The endothelial barrier and cancer metastasis : does the protective facet of platelet function matter?

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Commentary

The endothelial barrier and cancer metastasis: Does the protective facet of platelet function matter?

Marta Smeda

^a

, Kamil Przyborowski

^a

, Marta Stojak

^a

, Stefan Chlopicki

^a,b,⁎

aJagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland

bDepartment of Pharmacology, Jagiellonian University Medical College, Krakow, Poland

A R T I C L E I N F O Keywords:

Platelet inhibition Cancer metastasis

Platelet-cancer cell interaction Inflammation-associated hemostasis

A B S T R A C T

Overwhelming evidence suggests that platelets have a detrimental role in promoting cancer spread via platelet–cancer cell interactions linked to thrombotic mechanisms. On the other hand, a beneficial role of platelets in the preservation of the endothelial barrier in inflammatory conditions has been recently described, a phenomenon that could also operate in cancer–related inflammation. It is tempting to speculate that some antiplatelet strategies to combat cancer metastasis may impair the endogenous platelet–dependent mechanisms preserving endothelial barrier function. If the protective function of platelets is impaired, it may lead to increased endothelial permeability and more efficient cancer cell intravasation in the primary tumor and cancer cell extravasation at metastatic sites. In this commentary, we discuss current evidence that could support this hypothesis.

1. Introduction

It has been repeatedly demonstrated that platelets are involved in cancer metastasis, and a number of platelet–dependent mechanisms promoting metastatic processes have been identified. Therefore, anti- platelet therapy has been considered for decades as an attractive strategy to treat cancer. However, even though a number of platelet–dependent prometastatic mechanisms have been identified in experimental studies, no effective therapeutic strategy based on an antiplatelet regimen has yet been introduced into the clinic to prevent or treat cancer metastasis.

Moreover, in some studies, long–term treatment with antiplatelet agents including aspirin

[1]

and P2Y12 or PAR-1 receptor antagonists

[2]

had detrimental effects on cancer progression, indicating that our under- standing of the role of platelets in the context of cancer biology is still incomplete. Recently, it has been appreciated that, apart from their classical role in thrombosis, platelets also prevent the deleterious con- sequences of inflammation on vasculature by maintaining vascular in- tegrity. These mechanisms have not yet been studied in the context of cancer progression and metastasis, the pathophysiology of which is clo- sely linked to inflammation and destabilization of the endothelial barrier.

Therefore, it seems likely that in the pro–inflammatory environment of cancer, platelet–dependent mechanisms are important to support

endothelial barrier integrity and, accordingly, their inhibition could impair endothelial barrier function and favor metastatic spread. In this commentary, we briefly summarize experimental and clinical evidence that could support this hypothesis.

2. Detrimental role of platelets in cancer

The association between thrombosis and cancer was initially ob- served in ancient times. However, it was not until 1865 that Armand Trousseau in his famous lecture indicated that migratory deep vein thrombosis is the prognostic indicator of a “deep–seated concealed cancer”

[3], a phenomenon later confirmed by numerous researchers [4–6]. Subsequently, a number of studies pointed to the positive cor-

relation between increased platelet counts and worse prognosis in various types of cancers, with up to 20% of cancer patients found to have vascular thromboembolism

[7].

Since the early 1970s, a number of platelet–dependent mechanisms favoring metastatic spread have been identified, highlighting the role of platelets as active players in cancer growth and spread. Moreover, pla- telets have been acknowledged as “first responders” to the presence of cancer cells in the circulation

[3]. Indeed, tumor cells injected in-

travenously lead to immediate platelet activation in a process of tumor

https://doi.org/10.1016/j.bcp.2020.113886

Received 26 November 2019; Accepted 24 February 2020

⁎Corresponding author at: Jagiellonian Centre for Experimental Therapeutics, Jagiellonian University, Krakow, Poland.

E-mail address:stefan.chlopicki@jcet.eu(S. Chlopicki).

Available online 28 February 2020

T

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Fig. 1. Two facets of platelet function in cancer. (A) Detrimental role of platelet activation: The scheme represents selected platelet receptors and factors secreted from their α and dense granules inducing endothelium inflammation and promoting cancer metastasis[11,16–19,31,122]; (TF-: tissue factor; PAR-: Protease-activated receptor; CD97-: G protein-coupled receptor CD97; LPAR-: lysophosphatidic acid receptor; sLEx-: Sialyl LewisX receptor; GPIIb/IIIa-: glycoprotein IIb/IIIa (integrin complex); ADAM9-:

metalloproteinase domain-containing protein 9; integrins: α2β1, α5β1, αVβ1, α6β1; HMGB1-: high-mobility group protein 1; vWF-: von Willebrand Factor; P2Y1, P2Y12-:

platelet purinergic receptors; Psel-: P-selectin; TLR4-: Toll-like receptor 4; GPIb-IX-V-: Glycoprotein Ib-IX-V Receptor Complex; GPVI-: Glycoprotein VI; TXA2-: thromboxane A2; PS-: phosphatidylserine; TP-: TXA2 receptor). (B) Protective role of platelet activation: The scheme represents platelet-dependent mechanisms supporting endothelial integrity via C-type lectin-like receptor (CLEC-2)/podoplanin and GPVI-dependent mechanisms that induce secretion of endothelium-protective factors from platelets as well as participate in the plugging of endothelial holes by single platelets[76,87,89,123,124]. (PLCγ2-phosphoinositide-specific phospholipase C; SLP-76-: Lymphocyte lymphocyte cytosolic protein 2; Syk-: spleen tyrosine kinase).

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cell–induced platelet aggregation, contributing to the release of various pro–tumorigenic factors from platelets

[3]. For example, platelet–derived

transforming growth factor β1 (TGFβ1) has been shown to downregulate NK cell anti–tumor reactivity

[8], while simultaneously promoting cancer

malignancy by activating the epithelial–mesenchymal transition (EMT) of cancer cells

[9,10]. Furthermore, platelets increase the survival of circu-

lating tumour cells shielding them inside platelet–cancer cell hetero- aggregates from immune surveillance

[10–13]. Platelets have also been

shown to protect cancer cells against chemotherapy–induced apoptosis

[14,15]. Over the years, a plethora of other molecular mechanisms of

platelet–cancer cell interactions have been identified that promote cancer progression, including adenine nucleotides

[16], integrin α6

β

1[17], TLR4 [18], CD97[19]

clusterin and thrombospondin-1

[20], and many others

reviewed elsewhere

[10,11,21].

Fig. 1A

and

Table 1

present selected mechanisms of platelet–de- pendent processes that promote cancer progression. The complexity and various cross–talk mechanisms between platelets and cancer cells alongside cancer development are surprisingly well–orchestrated and render tumour–educated platelets that effectively support cancer growth and metastatic spread

[9,22].

3. Antiplatelet strategies in cancer

A possible efficacy of an antiplatelet regimen to combat cancer was first experimentally supported in 1968, when Gasic et al. found that neuraminidase–induced thrombocytopenia resulted in reduced experi- mental metastasis

[29]. Later, the effects of pharmacological inhibition

of platelet aggregation by aspirin

[30]

and of other antiplatelet stra- tegies

[9,21]

on cancer spread were investigated, and many were shown to decrease cancer progression and metastasis (see

Table 2).

These strategies included inhibition of glycoprotein platelet receptors GPIIb/IIIa, P-selectin

[31,32], GPIbα [33]

and GPVI

[34]; selective

inhibition of platelet storage granule release

[35]; the use of ADP

P2Y12 receptor antagonists

[26]; prostacyclin receptor agonist[36];

thromboxane receptor antagonists

[37]; thromboxane synthase inhibi-

tion

[38]; 12 –lipoxyganase inhibitor[39]; heparins and other thrombin

inhibitors

[40]; and PAR-1 receptor antagonists [41]. Furthermore,

detergent–extracted modified human platelets (platelet decoys) that retained platelet binding functions but were incapable of functional aggregation also inhibited metastasis, suggesting that antithrombotic and antimetastatic effects are closely linked

[42].

4. Emerging evidence for the lack of anticancer effects or even detrimental effects of antiplatelet treatment in cancer

Mounting experimental evidence has documented the involvement of platelets in the promotion of cancer progression, and resulted in a rather accepted view of platelet inhibition as a promising strategy to combat cancer. However, in recent years, data have been gathered from a number of experiments showing neutral or even negative effects of anticancer or antimetastatic strategies based on platelet inhibition (Table 2).

4.1. Aspirin

As early as the antimetastatic effectiveness of aspirin was evidenced by Gasic et al., this effect was already questioned by his contemporaries

[55]. Recently, a study elegantly demonstrated that inhibition of pla-

telet COX-1-derived TXA

2

by aspirin effectively reduced metastasis only when aspirin was applied in the intravascular phase of cancer, but the treatment was not effective when aspirin was administered in the ex- travasation or extravascular phases of metastasis

[44]. These results

underscore the link between the antimetastatic effect of aspirin and the acute inhibition of TXA

₂

-induced intravascular inflammation associated with cancer spread, simultaneously questioning the long–term anti- cancer efficacy of aspirin.

Indeed, although initial evidence from clinical studies suggested that aspirin prevented cancer metastasis

[45–47], this issue raised some

doubts later

[50–52]

and is currently being tested in the on-going ADD- ASPIRIN Trial

[56]. Moreover, the results of the recent Aspirin in Re-

ducing Events in the Elderly (ASPREE) trial revealed that long–term platelet inhibition with low–dose aspirin in healthy older adults was associated with a significantly increased number of cancer–related deaths

[1]. The latter data are especially alarming since aspirin has

been already recommended for primary prevention of colorectal cancer in healthy adults older than 76 years

[57].

