Mechanisms of cancer-associated thrombosis.

Introduction

Patients with cancer have a 4-fold to 9-fold increased incidence of venous thromboembolism (VTE) compared with the general population which is called cancer-associated thrombosis (CAT) [ [1] , [2] , [3] ]. Several studies have found that 20%–30% of all first VTE events are associated with cancer [ 1 ]. Patients with cancer and VTE have ≥2-fold increased mortality compared with the patients with cancer without VTE [ 3 , 4 ]. Additionally, CAT can delay or interfere with anticancer therapy, prolong hospitalization, and worsen the quality of life [ 5 , 6 ]. Interestingly, the incidence of VTE in different types of cancer varies widely. For instance, the highest incidences are observed in pancreatic and brain cancer followed by lung and colorectal cancer, whereas a low incidence is observed in breast and prostate cancer [ 1 ]. This suggests that there are cancer-type-specific mechanisms for VTE.

Clinical studies have measured a variety of different circulating biomarkers that may be useful in identifying patients with cancer at a high risk for VTE [ 7 ]. Most of these studies measured the levels of the different biomarkers in populations of patients with cancer that included different types of cancer. This study design allows the inclusion of larger numbers of patients. Alternatively, one could study a specific type of cancer because there may be cancer-type specific biomarkers of VTE [ 8 ]. The disadvantage of this approach is that there are fewer patients in the study. Preclinical studies using animal models can be used to analyze the mechanisms of thrombosis with different types of cancer [ 9 ]. However, as with all the other models, animal cancer models have limitations and may not reproduce the complexity of CAT in patients.

In this review, we summarize four specific pathways that have been shown to contribute to CAT: tissue factor (TF), podoplanin (PDPN), neutrophil extracellular traps (NETs), and plasminogen activator inhibitor-1 (PAI-1) ( Figure ). Figure Pathways of cancer-associated thrombosis. Pathway 1: tumors release TF-positive extracellular vesicles that activate the coagulation cascade and platelets and increase the risk of VTE. Pathway 2: tumors release podoplanin (PDPN)-positive EVs that activate platelets and increase the risk of VTE. Pathway 3: tumors release granulocyte-colony stimulating factor (G-CSF) that increases the number of neutrophils in the circulation and enhances NET formation and the risk of VTE. Pathway 4: tumors release PAI-1 that increases the risk of VTE by inhibiting plasmin generation and fibrinolysis. The figure is created with BioRender.com . MPN, myeloproliferative neoplasm; NET, neutrophil extracellular trap; PAI-1, plasminogen activator inhibitor-1; PDAC, pancreatic ductal adenocarcinoma; TF, tissue factor; VTE, venous thromboembolism.

Tissue Factor

TF is a glycoprotein receptor and cofactor for factors (F)VII and FVIIa. The TF/FVIIa complex is the physiological initiator of the extrinsic pathway of blood coagulation [ 10 ].

TF and VTE in patients with cancer

TF is expressed by most types of tumors, particularly in pancreatic ductal adenocarcinoma (PDAC) and glioblastoma [ 11 , 12 ].

Gene mutations are associated with increased TF expression in some types of cancer. For example, KRAS and tumor protein 53 ( TP53) gene mutations increase TF expression in human colorectal cancer cell lines [ 13 ]. Additionally, KRAS , TP53, and phosphatase and tensin homolog ( PTEN) gene mutations are associated with increased TF mRNA expression in tumors in patients with nonsmall cell lung carcinoma (NSCLC) [ 14 , 15 ]. A recent study found that anaplastic lymphoma kinase ( ALK) rearrangement is also associated with increased TF protein expression in patients with NSCLC [ 16 ]. Additionally, several retrospective studies [ [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] ] and clinical trials [ [24] , [25] , [26] , [27] ] have reported higher rates of VTE (8%–47% and 1.1%–6.4%, respectively) in patients with lung cancer with ALK rearrangement compared with those without ALK rearrangement. A recent study from Dunbar et al. [ 28 ] showed that the somatic mutation of serine/threonine kinase 11 ( STK11) is associated with increased TF mRNA expression in patients with lung adenocarcinoma. The STK11 gene mutation is also associated with VTE in patients with lung cancer [ 28 ]. Somatic mutations in isocitrate dehydrogenase 1 or 2 ( IDH1/2 ) commonly occur in glioma [ 29 ]. One study found that the F3 (the gene name for TF) gene is ranked in the top 50 most differentially hypermethylated and downregulated genes in the proneural subtype of glioblastoma/glioma-CpG island methylator phenotype [ 30 ]. Indeed, Unruh et al. [ 31 ] found that IDH1/2 mutations are associated with decreased TF mRNA and protein expression in glioma because of F3 (TF) gene hypermethylation. Strikingly, the study found that glioma patients with mutated IDH1/2 had a 0% incidence of VTE, whereas glioma patients with wild-type IDH1/2 showed a 26%–30% incidence of VTE [ 31 ]. More studies are needed to determine the relationship between gene mutations, TF expression, and VTE in patients with different types of cancer.

The Cancer Genome Atlas Network classified glioblastoma into 4 subtypes based on the gene expression patterns: proneural, neural, classical, and mesenchymal [ 32 ]. One study found that levels of F3 (TF) gene expression were different in 4 subtypes of glioblastoma with the highest in the classical type and the lowest in the proneural type [ 33 ]. More studies are needed to determine the associations between F3 (TF) gene expression, glioblastoma subtypes, and VTE.

Extracellular vesicles (EVs) are submicron vesicles released from cells, particularly tumor cells [ 34 ]. Active TF is released from tumors into circulation in the form of EVs. We and other investigators have developed in-house assays that measure EVTF activity [ [35] , [36] , [37] , [38] , [39] ]. There is also a commercial assay called the Zymuphen MP-TF assay that measures FXa activity of EVs captured using an anti-TF antibody [ 40 , 41 ]. These EVTF activity assays are more specific and sensitive than commercial TF assays [ 37 , 40 , 42 ].

We and other investigators found that the patients with cancer have increased levels of EVTF activity compared with healthy controls [ 35 , 36 , [43] , [44] , [45] , [46] ]. Of note, patients with adenocarcinoma have higher levels of EVTF activity compared with patients with other histological types of tumors [ 46 ]. Moreover, increased levels of EVTF activity are also associated with an increased risk of VTE in populations of patients with different types of cancer [ 35 , 47 , 48 ].

Several studies have investigated if there is an association between levels of EVTF activity and VTE and survival in different types of cancer ( Table 1 ). We and other found an association between EVTF activity and VTE in PDAC patients in both longitudinal and prospective studies [ 36 , 45 , 49 , 50 ]. In contrast, 1 study concluded that there was no association between EVTF activity and VTE in patients with PDAC [ 51 ]. However, this study had a much longer follow-up time (2 years) compared with the other studies. Together, these studies suggest that tumor-derived TF+ EVs contribute to VTE in patients with PDAC. Table 1 Association between EVTF activity and VTE and survival in different types of cancer. Tumor type Number of VTEs and patients Association of EVTF activity with VTE Association of EVTF activity with survival Reference Pancreaticobiliary 52/117 (44%) Yes Yes [ 49 ] PDAC 20/79 (25%) Yes Yes [ 45 ] PDAC 12/69 (17%) No Yes [ 51 ] Brain 19/119 (16%) No No [ 51 ] Ovarian 35/105 (33%) No No [ 55 ] Colorectal 12/126 (10%) No No [ 51 ] Gastric 6/43 (14%) No No [ 51 ] SCLC 15/242 (6%) No No [ 56 ] Multiple myeloma 15/122 (12%) No No [ 43 ] AML 30/253 (12%) No Not tested unpublished data AML, acute myeloid leukemia; EVTF, extracellular vesicle tissue factor; PDAC, pancreatic ductal adenocarcinoma; SCLC, small cell lung carcinoma; VTE, venous thromboembolism.

The role of EVTF activity in VTE in patients with glioblastoma has also been investigated. One study found no association between EVTF activity and VTE in patients with glioblastoma, but the study had a long follow-up period (2 years) [ 51 ]. Interestingly, glioblastoma patients with wild-type IDH1/2 express higher levels of tumor TF and have a higher rate of VTE compared with patients with mutated IDH1/2 [ 31 ]. Further studies are needed to determine if tumor-derived TF+ EVs contribute to VTE in patients with glioblastoma.

