Thrombin Generation and Cancer: Contributors and Consequences

The high occurrence of cancer-associated thrombosis is associated with elevated thrombin generation. Tumour cells increase the potential for thrombin generation both directly, through the expression and release of procoagulant factors, and indirectly, through signals that activate other cell types (including platelets, leukocytes and erythrocytes). Furthermore, cancer treatments can worsen these effects. Coagulation factors, including tissue factor, and inhibitors of coagulation are altered and extracellular vesicles (EVs), which can promote and support thrombin generation, are released by tumour and other cells. Some phosphatidylserine-expressing platelet subsets and platelet-derived EVs provide the surface required for the assembly of coagulation factors essential for thrombin generation in vivo. This review will explore the causes of increased thrombin production in cancer, and the availability and utility of tests and biomarkers. Increased thrombin production not only increases blood coagulation, but also promotes tumour growth and metastasis and as a consequence, thrombin and its contributors present opportunities for treatment of cancer-associated thrombosis and cancer itself.

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Glioblastoma cell populations with distinct oncogenic programs release podoplanin as procoagulant extracellular vesicles

Blood Advances ◽

10.1182/bloodadvances.2020002998 ◽

2021 ◽

Vol 5 (6) ◽

pp. 1682-1694

Author(s):

Nadim Tawil ◽

Rayhaan Bassawon ◽

Brian Meehan ◽

Ali Nehme ◽

Laura Montermini ◽

...

Keyword(s):

Cancer Cells ◽

Tissue Factor ◽

Extracellular Vesicles ◽

Coagulation System ◽

Platelet Factor ◽

Increased Risk ◽

Cell Subpopulations ◽

Cancer Associated Thrombosis

Abstract Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type–based diagnosis and antithrombotic intervention.

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Comparison of natural and recombinant tissue factor proteins: new insights

Biological Chemistry ◽

10.1515/hsz-2012-0350 ◽

2013 ◽

Vol 394 (7) ◽

pp. 819-829 ◽

Cited By ~ 4

Author(s):

Saulius Butenas

Keyword(s):

Tissue Factor ◽

Blood Coagulation ◽

Recombinant Proteins ◽

Thrombin Generation ◽

Extracellular Domain ◽

Full Length ◽

Post Translational Modifications ◽

Limited Availability ◽

Clone And Express

AbstractTissue factor (TF), an initiator of blood coagulationin vivo, is expressed in a variety of cells. Sufficient natural TF has been isolated to clone and express recombinant proteins ranging from full-length TF to its extracellular domain. Because of the limited availability of natural TF, recombinant proteins have been used as surrogates. Despite the differences in their post-translational modifications, it has been accepted that membrane-anchored recombinant TFs are quite similar to the natural TF. Recent studies, however, have shown that post-translational modifications play an important role in TF-triggered thrombin generation.

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Demystifying Tissue Factor.

Blood ◽

10.1182/blood.v104.11.1936.1936 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1936-1936 ◽

Cited By ~ 2

Author(s):

Saulius Butenas ◽

Matthew T. Gissel ◽

Beth A. Bouchard ◽

Kathleen E. Brummel ◽

Behnaz Parhami-Seren ◽

...

Keyword(s):

Tissue Factor ◽

Blood Coagulation ◽

Whole Blood ◽

Thrombin Generation ◽

Factor Viia ◽

Integral Membrane Protein ◽

Factor X ◽

Initiation Phase

Abstract Tissue factor (TF) is an integral membrane protein, which is the key initiator of blood coagulation in vivo. Due to the limited availability of natural TF, recombinant proteins of various lengths and origins have been extensively used in research and clinical laboratories worldwide. Experimental results acquired with recombinant TF proteins are frequently used for the understanding of the coagulation processes occurring in vivo, although there is a lack of data confirming the structural and functional identity of natural TF proteins from various sources and recombinant ones. In the current study, human TF from cultured monocytes and purified from placenta were compared with three different species of recombinant TF: 1–218 (extracellular domain only), 1–242 (lacking cytoplasmic domain) and 1–263 (full-length). Anti-TF mAbs gave 93–98% inhibition of TF activity for all TF species tested, in both natural and relipidated preparations. It was established that purified placental TF has a higher affinity for factor VIIa (Kd 0.13 nM) than recombinant counterparts 1–242 and 1–263 (Kd 0.50–0.80 nM). Similarly, placental TF is more efficient in factor X activation by the extrinsic Xase than recombinant TF 1–242 (the second order rate constants are 3.0x107 and 0.7x107 M−1s−1, respectively). We explored the use of these TF species as well as monocyte TF (purified/relipidated and present on LPS-stimulated monocytes) for the initiation of thrombin generation in two in vitro models of blood coagulation. At equimolar concentrations (5 pM; determined by immunoassay), when evaluated in synthetic plasma reconstituted with 2x108/ml platelets, recombinant TF 1-263 provided an initiation phase of ~4 min. Placental TF and relipidated monocyte TF had similar profiles of thrombin generation with an initiation phase of ~3 min. In contrast, 0.5 pM TF on LPS-stimulated monocytes gave an initiation phase of ~1 min. Even at 0.05 pM concentration, monocyte TF was as active as any relipidated protein at 5.0 pM. A similar pattern of relative TF activity was observed in whole blood and plasma PT clotting assays. TF on stimulated monocytes gave the highest activity, exceeding that of any relipidated protein by 100–200-fold. Recombinant TF 1–242 was more active than recombinant TF 1–263 and placental TF in the PT assay but less active in synthetic plasma and whole blood. The lowest overall activity was observed for relipidated monocyte TF. Our data suggest that TF proteins from different sources are different with respect to their functional properties.