4.2. Thrombin inhibitors and PAR-1 antagonists

In an experimental study, Niers et al. demonstrated that mice treated with ximelagatran (a direct reversible thrombin inhibitor) or hirudin (a bivalent direct thrombin inhibitor) for seven consecutive days up to 24 h before intravenous injection of melanoma cells

Table 1

Selected platelet-dependent mechanisms favoring cancer progression.

Platelet-derived factor/platelet receptor Mechanism(s) of action References

Transforming growth factor β1 (TGFβ1) Downregulation of NK cell anti–tumor reactivity, activation of epithelial–mesenchymal transition (EMT), primary tumor

growth [8–10]

ATP Triggering endothelium permeability by activation of P2Y2 receptor on endothelial cells, induction of EMT and

chemoresistance [16,23]

ADP Promotion of tumor cell survival via favoring the formation of platelet–tumor cell aggregates, activation of EMT and

chemoresistance, activation of VEGF release, increase in vascular permeability [10,23]

Integrin α6β1 Supporting platelet adhesion to various types of cancer cells via metalloproteinase domain–containing protein 9

(ADAM9) [17]

Toll-like receptor 4 (TLR4) Supporting platelet–tumour cell interaction via high-mobility group box1 (HMGB1) released by cancer cells [18]

Lysophosphatidic acid (LPA) Increasing tumor invasiveness and vascular permeability via proximal CD97-LPAR heterodimer signaling, increasing

tumor cell proliferation and secretion of pro-inflammatory IL-6 and IL-8 [19,24]

clusterin Promotion of cancer invasiveness by upregulation of metalloproteinase 9 (MMP9) expression in cancer cells [20]

Trombospondin-1 Promotion of cancer invasiveness by upregulation of MMP-9 expression in cancer cells [20]

Vascular endothelial growth factor

(VEGF) Activation of angiogenesis and increase in vascular permeability [10]

Glycoprotein VI (GPVI) Upregulating expression of cyclooxygenase 2 (COX-2) and EMT markers in cancer cells via interacting with galectin 3

containing collagen–like domain on cancer cells [25]

Platelet purinergic receptor P2Y12 Supporting primary tumor growth [26]

GPIIb/IIIa, GPIb-IX-V P-selectin Favoring platelet adhesion to cancer cells and formation of tumor cell–platelet aggregates [10]

Thromboxane A2(TXA2) Promotion of tumor cell survival in the circulation via favoring formation of platelet–tumor cell aggregates [10]

Chemokines CXCL5 and CXCL7 Recruitment of granulocytes to tumor cells and formation of early metastatic niche [27]

Protease-activated receptor (PAR-1) Selective release of pro-angiogenic VEGF with simultaneous suppression of antiangiogenic endostatin release from

platelets, increase in vascular permeability [28]

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displayed significantly more pulmonary metastases compared with control and acute one-dose or uninterrupted short (up to 24–h) treat- ment

[40]. These results show that long–term inhibition of thrombin

negatively affected metastatic spread and the authors ascribed the in- creased metastatic loads to more effective extravasation of cancer cells, though without mechanistically–oriented studies that would explain these findings. Clinically, safety monitoring of the PAR-1 antagonist vorapaxar use in cardiovascular patients in the Thrombin Receptor Antagonist for Clinical Event Reduction in Acute Coronary Syndrome (TRACER) trial also revealed a significant excess of solid cancers

[2].

4.3. P2Y12 receptor antagonists

Some alarming data have been also recently gathered from cardio- vascular patients participating in the Dual Antiplatelet Therapy (DAPT) trial. They were treated with the platelet purinergic receptor antago- nists prasugrel or clopidogrel on top of aspirin for 12 and 30 months, and the longer treatment increased incidence of cancer deaths

[2].

These data confirmed the earlier results of the TRITON trial (prasugrel vs clopidogrel) that also reported an increased number of cancer deaths following the treatment

[2]. Notably, the deleterious effects of platelet

inhibition with P2Y12 antagonists in the DAPT trial were particularly observed in cases of prolonged treatment and were more prominent in cases of a more potent platelet P2Y12 receptor antagonist. Based on these results indicating a possible cancer–favoring effects of potent and long–term antiplatelet regimens in patients with cardiovascular dis- eases, the authors suggested that more delicate platelet inhibition (i.e., aspirin alone) or, possibly, shorter exposure to dual oral antiplatelet agents should be used

[2].

In our recent experimental study we showed that clopidogrel used on top of aspirin increased breast cancer malignancy in mice

[53]. In-

terestingly, the substitution of the irreversible P2Y12 antagonists pra- sugrel or clopidogrel with their reversible counterpart ticagrelor on top of aspirin was not associated with substantially increased risk of cancer death as compared to prasugrel or clopidogrel

[58]. Therefore, based on

current evidence, prolonged DAPT in patients should be rather based on

ticagrelor and aspirin as an effective therapy reducing the number of cardiovascular events and, at the same time, being safe as regards the cancer risk (PEGASUS-TIMI 54 trial).

4.4. In search of the mechanism(s) underlying negative effects of antiplatelet and anti-thrombotic drugs on cancer progression

The mechanisms of the possible cancer–promoting effects of these antiplatelet and antithrombotic strategies have not been explained. The results suggest that targeting platelets for cancer treatment still re- presents a challenge and we need a better understanding of the me- chanisms controlling metastasis to develop specific antiplatelet thera- pies to impede cancer spread without possible unwanted side effects.

Especially puzzling are the results suggesting paradoxical prome- tastatic effects of vorapaxar and ximelagatran, as it is well known that cancer cells increase thrombin generation

[59], promoting plate-

let–cancer cell interactions and disrupting the endothelial barrier via PAR-1 receptor signaling. However, it has been reported that low doses of thrombin elicit the opposite barrier–protective effect via throm- bin–dependent activation of protective activated protein C signaling through PAR1-dependent sphingosine 1-phosphate receptor-1 (S1P1) crossactivation

[60,61]. Thrombin–dependent protection of the en-

dothelial barrier can be also elicited by activation of thrombin receptor PAR-4 expressed on platelets

[62,63]

leading to selective release of antiangiogenic endostatin (without an effect on proangiogenic VEGF release)

[28,64]. Therefore, paradoxically, inhibition of thrombin

generation or PAR-1 antagonism by antiplatelet agents can negatively affect endothelial barrier function. Furthermore, inhibition of platelets by antiplatelet agents can inhibit platelet–dependent mechanisms maintaining endothelial barrier function in the cancer inflammatory microenvironment, recently referred to as inflammation-associated hemostasis

[65–68]. In fact, it is tempting to speculate that long-term

inhibition of platelet-dependent mechanisms maintaining endothelial barrier integrity might paradoxically favor metastatic spread, as the endothelial barrier and endothelial NO–dependent function represent key elements of cancer cell extravasation and formation of metastases

Table 2

Effects of antiplatelet therapy on cancer progression.

Therapeutic target Antiplatelet approach References

INHIBITION OF CANCER PROGRESSION BY ANTIPLATELET/ANTITHROMBOTIC REGIMEN

Thrombin Hirudin, dabigatran etexilate nadroparin, Succ-100-LMWH [40,43]

Platelet COX-1 Aspirin [30,44–47]

Thrombocytopenia Neuraminidase, antibody–mediated platelet depletion [29,31]

GPIIb/IIIa Function blocking by Fab antibody fragment [31]

P-selectin P-selectin knockout mice [31]

GPIbα Function blocking by antibody [33]

GPVI Function blocking by F(ab)2 [34]

Platelet purinergic receptor P2Y12 Ticagrelor, P2Y12 knockout mice [26]

PGI2/iloprost Platelet inhibition with endogenous platelet inhibitor prostacyclin (PGI2) or its stable analog [36,37]

TXA2 1-(7-carboxyheptyl)imidazole, TBXAS1 (thromboxane A2synthase) knockdown mice [37,38]

PAR-1 PAR-1 (protease-activated receptor-1) knockdown mice [41]

Platelet decoys Detergent-extracted modified human platelets that retained platelet binding functions but were incapable of functional

activation and aggregation [42]

NEUTRAL EFFECT OF ANTIPLATELET/ANTITHROMBOTIC REGIMEN

Thrombin Dabigatran etexilate, rivaroxaban [48,49]

Platelet COX-1 Aspirin [44,50–52]

GPVI Antibody-mediated blockade [31]

Platelet purinergic receptor P2Y12 P2Y1 knockout mice [26]

PROMOTION OF CANCER PROGRESSION BY ANTIPLATELET/ANTITHROMBOTIC REGIMEN

Platelet purinergic receptor P2Y12 Clopidogrel and prasugrel on top of aspirin [2,53]

Thrombin Ximelagatran, hirudin [2,40]

PAR-1 Vorapaxar [2]

Platelet COX-1 Aspirin [1,51]

Thrombocytopenia Tpo (thrombopoetin) knockout mice [54]

GPIbα Function blocking by Fab antibody fragment [31]

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in the targeted organ

[69,70]. Similarly, inhibition of platelet–depen-

dent mechanisms might also impair the microvasculature of the pri- mary tumor and promote intravasation of cancer cells

[71]. Impairment

of these protective platelet–dependent mechanisms may explain some of the detrimental effects of antiplatelet strategies on metastatic spread reported in the literature. Furthermore, it may well be that, depending on the environment (local or bloodstream), the consequences of the interactions between platelets and tumor may promote or prevent cancer progression

[72].

