remains to be firmly established.

Platelet thrombi and the progression of atherosclerotic lesions. Although the importance of platelet proinflammatory function in vascular disease in humans is not clearly defined, the role of platelet-rich thrombi in accelerating plaque progression and increasing the risk of an acute coronary syndrome is well established. Fortunately, the majority of atherosclerotic lesions progress slowly over decades, and the transition to an abrupt life-threatening thrombosis occurs infrequently. Thus, the majority of individuals with atherosclerosis have an indolent clinical disease, and the impact of slow progres-sive vessel narrowing and chronic ischemia is partly compensated by the development of collateral vessels. Postmortem studies have suggested that plaque disruption in individuals with this type of disease typically resolves spontaneously and asymptomatically through a repair process similar to that which occurs during wound healing10,45,46. Thus, thrombi that form on disrupted plaques, for example, in asymptomatic coronary disease, often appear small and nonocclusive47, a finding that has been reproduced in mouse models of ruptured atherosclerotic plaques48. Notably, researchers from one study suggested that up to 14% of individuals with stable coronary disease that died of noncardiac causes have evidence of nonocclusive coronary thrombi49, indicating that asymptomatic arterial thrombus formation is a relatively common event.

Rapid progression of coronary disease occurring over months or a few years, rather than decades, seems to be a more aggressive clinical

course that is associated with unstable coronary lesions and a high risk of developing an acute coronary syndrome10,11,46. Thrombotic occlusion of coronary arteries in this situation may lead to more severe cardiac damage, caused in part by the reduced time frame available to develop an effective collateral circulation. Insights into the processes that lead to rapidly progressive coronary disease have been obtained from autopsy studies. Notably, it is not often that a single acute rupturing event of a coronary lesion causes occlusive thrombus formation47. More com-monly, occlusive thrombi form on lesions that have undergone multiple stages of rupture and repair14. Recurrent plaque rupture with subse-quent mural thrombus formation is considered to be a common cause of rapid progression of coronary lesions, which may partly explain angi-ographic findings of intermittent, rapid phases of plaque growth50,51 (Fig. 4). Thus, a dangerous cycle of plaque instability and intermittent thrombus formation may underlie the rapid progression of coronary artery lesions and the dynamic and rapidly evolving clinical features of acute coronary syndromes14. Of note, intraplaque hemorrhage and the associated development of a fibrin-rich clot within the lesion is also considered to be an important mechanism underlying rapid plaque pro-gression8,9, further highlighting the potential importance of hemostatic components in propagating advanced atherosclerotic lesions.

Factors promoting an exaggerated platelet aggregation responsePostmortem studies have shown that plaque rupture, in combina-tion with a severe preexisting arterial stenosis, is a situation that puts

Box 2 Hemostasis and thrombosis: two sides of the same coin Arterial thrombosis is primarily an exaggerated hemostatic response at sites of vascular injury. Guilio Bizzozero, the Italian physician who first defined the role of platelets in hemostasis in 1881, quickly recognized their central contribution to the development of thrombosis173,174. Thus, an inherent weakness with all currently used antiplatelet agents is their deleterious impact on hemostasis, with the most potent antithrombotic drugs typically conferring the greatest bleeding risk. Whether there are molecular targets that are truly ‘thrombosis specific’ remains unclear. Nevertheless, there are important differences between arterial thrombi and hemostatic plugs that need to be considered when targeting arterial thrombosis therapeutically. One of these differences relates to their relative localization in the vessel wall; whereas arterial thrombi form exclusively within the vessel  lumen, hemostatic plugs primarily form in the vessel wall and  extravascular space (Fig. 2). As a consequence, the blood flow conditions operating around forming thrombi and hemostatic plugs are quite distinct. Furthermore, the time frame of  hemostatic plug formation is usually different from that of  arterial thrombi. Hemostatic plugs typically form within  minutes of vascular injury to quickly stop bleeding, whereas  arterial thrombi can form over hours or days.

Box 3 Further information Further information on the treatment of thrombosis using brilinta (ticagrelor) can be found at the AstraZeneca website  (http://www.astrazeneca.com/Media/Press-releases/Article/ 20110720-fda-approves-brilinta-us), as well as at http:// cardiobrief.files.wordpress.com/2011/07/brilinta-label.pdf and http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ ucm263964.htm.

Information about the cangrelor clinical trials can be found at http://www.theheart.org/article/969693.do, http://www.medscape.com/viewarticle/702828, http://ir.themedicinescompany.com/phoenix.zhtml?c=122204&p=irol-newsArticle&ID=1287788 &highlight and http://seekingalpha.com/article/137521-what-cangrelor-failure-means-to-medicines.

Information about the elinogrel clinical trials can be found at http://www.pharmanews.eu/novartis/122-novartis-gains- worldwide-rights-to-elinogrel-a-phase-ii-anti-clotting-compound,  http://www.manufacturingchemist.com/technical/article_page/Antiplatelet_therapy__elinogrel/60859 and http://www. bioportfolio.com/news/article/757762/Novartis-Suspends- 24-000-patient-Elinogrel-Trial-In-Chronic-Chd.html. 