Three studies have concluded that tumor TF expression was associated with VTE in patients with ovarian cancer [ [52] , [53] , [54] ]. In contrast to these studies, we found no association between EVTF activity and VTE in patients with ovarian cancer [ 55 ]. We studied another cohort of patients with ovarian cancer and again found no association between EVTF activity and VTE (Hisada Y, Archibald SJ, Chinthapatla R, Newton M, Paraghamian S, Bae-Jump VL, and Mackman N, manuscript in press). We suggest that the measurement of EVTF activity in plasma is a more accurate way to assess thrombotic risk than measuring TF expression in the tumor because thrombi occur at a separate site from the tumor.

We and other investigators have also not observed an association between EVTF activity and VTE in patients with other types of cancer, including colorectal, stomach, small cell lung carcinoma, multiple myeloma, and acute myeloid leukemia [ 43 , 50 , 51 , 56 ] ( Table 1 ) (Hisada, Mackman, Gangaraju unpublished data).

Recently, Gi et al. [ 57 ] characterized venous thrombi collected postmortem from patients with (n=114) and without cancer (n=66). Interestingly, they observed 26.6% of the thrombi from patients with cancer contained cancer cells. There were 2 patterns of venous thrombi: those with cancer cells invading the venous or pulmonary artery wall and those with clusters of cancer cells without invasion [ 57 , 58 ]. Importantly, cancer cells in the majority (76%) of the venous thrombi expressed TF. Hence, these TF-expressing cancer cells might have triggered thrombosis in these patients [ 58 ].

TF and thrombosis in preclinical studies

Two different mouse models have been used to investigate the role of TF+ EVs in venous thrombosis. First, exogenous TF+ EVs can be injected into wild-type mice without tumors. Second, endogenous TF+ EVs can be released into circulation from the tumors grown in mice. In the TF+ EVs injection model, TF+ EVs from cancer cell culture supernatants are injected into mice. This model is fast and easy. A series of studies showed that the injection of TF+ EVs into wild-type mice increased venous and arterial thrombosis [ [59] , [60] , [61] , [62] , [63] ]. Importantly, pretreatment of TF+ EVs with anti-TF antibodies abolished the increase in thrombus size and weight and reduced the incidence of thrombosis in an inferior vena cava (IVC) stenosis model [ 61 , 63 ]. Similarly, the injection of TF+ EVs derived from the murine pancreatic cancer cell line Panc02 in which the F3 (TF) gene was knocked down reduced thrombosis in an IVC stenosis model compared with the injection of TF+ EVs derived from wild-type Panc02 cells [ 62 ]. Injection of TF+ EVs derived from a human ovarian cancer cell line A2780 overexpressing TF enhanced thrombosis in an IVC stenosis model compared with an injection of TF+ EVs derived from wild-type A2780 cells [ 64 ]. These data demonstrate that TF+ EVs are highly procoagulant and enhance thrombosis in mice.

It should be noted that we found that the level of exogenous TF+ EVs derived from the pancreatic cancer cell line HPAF-II required to increase levels of thrombin–antithrombin complexes (TAT), a marker of activation of coagulation, was 100-fold higher than the level of endogenous TF+ EVs observed in HPAF-II tumor-bearing mice [ 60 ]. We and others have found that exogenous TF+ EVs are rapidly removed from the circulation, which may explain, in part, the reason for high levels of EVs to activate coagulation [ 59 , 60 , 65 ]. We hypothesize that levels of tumor-derived EVs in the circulation reach a steady state in tumor-bearing mice and may be cleared more slowly than in mice without tumors.

Tumor-bearing mouse models more closely mimic patients with cancer compared with the injection of EVs into wild-type mice because it includes other prothrombotic pathways present in cancer patients. However, it takes time (up to several months) to grow tumors in mice. Early studies detected tumor-derived TF protein and activity in plasma from mice bearing human tumors [ 13 , 65 ]. Later, we detected tumor-derived TF+ EVs in plasma from mice bearing human pancreatic tumors [ 60 ]. Importantly, the presence of TF-expressing tumors was associated with increased activation of coagulation, as measured by increased TAT, and thrombosis was compared with mice without tumors ( Table 2 ) [ [59] , [60] , [61] , [62] , 66 ]. Furthermore, we found that the administration of an anti-human TF antibody reduced thrombus weight in mice bearing human pancreatic BxPC-3 tumors compared with a control antibody [ 66 ]. The human TF derived from the tumor was also detected in thrombi from BxPC-3 tumor-bearing mice [ 66 ]. These data suggest that tumors release TF+ EVs into circulation and these enhance venous thrombosis in mice. Table 2 Tissue factor and increased thrombosis in different mouse cancer models. Model Tumor Site of tumor Phenotype Reference Allograft Murine pancreatic cancer cell line Panc02 Subcutaneous Reduced vessel occlusion times in tumor-bearing mice in FeCl 3 mesenteric arteriole and venule occlusion models [ 59 ] Allograft Murine pancreatic cancer cell line Panc02 Subcutaneous Increased venous thrombosis in tumor-bearing mice in an IVC stenosis model (3 h) [ 62 ] Xenograft Human pancreatic cancer cell line HPAF-II Pancreas Reduced vessel occlusion times in tumor-bearing mice in a FeCl 3 saphenous vein occlusion model [ 60 ] Xenograft Human pancreatic cancer cell line BxPC-3 Pancreas Increased venous thrombosis in tumor-bearing mice in an IVC stenosis model (3–24 h) [ 61 ] Xenograft Human pancreatic cancer cell line BxPC-3 Pancreas Increased venous thrombosis in tumor-bearing mice in an IVC stasis model (48 h) [ 66 ] IVC, inferior vena cava.

Podoplanin

PDPN is a transmembrane glycoprotein that induces platelet aggregation by binding to C-type lectin-like receptor 2 (CLEC-2) [ 67 ].

PDPN and VTE in patients with cancer

PDPN is expressed in different types of cancer, including mesothelioma, seminoma, and glioma [ [68] , [69] , [70] ]. Interestingly, squamous cell carcinoma is associated with high PDPN expression [ 67 ]. Two studies reported that there is a strong inverse association between PDPN protein expression levels and IDH1 mutation in patients with glioma [ 71 , 72 ]. One study found that higher PDPN hypermethylation was associated with lower PDPN mRNA expression in patients with low-grade glioma but not in patients with glioblastoma multiforme [ 71 ]. Another study found that there is a glioma-CpG island methylator phenotype that is prevalent in lower-grade gliomas and is associated with IDH1 somatic mutation [ 30 ]. These data indicate that regulation of PDPN gene expression by IDH1 mutation may be dependent on the glioma grade.

The levels of PDPN gene expression were different in different subtypes of glioblastoma with the highest expression in the mesenchymal type and the lowest expression in the proneural type [ 73 ]. Additional studies are needed to determine the associations between PDPN gene expression, glioblastoma subtype, and VTE.

Although an early study found that PDPN induces platelet aggregation [ 74 ], the association between levels of PDPN and CAT was not recognized until recently. Thaler et al. [ 75 ] found that a low platelet count was associated with a high risk of VTE in patients with high-grade gliomas. In a follow-up study, they found that there was a significant association between high PDPN protein expression in tumors and a low platelet count in patients with glioma [ 68 ]. Additionally, they found that tumor PDPN protein expression levels were associated with an increased risk of VTE in patients with glioma [ 68 ]. A recent study also found that there was an association between tumor PDPN protein expression and VTE in patients with pulmonary squamous cell carcinoma [ 76 ]. Interestingly, the risk of VTE in patients with IDH1 wild-type glioma tumors was found to be strongly associated with increased tumor PDPN mRNA expression levels [ 71 ]. Thus, these data suggest that PDPN induces platelet aggregation that causes VTE in these patients with cancer.

Like TF, PDPN is released from cells in the form of EVs. One study detected PDPN+ EVs in plasma from patients with pancreatic and colorectal cancer using flow cytometry [ 77 ]. However, the association between levels of PDPN+ EVs and VTE in patients with cancer was not determined. Further studies are needed to measure the levels of PDPN+ EVs in circulation and determine their association with VTE.

A recent study from Gi et al. [ 57 ] evaluated PDPN expression in postmortem venous thrombi in patients with cancer. They found that 44% of the cancer cells in the venous thrombi expressed PDPN [ 57 ]. These cancer cells may activate platelets and initiate thrombosis in patients with cancer.