5. Protective role of platelets in maintenance of endothelial barrier integrity

Platelets support the integrity of the endothelial barrier by the constitutive release of a plethora of mediators stabilizing adherens junctions between the neighboring endothelial cells and preventing extravasation of erythrocytes into the surrounding tissues, such as platelet factor 4 (PF-4), endostatin, or thrombospondin-1 (TSP-1)

[65,67,73]. This facet of platelet function is less stressed in the litera-

ture than is the ability of platelets to release factors increasing en- dothelium permeability such as vascular endothelial growth factor (VEGF), IL-1β or serotonin

[74–76]

The importance of platelets in maintaining the endothelial barrier has first been recognized in patients with immune thrombocytopenia

[65]

(also known as idiopathic thrombocytopenic purpura (ITP)). This disease features spontaneous bleeding into the skin that results in a characteristic petechiae due to postcapillary venular extravasation of red blood cells

[65]. Mechanisms of ITP have been ascribed to low

numbers of platelets, diminished production of platelet–derived factors, and impaired trophic signaling via various endothelial receptors, such as vascular endothelial growth factor receptor (VEGFR2), platele- t–activating factor receptor (PAFR), tyrosine–protein kinase receptor (Tie-2), endothelial differentiation gene 1 (EDG-1), epidermal growth factor receptor (EGFR), brain–derived neurotrophic factor receptor (BDNFR)

[65]. Further studies have demonstrated that classical factors

increasing endothelial permeability such as VEGF, when given alone, did not induce skin bleeding even in severely thrombocytopenic mice, whereas induction of skin inflammation by UVB irradiation caused cutaneous petechiae in thrombocytopenic patients but not in control subjects

[77]

pointing to the important role of platelets in preventing bleeding in inflamed organs

[78].

In fact, the important pathophysiological context for the role of pla- telets in preventing bleeding has been linked to inflammation–associated hemostasis, whereby platelets coordinate a host defense, serving as gatekeepers of the vascular wall and protecting the endothelial barrier against leaks, subsequent bleeding and excessive inflammatory damage

[66]. In recent years, the ability of inflammation to induce bleeding in

thrombocytopenia has been confirmed in a variety of models of in- flammation induced by a wide range of factors such as viruses, bacteria, injections, immunization, immune complexes, irritants, ischemia–r- eperfusion, and irradiation

[79], underscoring the important role of

platelets in the protection of vascular integrity in inflammation, in- dependent of the inflammatory stimulus.

5.1. Mechanisms of classical hemostasis and non-classical inflammation- associated hemostasis

Mechanisms of classical hemostasis are based on thrombus forma- tion under high shear stress conditions in response to vascular injury.

They are controlled by binding of the GPIb-IX-V complex on platelets to immobilized von Willebrand factor, followed by firm platelet adhesion mediated by direct binding of platelet GPVI and α2β1 receptors to collagen and von Willebrand factor to platelet GPIIb/IIIa receptor

[80].

Other subendothelial matrix components are also involved, including laminin and fibronectin binding to α6β1 and α5β1 integrins, respec- tively

[81]. Signaling via the GPVI receptor induces platelet activation,

degranulation, aggregation, and procoagulant activity in response to collagen

[82], a response involving thromboxane A2

production and ADP release from dense granules as major autocrine mechanisms re- inforcing platelet activation. The subsequent conversion of integrin GPIIb/IIIa to a high–affinity fibrinogen receptor

[81,82]

represents the final major step in platelet aggregation and thrombus formation

[83]. It

is tightly linked to phosphatidylserine (PS) exposure on the platelet surface and assembly of the tenase and prothrombinase complex with subsequent thrombin generation converting soluble fibrinogen into fi- brin, as well as the induction of the anticoagulant pathway mediated by activated protein C activity

[84].

In contrast to the classical mechanisms of thrombus formation, platelet–dependent inflammation–associated hemostasis and preven- tion of bleeding have been shown to be mediated by GPIIb/

IIIa–independent mechanisms in a number of models, such as IgG complex–mediated cutaneous RPA reaction

[85–88], LPS–induced lung

inflammation

[86,87], immunization with ovalbumin or complex

Freund’s adjuvant

[89], and syngeneic tumors grafted subcutaneously [66,68,90,91]. Furthermore, GPIbα, calcium, diacylglycerol–regulated

guanine nucleotide exchange factor I

[85], and G protein-coupled re-

ceptors (GCPRs)

[86]

are also not involved in the maintenance of pla- telet–dependent inflammation–associated hemostasis.

The major mechanism regulating non–classical platelet–dependent inflammation–associated hemostasis has been identified to depend on signaling from platelet immunoreceptor tyrosine activation motif (ITAM) –containing receptors

[66,86]. As reviewed by Lee and Berg-

meier

[92], the group of ITAM–containing receptors in mice comprises

CLEC-2 and Fcγ- chain receptors (the latter is functionally associated with the classical collagen receptor glycoprotein GPVI). Human plate- lets express an additional ITAM receptor:Fcγ RIIA. Podoplanin has been considered for a long time as the only known endogenous receptor for CLEC-2 although recently S100A13 expressed by vascular smooth muscle cells has also been found to bind platelet CLEC-2

[93]

while collagen and fibrin are well-known ligands for GPVI.

The mechanisms by which platelets limit bleeding and preserve endothelial barrier integrity in inflammation seem to be stimulus– and organ–dependent

[76]. They involve GPVI and CLEC-2 in the skin as

well as GPVI, CLEC-2, and GPIb in the lungs and the ischemic brain

[87]. Involvement of GPIIb/IIIa integrin and platelet secretion from α

and dense granules has also been shown to prevent bleeding in various models of inflammation

[87]. It is worth noting that platelet CLEC-2

also protected organs from the injury exerted by infiltrating in- flammatory cells in sepsis

[94]

and, thus, CLEC-2-dependent mechan- isms could hamper monocyte recruitment, lung injury, and, conse- quently, increased pulmonary permeability during cancer metastasis

[95]. To add to the complexity of mechanisms governing platelet–de-

pendent inflammation–associated hemostasis, these signaling pathways might simultaneously regulate other processes, for example, by exerting anti–inflammatory action as shown for the CLEC-2/podoplanin axis

[96]

or anticoagulant action as shown for PF4

[97]. It was surprising to

find out in the literature that the mechanisms regulating in- flammation–associated hemostasis seem to be also involved in the platelet–cancer cell interactions and classical thrombosis. For instance, the ITAM motif–containing CLEC-2/podoplanin pathway, identified as a major pathway limiting inflammation–associated bleeding

[89], is

also involved in classical thrombosis, as CLEC-2–deficient mice and inhibition of podoplanin have been shown to limit the number of thrombotic events

[98]. Furthermore, podoplanin expressed on cancer

cells and CLEC-2/podoplanin interactions have been shown to stimulate cancer progression

[99,100]. Similarly, ITAM motif–containing platelet

GPVI, also involved in inflammation–associated hemostasis

[34,88],

additionally plays a classical role in thrombosis

[101]

and stimulation of cancer growth and spread via its binding to galectin 3 expressed on tumor cells

[11].

Initially, platelet–dependent mechanisms involved in in-

flammation–associated hemostasis were suggested to be distinct from

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the classical mechanisms regulating platelet aggregation and thrombus formation

[66]. However, recent data indicate that platelet–dependent

mechanisms governing platelet aggregation and thrombus formation (classical hemostasis) and those of non–classical hemostasis depending on CLEC-2 are rather interlinked than distinct

[102]. If so, targeting

platelet aggregation and thrombus formation by antiplatelet regimens in cancer might simultaneously impair platelet–dependent non–- classical inflammation–associated hemostasis.

6. Endothelial barrier integrity and cancer cell intravasation in the primary tumor and extravasation in the metastatic organ

The endothelial barrier is crucial for controlling the rates of cancer spread both in primary tumors and in metastatic organs. The metastatic process starts from losing tumor cell–cell adhesion capacity and changes in the tumor cell–matrix interactions. During this step, cancer cells downregulate E-cadherin expression and activate mechanisms al- lowing for degradation of the extracellular matrix (e.g., metalloprotei- nases, proteolytic urokinase–type plasminogen activator system)

[103].

To form distant metastases, cancer cells must cross the endothelial barrier twice, firstly entering the circulation in the primary tumor (intravasation) and secondly leaving the bloodstream at the metastatic site in the process of extravasation via transendothelial or paracellular routes

[104]. Both processes are tightly linked to local vascular in-

flammation in the primary tumor and the metastatic organ, as well as with systemic inflammation

[105,106]. Therefore, it is tempting to

speculate on the important role of platelets in the maintenance of en- dothelium integrity in the pathophysiological context of intravasation and extravasation of cancer cells (Fig. 1B).

6.1. Platelets and endothelial barrier integrity in the primary tumor

As regards the primary tumor, it has been shown that thrombocy- topenia consistently results in massive bleeding in the surroundings of the tumor, without affecting vascular integrity elsewhere

[90,107].

This suggests that its dysfunctional microvasculature with an in- flammatory microenvironment might be particularly prone to defi- ciency of the platelet–dependent safeguarding role and maintenance of the endothelial barrier. Indeed, platelet–derived factors have prevented β2 and β3–dependent macrophage and neutrophil infiltration into the primary tumor as well as destruction of the primary tumor vasculature

[90,91]. Similarly, it has been recently shown that blocking of platelet

GPVI function increased intratumor bleeding

[34]. In addition, we have

also shown that pharmacological platelet inhibition by the combination of aspirin and clopidogrel promoted dysfunction of the primary tumor vasculature triggering induction of vascular mimicry which increased cancer malignancy

[53]. In this study, an antiplatelet regimen resulted

in lower expression of eNOS and higher expression of Ang-2 in the primary tumor in response to platelet inhibition, in line with the emerging role of platelets in the regulation of the vascular network of the malignant tissue

[66,90].