Information about the vorapaxar clinical trials can be found  at http://www.reuters.com/article/2011/01/13/us-merck-idUST RE70C4EH20110113 and http://online.wsj.com/article/SB 10001424052748703583404576079780771781652.html. Further information on the design of stroke-related clinical trials is at http://bd-d7000.info/category/11-worthington-library- medical-knowledge.

More information about clinical trials conducted by  AstraZeneca can be found at http://www.astrazenecaclinicaltrials.com/therapy-areas/cardiovascular/?itemId=8595603 and http://www.astrazenecaclinicaltrials.com/therapy-areas/cardiovascular/?itemId=8595601.

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Table 2 Thrombosis protection in mice with preserved hemostatic functionGenotype Injury model Consequences for thrombus formation in vivo

Cell surface receptors

Itga2−/− (α2)  • Pulmonary embolism model (collagen and epinephrine)  • Photochemical injury (Rose bengal), carotid artery

• No effect65 • Prolonged time to occlusion (~twofold)65

Fcgr1−/− (FcRg) or  Gp6−/− (GPVI)

Fcgr1−/−  •  Folts-type arterial thrombosis model (repetitive  

crush injury to stenosed carotid artery) • Electrolytic injury, carotid artery  • Laser injury, mesenteric120 and cremaster142 arterioles   • FeCl3 injury (8–10%): mesenteric and cremaster arterioles  •  Pulmonary embolism model (collagen and epinephrine) 

Gp6−/− • FeCl3 injury (10%), carotid artery

• No effect120 

• No effect120 •  Severe injury: no effect120,142. Moderate injury: 30% reduction in thrombus size120. 

Enhanced antithrombotic effect in the presence of anticoagulant120

• Delayed thrombus formation, delayed or absent vessel occlusion142 • Reduced pulmonary thrombi65  •  Delayed occlusion time associated with instability and frequent thrombus embolization143

Selp−/− (P-selectin)   •  Laser injury, cremaster muscle arterioles of Selp−/− or  Selp−/− mice

•  No antithrombotic effect tested. Developing thrombi contained minimal  tissue factor and fibrin144

Selplg −/− (PSGL-1)

•  Pulmonary embolism model (collagen and epinephrine), Selplg−/−

•   Milder thrombocytopenia, less fibrin deposition and lower number of  thrombosed vessels145

P2rx1−/− (P2X1) • Pulmonary embolism model (collagen and epinephrine)  • Laser injury, mesenteric arterioles

•  Reduced mortality, reduced vessel obstruction with rare incidence of  occlusive thrombi146

• Reduced size of mural thrombi146

Scarb1−/− (SRB-1) • FeCl3 (10%) injury, carotid artery •  Thromboprotective under dyslipidemic and hyperlipidemic conditions with delayed thrombus formation and vessel occlusion84

Cd36−/−  •  FeCl3 (12.5%) injury, mesenteric arterioles and  venules, carotid artery 

•  FeCl3 injury (7.5%), mesenteric arterioles and venules,  carotid artery

•  Prolonged time to occlusion under hyperlipidemic but not normolipidemic conditions85

•  Prolonged time to occlusion under normolipidemic conditions (note: reduced injury conditions)147

Slamf −/− (CD150  or SLAM)

• FeCl3 (10%) injury, mesenteric arterioles •  Delay in initial platelet deposition, prolonged occlusion time and increased incidence of embolization (apparent only in female mice)148

Sema4d −/−  (semaphorin 4D)

• FeCl3 injury, carotid artery  • Photochemical injury (Rose bengal), cremaster arteriole

• Moderately delayed occlusion time149 • No stable occlusion apparent in Sema4D−/− mice149

Adhesive ligands

Fibronectin  (conditional knockout)150; Fn1+/− (ref. 165)

• FeCl3 injury, mesenteric arterioles •  Delayed platelet-platelet aggregation and thrombus growth, increased incidence of embolization (small aggregates). Delayed vessel occlusion150,151

Cd40lg −/− • FeCl3 injury, mesenteric arterioles •  Affects stability of arterial thrombi (increased incidence of large emboli), prolonged time to occlusion152

Gas6 −/− • Ligation of inferior vena cava  • Photochemical injury (Rose bengal), carotid artery  •  Pulmonary embolism model (collagen and  

epinephrine)

• Reduced thrombus size (85% smaller)153 • Reduced thrombus size (60% smaller)153 •  Reduced mortality, lack of histological evidence of pulmonary embolization in 

surviving mice153

Intracellular signaling pathways

Arrb2−/−  (arrestin 2) • FeCl3 (5–10%) injury, carotid artery •  Delayed time to occlusion, reduced rate of occlusion and less stable arterial occlusion154

Pld1−/−  (phospholipase D1)

• Pulmonary embolism model (collagen and epinephrine)  • FeCl3 (15%) injury, carotid artery  • Abdominal aorta crush injury