PDPN and thrombosis in preclinical studies

Recent studies have reported the role of PDPN in CAT in mouse models ( Table 3 ). One study used murine melanoma B16F10 tumors, which expressed PDPN. Intratumoral vascular thrombosis was reduced in tumor-bearing CLEC-2 deficient mice compared with tumor-bearing wild-type mice [ 78 ]. Moreover, the administration of an anti-CLEC-2 antibody reduced fibrin in the lungs of tumor-bearing mice compared with tumor-bearing mice that received a control antibody [ 78 ]. Lee et al. [ 79 ] used the human oral squamous cell carcinoma cell line OECM-1 that expresses high levels of PDPN. They found that OECM-1 cells activated platelets. Moreover, higher levels of platelets were present in OECM-1 tumors grown in the neck of nude mice compared with tumors with PNPD gene knockdown. In addition, mice-bearing OECM-1 tumors had higher levels of plasma TAT and fibrin in the small vessels of the pulmonary vasculature and kidney glomeruli compared with mice-bearing OECM-1 tumors with reduced PDPN expression [ 79 ]. More recently, Wang et al. [ 76 ] found that the podoplanin expressing human melanoma cell line C8161 induced platelet aggregation in vitro. Furthermore, the administration of an anti-PDPN antibody significantly reduced thrombosis in an IVC stenosis model in mice-bearing C8161 tumors [ 76 ]. These data indicate that PDPN expression by tumors enhances thrombosis in mice. Table 3 Podoplanin and platelet activation and thrombosis in different mouse cancer models. Model Tumor Site of tumor Phenotype Reference Allograft Murine melanoma cell line B16F10 Subcutaneous Reduced intratumoral vascular thrombosis in CLEC-2 deficient mice. An anti-CLEC-2 Ab reduced fibrin in the lung. [ 78 ] Xenograft Human oral squamous cell carcinoma cell line OECM-1 Neck Knockdown of PDPN in the tumor reduced platelets in the tumor, plasma TAT, and fibrin in the lung and kidney. [ 79 ] Xenograft Human glioma cell line U373 Subcutaneous Tumor-bearing mice had increased plasma PF4 and decreased platelet count. Tumors expressing both PDPN and TF had increased platelets and fibrin in tumor vessels. [ 80 ] Xenograft Human melanoma cell line C8161 Subcutaneous An anti-PDPN Ab reduced thrombosis in an IVC stenosis model. [ 76 ] Ab, antibody, CLEC-2, C-type lectin-like receptor 2, IVC, inferior vena cava; PDPN, podoplanin; PF4, platelet factor 4; TAT, thrombin-antithrombin complexes; TF, tissue factor.

Other studies reported that PDPN is expressed on EVs released from the cancer cells [ 80 , 81 ]. Sasano et al. [ 81 ] found that the incidence of venous thrombosis in an IVC stenosis model was lower in mice injected with EVs derived from the human ovarian cancer cell line HeyA8 in which the PDPN gene was knocked down compared with mice injected with EVs derived from wild-type HeyA8 cells expressing PDPN. These data indicate that PDPN+ EVs enhance venous thrombosis in mice.

In our recent study, we used several sublines of a human glioma cell line U373P, which express either TF or PDPN or both [ 80 ]. Mice-bearing PDPN-expressing tumors had increased levels of the platelet activation marker PF4 and a decreased platelet count compared with controls [ 80 ]. Interestingly, mice-bearing U373P tumors co-expressing TF and PDPN had significantly increased fibrin and platelet deposition in tumor vessels compared with the mice-bearing U373P tumors, expressing either TF or PDPN [ 80 ]. These data suggest that TF and PDPN may cooperate to enhance microthrombosis in tumors and possibly at distal sites in mice.

Neutrophil Extracellular Traps

NETs released from activated neutrophils are composed of extracellular chromatin and granule proteins [ 82 ]. NETs were originally identified as a part of the innate immune system to help neutrophils to kill bacteria [ 82 ]. However, more recently it was shown that NETs also enhance thrombosis by capturing platelets and EVs, including TF+ EVs [ 62 , [83] , [84] , [85] ]. A variety of cytokines, proteins, and other agents promote NET formation in cancer, including granulocyte-colony stimulating factor (G-CSF) [ 86 ], lipopolysaccharide (LPS) [ 86 ], interleukin (IL)-8 [ 87 , 88 ], inflammasomes [ 89 ], IL-1β [ 89 ], IL-17 [ 90 ], cathepsin C [ 91 ], and high mobility group box 1 (HMGB-1) protein [ 92 , 93 ].

NETs and VTE in patients with cancer

There are a variety of biomarkers that have been used to assess NET formation [ 93 ]. Several studies have reported that NET biomarkers are increased in patients with cancer compared with healthy controls [ [94] , [95] , [96] , [97] , [98] , [99] , [100] ]. However, it is important to note that citrullinated histone H3 (H3Cit) and H3Cit-DNA complexes are the best biomarkers for NETs. Myeloperoxidase (MPO)-DNA complexes can also be used, but a study questioned the specificity of the commercial assay [ 101 ]. Other biomarkers, such as neutrophil elastase, cell-free (cf) DNA, and nucleosomes are not specific for NETs.

NETs have been detected in the thrombi of patients with cancer. Thalin et al. [ 95 ] detected the colocalization of H3Cit and DNA in arterial microthrombi in patients with cancer. Similarly, Oklu et al. [ 96 ] observed histone-DNA complexes in venous thrombi in patients with cancer.

Several studies have examined the association between NET biomarkers and VTE and arterial thromboembolic events (ATE) in patients with cancer. Mauracher et al. [ 102 ] measured plasma levels of cfDNA, nucleosomes, and H3Cit in 984 cancer patients, of which 89 (9%) had a VTE in the 2-year follow-up. Importantly, H3Cit was associated with VTE. Moreover, cfDNA and nucleosomes were associated only with VTE in the high-risk period VTE (3–6 mo after the diagnosis). Although the study was underpowered for sub-analyses, Mauracher et al. [ 102 ] found strong associations between plasma H3Cit levels and VTE in patients with lung and pancreatic patients, a borderline association in patients with breast cancer, and no association in patients with brain or colon/rectum cancer. A small study of 27 patients with cancer found no association between cfDNA or nucleosomes and venous thrombosis in patients with cancer [ 96 ]. In a study of 177 patients with hepatocellular carcinoma, of which 77 (43%) had portal vein thrombosis, cfDNA, neutrophil elastase, and DNA-histone complexes were all associated with thrombosis [ 103 ]. In a study of 52 patients with myeloproliferative neoplasms (MPN) half of which have a history of thrombosis, MPO-DNA complexes were associated with thrombosis [ 104 ]. Interestingly, a recent study did not find any difference in the levels of H3Cit in thrombi from patients with cancer compared with thrombi from patients without cancer [ 57 ]. These studies suggest that NETs play a role in CAT in a cancer-type-specific manner.

Grilz et al. [ 105 ] measured plasma levels of cfDNA, nucleosomes, and H3Cit in 957 cancer patients, of which 22 (2%) had an ATE in the 2-year follow-up. None of the NET biomarkers were associated with ATE, but H3Cit and cfDNA were associated with higher mortality. Another smaller study measured cfDNA and nucleosomes in 138 patients with cancer 38 (27%) of which had a stroke and found that cfDNA was associated with stroke [ 106 ].

NETs and thrombosis in preclinical studies

NET formation is increased in tumor-bearing mouse models ( Table 4 ) [ 86 , [107] , [108] , [109] , [110] ]. Therefore, mouse models have been used to investigate the roles of NETs in thrombosis in tumor-bearing mice. An early study found microthrombi in the lungs of mice bearing murine mammary 4T1 tumors, which were associated with plasma levels of cfDNA [ 86 ]. Leal et al. [ 108 ] demonstrated that 4T1 tumor-bearing mice had shortened venous occlusion times in a Rose Bengal laser-induced injury model of the jugular vein and FeCl 3 -induced injury of the carotid artery compared with control mice. Interestingly, the shortened occlusion times in the jugular vein but not the carotid artery in 4T1 tumor-bearing mice were normalized when the mice were pretreated with DNase I [ 108 ]. Similarly, we and other investigators found that increased venous thrombosis in tumor-bearing mice was abolished when mice were treated with DNase I [ 110 , 111 ]. We also found that the depletion of neutrophils abolished the increased venous thrombosis in BxPC-3 tumor-bearing mice compared with the mice without tumors [ 110 ]. Importantly, DNase I treatment and neutrophil depletion did not affect venous thrombosis in control mice [ 110 ]. Wolach et al. [ 109 ] found the increased NET formation and enhanced venous thrombosis in a mouse model of MPN ( JAK2 gene V617F mutation) compared with wild-type controls. Increased NET formation and enhanced venous thrombosis were abolished when the mice were treated with a JAK2 inhibitor [ 109 ]. These data suggest that NETs enhance venous thrombosis in these tumor-bearing mice. Table 4 Neutrophils and NETs in thrombosis in different mouse cancer models. Model Tumor Site of tumor Phenotype Reference Allograft Murine breast cancer cell line 4T1 Mammary fat pad Tumor-bearing mice had increased levels of neutrophils, cfDNA, and H3Cit, and had vWF-and fibrin-rich thrombi in the veins in the lung. [ 86 ] Allograft Murine breast cancer cell line 4T1 Mammary fat pad Tumor-bearing mice had increased levels of neutrophils, cfDNA, and MPO, and had shortened occlusion times in jugular vein and carotid artery injury models. DNase I normalized the occlusion time in the jugular vein but not the carotid artery in tumor-bearing mice. [ 108 ] Allograft Murine forestomach squamous cell carcinoma cell line MFC Subcutaneous Tumor-bearing mice had increased thrombosis in an IVC stenosis model that was inhibited with DNase I. [ 111 ] Xenograft Human pancreatic cancer cell line BxPC-3 Pancreas Tumor-bearing mice had increased levels of neutrophils, neutrophil elastase, cfDNA, and H3Cit, and increased thrombosis in an IVC stasis model (48 h). DNase I reduced in tumor-bearing mice. [ 110 ] Conditional knock-in of Jak2 V617F NA NA Tumor-bearing mice had increased cfDNA and thrombosis at 3 h but not 4 h thrombosis in an IVC stenosis model. The JAK2 inhibitor ruxolitinib reduced thrombosis. [ 109 ] cf, cell-free; H3Cit, citrullinated histone H3; IVC, inferior vena cava; MPO, myeloperoxidase; NA, not applicable; vWF, von Willebrand factor.