6.2. Platelets and endothelial barrier integrity in metastasis to the lungs

In contrast to the accumulating evidence on the platelet–dependent regulation of endothelial barrier integrity in the primary tumor vascu- lature affecting cancer cell intravasation, less is known about the me- chanisms involved in the regulation of cancer cell extravasation in the metastatic organ. Obviously, the mechanisms of extravasation are or- gan–specific to some extent. It could well be that the protective role of platelets is of particular importance in the regulation of metastasis to the lungs since thrombocytopenia significantly increases pulmonary vascular permeability, reversed by restoring the circulating platelet population

[108].

Adhesion of cancer cells to the pulmonary endothelium, followed by extravasation and subsequent colonization of the lungs

[109,110],

involves many cross–talk mechanisms between cancer cells, platelets, and leukocytes in heterocellular aggregates. Early pulmonary en- dothelial dysfunction, characterized by impaired NO–dependent func- tion and increased endothelial permeability, also involves glycocalyx disruption and endothelial inflammation

[106,111]. Altogether, the

major mechanisms of early pulmonary metastasis involve tumor–- induced host pulmonary endothelial response and increased endothelial permeability associated with the pro–thrombotic response of platelets and activation of leukocytes

[112–114]. Thus, targeting cancer cel-

l–induced local endothelial barrier dysfunction in the metastatic organ preceding metastatic spread could be an effective strategy to prevent or lower metastasis rates at an early stage of cancer, while factors that impair the endothelial barrier should be inhibited as they favor me- tastasis.

Good examples supporting the central role of the endothelial barrier in metastasis come from studies showing that atrial natriuretic peptide

[115,116]

and vasculotide

[117]

not only limited an increase of en- dothelial permeability but also decreased metastasis. On the other hand, inhibition of NO production intensified endothelial permeability both in vitro

[118]

and in vivo

[119]

indicating that NO is one of the important players regulating endothelial barrier integrity in addition to prostacyclin and the cAMP–mediated pathway

[120]. Similarly, co-

operation between NO and PGI

2

plays an important role in the en- dothelial–dependent regulation of platelet activity

[121].

Determination of the decisive factors maintaining the endothelial barrier in a metastatic organ – which involve multiple en- dothelial–dependent mechanisms far beyond NO and PGI

2

-depedent regulation as well as multiple blood cell–dependent mechanisms other than those linked to platelet–cancer cells interactions – represents a fundamental challenge to develop novel treatment strategies to prevent metastasis. In this context, it seems possible that platelet–dependent mechanisms of inflammation-associated hemostasis play a role in the maintenance of endothelial barrier function in the metastatic organ, and impairment of these mechanisms favors metastatic spread, parti- cularly to the lungs, with pulmonary vascular permeability regulated by platelets

[108].

7. Conclusions

The well–known role of platelet adhesion, aggregation, and thrombus formation in the promotion of cancer growth and spread has been widely investigated since the 1960s as a possible therapeutic target to reduce dissemination of cancer cells. In contrast still little is known about the vital role of platelets in maintenance of the en- dothelial barrier in the primary tumor and metastatic organ. Although initially the classical mechanisms of thrombosis and hemostasis were thought to be distinct from mechanisms of inflammation–associated hemostasis, they seem to be interlinked. Thus, at the current stage of knowledge, it is uncertain whether long-term anticancer or antimeta- static therapy based on the inhibition of platelet–dependent thrombotic mechanisms supporting cancer would not impair simultaneously the endogenous platelet–dependent mechanisms preserving endothelial barrier function leading to increased endothelial permeability and more efficient cancer cell intravasation in the primary tumor and their ex- travasation at metastatic sites. Clearly, the consequences of the activity of platelets may depend on the local environment, showing detrimental or protective effects on cancer progression

[72]. In conclusion, efficient

targeting of platelets to combat cancer requires better understanding of the platelet–dependent mechanisms involved in the preservation of the endothelial barrier (Fig. 1B) as compared with those of platelet–cancer cell interactions favoring metastasis (Fig. 1A) in order to target the latter mechanisms selectively.

Conflict of interest

None.

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CRediT authorship contribution statement

Marta Smeda: Conceptualization, Writing - original draft, Writing -

review & editing. Kamil Przyborowski: Writing - review & editing.

Marta Stojak: Writing - review & editing. Stefan Chlopicki:

Conceptualization, Writing - final manuscript, Writing - review &

editing.

Acknowledgements

The study was supported by The National Centre for Research and Development (STRATEGMED1/233226/11/NCBR/2015).

References

[1] J.J. McNeil, M.R. Nelson, R.L. Woods, J.E. Lockery, R. Wolfe, C.M. Reid, B. Kirpach, R.C. Shah, D.G. Ives, E. Storey, J. Ryan, A.M. Tonkin, A.B. Newman, J.D. Williamson, K.L. Margolis, M.E. Ernst, W.P. Abhayaratna, N. Stocks, S.M. Fitzgerald, S.G. Orchard, R.E. Trevaks, L.J. Beilin, G.A. Donnan, P. Gibbs, C.I. Johnston, B. Radziszewska, R. Grimm, A.M. Murray, Effect of aspirin on all- cause mortality in the healthy elderly, N. Engl. J. Med. 379 (2018) 1519–1528, https://doi.org/10.1056/NEJMoa1803955.

[2] V.L. Serebruany, V. Cherepanov, E.Z. Golukhova, M.H. Kim, The dual antiplatelet therapy trial after the FDA update: noncardiovascular deaths, cancer and optimal treatment duration, Cardiology 132 (2015) 74–80,https://doi.org/10.1159/

000431356.

[3] D.G. Menter, S.C. Tucker, S. Kopetz, A.K. Sood, J.D. Crissman, K.V. Honn, Platelets and cancer: a casual or causal relationship: revisited, Cancer Metastasis Rev. 33 (2014) 231–269,https://doi.org/10.1007/s10555-014-9498-0.

[4] E.A. Edwards, Migrating thrombophlebitis associated with carcinoma, N. Engl. J.

Med. 240 (1949) 1031–1035,https://doi.org/10.1056/NEJM194906302402601.

[5] M. Linquette, R. Mesmacque, P. Fossati, G. Luez, B. Beghin, Recurrent and migratory venous thromboses, Prog. Med. (Paris) 92 (1964) 689–698 (accessed November 14, 2019),http://www.ncbi.nlm.nih.gov/pubmed/14242585.

[6] S. Noble, J. Pasi, Epidemiology and pathophysiology of cancer-associated thrombosis, Br. J. Cancer. 102 (2010) S2–S9,https://doi.org/10.1038/sj.bjc.6605599.

[7] M. Haemmerle, R.L. Stone, D.G. Menter, V. Afshar-Kharghan, A.K. Sood, The platelet lifeline to cancer: challenges and opportunities, Cancer Cell. 33 (2018) 965–983,https://doi.org/10.1016/j.ccell.2018.03.002.

[8] H.-G. Kopp, T. Placke, H.R. Salih, Platelet-derived transforming growth factor-beta down-regulates NKG2D thereby inhibiting natural killer cell antitumor reactivity, Cancer Res. 69 (2009) 7775–7783,https://doi.org/10.1158/0008-5472.CAN-09- 2123.

[9] X.R. Xu, G.M. Yousef, H. Ni, Cancer and platelet crosstalk: opportunities and challenges for aspirin and other antiplatelet agents, Blood 131 (2018) 1777–1789, https://doi.org/10.1182/blood-2017-05-743187.

[10] B. Tesfamariam, Involvement of platelets in tumor cell metastasis, Pharmacol.

Ther. 157 (2016) 112–119,https://doi.org/10.1016/j.pharmthera.2015.11.005.

[11] M. Schlesinger, Role of platelets and platelet receptors in cancer metastasis, J.

Hematol. Oncol. 11 (2018) 125,https://doi.org/10.1186/s13045-018-0669-2.

[12] C. Philippe, B. Philippe, B. Fouqueray, J. Perez, M. Lebret, L. Baud, Protection from tumor necrosis factor-mediated cytolysis by platelets, Am J Pathol. 143 (1993) 1713–1723.

[13] B. Nieswandt, M. Hafner, B. Echtenacher, D.N. Männel, Lysis of tumor cells by natural killer cells in mice is impeded by platelets, Cancer Res. 59 (1999) 1295–1300.

[14] O. Elaskalani, M.C. Berndt, M. Falasca, P. Metharom, Targeting platelets for the treatment of cancer, Cancers (Basel) 9 (pii) (2017) E94,https://doi.org/10.3390/

cancers9070094.

[15] A. Radziwon-Balicka, C. Medina, L. O’Driscoll, A. Treumann, D. Bazou, I. Inkielewicz-Stepniak, A. Radomski, H. Jow, M.W. Radomski, Platelets increase survival of adenocarcinoma cells challenged with anticancer drugs: mechanisms and implications for chemoresistance, Br. J. Pharmacol. 167 (2012) 787–804, https://doi.org/10.1111/j.1476-5381.2012.01991.x.

[16] D. Schumacher, B. Strilic, K. Sivaraj, N. Wettschureck, S. Offermanns, Platelet- derived nucleotides promote tumor-cell transendothelial migration and metastasis via P2Y2 receptor, Cancer Cell. 24 (2013) 130–137,https://doi.org/10.1016/j.

ccr.2013.05.008.