• Reduced mortality at a lower dose of collagen and epinephrine155 • Prolonged time to occlusion, no stable occlusion155 •  Prolonged time to occlusion, reduced rate of occlusion and markedly reduced stable 

occlusion155

Pik3cb−/−  (PI 3-kinase p110β)

• Pulmonary embolism model (collagen and epinephrine)  • FeCl3 carotid artery injury

• No protection156 •  Ninety percent of mice showed partial occlusion (within this population, 30% 

presented with unstable thrombus)156

Prkcq−/− (protein  kinase C-θ)

• FeCl3 (10%), carotid artery • Prolonged time to occlusion and reduced stable occlusion157

Orai1−/− (CRACM1) • Pulmonary embolism model (collagen and epinephrine)  • Abdominal aorta mechanical injury  • FeCl3 (20%) injury, mesenteric arterioles  • Transient middle cerebral artery occlusion (tMCAO)

•  Significantly reduced mortality, thrombocytopenia and clot burden in pulmonary arteries110

• Reduced rate of occlusion and unstable occlusion110 • No difference110 • Reduced infarct size and improved neurological performance110

Coagulation factors

F11−/− (FXI) • FeCl3 (3.5–10.0%) injury, carotid artery • Protect against vessel occlusion158

F12−/− (FXII) • tMCAO  • Pulmonary embolism model (collagen and epinephrine)  • FeCl3 (20%) injury, mesenteric arterioles  • Abdominal aorta crush injury  • Injury caused by ligation of carotid artery

• Reduced cerebral infarct size and improved neurological performance98 •  Reduced mortality and significant reduction in thrombus burden in pulmonary arteries99

•  Normal initial platelet adhesion at site of injury, significantly impaired thrombus formation and stability99

• Reduced vessel occlusion, increased instability in all occlusive thrombi99 • Significantly reduced thrombus size with impaired stability99

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individuals at a very high risk for arterial thrombotic occlusion12. Nonetheless, angiographic studies of individuals who have undergone successful pharmacological thrombolysis have showed that >50% of the culprit lesions cause <50% of the coronary stenosis52,53. This indi-cates that in a high proportion of individuals with acute myocardial infarction, an exaggerated thrombotic response at sites of plaque disruption is probably a major factor in the development of disease. Multiple factors are known to contribute to an exaggerated plate-let response at sites of atherosclerotic plaque rupture including the presence of potent thrombogenic elements within the atherosclerotic plaque, heightened platelet reactivity and a loss of the normal control mechanisms dampening platelet activation, as well as localized blood flow disturbances at the atherothrombotic site.

Plaque thrombogenicity. Multiple factors contribute to the height-ened thrombogenic potential of ruptured atherosclerotic lesions, including the presence of tissue factor54,55 and fibrillar collagens in the lesion56, as well as the presence of platelet-activating lipids in the necrotic lipid core57,58. In advanced complicated lesions, tissue factor is thought to be derived from the shedding of procoagulant microparticles from the surface of apoptotic tissue macrophages and T lymphocytes59. Direct inhibition of tissue factor markedly reduces thrombus growth on ruptured atherosclerotic plaques54, providing a rational explanation for the benefits of anticoagulant agents in the management of the acute coronary syndromes. Type I and III collagens are among the most potent activators of platelets and support efficient platelet adhesion and aggregation under the rapid blood flow condi-tions that operate in stenosed arteries6. Blockade or deficiency of the platelet collagen receptors (GPVI and integrin α2β1) in humans60–64

and animals65,66 does not produce a marked bleeding tendency, raising the possibility that targeting platelet collagen receptors may be a well-tolerated approach to reduce excess platelet deposition on disrupted atherosclerotic plaques. However, this possibility is tempered by the observation that expression of collagen receptors on the surface of platelets varies widely, such that inhibition of a single collagen recep-tor can predispose to bleeding when the second collagen receptor is expressed at low levels67.

Loss of endothelial protective mechanisms. The endothelium has a major role in regulating the adhesiveness of platelets at the vessel wall. In addition to providing a strong negative charge that repels platelets from the vessel wall, the endothelium synthesizes a range of molecules that actively inhibit platelet activation and thrombin gen-eration. These include nitric oxide68, prostacyclin (PGI2)69 and CD39 (ecto-ADPase, also known as NTPDase)70–72, as well as the natural anticoagulant thrombomodulin73 (Fig. 3). Nitric oxide and PGI2 are powerful inhibitors of platelet activation that work by stimulating an increase in the intracellular levels of cyclic guanosine monophos-phate (cGMP) and cyclic adenosine monophosphate (cAMP), respec-tively. Agents that increase cAMP (cilostazol) and cGMP (nitrates) are used clinically and have potent antithrombotic and vasoactive effects. CD39 has a major role in metabolizing extracellular ADP, which is necessary to prevent premature activation at the vessel wall, whereas thrombomodulin rapidly inhibits the prothrombotic effects of α thrombin, thereby reducing platelet activation and fibrin genera-tion. Loss of these natural protective mechanisms at sites of vascu-lar injury has a major role in facilitating rapid platelet recruitment and activation.