Plasminogen Activator Inhibitor-1

PAI-1 is a serine protease inhibitor that inhibits tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) and thereby reduces the generation of plasmin. Increased levels of circulating PAI-1 are associated with thrombosis [ 112 ].

PAI-1 and VTE in patients with cancer

Levels of circulating PAI-1 are increased in different types of cancer, including melanoma, colorectal, breast, and pancreatic cancer [ [113] , [114] , [115] , [116] ]. There are limited numbers of studies that have analyzed the association between levels of PAI-1 and VTE in patients with cancer. These studies measured either PAI-1 antigen, PAI-1 activity, or both in patients with cancer. We believe that PAI-1 activity is superior to the PAI-1 antigen because the PAI-1 antigen includes both active and inactive forms of PAI-1. Indeed, a case-control study of biomarkers for VTE found that PAI-1 activity was superior to PAI-1 antigen for distinguishing patients with VTE from healthy controls [ 117 ].

We found an association between levels of PAI-1 activity and VTE in patients with pancreatic cancer, which suggested that PAI-1 contributes to VTE in patients with pancreatic cancer [ 118 ]. The strength of our study was that it was specifically designed to investigate biomarkers for VTE in patients with cancer. In contrast, Kondo et al. [ 119 ] found no association between PAI-1 antigen and VTE in patients with pancreatic cancer. Furthermore, no association was found between previous deep vein thrombosis and plasma levels of PAI-1 antigen and activity in patients with pancreatic cancer [ 116 ]. However, it is notable that this study found intermittent peaks of both PAI-1 antigen and PAI-1 activity in 11 out of 14 patients. This suggests that there may be transient hypercoagulable states in these patients. In a recent study, we observed an association between PAI-1 activity and VTE in patients with acute leukemia (Hisada, Mackman, Gangaradu, unpublished data). Additional studies are needed to determine the association between PAI-1 and VTE in patients with cancer.

PAI-1 and thrombosis in preclinical studies

We and other investigators found that tumor-bearing mice have increased levels of PAI-1 antigen and activity in plasma [ 118 , 120 ]. To date, there are only 2 studies that investigated the role of PAI-1 in CAT in mice. The first study showed that mice bearing human lung A549 tumors had increased thrombus weight in an IVC stenosis model compared with the mice without tumors [ 121 ]. Interestingly, the authors found that administration of the vascular endothelial growth factor inhibitor bevacizumab increased PAI-1 levels in tumors and plasma, and venous thrombosis in mice [ 121 ]. The increase in venous thrombosis by bevacizumab was abolished in mice-bearing A549 tumors when PAI-1 was inhibited with a small-molecule PAI-1 inhibitor PAI-039 [ 121 ]. However, the authors only evaluated venous thrombosis at 3 h after IVC stenosis, which is a very early time point for evaluating thrombosis in this model. Indeed, Obi et al. [ 122 ] found that the overexpression of PAI-1 in mice only affected thrombosis 6 and 14 d after IVC stasis. Consistent with this observation, we observed increased thrombus weight in mice bearing human pancreatic PANC-1 tumors that expressed high levels of PAI-1, 8 d after IVC stasis [ 118 ]. We did not observe any difference in levels of macrophages in thrombi from tumor-bearing mice and mice without tumors. This suggested that PAI-1 reduced thrombus resolution by inhibiting plasmin generation.

ISTH Congress Reports

Siddiqui et al. [ 123 ] analyzed biomarkers associated with pulmonary embolism (PE) in patients with cancer. They collected plasma samples from 122 PE patients with cancer as well as 260 PE patients without cancer and 50 healthy controls. Plasma samples from PE patients were collected within 24 to 72 h of the diagnosis. They measured plasma levels of D-dimer, PAI-1, tPA, thrombin activatable fibrinolysis inhibitor (TAFI), von Willebrand factor (vWF), C-reactive protein, interleukin-6, and EVs. They found that plasma levels of vWF, interleukin-6, and EVs were significantly increased in PE patients with cancer compared with PE patients without cancer. The limitation of this study was that it was a relatively small retrospective study, and there might be effects of treatment of the PE or the PE itself on the biomarkers.

Guy et al. [ 124 ] presented the mechanisms of NET formation in MPN using mouse models. They established 2 mouse models in which the JAK2 V617F mutation is present in either neutrophils (MRP8 Cre , JAK2 V617F/WT ) or platelets (PF4 Cre , JAK2 V617F/WT ). They found that NET formation and microthrombi in the lungs of mice increased in PF4 Cre , JAK2 V617F/WT but not in mMRP8 Cre , JAK2 V617F/WT mice. DNase treatment significantly reduced microthrombi in the lungs of PF4 Cre and JAK2 V617F/WT mice. Moreover, aspirin treatment significantly reduced NET formation and microthrombi in the lungs of PF4 Cre and JAK2 V617F/WT mice. Strikingly, MPN patients with the JAK2 V617F mutation treated with aspirin had significantly lower plasma levels of MPO-DNA complexes compared with MPN patients with the JAK2 V617F mutation without aspirin treatment.

Future Direction

Future studies should focus on identifying cancer-type-specific mechanisms of CAT. A better understanding of these mechanisms may identify patients with cancer with a high risk for VTE. Since patients with cancer have an increased risk of bleeding compared with the general population, new anticoagulants that cause less bleeding, such as FXIa inhibitors, may be safer than the conventional anticoagulants for the prevention of CAT.

References

1 Timp J.F. Braekkan S.K. Versteeg H.H. Cannegieter S.C. Epidemiology of cancer-associated venous thrombosis Blood 122 2013 1712 1723 23908465 10.1182/blood-2013-04-460121

2 Mulder F.I. Horvath-Puho E. van Es N. van Laarhoven H.W.M. Pedersen L. Moik F. Venous thromboembolism in cancer patients: a population-based cohort study Blood 137 2021 1959 1969 33171494 10.1182/blood.2020007338

3 Mahajan A. Brunson A. Adesina O. Keegan T.H.M. Wun T. The incidence of cancer-associated thrombosis is increasing over time Blood Adv 6 2022 307 320 34649273 10.1182/bloodadvances.2021005590 PMC8753193

4 Khorana A.A. Kuderer N.M. McCrae K. Milentijevic D. Germain G. Laliberte F. Cancer associated thrombosis and mortality in patients with cancer stratified by Khorana score risk levels Cancer Med 9 2020 8062 8073 32954653 10.1002/cam4.3437 PMC7643641

5 Hogg K. Carrier M. Prevention and treatment of venous thromboembolism in patients with cancer Ther Adv Hematol 3 2012 45 58 23556111 10.1177/2040620711422590 PMC3573425

6 Lyman G.H. Carrier M. Ay C. Di Nisio M. Hicks L.K. Khorana A.A. American Society of Hematology 2021 guidelines for management of venous thromboembolism: prevention and treatment in patients with cancer Blood Adv 5 2021 927 974 33570602 10.1182/bloodadvances.2020003442 PMC7903232

7 Pabinger I. Thaler J. Ay C. Biomarkers for prediction of venous thromboembolism in cancer Blood 122 2013 2011 2018 23908470 10.1182/blood-2013-04-460147

8 Hisada Y. Mackman N. Cancer-associated pathways and biomarkers of venous thrombosis Blood 130 2017 1499 1506 28807983 10.1182/blood-2017-03-743211 PMC5620413