[17] E. Mammadova-Bach, P. Zigrino, C. Brucker, C. Bourdon, M. Freund, A. De Arcangelis, S.I. Abrams, G. Orend, C. Gachet, P.H. Mangin, Platelet integrin α6β1 controls lung metastasis through direct binding to cancer cell-derived ADAM9, JCI Insight. 1 (2016) e88245, ,https://doi.org/10.1172/jci.insight.88245.

[18] L.X. Yu, L. Yan, W. Yang, F.Q. Wu, Y. Ling, S.Z. Chen, L. Tang, Y.X. Tan, D. Cao, M.C. Wu, H.X. Yan, H.Y. Wang, Platelets promote tumour metastasis via interaction between TLR4 and tumour cell-released high-mobility group box1 protein, Nat. Commun. 5 (2014) 5256,https://doi.org/10.1038/ncomms6256.

[19] Y. Ward, R. Lake, F. Faraji, J. Sperger, P. Martin, C. Gilliard, K.P. Ku, T. Rodems, D. Niles, H. Tillman, J. Yin, K. Hunter, A.G. Sowalsky, J. Lang, K. Kelly, Platelets promote metastasis via binding tumor CD97 leading to bidirectional signaling that coordinates transendothelial migration, Cell Rep. 23 (2018) 808–822,https://doi.

org/10.1016/j.celrep.2018.03.092.

[20] A. Radziwon-Balicka, M.J. Santos-Martinez, J.J. Corbalan, S. O’Sullivan, A. Treumann, J.F. Gilmer, M.W. Radomski, C. Medina, Mechanisms of platelet- stimulated colon cancer invasion: role of clusterin and thrombospondin 1 in regulation of the P38MAPK-MMP-9 pathway, Carcinogenesis 35 (2014) 324–332, https://doi.org/10.1093/carcin/bgt332.

[21] E. Sierko, M.Z. Wojtukiewicz, Inhibition of platelet function: does it offer a chance of better cancer progression control? Semin. Thromb. Hemost. 33 (2007) 712–721, https://doi.org/10.1055/s-2007-991540.

[22] M.G. Best, P. Wesseling, T. Wurdinger, Tumor-educated platelets as a noninvasive biomarker source for cancer detection and progression monitoring, Cancer Res. 78 (2018) 3407–3412,https://doi.org/10.1158/0008-5472.CAN-18-0887.

[23] O. Elaskalani, M. Falasca, N. Moran, M.C. Berndt, P. Metharom, The role of platelet-derived ADP and ATP in promoting pancreatic cancer cell survival and gemcitabine resistance, Cancers (Basel) 9 (2017),https://doi.org/10.3390/

cancers9100142.

[24] A. Boucharaba, C.-M. Serre, S. Grès, J.S. Saulnier-Blache, J.-C. Bordet, J. Guglielmi, P. Clézardin, O. Peyruchaud, Platelet-derived lysophosphatidic acid supports the progression of osteolytic bone metastases in breast cancer, J. Clin.

Invest. 114 (2004) 1714–1725,https://doi.org/10.1172/JCI22123.

[25] M. Dovizio, T.J. Maier, S. Alberti, L. Di Francesco, E. Marcantoni, G. Munch, C.M. John, B. Suess, A. Sgambato, D. Steinhilber, P. Patrignani, Pharmacological inhibition of platelet-tumor cell cross-talk prevents platelet-induced over- expression of cyclooxygenase-2 in HT29 human colon carcinoma cellss, Mol.

Pharmacol. 84 (2013) 25–40,https://doi.org/10.1124/mol.113.084988.

[26] M.S. Cho, K. Noh, M. Haemmerle, D. Li, H. Park, Q. Hu, T. Hisamatsu, T. Mitamura, S.L.C. Mak, S. Kunapuli, Q. Ma, A.K. Sood, V. Afshar-Kharghan, Role of ADP receptors on platelets in the growth of ovarian cancer, Blood 130 (2017) 1235–1242,https://doi.org/10.1182/blood-2017-02-769893.

[27] M. Labelle, S. Begum, R.O. Hynes, Platelets guide the formation of early metastatic niches, Proc. Natl. Acad. Sci. USA 111 (2014) E3053–E3061,https://doi.org/10.

1073/pnas.1411082111.

[28] L. Ma, R. Perini, W. McKnight, M. Dicay, A. Klein, M.D. Hollenberg, J.L. Wallace, Proteinase-activated receptors 1 and 4 counter-regulate endostatin and VEGF release from human platelets, Proc. Natl. Acad. Sci. USA 102 (2005) 216–220, https://doi.org/10.1073/pnas.0406682102.

[29] G.J. Gasic, T.B. Gasic, C.C. Stewart, Antimetastatic effects associated with platelet reduction, Proc. Natl. Acad. Sci. USA 61 (1968) 46–52,https://doi.org/10.1073/

pnas.61.1.46.

[30] G.J. Cask, T.B. Gasic, N. Galanti, T. Johnson, S. Murphy, Platelet-tumor-cell interactions in mice. The role of platelets in the spread of malignant disease, Int. J.

Cancer 11 (1973) 704–718,https://doi.org/10.1002/ijc.2910110322.

[31] L. Erpenbeck, M.P. Schön, Deadly allies: the fatal interplay between platelets and metastasizing cancer cells, Blood 115 (2010) 3427–3436,https://doi.org/10.

1182/blood-2009-10-247296.

[32] F.C.O.B. Teixeira, E.O. Kozlowski, K.V. de A. Micheli, A.C.E.S. Vilela-Silva, L. Borsig, M.S.G. Pavão, Sulfated fucans and a sulfated galactan from sea urchins as potent inhibitors of selectin-dependent hematogenous metastasis, Glycobiology 28 (2018) 427–434,https://doi.org/10.1093/glycob/cwy020.

[33] Y. Qi, W. Chen, X. Liang, K. Xu, X. Gu, F. Wu, X. Fan, S. Ren, J. Liu, J. Zhang, R. Li, J. Liu, X. Liang, Novel antibodies against GPIbα inhibit pulmonary metastasis by affecting vWF-GPIbα interaction, J. Hematol. Oncol. 11 (2018) 117,https://doi.

org/10.1186/s13045-018-0659-4.

[34] J. Volz, E. Mammadova-Bach, J. Gil-Pulido, R. Nandigama, K. Remer, L. Sorokin, A. Zernecke, S.I. Abrams, S. Ergün, E. Henke, B. Nieswandt, Inhibition of platelet GPVI induces intratumor hemorrhage and increases efficacy of chemotherapy in mice, Blood 133 (2019) 2696–2706,https://doi.org/10.1182/blood.2018877043.

[35] V. Martín-Granado, S. Ortiz-Rivero, R. Carmona, S. Gutiérrez-Herrero, M. Barrera, L. San-Segundo, C. Sequera, P. Perdiguero, F. Lozano, F. Martín-Herrero, J. Ramón González-Porras, R. Muñoz-Chápuli, A. Porras, C. Guerrero, C3G promotes a selective release of angiogenic factors from activated mouse platelets to regulate angiogenesis and tumor metastasis, Oncotarget 8 (2017) 110994–111011,https://

doi.org/10.18632/oncotarget.22339.

[36] J.-H. Ahn, K.-T. Lee, Y.S. Choi, J.-H. Choi, Iloprost, a prostacyclin analog, inhibits the invasion of ovarian cancer cells by downregulating matrix metallopeptidase-2 (MMP-2) through the IP-dependent pathway, Prostaglandins Other Lipid Mediat.

134 (2018) 47–56,https://doi.org/10.1016/j.prostaglandins.2017.12.002.

[37] K.V. Honk, Inhibition of tumor cell metastasis by modulation of the vascular prostacyclin/thromboxane A2 system, Clin. Exp. Metastasis 1 (1983) 103–114, https://doi.org/10.1007/BF00121490.

[38] H. Li, M.-H. Lee, K. Liu, T. Wang, M. Song, Y. Han, K. Yao, H. Xie, F. Zhu, M. Grossmann, M.P. Cleary, W. Chen, A.M. Bode, Z. Dong, Inhibiting breast cancer by targeting the thromboxane A2 pathway, Npj Precis. Oncol. 1 (2017) 8,https://

doi.org/10.1038/s41698-017-0011-4.

[39] B.C.Y. Wong, 12-Lipoxygenase inhibition induced apoptosis in human gastric cancer cells, Carcinogenesis 22 (2001) 1349–1354,https://doi.org/10.1093/

carcin/22.9.1349.

[40] T.M.H. Niers, L.W. Brüggemann, G.L. van Sluis, R.D. Liu, H.H. Versteeg, H.R. Büller, C.J.F. van Noorden, P.H. Reitsma, C.A. Spek, D.J. Richel, Long-term thrombin inhibition promotes cancer cell extravasation in a mouse model of experimental metastasis, J. Thromb. Haemost. 7 (2009) 1595–1597,https://doi.org/

10.1111/j.1538-7836.2009.03529.x.

[41] R. Auvergne, C. Wu, A. Connell, S. Au, A. Cornwell, M. Osipovitch, A. Benraiss, S. Dangelmajer, H. Guerrero-Cazares, A. Quinones-Hinojosa, S.A. Goldman, PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma pro- genitor cells and their derived gliomas in vivo, Oncogene 35 (2016) 3817–3828, https://doi.org/10.1038/onc.2015.452.

(8)

[42] A.L. Papa, A. Jiang, N. Korin, M.B. Chen, E.T. Langan, A. Waterhouse, E. Nash, J. Caroff, A. Graveline, A. Vernet, A. Mammoto, T. Mammoto, A. Jain, R.D. Kamm, M.J. Gounis, D.E. Ingber, Platelet decoys inhibit thrombosis and prevent metastatic tumor formation in preclinical models, Sci. Transl. Med. 11 (2019),https://

doi.org/10.1126/scitranslmed.aau5898.