Figure 3  The antiadhesive phenotype of endothelial cells is maintained through four intrinsic pathways: ecto-ADPase, prostaglandin I2 (PGI2), nitric oxide (NO) and the thrombomodulin (TM)-activated protein C (APC) pathways. In a hyperlipidemic milieu, the endothelium becomes inflamed and activated as a result of modified lipoprotein particles (cholesterol) and reactive oxygen species (ROS) accumulating in the intima.  This leads to the expression of adhesive ligands (including vWF and P-selectin (P-Sel)) on the endothelium that support platelet tethering  and rolling. Subsequent stable platelet adhesion occurs through the binding of  αIIbβ3-fibrinogen complexes with endothelial αvβ3 or intercellular adhesion molecule-1 (ICAM-1). Adherent platelets secrete numerous bioactive substances that alter the chemotactic and adhesive properties of the endothelium. Platelet-derived IL-1β induces endothelial secretion of IL-6 and IL-8 as well as surface expression of ICAM-1, αvβ3 and monocyte chemotactic protein-1 (MCP-1). Platelet CD40 ligand (CD40L) binds CD40 on endothelial cells, resulting in upregulation of adhesive molecules (ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), E-selectin and P-selectin), cytokine and tissue factor release and reduction in NO synthesis. In addition, platelet-leukocyte cross-talk promotes atherogenesis by recruiting leukocytes to endothelial-bound platelets through a multistep coordinated process. Initial tethering of leukocytes is mediated by the interaction of P-selectin expressed on the platelet surface with its cognate receptor leukocyte P-selectin glycoprotein ligand-1 (PSGL-1). Ligation of PSGL-1 promotes activation of leukocyte β2 integrins (Macrophage adhesion molecule-1 (Mac-1) and LFA-1), necessary for stable leukocyte adhesion. Bioactive mediators derived from α-granules of activated platelets, including platelet factor 4 (PF4), synergizes with RANTES (regulated upon activation, normal T-cell expressed and secreted) to enhance leukocyte adhesion and monocyte differentiation. Upregulation of COX-2 in monocytes via pathways involving IL-1β and P-selectin results in increased production of proinflammatory mediators including platelet-activating factor (PAF) and leukotrienes via transcellular eicosanoid metabolism.

APC

ICAM-1

ROS

ROSvWF

Protease

Cholesterol

Cholesterol

MacrophageFoam cell

DC

GPIb/V/IX

IL-6IL-8

Platelet RANTESPF4

Mac-1

FibrinogenADP

EctoADPase

IL-1β

NF-κB

PAFleukotrienes

P-Sel

PSGL-1

Cytokine

αvβ3

Intima

Inflamed endotheliumSmooth muscle cells

CD40LCD40 MCP-1

NO PrCPGl2

TM

Clot formation

Th

α granule

Antiplatelet action of healthy endothelium

Platelet-inflamed endothelial crosstalk

Platelet-leukocytecrosstalk

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flow on platelet adhesive function. Rheological disturbances have an important role in promoting occlusive thrombus formation6, as thrombus propagation and vessel occlusion will probably occur on plaques that markedly disturb laminar blood flow relative to non-stenotic lesions86. Experimental perfusion chambers incorporating fixed stenoses have highlighted the importance of disturbed blood flow conditions in accelerating platelet deposition onto the vessel wall87. High shear forces and rapid changes in blood flow accelerate platelet accumulation on thrombogenic substrates. Specific altera-tions in blood flow include flow acceleration at the apex of the ste-nosis as well as flow separation, eddy formation, flow reversal and turbulence at the post-stenotic region88–90. The decelerating low-shear zone that forms in the post-stenotic region provides an ideal flow environment for the progressive accrual and aggregation of platelets91. Experimental flow devices with fixed stenotic geometries have shown that shear gradients, such as regions of shear accelera-tion immediately followed by flow deceleration, can promote plate-let deposition onto thrombogenic surfaces27. Platelet aggregation induced by shear depends on the adhesive interaction between vWF, GPIb and GPIIb-IIIa92. Notably, platelet deposition onto reactive

Diapedesis

Leukocyte tethering and adhesion

Intima

Media

Internal elastic lamina

Unstable rapid progressionStable progressive disease

Fibrin

Fibrin

Subtotal: 70–90% stenosis (unstable angina)

Total occlusion: AMI

SMCs

Foamcells

Atherosclerotic plaquedevelopment

Fibrous cap(collagen)

Intimalthickening Necrotic

core

PlateletadhesionPlaque fisure

Plateletdebris

Increasing stenosis of vessel lumen

Layers of‘older’

thrombi

Figure 4  Arterial thrombosis and the rapid progression of atherosclerotic lesions. Leukocyte-platelet and leukocyte-endothelial interactions are crucial for initiating and propagating the development of atherosclerotic lesions. During stable progressive disease, the slow progressive recruitment of proatherogenic leukocytes and foam cells to sites of atherosclerosis and the development of a collagen-rich fibrous cap help stabilize the plaque. In contrast, during unstable rapid progression, there is development of a necrotic lipid core, inflammatory infiltrates and a thinning of the fibrous cap, which are all characteristic features of an unstable atherosclerotic plaque. Erosion or disruption of the fibrous cap exposes thrombogenic matrix proteins that promote platelet accumulation and local fibrin generation. Re-endothelialization of the lesion and incorporation of the organizing thrombus into the plaque leads to the progression of the atherosclerotic lesion. With repeated cycles of plaque injury and thrombus formation, progressive stenosis of the vessel lumen occurs, leading to marked reduction in blood flow and transient tissue ischemia (unstable angina) or total thrombotic occlusion of the artery and tissue infarction (acute myocardial infarction (AMI)).