9 Hisada Y. Mackman N. Mouse models of cancer-associated thrombosis Thromb Res 164 Supplement 1 2018 S48 S53 29306575 10.1016/j.thromres.2017.12.018 PMC5942599

10 Grover S.P. Mackman N. Tissue factor: an essential mediator of hemostasis and trigger of thrombosis Arterioscler Thromb Vasc Biol 38 2018 709 725 29437578 10.1161/ATVBAHA.117.309846

11 Khorana A.A. Ahrendt S.A. Ryan C.K. Francis C.W. Hruban R.H. Hu Y.C. Tissue factor expression, angiogenesis, and thrombosis in pancreatic cancer Clin Cancer Res 13 2007 2870 2875 17504985 10.1158/1078-0432.CCR-06-2351

12 Kasthuri R.S. Taubman M.B. Mackman N. Role of tissue factor in cancer J Clin Oncol 27 2009 4834 4838 19738116 10.1200/JCO.2009.22.6324 PMC2764391

13 Yu J.L. May L. Lhotak V. Shahrzad S. Shirasawa S. Weitz J.I. Oncogenic events regulate tissue factor expression in colorectal cancer cells: implications for tumor progression and angiogenesis Blood 105 2005 1734 1741 15494427 10.1182/blood-2004-05-2042

14 Regina S. Rollin J. Blechet C. Iochmann S. Reverdiau P. Gruel Y. Tissue factor expression in non-small cell lung cancer: relationship with vascular endothelial growth factor expression, microvascular density, and K-ras mutation J Thorac Oncol 3 2008 689 697 18594312 10.1097/JTO.0b013e31817c1b21

15 Regina S. Valentin J.B. Lachot S. Lemarie E. Rollin J. Gruel Y. Increased tissue factor expression is associated with reduced survival in non-small cell lung cancer and with mutations of TP53 and PTEN Clin Chem 55 2009 1834 1842 19661141 10.1373/clinchem.2009.123695

16 Yang S. Yang L. Wu Y. Zhang C. Wang S. Ma N. Anaplastic lymphoma kinase rearrangement may increase the incidence of venous thromboembolism by increasing tissue factor expression in advanced lung adenocarcinoma Ann Transl Med 8 2020 1307 33209887 10.21037/atm-20-6619 PMC7661863

17 Roopkumar J. Poudel S.K. Gervaso L. Reddy C.A. Velcheti V. Pennell N.A. Risk of thromboembolism in patients with ALK- and EGFR-mutant lung cancer: A cohort study J Thromb Haemost 19 2021 822 829 33314597 10.1111/jth.15215

18 Al-Samkari H. Leiva O. Dagogo-Jack I. Shaw A. Lennerz J. Iafrate A.J. Impact of ALK rearrangement on venous and arterial thrombotic risk in NSCLC J Thorac Oncol 15 2020 1497 1506 32437899 10.1016/j.jtho.2020.04.033

19 Zer A. Moskovitz M. Hwang D.M. Hershko-Klement A. Fridel L. Korpanty G.J. ALK-rearranged non-small-cell lung cancer is associated with a high rate of venous thromboembolism Clin Lung Cancer 18 2017 156 161 27913214 10.1016/j.cllc.2016.10.007

20 Alexander M. Solomon B. Burbury K. Thromboembolism in anaplastic lymphoma kinase-rearranged non-small cell lung cancer Clin Lung Cancer 19 2018 e71 e72 28818428 10.1016/j.cllc.2017.07.001

21 Davidsson E. Murgia N. Ortiz-Villalon C. Wiklundh E. Skold M. Kolbeck K.G. Mutational status predicts the risk of thromboembolic events in lung adenocarcinoma Multidiscip Respir Med 12 2017 16 28560038 10.1186/s40248-017-0097-0 PMC5447302

22 Lee Y.G. Kim I. Lee E. Bang S.M. Kang C.H. Kim Y.T. Risk factors and prognostic impact of venous thromboembolism in Asian patients with non-small cell lung cancer Thromb Haemost 111 2014 1112 1120 24477503 10.1160/TH13-11-0956

23 Verso M. Chiari R. Mosca S. Franco L. Fischer M. Paglialunga L. Incidence of CT scan-detected pulmonary embolism in patients with oncogene-addicted, advanced lung adenocarcinoma Thromb Res 136 2015 924 927 26377037 10.1016/j.thromres.2015.09.006

24 Solomon B.J. Mok T. Kim D.W. Wu Y.L. Nakagawa K. Mekhail T. First-line crizotinib versus chemotherapy in ALK-positive lung cancer N Engl J Med 371 2014 2167 2177 25470694 10.1056/NEJMoa1408440

25 Shaw A.T. Kim D.W. Mehra R. Tan D.S. Felip E. Chow L.Q. Ceritinib in ALK-rearranged non-small-cell lung cancer N Engl J Med 370 2014 1189 1197 24670165 10.1056/NEJMoa1311107 PMC4079055

26 Shaw A.T. Kim D.W. Nakagawa K. Seto T. Crino L. Ahn M.J. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer N Engl J Med 368 2013 2385 2394 23724913 10.1056/NEJMoa1214886

27 Gettinger S.N. Bazhenova L.A. Langer C.J. Salgia R. Gold K.A. Rosell R. Activity and safety of brigatinib in ALK-rearranged non-small-cell lung cancer and other malignancies: a single-arm, open-label, phase 1/2 trial Lancet Oncol 17 2016 1683 1696 27836716 10.1016/S1470-2045(16)30392-8

28 Dunbar A. Bolton K.L. Devlin S.M. Sanchez-Vega F. Gao J. Mones J.V. Genomic profiling identifies somatic mutations predicting thromboembolic risk in patients with solid tumors Blood 137 2021 2103 2113 33270827 10.1182/blood.2020007488 PMC8057259

29 Yan H. Parsons D.W. Jin G. McLendon R. Rasheed B.A. Yuan W. IDH1 and IDH2 mutations in gliomas N Engl J Med 360 2009 765 773 19228619 10.1056/NEJMoa0808710 PMC2820383

30 Noushmehr H. Weisenberger D.J. Diefes K. Phillips H.S. Pujara K. Berman B.P. Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma Cancer Cell 17 2010 510 522 20399149 10.1016/j.ccr.2010.03.017 PMC2872684

31 Unruh D. Schwarze S.R. Khoury L. Thomas C. Wu M. Chen L. Mutant IDH1 and thrombosis in gliomas Acta Neuropathol 132 2016 917 930 27664011 10.1007/s00401-016-1620-7 PMC5640980

32 Verhaak R.G. Hoadley K.A. Purdom E. Wang V. Qi Y. Wilkerson M.D. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1 Cancer Cell 17 2010 98 110 20129251 10.1016/j.ccr.2009.12.020 PMC2818769

33 Magnus N. Gerges N. Jabado N. Rak J. Coagulation-related gene expression profile in glioblastoma is defined by molecular disease subtype J Thromb Haemost 11 2013 1197 1200 23582031 10.1111/jth.12242

34 Gyorgy B. Szabo T.G. Pasztoi M. Pal Z. Misjak P. Aradi B. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles Cell Mol Life Sci 68 2011 2667 2688 21560073 10.1007/s00018-011-0689-3 PMC3142546

35 Tesselaar M.E. Romijn F.P. Van Der Linden I.K. Prins F.A. Bertina R.M. Osanto S. Microparticle-associated tissue factor activity: a link between cancer and thrombosis? J Thromb Haemost 5 2007 520 527 17166244 10.1111/j.1538-7836.2007.02369.x

36 Khorana A.A. Francis C.W. Menzies K.E. Wang J.G. Hyrien O. Hathcock J. Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer J Thromb Haemost 6 2008 1983 5 18795992 10.1111/j.1538-7836.2008.03156.x PMC2848502

37 Lee R.D. Barcel D.A. Williams J.C. Wang J.G. Boles J.C. Manly D.A. Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity Thromb Res 129 2012 80 85 21737126 10.1016/j.thromres.2011.06.004 PMC3272762

38 Vallier L. Bouriche T. Bonifay A. Judicone C. Bez J. Franco C. Increasing the sensitivity of the human microvesicle tissue factor activity assay Thromb Res 182 2019 64 74 31450010 10.1016/j.thromres.2019.07.011 PMC6825876

39 Franco C. Lacroix R. Vallier L. Judicone C. Bouriche T. Laroumagne S. A new hybrid immunocapture bioassay with improved reproducibility to measure tissue factor-dependent procoagulant activity of microvesicles from body fluids Thromb Res 196 2020 414 424 33038585 10.1016/j.thromres.2020.09.020