[43] K. DeFeo, C. Hayes, M. Chernick, J. Van Ryn, S.K. Gilmour, Use of dabigatran etexilate to reduce breast cancer progression, Cancer Biol. Ther. 10 (2010) 1001–1008,https://doi.org/10.4161/cbt.10.10.13236.

[44] S. Lucotti, C. Cerutti, M. Soyer, A.M. Gil-Bernabé, A.L. Gomes, P.D. Allen, S. Smart, B. Markelc, K. Watson, P.C. Armstrong, J.A. Mitchell, T.D. Warner, A.J. Ridley, R.J. Muschel, Aspirin blocks formation of metastatic intravascular niches by inhibiting platelet-derived COX-1/thromboxane A2, J. Clin. Invest. 129 (2019) 1845–1862,https://doi.org/10.1172/JCI121985.

[45] P.M. Rothwell, M. Wilson, C.E. Elwin, B. Norrving, A. Algra, C.P. Warlow, T.W. Meade, Long-term effect of aspirin on colorectal cancer incidence and mortality: 20-year follow-up of five randomised trials, Lancet 376 (2010) 1741–1750, https://doi.org/10.1016/S0140-6736(10)61543-7.

[46] P.M. Rothwell, F.G.R. Fowkes, J.F. Belch, H. Ogawa, C.P. Warlow, T.W. Meade, Effect of daily aspirin on long-term risk of death due to cancer: analysis of in- dividual patient data from randomised trials, Lancet 377 (2011) 31–41,https://

doi.org/10.1016/S0140-6736(10)62110-1.

[47] A.M. Algra, P.M. Rothwell, Effects of regular aspirin on long-term cancer incidence and metastasis: a systematic comparison of evidence from observational studies versus randomised trials, Lancet. Oncol. 13 (2012) 518–527,https://doi.org/10.

1016/S1470-2045(12)70112-2.

[48] E.T. Alexander, A.R. Minton, C.S. Hayes, A. Goss, J. Van Ryn, S.K. Gilmour, Thrombin inhibition and cyclophosphamide synergistically block tumor progression and metastasis, Cancer Biol. Ther. 16 (2015) 1802–1811,https://doi.org/10.

1080/15384047.2015.1078025.

[49] J.T. Buijs, E.H. Laghmani, R.F.P. van den Akker, C. Tieken, E.M. Vletter, K.M. van der Molen, J.J. Crooijmans, C. Kroone, S.E. Le Dévédec, G. van der Pluijm, H.H. Versteeg, The direct oral anticoagulants rivaroxaban and dabigatran do not inhibit orthotopic growth and metastasis of human breast cancer in mice, J.

Thromb. Haemost. 17 (2019) 951–963,https://doi.org/10.1111/jth.14443.

[50] N.R. Cook, I.M. Lee, J.M. Gaziano, D. Gordon, P.M. Ridker, J.A.E. Manson, C.H. Hennekens, J.E. Buring, Low-dose aspirin in the primary prevention of cancer. The women’s health study: a randomized controlled trial, J. Am. Med.

Assoc. 294 (2005) 47–55,https://doi.org/10.1001/jama.294.1.47.

[51] S.M. Zhang, N.R. Cook, J.E. Manson, I.-M. Lee, J.E. Buring, Low-dose aspirin and breast cancer risk: results by tumour characteristics from a randomised trial, Br. J.

Cancer. 98 (2008) 989–991,https://doi.org/10.1038/sj.bjc.6604240.

[52] P.C. Elwood, G. Morgan, J.E. Pickering, J. Galante, A.L. Weightman, D. Morris, M. Kelson, S. Dolwani, Aspirin in the treatment of cancer: reductions in metastatic spread and in mortality: a systematic review and meta-analyses of published studies, PLoS One 11 (2016) e0152402,https://doi.org/10.1371/journal.pone.

0152402.

[53] M. Smeda, A. Kieronska, B. Proniewski, A. Jasztal, A. Selmi, K. Wandzel, A. Zakrzewska, T. Wojcik, K. Przyborowski, K. Derszniak, M. Stojak, D. Kaczor, E. Buczek, C. Watala, J. Wietrzyk, S. Chlopicki, Dual antiplatelet therapy with clopidogrel and aspirin increases mortality in 4T1 metastatic breast cancer- bearing mice by inducing vascular mimicry in primary tumour, Oncotarget 9 (2018) 17810–17824,https://doi.org/10.18632/oncotarget.24891.

[54] W. Jackson, D.M. Sosnoski, S.E. Ohanessian, P. Chandler, A. Mobley, K.D. Meisel, A.M. Mastro, Role of megakaryocytes in breast cancer metastasis to bone, Cancer Res. 77 (2017) 1942–1954,https://doi.org/10.1158/0008-5472.CAN-16-1084.

[55] S. Wood, P. Hilgard, Aspirin and tumour metastasis, Lancet (London, England) 2 (1972) 1416–1417,https://doi.org/10.1016/s0140-6736(72)92982-0.

[56] C. Coyle, F.H. Cafferty, S. Rowley, M. MacKenzie, L. Berkman, S. Gupta, C.S. Pramesh, D. Gilbert, H. Kynaston, D. Cameron, R.H. Wilson, A. Ring, R.E. Langley, Add-Aspirin investigators, ADD-ASPIRIN: a phase III, double-blind, placebo controlled, randomised trial assessing the effects of aspirin on disease recurrence and survival after primary therapy in common non-metastatic solid tumours, Contemp. Clin. Trials 51 (2016) 56–64,https://doi.org/10.1016/j.cct.

2016.10.004.

[57] K. Bibbins-Domingo, D.C. Grossman, S.J. Curry, K.W. Davidson, J.W. Epling, F.A.R. García, M.W. Gillman, D.M. Harper, A.R. Kemper, A.H. Krist, A.E. Kurth, C.S. Landefeld, C.M. Mangione, D.K. Owens, W.R. Phillips, M.G. Phipps, M.P. Pignone, A.L. Siu, Screening for colorectal cancer: US preventive services task force recommendation statement, JAMA - J. Am. Med. Assoc. 315 (2016) 2564–2575,https://doi.org/10.1001/jama.2016.5989.

[58] S. Raposeiras-Roubín, E. Abu-Assi, I. Muñoz-Pousa, M. Cespón-Fernández, R. Cobas-Paz, B. Caneiro-Queija, E. López-Rodríguez, L. Pérez-Casares, K. Jamhour-Chelh, M. Castiñeira-Busto, C. Barreiro-Pardal, F. Calvo-Iglesias, A. Íñiguez-Romo, Risk of cancer after an acute coronary syndrome according to the type of P2Y12 inhibitor, Thromb. Res. 174 (2019) 51–58,https://doi.org/10.

1016/j.thromres.2018.12.014.

[59] C.J. Reddel, C.W. Tan, V.M. Chen, Thrombin generation and cancer: contributors and consequences, Cancers (Basel). 11 (pii) (2019) E100,https://doi.org/10.

3390/cancers11010100.

[60] C. Feistritzer, M. Riewald, Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation, Blood 105 (2005) 3178–3184,https://doi.org/10.1182/blood-2004-10-3985.

[61] J.S. Bae, Y.U. Kim, M.K. Park, A.R. Rezaie, Concentration dependent dual effect of thrombin in endothelial cells via Par-1 and Pi3 kinase, J. Cell. Physiol. 219 (2009) 744–751,https://doi.org/10.1002/jcp.21718.

[62] A. Arachiche, M. de la Fuente, M.T. Nieman, Calcium mobilization and protein

kinase C activation downstream of protease activated receptor 4 (PAR4) is negatively regulated by PAR3 in mouse platelets, PLoS One. 8 (2013) e55740,https://

doi.org/10.1371/journal.pone.0055740.

[63] M. Holinstat, B. Voss, M.L. Bilodeau, J.N. McLaughlin, J. Cleator, H.E. Hamm, PAR4, but not PAR1, signals human platelet aggregation via Ca2+ mobilization and synergistic P2Y12 receptor activation, J. Biol. Chem. 281 (2006) 26665–26674,https://doi.org/10.1074/jbc.M602174200.

[64] N.M. Bambace, J.E. Levis, C.E. Holmes, The effect of P2Y-mediated platelet activation on the release of VEGF and endostatin from platelets, Platelets 21 (2010) 85–93,https://doi.org/10.3109/09537100903470298.

[65] R.L. Nachman, S. Rafii, Platelets, petechiae, and preservation of the vascular wall, N. Engl. J. Med. 359 (2008) 1261–1270,https://doi.org/10.1056/

NEJMra0800887.

[66] B. Ho-Tin-Noé, Y. Boulaftali, E. Camerer, Platelets and vascular integrity: How platelets prevent bleeding in inflammation, Blood 131 (2018) 277–288,https://

doi.org/10.1182/blood-2017-06-742676.

[67] E.M. Golebiewska, A.W. Poole, Platelet secretion: from haemostasis to wound healing and beyond, Blood Rev. 29 (2015) 153–162,https://doi.org/10.1016/j.

blre.2014.10.003.

[68] B. Ho-Tin-Noé, T. Goerge, D.D. Wagner, Platelets: guardians of tumor vasculature, Cancer Res. 69 (2009) 5623–5626,https://doi.org/10.1158/0008-5472.CAN-09- 1370.

[69] C.T. Mierke, D.P. Zitterbart, P. Kollmannsberger, C. Raupach, U. Schlötzer- Schrehardt, T.W. Goecke, J. Behrens, B. Fabry, Breakdown of the endothelial barrier function in tumor cell transmigration, Biophys. J. 94 (2008) 2832–2846, https://doi.org/10.1529/biophysj.107.113613.