Heightened platelet reactivity. Increased platelet reactivity is increasingly recognized as an important risk factor for arterial thrombosis, as individuals with hyper-reactive platelets have more chances to suffer acute coronary events74,75. For example, platelets from people with type 1 and type 2 diabetes are more sensitive to stimulation by soluble platelet agonists and form larger thrombi on thrombogenic surfaces76. The importance of excessive platelet reactivity in diabetes is underscored by the resistance to antiplatelet therapy observed in individuals with diabetes and the greater benefits derived from more intensive antiplatelet regimens when compared to individuals without diabetes77,78. Platelet hyperactivity is also clear in other thrombosis-prone groups, including individuals with hyper-tension79,80, those with hypercholesterolemia81, cigarette smokers82 and the elderly83.

Important new insights into the mechanisms of platelet hyperactiv-ity induced by hypercholesterolemia have recently been delineated from mouse models of dyslipidemia84,85. Platelets express scavenger receptors, including CD36 and scavenger receptor class B member 1 (SR-BI), that recognize modified proatherogenic lipoproteins. A spe-cific new class of oxidized choline phospholipids, termed oxPCCD36, accumulates in the plasma of hyperlipidemic mice and in humans with low high-density–lipoprotein levels. oxPCCD36 binds CD36 and stimulates platelet activation, leading to a prothrombotic phenotype85. Similarly, SR-BI can also influence platelet reactivity by scavenging plasma cholesterol and enhancing the cholesterol content of platelet membranes84. Although these findings were obtained primarily from the study of mice with severe plasma lipid disturbances (and remain to be substantiated in humans), they nonetheless raise the possibility that antagonism of platelet scavenger receptors may be an innova-tive approach to dampen the prothrombotic effects of proatherogenic lipids with minimal impact on hemostasis.

Disturbed blood flow. An important, yet incompletely under-stood, aspect of thrombogenesis is the impact of disturbed blood

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(and hemostatic plug), thereby increasing the risk of bleeding. Recent progress in understanding the mechanisms by which platelets sup-port blood coagulation have raised the possibility that selective inhibition of platelet procoagulant function may specifically reduce thrombin generation within a thrombus with the possibility of less bleeding (Fig. 5).

Platelet regulation of the contact phase of blood coagulation. Recent studies in mice have revealed that the contact phase of blood coagulation and, particularly, the coagulation factors FXII and FXI are involved in promoting arterial thrombus formation98–102. These studies have suggested that FXII activation on the surface of platelets can promote α-thrombin generation and fibrin accumulation during thrombus propagation. The mechanism by which platelets activate the contact phase of coagulation has remained elusive103, although recent studies have suggested a major role for inorganic polyphos-phates in this process104. Notably, inorganic polyphosphates and FXII seem to preferentially facilitate fibrin generation on the surface of growing thrombi and may be less important for fibrin generation in wounds. Pharmacologic antagonism of platelet polyphosphate by phosphatases or FXII (ref. 105) may therefore be an innovative anti-thrombotic approach that does not increase bleeding risk. But there is no clear evidence showing that humans with FXII deficiency are protected from arterial thrombosis.

surfaces induced by shear gradients is not prevented by aspirin, clopidogrel or thrombin inhibitors27, highlighting a prothrombotic mechanism that can undermine the benefits of commonly used anti-thrombotic drugs.

Flow stagnation, blood coagulation and thrombotic occlusion. An important consideration for arterial thrombotic occlusion is the effect of flow deceleration on the blood clotting process. Once a critical ste-nosis is reached as a result of the atherothrombotic process and super-imposed vasoconstriction, blood flow recirculation and stagnation downstream from the site of plaque injury becomes more prominent. Blood coagulation is favored under such conditions, leading to the propagation of a fibrin-rich and red-cell–rich thrombus (known as a fibrin tail)46,93. Angioscopic examination of the coronary vessels of individuals who have had an acute coronary event confirmed that platelet-rich thrombi are prominent in individuals with unstable angina94, whereas total vascular occlusion leading to an acute myocar-dial infarction is typically dependent on the subsequent development of a fibrin-rich and red-cell–rich thrombus95. Successful pharmaco-logical thrombolysis typically leads to the dissolution of the fibrin and red-cell component of the thrombus and a restoration of blood flow. These findings provide a rational explanation for the benefit of pharmacological thrombolysis in acute myocardial infarction and for its relative ineffectiveness in unstable angina.