40 Tatsumi K. Antoniak S. Monroe D.M. 3rd Khorana A.A. Mackman N. Subcommittee on Hemostasis and Malignancy of the Scientific and Standardization Committee of the International Society on Thrombosis and Hemostasis. Evaluation of a new commercial assay to measure microparticle tissue factor activity in plasma: communication from the SSC of the ISTH J Thromb Haemost 12 2014 1932 1934 25186801 10.1111/jth.12718

41 Hellum M. Ovstebo R. Troseid A.M. Berg J.P. Brandtzaeg P. Henriksson C.E. Microparticle-associated tissue factor activity measured with the Zymuphen MP-TF kit and the calibrated automated thrombogram assay Blood Coagul Fibrinolysis 23 2012 520 526 22732249 10.1097/MBC.0b013e328354a256

42 Mackman N. Hisada Y. Archibald S.J. Tissue factor and its procoagulant activity on cancer-associated thromboembolism in pancreatic cancer: comment by Mackman et al Cancer Sci 113 2022 1885 1887 35132733 10.1111/cas.15276 PMC9128168

43 Auwerda J.J. Yuana Y. Osanto S. de Maat M.P. Sonneveld P. Bertina R.M. Microparticle-associated tissue factor activity and venous thrombosis in multiple myeloma Thromb Haemost 105 2011 14 20 21057704 10.1160/TH10-03-0187

44 Thaler J. Ay C. Mackman N. Metz-Schimmerl S. Stift J. Kaider A. Microparticle-associated tissue factor activity in patients with pancreatic cancer: correlation with clinicopathological features Eur J Clin Investig 43 2013 277 285 23398637 10.1111/eci.12042

45 Woei-A-Jin F.J. Tesselaar M.E. Garcia Rodriguez P. Romijn F.P. Bertina R.M. Osanto S. Tissue factor-bearing microparticles and CA19.9: two players in pancreatic cancer-associated thrombosis? Br J Cancer 115 2016 332 338 27404454 10.1038/bjc.2016.170 PMC4973148

46 Hisada Y. Thalin C. Lundstrom S. Wallen H. Mackman N. Comparison of microvesicle tissue factor activity in non-cancer severely ill patients and cancer patients Thromb Res 165 2018 1 5 29539580 10.1016/j.thromres.2018.03.001 PMC5943147

47 Tesselaar M.E. Romijn F.P. van der Linden I.K. Bertina R.M. Osanto S. Microparticle-associated tissue factor activity in cancer patients with and without thrombosis J Thromb Haemost 7 2009 1421 1423 19500241 10.1111/j.1538-7836.2009.03504.x

48 van Doormaal F. Kleinjan A. Berckmans R.J. Mackman N. Manly D. Kamphuisen P.W. Coagulation activation and microparticle-associated coagulant activity in cancer patients. An exploratory prospective study Thromb Haemost 108 2012 160 165 22535219 10.1160/TH12-02-0099

49 Bharthuar A. Khorana A.A. Hutson A. Wang J.G. Key N.S. Mackman N. Circulating microparticle tissue factor, thromboembolism and survival in pancreaticobiliary cancers Thromb Res 132 2013 180 184 23856554 10.1016/j.thromres.2013.06.026

50 Kasthuri R.S. Hisada Y. Ilich A. Key N.S. Mackman N. Effect of chemotherapy and longitudinal analysis of circulating extracellular vesicle tissue factor activity in patients with pancreatic and colorectal cancer Res Pract Thromb Haemost 4 2020 636 643 32548563 10.1002/rth2.12317 PMC7292676

51 Thaler J. Ay C. Mackman N. Bertina R.M. Kaider A. Marosi C. Microparticle-associated tissue factor activity, venous thromboembolism and mortality in pancreatic, gastric, colorectal and brain cancer patients J Thromb Haemost 10 2012 1363 1370 22520016 10.1111/j.1538-7836.2012.04754.x

52 Uno K. Homma S. Satoh T. Nakanishi K. Abe D. Matsumoto K. Tissue factor expression as a possible determinant of thromboembolism in ovarian cancer Br J Cancer 96 2007 290 295 17211468 10.1038/sj.bjc.6603552 PMC2359996

53 Sakurai M. Matsumoto K. Gosho M. Sakata A. Hosokawa Y. Tenjimbayashi Y. Expression of tissue factor in epithelial ovarian carcinoma is involved in the development of venous thromboembolism Int J Gynecol Cancer 27 2017 37 43 27755234 10.1097/IGC.0000000000000848 PMC5181121

54 Abu Saadeh F. Norris L. O'Toole S. Mohamed B.M. Langhe R. O'Leary J. Tumour expresion of tissue factor and tissue factor pathway inhibitor in ovarian cancer- relationship with venous thrombosis risk Thromb Res 132 2013 627 634 24094893 10.1016/j.thromres.2013.09.016

55 Cohen J.G. Prendergast E. Geddings J.E. Walts A.E. Agadjanian H. Hisada Y. Evaluation of venous thrombosis and tissue factor in epithelial ovarian cancer Gynecol Oncol 146 2017 146 152 28501328 10.1016/j.ygyno.2017.04.021

56 Gezelius E. Flou Kristensen A. Bendahl P.O. Hisada Y. Risom Kristensen S. Ek L. Coagulation biomarkers and prediction of venous thromboembolism and survival in small cell lung cancer: A sub-study of RASTEN - A randomized trial with low molecular weight heparin PLoS One 13 2018 e0207387 10.1371/journal.pone.0207387 PMC6226210 30412630

57 Gi T. Kuwahara A. Yamashita A. Matsuda S. Maekawa K. Moriguchi-Goto S. Histopathological features of cancer-associated venous thromboembolism: presence of intrathrombus cancer cells and prothrombotic factors Arterioscler Thromb Vasc Biol 43 2023 146 159 36384269 10.1161/ATVBAHA.122.318463 PMC9762717

58 Mackman N. Hisada Y. Circulating tumor cells and cancer-associated venous thrombosis: a missing link Arterioscler Thromb Vasc Biol 43 2023 160 162 36453278 10.1161/ATVBAHA.122.318715 PMC10234083

59 Thomas G.M. Panicot-Dubois L. Lacroix R. Dignat-George F. Lombardo D. Dubois C. Cancer cell-derived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo J Exp Med 206 2009 1913 1927 19667060 10.1084/jem.20082297 PMC2737159

60 Wang J.G. Geddings J.E. Aleman M.M. Cardenas J.C. Chantrathammachart P. Williams J.C. Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer Blood 119 2012 5543 5552 22547577 10.1182/blood-2012-01-402156 PMC3369688

61 Geddings J.E. Hisada Y. Boulaftali Y. Getz T.M. Whelihan M. Fuentes R. Tissue factor-positive tumor microvesicles activate platelets and enhance thrombosis in mice J Thromb Haemost 14 2016 153 166 26516108 10.1111/jth.13181 PMC4715578

62 Thomas G.M. Brill A. Mezouar S. Crescence L. Gallant M. Dubois C. Tissue factor expressed by circulating cancer cell-derived microparticles drastically increases the incidence of deep vein thrombosis in mice J Thromb Haemost 13 2015 1310 1319 25955268 10.1111/jth.13002 PMC4496280

63 Stark K. Schubert I. Joshi U. Kilani B. Hoseinpour P. Thakur M. Distinct pathogenesis of pancreatic cancer microvesicle-associated venous thrombosis identifies new antithrombotic targets in vivo Arterioscler Thromb Vasc Biol 38 2018 772 786 29419408 10.1161/ATVBAHA.117.310262

64 Sasano T. Cho M.S. Rodriguez-Aguayo C. Bayraktar E. Taki M. Afshar-Kharghan V. Role of tissue-factor bearing extracellular vesicles released from ovarian cancer cells in platelet aggregation in vitro and venous thrombosis in mice Thromb Update 2 2021 100020

65 Davila M. Amirkhosravi A. Coll E. Desai H. Robles L. Colon J. Tissue factor-bearing microparticles derived from tumor cells: impact on coagulation activation J Thromb Haemost 6 2008 1517 1524 18433463 10.1111/j.1538-7836.2008.02987.x

66 Hisada Y. Ay C. Auriemma A.C. Cooley B.C. Mackman N. Human pancreatic tumors grown in mice release tissue factor-positive microvesicles that increase venous clot size J Thromb Haemost 15 2017 2208 2217 28834179 10.1111/jth.13809

67 Suzuki-Inoue K. Platelets and cancer-associated thrombosis: focusing on the platelet activation receptor CLEC-2 and podoplanin Blood 134 2019 1912 1918 31778548 10.1182/blood.2019001388