[70] J.W. Franses, N.C. Drosu, W.J. Gibson, V.C. Chitalia, E.R. Edelman, Dysfunctional endothelial cells directly stimulate cancer inflammation and metastasis, Int. J.

Cancer. 133 (2013) 1334–1344,https://doi.org/10.1002/ijc.28146.

[71] J.D. Martin, G. Seano, R.K. Jain, Normalizing function of tumor vessels: progress, opportunities, and challenges, Annu. Rev. Physiol. 81 (2019) 505–534,https://

doi.org/10.1146/annurev-physiol-020518-114700.

[72] L. Plantureux, D. Mège, L. Crescence, E. Carminita, S. Robert, S. Cointe, N. Brouilly, W. Ezzedine, F. Dignat-George, C. Dubois, L. Panicot-Dubois, The interaction of platelets with colorectal cancer cells inhibits tumor growth but promotes metastasis, Cancer Res. 80 (2019) 291–303,https://doi.org/10.1158/

0008-5472.CAN-19-1181.

[73] J. Lawler, Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth, J. Cell. Mol. Med. 6 (2002) 1–12,https://doi.org/10.1111/j.1582- 4934.2002.tb00307.x.

[74] J.P. Maloney, C.C. Silliman, D.R. Ambruso, J. Wang, R.M. Tuder, N.F. Voelkel, In vitro release of vascular endothelial growth factor during platelet aggregation, Am. J. Physiol. - Hear. Circ. Physiol. 275 (1998) H1054–H1061,https://doi.org/

10.1152/ajpheart.1998.275.3.h1054.

[75] E.D. Hottz, J.F. Lopes, C. Freitas, R. Valls-De-Souza, M.F. Oliveira, M.T. Bozza, A.T. Da Poian, A.S. Weyrich, G.A. Zimmerman, F.A. Bozza, P.T. Bozza, Platelets mediate increased endothelium permeability in dengue through NLRP3-in- flammasome activation, Blood 122 (2013) 3405–3414,https://doi.org/10.1182/

blood-2013-05-504449.

[76] A. Gros, V. Ollivier, B. Ho-Tin-Noé, Platelets in inflammation: regulation of leu- kocyte activities and vascular repair, Front. Immunol. 5 (2014) 678,https://doi.

org/10.3389/fimmu.2014.00678.

[77] C. Hillgruber, B. Pöppelmann, C. Weishaupt, A.K. athri, F. Steingräber, W.E. Wessel, J.E. Berdel, B. Gessner, D. Ho-Tin-Noé, T Goerge Vestweber, Blocking neutrophil diapedesis prevents hemorrhage during thrombocytopenia, J. Exp.

Med. 212 (2015) 1255–1266,https://doi.org/10.1084/jem.20142076.

[78] C. Deppermann, Platelets and vascular integrity, Platelets 29 (2018) 549–555, https://doi.org/10.1080/09537104.2018.1428739.

[79] B. Ho-Tin-Noé, S. Jadoui, Spontaneous bleeding in thrombocytopenia: is it really spontaneous? Transfus. Clin. Biol. 25 (2018) 210–216,https://doi.org/10.1016/j.

tracli.2018.06.005.

[80] G.L. Mendolicchio, Z.M. Ruggeri, New perspectives on von Willebrand factor functions in hemostasis and thrombosis, Semin. Hematol. 42 (2005) 5–14,https://

doi.org/10.1053/j.seminhematol.2004.09.006.

[81] Z.M. Ruggeri, G.L. Mendolicchio, Adhesion mechanisms in platelet function, Circ.

Res. 100 (2007) 1673–1685,https://doi.org/10.1161/01.RES.0000267878.

97021.ab.

[82] B. Nieswandt, S.P. Watson, Platelet-collagen interaction: Is GPVI the central receptor? Blood 102 (2003) 449–461,https://doi.org/10.1182/blood-2002-12- 3882.

[83] D.J. Schneider, Anti-platelet therapy: glycoprotein IIb-IIIa antagonists, British J.

Clin. Pharmacol. 72 (2011) 672–682,https://doi.org/10.1111/j.1365-2125.2010.

03879.x.

[84] H.H. Versteeg, J.W.M. Heemskerk, M. Levi, P.H. Reitsma, New fundamentals in hemostasis, Physiol. Rev. 93 (2013) 327–358,https://doi.org/10.1152/physrev.

00016.2011.

[85] T. Goerge, B. Ho-Tin-Noe, C. Carbo, C. Benarafa, E. Remold-O’Donnell, B.Q. Zhao, S.M. Cifuni, D.D. Wagner, Inflammation induces hemorrhage in thrombocytopenia, Blood 111 (2008) 4958–4964,https://doi.org/10.1182/blood-2007-11- 123620.

[86] Y. Boulaftali, P.R. Hess, T.M. Getz, A. Cholka, M. Stolla, N. Mackman, A.P. Owens, J. Ware, M.L. Kahn, W. Bergmeier, Platelet ITAM signaling is critical for vascular integrity in infammation, J. Clin. Invest. 123 (2013) 908–916,https://doi.org/10.

1172/JCI65154.

[87] C. Deppermann, P. Kraft, J. Volz, M.K. Schuhmann, S. Beck, K. Wolf, D. Stegner, G. Stoll, B. Nieswandt, Platelet secretion is crucial to prevent bleeding in the

(9)

ischemic brain but not in the inflamed skin or lung in mice, Blood 129 (2017) 1702–1706,https://doi.org/10.1182/blood-2016-12-750711.

[88] A. Gros, V. Syvannarath, L. Lamrani, V. Ollivier, S. Loyau, T. Goerge, B. Nieswandt, M. Jandrot-Perrus, B. Ho-Tin-Noé, Single platelets seal neutrophil- induced vascular breaches via GPVI during immune-complex-mediated inflammation in mice, Blood 126 (2015) 1017–1026,https://doi.org/10.1182/

blood-2014-12-617159.

[89] B.H. Herzog, J. Fu, S.J. Wilson, P.R. Hess, A. Sen, J.M. McDaniel, Y. Pan, M. Sheng, T. Yago, R. Silasi-Mansat, S. McGee, F. May, B. Nieswandt, A.J. Morris, F. Lupu, S.R. Coughlin, R.P. McEver, H. Chen, M.L. Kahn, L. Xia, Podoplanin maintains high endothelial venule integrity by interacting with platelet CLEC-2, Nature 502 (2013) 105–109,https://doi.org/10.1038/nature12501.

[90] B. Ho-Tin-Noé, T. Goerge, S.M. Cifuni, D. Duerschmied, D.D. Wagner, Platelet granule secretion continuously prevents intratumor hemorrhage, Cancer Res. 68 (2008) 6851–6858,https://doi.org/10.1158/0008-5472.CAN-08-0718.

[91] B. Ho-Tin-Noé, C. Carbo, M. Demers, S.M. Cifuni, T. Goerge, D.D. Wagner, Innate immune cells induce hemorrhage in tumors during thrombocytopenia, Am. J.

Pathol. 175 (2009) 1699–1708,https://doi.org/10.2353/ajpath.2009.090460.

[92] R.H. Lee, W. Bergmeier, Platelet immunoreceptor tyrosine-based activation motif (ITAM) and hemITAM signaling and vascular integrity in inflammation and development, J. Thromb. Haemost. 14 (2016) 645–654,https://doi.org/10.1111/

jth.13250.

[93] Y. Ozaki, S. Tamura, K. Suzuki-Inoue, New horizon in platelet function: with special reference to a recently-found molecule, CLEC-2, Thromb. J. 14 (2016) 27, https://doi.org/10.1186/s12959-016-0099-8.

[94] J. Rayes, S. Jadoui, S. Lax, A. Gros, S. Wichaiyo, V. Ollivier, C.V. Denis, J. Ware, B. Nieswandt, M. Jandrot-Perrus, S.P. Watson, B. Ho-Tin-Noé, The contribution of platelet glycoprotein receptors to inflammatory bleeding prevention is stimulus and organ dependent, Haematologica 103 (2018) e256–e258,https://doi.org/10.

3324/haematol.2017.182162.

[95] I. Häuselmann, M. Roblek, D. Protsyuk, V. Huck, L. Knopfova, S. Grässle, A.T. Bauer, S.W. Schneider, L. Borsig, Monocyte induction of E-selectin-mediated endothelial activation releases VE-cadherin junctions to promote tumor cell extravasation in the metastasis cascade, Cancer Res. 76 (2016) 5302–5312,https://

doi.org/10.1158/0008-5472.CAN-16-0784.

[96] J. Rayes, S. Lax, S. Wichaiyo, S.K. Watson, Y. Di, S. Lombard, B. Grygielska, S.W. Smith, K. Skordilis, S.P. Watson, The podoplanin-CLEC-2 axis inhibits inflammation in sepsis, Nat Commun. 8 (2017) 2239,https://doi.org/10.1038/

s41467-017-02402-6.

[97] M.A. Kowalska, S.A. Mahmud, M.P. Lambert, M. Poncz, A. Slungaard, Endogenous platelet factor 4 stimulates activated protein C generation in vivo and improves survival after thrombin or lipopolysaccharide challenge, Blood 110 (2007) 1903–1905,https://doi.org/10.1182/blood-2007-03-081901.

[98] H. Payne, T. Ponomaryov, S.P. Watson, A. Brill, Mice with a deficiency in CLEC-2 are protected against deep vein thrombosis, Blood 129 (2017) 2013–2020, https://doi.org/10.1182/blood-2016-09-742999.