Procoagulant function of platelets: emerging conceptsBlood coagulation typically commences at sites of vascular injury and propagates throughout the body of a thrombus, promoting throm-bus growth and stability. The development of a fibrin and red-cell thrombus on the surface of platelet-rich thrombi is dependent on the procoagulant function of the platelets and, potentially, of blood-borne microparticles96,97. All currently used anticoagulant agents indiscriminately inhibit coagulation reactions at the injured vessel wall and throughout the body of a developing platelet thrombus

Thrombin

Thrombin

Collagen

Poly P

XII

Va Xa

TF

?

Thrombin

Blood flow

Fibrin

Fibrin tail

Injured vessel wallSMCs

Membraneblebbing

Substrateproteolysis Bak/Bax

pore

Caspases

Apoptosis

↑ Intracellularcalcium

MPTP

ROSδ granule

PS exposure

Scramblase

PSPC

FactorXIIProcoagulant

activityFibrin

Clearance

Platelet

Cytochrome C

Necrosis

Prothrombin

Figure 5  Platelet procoagulant activity necessary for α-thrombin generation, fibrin formation and thrombus stability. Thrombin generation and fibrin accumulation occur at spatially discrete sites during thrombus formation (top). Fibrin generation at the site of vascular injury has a crucial role in anchoring the thrombus to the vessel and depends on tissue factor (TF) expression on the surface of smooth muscle cells and fibroblasts. Endothelial- and monocyte-derived tissue factor may also contribute to this process. Fibrin generation throughout the body of the thrombus, and in fibrin tails that form on the downstream margin of the thrombus, is thought to be primarily dependent on the procoagulant function of platelets. Tissue factor–bearing microparticles and leukocytes incorporated into the body of the thrombus may also enhance local thrombin generation. The regulation of platelet-derived procoagulant activity involves different factors and pathways (bottom). Platelets support the initiation of the contact phase of blood coagulation, particularly the activation of FXII, by releasing inorganic polyphosphates (Poly P) from the dense granules of activated platelets, which results in the production of thrombin and, ultimately, fibrin. The platelet provides a suitable surface for the assembly of coagulation protein complexes by expressing phosphatidylserine (PS). The exteriorization of phosphatidylserine is mediated by the calcium-dependent regulation of a phospholipid scramblase called TMEM16F. Platelet procoagulant function is tightly linked to the induction of platelet cell death pathways, including apoptotic and necrotic cell death. Both pathways perturb mitochondrial function through distinct mechanisms that ultimately lead to marked alterations in the platelet ultrastructure and surface membranes, leading to phosphatidylserine exposure. PC, phosphatidylcholine; Xa, coagulation factor Xa; Va, coagulation factor Va; Bak, Bcl-2 homologous antagonist/killer; Bax, Bcl-2-associated X protein.

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Identification of the elusive phospholipid scramblase. The contri-bution of platelet-derived phospholipids to thrombin generation has long been recognized. Central to this is the expression of phosphati-dylserine on the platelet surface by the poorly defined phospholi-pid scramblase106 (Fig. 5). Functional deficiency of scramblase has been described in individuals with Scott syndrome, a rare bleeding disorder107. The identity of the phospholipid scramblase has recently been elucidated with the discovery of a plasma membrane protein, TMEM16F, that confers calcium-dependent scrambling of phos-pholipids108. Notably, results from recent studies of mice deficient in the platelet calcium channel Orai1, which leads to reduced platelet cytosolic calcium flux, phosphatidylserine expression and platelet procoagulant function109,110, have shown protection from thrombosis without a substantial increase in bleeding110. These findings provide proof-of-concept evidence that dampening, but not eliminating, the calcium-dependent expression of platelet phosphatidylserine may be an effective and safe antithrombotic strategy.

Coagulation and the dying platelet. A unique feature of procoagulant platelets is that they have morphologic and biochemical features of dead or dying cells. For example, phosphatidylserine-expressing plate-lets undergo membrane blebbing, microvesiculation and cytoskel-etal disruption, they lose their intracellular granules, and they show marked alterations in their mitochondrial membrane potential111. This suggests that the pathways regulating cell death in platelets also regulate the platelet procoagulant phenotype. Programmed cell death is orchestrated by two classical pathways, apoptosis and necrosis, with recent evidence suggesting that activation of either pathway can pro-mote platelet procoagulant function in vitro112 (Fig. 5). Notably, most of the morphological and biochemical changes in platelets that are stimulated by potent platelet agonists, such as collagen and thrombin, are consistent with necrotic cell death111,112, raising the possibility that pharmacological modulation of this death pathway may be a new approach to modulate platelet procoagulant function in vivo.

Identifying new antithrombotic targetsMuch of the progress over the last two decades in the understand-ing of the molecular mechanisms regulating arterial thrombosis has come from the use of genetically engineered mice lacking one or more hemostatic components (Table 2). These mouse models, in combina-tion with sophisticated in vivo thrombosis models, have shed light on the complexities underlying the dynamics of thrombus growth and have led to the identification of potentially new antithrombotic targets113–115. In general, mouse models have faithfully reproduced the bleeding disorders identified in humans and have validated the antithrombotic targets of antithrombotic drugs that are convention-ally used116. In the future, these models may be increasingly used as screens for the identification of safer and more effective antithrom-botic targets.