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Inhibitors of blood coagulation factors Xa and IIa synergize to reduce thrombus weight and thrombin generation in vivo and in vitro

Journal of Thrombosis and Haemostasis ◽

10.1111/j.1538-7836.2006.01830.x ◽

2006 ◽

Vol 4 (4) ◽

pp. 834-841 ◽

Cited By ~ 14

Author(s):

W. R. GOULD ◽

T. B. MCCLANAHAN ◽

K. M. WELCH ◽

S. M. BAXI ◽

K. SAIYA-CORK ◽

...

Keyword(s):

Blood Coagulation ◽

Thrombin Generation ◽

Coagulation Factors ◽

Blood Coagulation Factors

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The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657715 ◽

1997 ◽

Vol 78 (04) ◽

pp. 1202-1208 ◽

Cited By ~ 18

Author(s):

Marianne Kjalke ◽

Julie A Oliver ◽

Dougald M Monroe ◽

Maureane Hoffman ◽

Mirella Ezban ◽

...

Keyword(s):

Tissue Factor ◽

Active Site ◽

Large Scale ◽

Thrombin Generation ◽

Factor Viia ◽

Dependent Manner ◽

Antithrombotic Agent ◽

Before And After ◽

Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

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Postprandial transfer of colostral extracellular vesicles and their protein and miRNA cargo in neonatal calves

10.21203/rs.2.10032/v1 ◽

2019 ◽

Author(s):

Benedikt Kirchner ◽

Dominik Buschmann ◽

Vijay Paul ◽

Michael W. Pfaffl

Keyword(s):

Extracellular Vesicles ◽

Expression Profiles ◽

Bovine Milk ◽

Cell Types ◽

Specific Protein ◽

In Vivo Experiments ◽

Neonatal Calves ◽

Almost All

Abstract Background Extracellular vesicles (EVs) such as exosomes are key regulators of intercellular communication that can be found in almost all bio fluids. Although studies in the last decade have made great headway in discerning the role of EVs in many physiological and pathophysiological processes, the bioavailability and impact of dietary EVs and their cargo still remain to be elucidated. Due to its widespread consumption and high content of EV-associated microRNAs and proteins, a major focus in this field has been set on EVs in bovine milk and colostrum. Despite promising in vitro studies in recent years that show high resiliency of milk EVs to degradation and uptake of milk EV cargo in a variety of intestinal and blood cell types, in vivo experiments continue to be inconclusive and sometimes outright contradictive. Results To resolve this discrepancy, we assessed the potential postprandial transfer of colostral EVs to the circulation of newborn calves by analysing colostrum-specific protein and miRNAs, including specific isoforms (isomiRs) in cells, EV isolations and unfractionated samples from blood and colostrum. Our findings reveal distinct populations of EVs in colostrum and blood from cows that can be clearly separated by density, particle concentration and protein content (BTN1A1, MFGE8). Postprandial blood samples of calves show a time-dependent increase in EVs that share morphological and protein characteristics of colostral EVs. Analysis of miRNA expression profiles by Next-Generation Sequencing gave a different picture however. Although significant postprandial expression changes could only be detected for calf EV samples, expression profiles show very limited overlap with highly expressed miRNAs in colostral EVs or colostrum in general. Conclusions Taken together our results indicate a selective uptake of membrane-associated protein cargo but not luminal miRNAs from colostral EVs into the circulation of neonatal calves.

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Accumulation of Tissue Factor into Developing Thrombi In Vivo Is Dependent upon Microparticle P-Selectin Glycoprotein Ligand 1 and Platelet P-Selectin

Journal of Experimental Medicine ◽

10.1084/jem.20021868 ◽

2003 ◽

Vol 197 (11) ◽

pp. 1585-1598 ◽

Cited By ~ 501

Author(s):

Shahrokh Falati ◽

Qingde Liu ◽

Peter Gross ◽

Glenn Merrill-Skoloff ◽

Janet Chou ◽

...

Keyword(s):

Tissue Factor ◽

Blood Coagulation ◽

Thrombus Formation ◽

Recipient Mouse ◽

Wild Type ◽

Activated Platelets ◽

Injury Model ◽

Platelet Thrombus ◽

Flowing Blood

Using a laser-induced endothelial injury model, we examined thrombus formation in the microcirculation of wild-type and genetically altered mice by real-time in vivo microscopy to analyze this complex physiologic process in a system that includes the vessel wall, the presence of flowing blood, and the absence of anticoagulants. We observe P-selectin expression, tissue factor accumulation, and fibrin generation after platelet localization in the developing thrombus in arterioles of wild-type mice. However, mice lacking P-selectin glycoprotein ligand 1 (PSGL-1) or P-selectin, or wild-type mice infused with blocking P-selectin antibodies, developed platelet thrombi containing minimal tissue factor and fibrin. To explore the delivery of tissue factor into a developing thrombus, we identified monocyte-derived microparticles in human platelet–poor plasma that express tissue factor, PSGL-1, and CD14. Fluorescently labeled mouse microparticles infused into a recipient mouse localized within the developing thrombus, indicating that one pathway for the initiation of blood coagulation in vivo involves the accumulation of tissue factor– and PSGL-1–containing microparticles in the platelet thrombus expressing P-selectin. These monocyte-derived microparticles bind to activated platelets in an interaction mediated by platelet P-selectin and microparticle PSGL-1. We propose that PSGL-1 plays a role in blood coagulation in addition to its known role in leukocyte trafficking.

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