[99] K. Miyata, A. Takemoto, S. Okumura, M. Nishio, N. Fujita, Podoplanin enhances lung cancer cell growth in vivo by inducing platelet aggregation, Sci. Rep. 7 (2017) 4059,https://doi.org/10.1038/s41598-017-04324-1.

[100] S. Takagi, S. Sato, T. Oh-hara, M. Takami, S. Koike, Y. Mishima, K. Hatake, N. Fujita, Platelets promote tumor growth and metastasis via direct interaction between aggrus/podoplanin and CLEC-2, PLoS One 8 (2013) e73609,https://doi.

org/10.1371/journal.pone.0073609.

[101] B. Nieswandt, V. Schulte, W. Bergmeier, R. Mokhtari-Nejad, K. Rackebrandt, J.P. Cazenave, P. Ohlmann, C. Gachet, H. Zirngibl, Long-term antithrombotic protection by in vivo depletion of platelet glycoprotein VI in mice, J. Exp. Med.

193 (2001) 459–469,https://doi.org/10.1084/jem.193.4.459.

[102] R. Badolia, V. Inamdar, B.K. Manne, C. Dangelmaier, J.A. Eble, S.P. Kunapuli, Gq pathway regulates proximal C-type lectin-like receptor-2 (CLEC-2) signaling in platelets, J. Biol. Chem. 292 (2017) 14516–14531,https://doi.org/10.1074/jbc.

M117.791012.

[103] E. Pachmayr, C. Treese, U. Stein, Underlying mechanisms for distant metastasis - molecular biology, Visc. Med. 33 (2017) 11–20,https://doi.org/10.1159/

000454696.

[104] H. Herman, C. Fazakas, J. Haskó, K. Molnár, Á. Mészáros, Á. Nyúl-Tóth, G. Szabó, F. Erdélyi, A. Ardelean, A. Hermenean, I.A. Krizbai, I. Wilhelm, Paracellular and transcellular migration of metastatic cells through the cerebral endothelium, J.

Cell. Mol. Med. 23 (2019) 2619–2631,https://doi.org/10.1111/jcmm.14156.

[105] E. Buczek, A. Denslow, L. Mateuszuk, B. Proniewski, T. Wojcik, B. Sitek, A. Fedorowicz, A. Jasztal, E. Kus, A. Chmura-Skirlinska, R. Gurbiel, J. Wietrzyk, S. Chlopicki, Alterations in NO- and PGI2- dependent function in aorta in the orthotopic murine model of metastatic 4T1 breast cancer: relationship with pulmonary endothelial dysfunction and systemic inflammation, BMC Cancer 18 (2018) 582,https://doi.org/10.1186/s12885-018-4445-z.

[106] M. Smeda, A. Kieronska, M.G. Adamski, B. Proniewski, M. Sternak, T. Mohaissen,

K. Przyborowski, K. Derszniak, D. Kaczor, M. Stojak, E. Buczek, A. Jasztal, J. Wietrzyk, S. Chlopicki, Nitric oxide deficiency and endothelial-mesenchymal transition of pulmonary endothelium in the progression of 4T1 metastatic breast cancer in mice, Breast Cancer Res. 20 (2018) 86,https://doi.org/10.1186/s13058- 018-1013-z.

[107] B. Ho-Tin-Noé, M. Demers, D.D. Wagner, How platelets safeguard vascular integrity, J. Thromb. Haemost. 9 (2011) 56–65,https://doi.org/10.1111/j.1538- 7836.2011.04317.x.

[108] S.K. Lo, K.E. Burhop, J.E. Kaplan, A.B. Malik, Role of platelets in maintenance of pulmonary vascular permeability to protein, Am. J. Physiol. - Hear. Circ. Physiol.

254 (1988) H763–H771,https://doi.org/10.1152/ajpheart.1988.254.4.h763.

[109] C.L. Chaffer, R.A. Weinberg, A perspective on cancer cell metastasis, Science 331 (2011) 1559–1564,https://doi.org/10.1126/science.1203543.

[110] A.S. Azevedo, G. Follain, S. Patthabhiraman, S. Harlepp, J.G. Goetz, Metastasis of circulating tumor cells: Favorable soil or suitable biomechanics, or both? Cell Adhes. Migr. 9 (2015) 345–356,https://doi.org/10.1080/19336918.2015.

1059563.

[111] Joanna Suraj, Anna Kurpińska, Agnieszka Zakrzewska, Magdalena Sternak, Marta Stojak, Agnieszka Jasztal, Maria Walczak, Stefan Chlopicki, Early and late endothelial response in breast cancer metastasis in mice: simultaneous quantifi- cation of endothelial biomarkers using a mass spectrometry-based method, Dis.

Model. Mech. 12 (3) (2019) dmm036269,https://doi.org/10.1242/dmm.036269.

[112] J. Molnár, C. Fazakas, J. Haskó, O. Sipos, K. Nagy, Á. Nyúl-Tóth, A.E. Farkas, A.G. Végh, G. Váró, P. Galajda, I.A. Krizbai, I. Wilhelm, Transmigration characteristics of breast cancer and melanoma cells through the brain endothelium:

role of Rac and PI3K, Cell Adh. Migr. 10 (2016) 269–281,https://doi.org/10.

1080/19336918.2015.1122156.

[113] A. Bartolini, S. Cardaci, S. Lamba, D. Oddo, C. Marchiò, P. Cassoni, C.A. Amoreo, G. Corti, A. Testori, F. Bussolino, R. Pasqualini, W. Arap, D. Corà, F. Di Nicolantonio, S. Marchiò, BCAM and LAMA5 mediate the recognition between tumor cells and the endothelium in the metastatic spreading of KRAS-mutant colorectal cancer, Clin. Cancer Res. 22 (2016) 4923–4933,https://doi.org/10.

1158/1078-0432.CCR-15-2664.

[114] F. Gao, A. Alwhaibi, S. Artham, A. Verma, P.R. Somanath, Endothelial Akt1 loss promotes prostate cancer metastasis via β-catenin-regulated tight-junction protein turnover, Br. J. Cancer. 118 (2018) 1464–1475,https://doi.org/10.1038/s41416- 018-0110-1.

[115] J. Xing, A.A. Birukova, ANP attenuates inflammatory signaling and Rho pathway of lung endothelial permeability induced by LPS and TNFalpha, Microvasc. Res. 79 (2010) 56–62,https://doi.org/10.1016/j.mvr.2009.11.006.

[116] T. Nojiri, H. Hosoda, T. Tokudome, K. Miura, S. Ishikane, K. Otani, I. Kishimoto, Y. Shintani, M. Inoue, T. Kimura, N. Sawabata, M. Minami, T. Nakagiri, S. Funaki, Y. Takeuchi, H. Maeda, H. Kidoya, H. Kiyonari, Y. Arai, T. Hasegawa, N. Takakura, M. Hori, Y. Ohno, M. Miyazato, N. Mochizuki, M. Okumura, K. Kangawa, M. Yanagisawa, Atrial natriuretic peptide prevents cancer metastasis through vascular endothelial cells, Proc. Natl. Acad. Sci. USA 112 (2015) 4086–4089, https://doi.org/10.1073/pnas.1417273112.

[117] F.T. Wu, C.R. Lee, E. Bogdanovic, A. Prodeus, J. Gariépy, R.S. Kerbel, Vasculotide reduces endothelial permeability and tumor cell extravasation in the absence of binding to or agonistic activation of Tie2, EMBO Mol. Med. 7 (2015) 770–787, https://doi.org/10.15252/emmm.201404193.

[118] R. Draijer, D.E. Atsma, A. Van Der Laarse, V.W.M. Van Hinsbergh, cGMP and nitric oxide modulate thrombin-induced endothelial permeability: regulation via dif- ferent pathways in human aortic and umbilical vein endothelial cells, Circ. Res. 76 (1995) 199–208,https://doi.org/10.1161/01.RES.76.2.199.

[119] J.G. Filep, É. Földes-Filep, P. Sirois, Nitric oxide modulates vascular permeability in the rat coronary circulation, Br. J. Pharmacol. 108 (1993) 323–326,https://doi.

org/10.1111/j.1476-5381.1993.tb12803.x.

[120] A.A. Birukova, F. Meng, Y. Tian, A. Meliton, N. Sarich, L.A. Quilliam, K.G. Birukov, Prostacyclin post-treatment improves LPS-induced acute lung injury and endothelial barrier recovery via Rap1, Biochim. Biophys. Acta 2015 (1852) 778–791, https://doi.org/10.1016/j.bbadis.2014.12.016.

[121] M.W. Radomski, R.M. Palmer, S. Moncada, The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide, Br. J.

Pharmacol. 92 (1987) 639–646,https://doi.org/10.1111/j.1476-5381.1987.

tb11367.x.

[122] B. Estevez, B. Shen, X. Du, Targeting integrin and integrin signaling in treating thrombosis, Arterioscler. Thromb. Vasc. Biol. 35 (2015) 24–29,https://doi.org/

10.1161/ATVBAHA.114.303411.

[123] F. May, I. Hagedorn, I. Pleines, M. Bender, T. Vögtle, J. Eble, M. Elvers, B. Nieswandt, CLEC-2 is an essential platelet-activating receptor in hemostasis and thrombosis, Blood 114 (2009) 3464–3472,https://doi.org/10.1182/blood-2009- 05-222273.

[124] W. Bergmeier, L. Stefanini, Platelet ITAM signaling, Curr. Opin. Hematol. 20 (2013) 445–450,https://doi.org/10.1097/MOH.0b013e3283642267.