Studies in mice confirmed the importance of subendothelial vWF, collagen and their platelet receptors (GPIb-V-IX, GPVI and integrin α2β1) in mediating platelet adhesion to the injured vessel wall66,113,117–123 (Fig. 1). Similarly, the central importance of integrin αIIbβ3 in mediating platelet aggregation and thrombus growth has been confirmed124–126. Perhaps slightly unexpected has been that mul-tiple αIIbβ3 ligands, including vWF, fibrinogen and fibronectin, con-tribute to distinct phases of the thrombotic process16. A great deal of insight has been gained into the contribution of thrombin, ADP, TxA2 and their receptors in promoting platelet activation, thrombus growth and stability127. Perhaps most notable from a clinical perspective is

the growing list of platelet molecules that seem to have an impor-tant role in promoting platelet thrombus formation and stability but which may be less important for hemostasis (Table 2 and Box 2). For some of these molecules, the defect in thrombus growth and/or stability depends on the thrombogenic stimulus and is more obvious in the arterial circulation than in veins, for example, FcRg and GPVI, α2-adrenergic receptor, PI 3-kinase p110β and Orai1. However, other molecules are probably more disease specific, such as CD36 and SRB-1, which are antithrombotic targets in the setting of hyperlipidemia, and other molecules seem to cause defects in both arterial and venous thrombi, for example, phospholipase D1, Gas6 and FXII. Notably, in most of the reported knockout mouse models, the level of anti-thrombotic protection conferred by the gene deletions has not been established. For example, it is not clear which of these targets would provide a relatively weak antithrombotic protection (an ‘aspirin-like’ effect), moderate protection (a ‘prasugrel-like’ effect) or potent pro-tection (‘GPIIb-IIIa–like’ effect). Given the growing clinical use of combination antithrombotic therapies, it will also be important to determine the amount of antithrombotic synergy when these specific knockout mice are administered antithrombotic drugs and at what cost this imposes from a bleeding perspective.

Although mouse models are informative, there are important limi-tations with this approach. For example, much of the progress in the understanding of arterial thrombosis has been based on the use of in vivo thrombosis models that do not accurately reproduce the con-ditions of arterial thrombosis in diseased atherosclerotic arteries in humans. Most thrombosis studies are performed on healthy vessels and involve injury techniques that have no direct relevance to plaque rupture. Moreover, the rheological conditions in the mouse micro-circulation that are used for intravital studies are markedly different from those operating in stenosed human arteries. Nonetheless, these models are informative in terms of defining the mechanisms that regulate thrombus initiation, growth and stability.

Clinical perspectiveOver the last two decades, we have witnessed remarkable progress in the treatment of arterial thrombosis and the acute coronary syndromes (Table 1). Much of the recent progress in antiplatelet therapy has been through the development of new inhibitors against the P2Y12 receptor. Some of these therapies have greater potency128,129, more predict-able pharmacokinetic and pharmacodynamic effects129, more rapid onset of action128,129 and/or a more rapidly reversible antithrombotic effect130 relative to clopidogrel. Two of these agents (prasugrel and ticagrelor) have been approved for use in the clinic in individuals with an acute coronary syndrome (Table 1).

Intensification of antithrombotic therapy. With the recognition that a high frequency of treatment failures occur with single antiplatelet therapy, there has been a strong push for the routine use of more intensive antiplatelet therapy. Thus, dual antiplatelet therapy with aspirin and P2Y12 antagonists has become the standard of care after an acute coronary event or after percutaneous coronary interven-tion42,131,132. In people with multiple cardiac pathologies, such as coronary disease with associated atrial fibrillation, left ventricular dysfunction or prosthethic heart valves, ‘triple therapy’ (anticoagulant warfarin in combination with dual antiplatelet therapy) is becoming increasingly used. Although the use of such an intense antithrom-botic regimen can be beneficial in groups of high-risk individuals, their widespread use cannot be justified because of the potential high incidence of bleeding42,133. For example, dual antiplatelet therapy is

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associated with a 4% annual incidence of major bleeding in individuals receiving the therapy, whereas triple therapy has been reported to cause a 12% incidence of bleeding episodes that require hospitali-zation134. Whether newer anticoagulant agents (such as dabigatran and rivaroxaban), which have a more consistent antithrombotic effect compared to warfarin, will cause less bleeding complications when combined with antiplatelet agents remains to be established.