68 Riedl J. Preusser M. Nazari P.M. Posch F. Panzer S. Marosi C. Podoplanin expression in primary brain tumors induces platelet aggregation and increases risk of venous thromboembolism Blood 129 2017 1831 1839 28073783 10.1182/blood-2016-06-720714 PMC5823234

69 Kimura N. Kimura I. Podoplanin as a marker for mesothelioma Pathol Int 55 2005 83 86 15693854 10.1111/j.1440-1827.2005.01791.x

70 Kato Y. Sasagawa I. Kaneko M. Osawa M. Fujita N. Tsuruo T. Aggrus: a diagnostic marker that distinguishes seminoma from embryonal carcinoma in testicular germ cell tumors Oncogene 23 2004 8552 8556 15361850 10.1038/sj.onc.1207869

71 Mir Seyed Nazari P. Riedl J. Preusser M. Posch F. Thaler J. Marosi C. Combination of isocitrate dehydrogenase 1 (IDH1) mutation and podoplanin expression in brain tumors identifies patients at high or low risk of venous thromboembolism J Thromb Haemost 16 2018 1121 1127 29676036 10.1111/jth.14129 PMC6099350

72 Birner P. Pusch S. Christov C. Mihaylova S. Toumangelova-Uzeir K. Natchev S. Mutant IDH1 inhibits PI3K/Akt signaling in human glioma Cancer 120 2014 2440 2447 24771584 10.1002/cncr.28732

73 Tawil N. Chennakrishnaiah S. Bassawon R. Johnson R. D'Asti E. Rak J. Single cell coagulomes as constituents of the oncogene-driven coagulant phenotype in brain tumours Thromb Res 164 2018 S136 S142 29703472 10.1016/j.thromres.2018.01.021

74 Toyoshima M. Nakajima M. Yamori T. Tsuruo T. Purification and characterization of the platelet-aggregating sialoglycoprotein gp44 expressed by highly metastatic variant cells of mouse colon adenocarcinoma 26 Cancer Res 55 1995 767 773 7850787

75 Thaler J. Ay C. Kaider A. Reitter E.M. Haselbock J. Mannhalter C. Biomarkers predictive of venous thromboembolism in patients with newly diagnosed high-grade gliomas Neuro Oncol 16 2014 1645 1651 24987133 10.1093/neuonc/nou106 PMC4232082

76 Wang X. Liu B. Xu M. Jiang Y. Zhou J. Yang J. Blocking podoplanin inhibits platelet activation and decreases cancer-associated venous thrombosis Thromb Res 200 2021 72 80 33548843 10.1016/j.thromres.2021.01.008

77 Mege D. Panicot-Dubois L. Ouaissi M. Robert S. Sielezneff I. Sastre B. The origin and concentration of circulating microparticles differ according to cancer type and evolution: A prospective single-center study Int J Cancer 138 2016 939 948 26341361 10.1002/ijc.29837

78 Shirai T. Inoue O. Tamura S. Tsukiji N. Sasaki T. Endo H. C-type lectin-like receptor 2 promotes hematogenous tumor metastasis and prothrombotic state in tumor-bearing mice J Thromb Haemost 15 2017 513 525 28028907 10.1111/jth.13604

79 Lee H.Y. Yu N.Y. Lee S.H. Tsai H.J. Wu C.C. Cheng J.C. Podoplanin promotes cancer-associated thrombosis and contributes to the unfavorable overall survival in an ectopic xenograft mouse model of oral cancer Biomed J 43 2020 146 162 32441651 10.1016/j.bj.2019.07.001 PMC7283562

80 Tawil N. Bassawon R. Meehan B. Nehme A. Montermini L. Gayden T. Glioblastoma cell populations with distinct oncogenic programs release podoplanin as procoagulant extracellular vesicles Blood Adv 5 2021 1682 1694 33720339 10.1182/bloodadvances.2020002998 PMC7993100

81 Sasano T. Gonzalez-Delgado R. Munoz N.M. Carlos-Alcade W. Cho M.S. Sheth R.A. Podoplanin promotes tumor growth, platelet aggregation, and venous thrombosis in murine models of ovarian cancer J Thromb Haemost 20 2022 104 114 34608736 10.1111/jth.15544 PMC8712373

82 Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Neutrophil extracellular traps kill bacteria Science 303 2004 1532 1535 15001782 10.1126/science.1092385

83 Fuchs T.A. Brill A. Duerschmied D. Schatzberg D. Monestier M. Myers D.D. Jr. Extracellular DNA traps promote thrombosis Proc Natl Acad Sci U S A 107 2010 15880 15885 20798043 10.1073/pnas.1005743107 PMC2936604

84 Petersen L.C. Bjorn S.E. Nordfang O. Effect of leukocyte proteinases on tissue factor pathway inhibitor Thromb Haemost 67 1992 537 541 1519213

85 Massberg S. Grahl L. von Bruehl M.L. Manukyan D. Pfeiler S. Goosmann C. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases Nat Med 16 2010 887 896 20676107 10.1038/nm.2184

86 Demers M. Krause D.S. Schatzberg D. Martinod K. Voorhees J.R. Fuchs T.A. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis Proc Natl Acad Sci U S A 109 2012 13076 13081 22826226 10.1073/pnas.1200419109 PMC3420209

87 Alfaro C. Teijeira A. Onate C. Perez G. Sanmamed M.F. Andueza M.P. Tumor-produced interleukin-8 attracts human myeloid-derived suppressor cells and elicits extrusion of neutrophil extracellular traps (NETs) Clin Cancer Res 22 2016 3924 3936 26957562 10.1158/1078-0432.CCR-15-2463

88 de Andrea C.E. Ochoa M.C. Villalba-Esparza M. Teijeira A. Schalper K.A. Abengozar-Muela M. Heterogenous presence of neutrophil extracellular traps in human solid tumours is partially dependent on IL-8 J Pathol 255 2021 190 201 34184758 10.1002/path.5753

89 Gomes T. Varady C.B.S. Lourenco A.L. Mizurini D.M. Rondon A.M.R. Leal A.C. IL-1beta blockade attenuates thrombosis in a neutrophil extracellular trap-dependent breast cancer model Front Immunol 10 2019 2088 31552036 10.3389/fimmu.2019.02088 PMC6737452

90 Zhang Y. Chandra V. Riquelme Sanchez E. Dutta P. Quesada P.R. Rakoski A. Interleukin-17-induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer J Exp Med 217 2020 e20190354 10.1084/jem.20190354 PMC7953739 32860704

91 Xiao Y. Cong M. Li J. He D. Wu Q. Tian P. Cathepsin C promotes breast cancer lung metastasis by modulating neutrophil infiltration and neutrophil extracellular trap formation Cancer Cell 39 2021 423 437.e7 33450198 10.1016/j.ccell.2020.12.012

92 Shinde-Jadhav S. Mansure J.J. Rayes R.F. Marcq G. Ayoub M. Skowronski R. Role of neutrophil extracellular traps in radiation resistance of invasive bladder cancer Nat Commun 12 2021 2776 33986291 10.1038/s41467-021-23086-z PMC8119713

93 Herre M. Cedervall J. Mackman N. Olsson A.K. Neutrophil extracellular traps in the pathology of cancer and other inflammatory diseases Physiol Rev 103 2023 277 312 35951483 10.1152/physrev.00062.2021 PMC9576172

94 Yang C. Sun W. Cui W. Li X. Yao J. Jia X. Procoagulant role of neutrophil extracellular traps in patients with gastric cancer Int J Clin Exp Pathol 8 2015 14075 14086 26823721 PMC4713507

95 Thalin C. Demers M. Blomgren B. Wong S.L. von Arbin M. von Heijne A. NETosis promotes cancer-associated arterial microthrombosis presenting as ischemic stroke with troponin elevation Thromb Res 139 2016 56 64 26916297 10.1016/j.thromres.2016.01.009 PMC4769435

96 Oklu R. Sheth R.A. Wong K.H.K. Jahromi A.H. Albadawi H. Neutrophil extracellular traps are increased in cancer patients but does not associate with venous thrombosis Cardiovasc Diagn Ther 7 Supplement 3 2017 S140 S149 29399517 10.21037/cdt.2017.08.01 PMC5778521

97 Thalin C. Lundstrom S. Seignez C. Daleskog M. Lundstrom A. Henriksson P. Citrullinated histone H3 as a novel prognostic blood marker in patients with advanced cancer PLoS One 13 2018 e0191231 10.1371/journal.pone.0191231 PMC5764486 29324871

98 Thalin C. Aguilera K. Hall N.W. Marunde M.R. Burg J.M. Rosell A. Quantification of citrullinated histones: development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma J Thromb Haemost 18 2020 2732 2743 32654410 10.1111/jth.15003 PMC8722705