We may be reaching a crossroad in terms of improving the out-come from high-intensity antithrombotic therapy, with any addi-tional clinical benefit being counteracted by an increased incidence of severe bleeding. There is a growing body of evidence indicating that bleeding is associated with adverse cardiovascular outcomes and carries a similar risk of death as recurrent ischemia135. Bleeding is a particular problem in older people with cardiovascular disease and associated comorbidities. In addition to the direct mortality caused by severe bleeding, blood transfusion is also associated with a worse outcome in individuals with acute coronary syndrome136,137. It is also possible that more intense antithrombotic treatment could lead to a greater risk of intraplaque hemorrhage. Blood vessels in atheroscle-rotic plaques are more fragile than normal vessels, and, as discussed earlier, plaque hemorrhage is a well known contributor to plaque inflammation and rapid plaque progression; the effect of potent anti-thrombotic regimens on this important complication therefore needs close scrutiny. Regardless of the underlying mechanisms that cause bleeding, the increasing awareness of the adverse effect of bleeding is prompting a reevaluation of antithrombotic regimens that maximize efficacy without increasing the risk of bleeding.

Future considerationsOptimizing antithrombotic approaches. Given the relevance of α-thrombin and platelets in the pathogenesis of arterial thrombosis, future developments in antithrombotic therapies will increasingly focus on the identification of antiplatelet and anticoagulant agents that can be used safely and effectively in combination. In this context, there is considerable interest in thrombin receptor antagonists (PAR1 antagonists), which reduce thrombin activation of platelets without affecting thrombin-induced fibrin generation, potentially causing less bleeding complications138. PAR1 antagonists have recently been tested in phase 2 clinical trials in combination with aspirin and clopidogrel and seemed to be well tolerated without increasing the risk of major hemorrhage139. But in a recent phase 3 trial (Thrombin Receptor Antagonist for Clinical Events Reduction), PAR1 antagonism in com-bination with dual antiplatelet therapy was associated with increased incidence of intracerebral hemorrhage in subjects with a past history of stroke, leading to premature termination of the study (Table 1). Therefore, careful subject selection will be required when combining PAR1 antagonists with dual antiplatelet therapy. It is noteworthy that anticoagulant therapy with warfarin has been shown to be as effec-tive as aspirin in reducing coronary thrombotic events, albeit with an increased risk of bleeding134,140. Safer combinations of antiplatelet and anticoagulant therapies may only be discovered through an improved understanding of the molecular pathways regulating platelet adhesive function and coagulation during the various stages of thrombus devel-opment. More targeted therapies, rather than the global inhibition of platelet function or coagulation, promises to offer a more rational and safer approach. In the case of platelets, a better understanding of the mechanisms regulating platelet adhesion and activation under the high shear conditions operating in stenosed arteries has the best chance of identifying a target that preferentially inhibits thrombo-sis growth without affecting physiological hemostatic responses.

Similarly, targeting processes that support thrombin generation on the surface of platelet thrombi, rather than globally inhibiting thrombin generation, may lead to therapies with a wider therapeutic window that can be used more freely with antiplatelet agents.

The case for tailored antiplatelet therapies. It has become increas-ingly clear that there is wide variability in platelet responsiveness to antiplatelet therapy and that a one size fits all approach to the manage-ment of arterial thrombosis is suboptimal141. Current antithrombotic approaches inhibit platelet function without regard to the underlying mechanisms promoting platelet hyperactivity. Subjects with diabe-tes, in particular, are more resistant to the antithrombotic benefits of standard doses of aspirin or clopidogrel. Major advances in the management of these individuals will probably require identification of the underlying pathogenic mechanisms leading to platelet hyper-activity and the development of specific tailored therapies. Central to this tailored approach will be the need to develop robust assays that can accurately assess the reactivity of platelets in each individual and monitor the response to therapy.

Preventing rapid plaque progression. All current antithrombotic therapies aim to reduce thrombotic occlusion of atherosclerotic arter-ies and thereby prevent ischemic injury to vital organs. But, as out-lined above, the formation of nonocclusive arterial thrombi at sites of plaque disruption can also accelerate the progression of atheroscle-rotic lesions, promoting adverse cardiovascular events. To date, there has been limited information on the impact of antiplatelet therapies on rapid plaque progression. There has also not been a clear under-standing of the pathways that should be targeted in order to minimize the impact of platelet thrombi on atherosclerotic lesions. Progress in this area will require the development of preclinical animal models that enable the investigation of thrombosis on rapid plaque progres-sion as well as the identification of biomarkers that can predict coro-nary instability and have sufficient reliability to monitor the effects of antiplatelet therapy. The development of more sophisticated imaging modalities that can detect rapid plaque progression, instability and subclinical thrombus formation will also be required. The dynamic nature of accelerated atherosclerosis and superimposed thrombosis provides a window for therapeutic intervention. Nevertheless, this intervention can only be successful with improvements in methods to noninvasively monitor the atherothrombotic process in humans in combination with the development of more effective targeted anti-thrombotic therapies.

AcknowledgmentsI would like to thank S. Schoenwaelder for preparation of the figures, constructive advice and for assistance with the manuscript. I also acknowledge H. Salem, M. Cooper and Z. Kaplan for their constructive advice and assistance with the manuscript. This work was supported by the National Health and Medical Research Council of Australia (NHMRC). I am an NHMRC Australia Fellow.

comPetIng FInAncIAl InteRestsThe author declares no competing financial interests.

Published online at http://www.nature.com/naturemedicine/. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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