99 Rosell A. Aguilera K. Hisada Y. Schmedes C. Mackman N. Wallen H. Prognostic value of circulating markers of neutrophil activation, neutrophil extracellular traps, coagulation and fibrinolysis in patients with terminal cancer Sci Rep 11 2021 5074 33658563 10.1038/s41598-021-84476-3 PMC7930088

100 Cedervall J. Herre M. Dragomir A. Rabelo-Melo F. Svensson A. Thalin C. Neutrophil extracellular traps promote cancer-associated inflammation and myocardial stress Oncoimmunology 11 2022 2049487 10.1080/2162402X.2022.2049487 PMC8928831 35309730

101 Hayden H. Ibrahim N. Klopf J. Zagrapan B. Mauracher L.M. Hell L. ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo PLoS One 16 2021 e0250265 10.1371/journal.pone.0250265 PMC8062102 33886636

102 Mauracher L.M. Posch F. Martinod K. Grilz E. Daullary T. Hell L. Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation, predicts the risk of venous thromboembolism in cancer patients J Thromb Haemost 16 2018 508 518 29325226 10.1111/jth.13951 PMC6294121

103 Seo J.D. Gu J.Y. Jung H.S. Kim Y.J. Kim H.K. Contact system activation and neutrophil extracellular trap markers: risk factors for portal vein thrombosis in patients with hepatocellular carcinoma Clin Appl Thromb Hemost 25 2019 1076029618825310 10.1177/1076029618825310 PMC6715110 30808222

104 Guy A. Favre S. Labrouche-Colomer S. Deloison L. Gourdou-Latyszenok V. Renault M.A. High circulating levels of MPO-DNA are associated with thrombosis in patients with MPN Leukemia 33 2019 2544 2548 31175322 10.1038/s41375-019-0500-2

105 Grilz E. Mauracher L.M. Posch F. Konigsbrugge O. Zochbauer-Muller S. Marosi C. Citrullinated histone H3, a biomarker for neutrophil extracellular trap formation, predicts the risk of mortality in patients with cancer Br J Haematol 186 2019 311 320 30968400 10.1111/bjh.15906 PMC6618331

106 Bang O.Y. Chung J.W. Cho Y.H. Oh M.J. Seo W.K. Kim G.M. Circulating DNAs, a marker of neutrophil extracellular traposis and cancer-related stroke: the OASIS-cancer study Stroke 50 2019 2944 2947 31394991 10.1161/STROKEAHA.119.026373

107 Cedervall J. Zhang Y. Huang H. Zhang L. Femel J. Dimberg A. Neutrophil extracellular traps accumulate in peripheral blood vessels and compromise organ function in tumor-bearing animals Cancer Res 75 2015 2653 2662 26071254 10.1158/0008-5472.CAN-14-3299

108 Leal A.C. Mizurini D.M. Gomes T. Rochael N.C. Saraiva E.M. Dias M.S. Tumor-derived exosomes induce the formation of neutrophil extracellular traps: implications for the establishment of cancer-associated thrombosis Sci Rep 7 2017 6438 28743887 10.1038/s41598-017-06893-7 PMC5526939

109 Wolach O. Sellar R.S. Martinod K. Cherpokova D. McConkey M. Chappell R.J. Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms Sci Transl Med 10 2018 eaan8292 10.1126/scitranslmed.aan8292 PMC6442466 29643232

110 Hisada Y. Grover S.P. Maqsood A. Houston R. Ay C. Noubouossie D.F. Neutrophils and neutrophil extracellular traps enhance venous thrombosis in mice bearing human pancreatic tumors Haematologica 105 2020 218 225 31048354 10.3324/haematol.2019.217083 PMC6939515

111 Li J.C. Zou X.M. Yang S.F. Jin J.Q. Zhu L. Li C.J. Neutrophil extracellular traps participate in the development of cancer-associated thrombosis in patients with gastric cancer World J Gastroenterol 28 2022 3132 3149 36051331 10.3748/wjg.v28.i26.3132 PMC9331535

112 Meltzer M.E. Lisman T. de Groot P.G. Meijers J.C. le Cessie S. Doggen C.J. Venous thrombosis risk associated with plasma hypofibrinolysis is explained by elevated plasma levels of TAFI and PAI-1 Blood 116 2010 113 121 20385790 10.1182/blood-2010-02-267740

113 Hanekom G.S. Stubbings H.M. Kidson S.H. The active fraction of plasmatic plasminogen activator inhibitor type 1 as a possible indicator of increased risk for metastatic melanoma Cancer Detect Prev 26 2002 50 59 12088203 10.1016/s0361-090x(02)00002-8

114 Herszenyi L. Plebani M. Carraro P. De Paoli M. Roveroni G. Cardin R. The role of cysteine and serine proteases in colorectal carcinoma Cancer 86 1999 1135 1142 10506696 10.1002/(sici)1097-0142(19991001)86:7<1135::aid-cncr6>3.0.co;2-2

115 Ferroni P. Roselli M. Portarena I. Formica V. Riondino S. Farina L.A.F. Plasma plasminogen activator inhibitor-1 (PAI-1) levels in breast cancer - relationship with clinical outcome Anticancer Res 34 2014 1153 1161 24596353

116 Andren-Sandberg A. Lecander I. Martinsson G. Astedt B. Peaks in plasma plasminogen activator inhibitor-1 concentration may explain thrombotic events in cases of pancreatic carcinoma Cancer 69 1992 2884 2887 1591681 10.1002/1097-0142(19920615)69:12<2884::aid-cncr2820691204>3.0.co;2-s

117 Bollen L. Peetermans M. Peeters M. Van Steen K. Hoylaerts M.F. Declerck P.J. Active PAI-1 as marker for venous thromboembolism: case-control study using a comprehensive panel of PAI-1 and TAFI assays Thromb Res 134 2014 1097 1102 25193405 10.1016/j.thromres.2014.08.007

118 Hisada Y. Garratt K.B. Maqsood A. Grover S.P. Kawano T. Cooley B.C. Plasminogen activator inhibitor 1 and venous thrombosis in pancreatic cancer Blood Adv 5 2021 487 495 33496742 10.1182/bloodadvances.2020003149 PMC7839372

119 Kondo S. Sasaki M. Hosoi H. Sakamoto Y. Morizane C. Ueno H. Incidence and risk factors for venous thromboembolism in patients with pretreated advanced pancreatic carcinoma Oncotarget 9 2018 16883 16890 29682191 10.18632/oncotarget.24721 PMC5908292

120 Furuya H. Hayashi K. Shimizu Y. Kim N. Tsukikawa Y. Chen R. Plasminogen activator inhibitor-2 (PAI-2) overexpression supports bladder cancer development in PAI-1 knockout mice in N-butyl-N- (4-hydroxybutyl)-nitrosamine- induced bladder cancer mouse model J Transl Med 18 2020 57 32024545 10.1186/s12967-020-02239-6 PMC7003426

121 Chen N. Ren M. Li R. Deng X. Li Y. Yan K. Bevacizumab promotes venous thromboembolism through the induction of PAI-1 in a mouse xenograft model of human lung carcinoma Mol Cancer 14 2015 140 26215730 10.1186/s12943-015-0418-x PMC4517418

122 Obi A.T. Diaz J.A. Ballard-Lipka N.L. Roelofs K.J. Farris D.M. Lawrence D.A. Plasminogen activator-1 overexpression decreases experimental postthrombotic vein wall fibrosis by a non-vitronectin-dependent mechanism J Thromb Haemost 12 2014 1353 1363 24943740 10.1111/jth.12644 PMC4127110

123 Siddiqui F. Darki A. Hoppensteadt D. Darwish B. Kantarcioglu A. Tafur A. Thrombo-inflammatory biomarkers in pulmonary embolism as stratified in cancer and non-cancer patient groups Res Pract Thromb Haemost 6 2022 183

124 Guy A. Gourdou-Latyszenok V. Wolff-Thrombini L. Favre S. Labrouche-Colomer S. Renault M. Neutrophils and platelets collaborate to induce an increase NET formation during JAK2V617F positive myeloproliferative neoplasm Res Pract Thromb Haemost 6 2022 170

Funding

This study was supported by a grant from the National Institutes of Health (N.M. 1R35HL155657) and the John C. Parker Professorship.

Author Contributions

Y.H. and N.M. drafted the manuscript.

Relationship Disclosure

There are no competing interests to disclose.

Funding information This study was supported by a grant from the 10.13039/100000002 National Institutes of Health (N.M. 1R35HL155657) and the John C. Parker Professorship.

Handling Editor: Dr Cihan Ay