**2.2. Endothelial activation**

myeloma and breast cancer at the time of diagnosis and during follow-up compared to healthy

Procoagulant microparticles (MP)—submicron extracellular vesicles. Their procoagulant capacity is determined by the expression of negatively charged phosphatidylserine and functionally active TF [33]. Increased MP-associated procoagulant activity was detected in patients with myeloproliferative neoplasms [34]. By intracellular exchange of MP, tumor cells

Heparanase—endoglucuronidase that degrades heparan sulfate. Overexpressed in almost all known malignancies but has not been detected in normal tissue adjacent to the tumor. Tumor cells secrete heparanase, which induces the expression of TF, activates directly f.X, and

Protein C (PrC)—inactivates Va and VIIIa in the presence of thrombin, and its functions are mediated by specific endothelial receptor. PrC performs cytoprotective and antimetastatic effects independently of coagulation [37]. Association between increased incidence of throm‐ botic events and acquired resistance to PrC (APC), unrelated to factor V Leiden, has been observed in multiple myeloma and colorectal cancer [38, 39]. There could be mechanisms of cell resistance due to the modification of the endothelial receptor and induction of APC under

Protein S—cofactor PrC whose activity can be inhibited by circulating paraproteins [24].

Antithrombin III (ATIII)—a major serpin that inactivates factors IIa, IXa, Xa, XIa, XIIa, kallikrein, plasmin. Decreased levels of ATIII are found in patients with malignant diseases

Tissue factor pathway inhibitor (TFPI)—main inhibitor of the complex TF:VIIa. Overexpres‐ sion of heparanase induces increase of functionally inactive TFPI in plasma (36), a putative

Tumor cells expressed on their cellular surface all proteins necessary for regulation of the fibrinolytic pathways. Deregulation in generating normal fibrinolytic activity was observed in patients with solid tumors and is a mechanism for the development of thrombotic tendency

Plasminogen activator inhibitor-1 (PAI-1)—important regulator of plasminogen activity. PAI-1 is overexpressed in different types of tumors [21]. Genomic sequencing of hepatocytes expressing the MET oncogene demonstrated significantly increased expression of the gene for PAI-1 and COX-2 that corresponded with triple increase in levels of circulating plasma proteins

transfer oncogenic signal and amplify the angiogenic phenotype [35].

inactivates tissue factor pathway inhibitor [36].

*2.1.2. Suppression of natural anticoagulant mechanisms*

the influence of tumor proinflammatory cytokines [39, 40].

and decreased survival [41, 42].

[45].

[46].

role of a tumor suppressor gene [43, 44].

*2.1.3. Defective fibrinolysis—hypofibrinolysis*

controls [31, 32].

6 Tumor Metastasis

Blood vessels and endothelial cells play a major role in the control of the processes of hemo‐ stasis, thrombosis, and inflammation. Endothelial tromboregulation is accomplished by selective expression of mediators (autacoids and cell adhesion molecules) in response to specific agonists. The synthesized mediators are involved in all phases of the hemostasic process and regulate/maintain vascular reactivity. Intact endothelium is anticoagulant and profibrinolytic under physiologic conditions. From the perspective of the Virchow's triad, endothelium loses its tromboresistive properties upon damage—for example, stretching of the vessel wall, mechanical and chemical injury, turbulent flow, inflammation. Malignant process causes deregulation of endothelial homeostasis, which can be defined more precisely as activation rather than damage as endothelial cells alter their functional capacity and acquire new properties in the absence of violation of tissue integrity [51]. Factors of endothelial activation in the presence of malignant process include the following: (a) dysfunctional endothelium—overexpression of adhesion molecules; (b) loss of anticoagulant and acquisition of procoagulant properties; and (c) switch to proangiogenic phenotype [52].

#### *2.2.1. Overexpression of adhesion molecules*

Endothelial expression of selectins and ligands from the immunoglobulin superfamily is increased under the effect of tumor-induced cytokine and cellular interactions. Tumorassociated macrophages secrete TNF-α, IL-1, IL-6, IFN-γ, which increase the expression of adhesion molecules through activation of de novo synthesis of mRNA [22]. Increased expres‐ sion of adhesion molecules ICAM-1, VCAM-1, and E-selectin mediated by NF-κB transcrip‐ tional activation was found when co-culturing endothelial and tumor cells [53]. Gene profiling of bone marrow cells from patients with multiple myeloma shows increased expression of genes BNIP3, IER3, and SEPW1. Through their silencing by small interfering RNA processes such as endothelial proliferation, adhesion and capillary formation are influenced [54].

#### *2.2.2. Procoagulant conversion and loss of anticoagulant properties*

Thrombomodulin (TM)—important endothelial cell-associated receptor that acts as direct anticoagulant. Binding of thrombin activates PrC system and inactivates the proteolytic degradation of procoagulant substrates [55]. Circulating tumor cytokines decreases TM levels causing degradation of its molecule and increased endothelial expression of TF [56]. There is inverse proportional relationship between TM expression and cellular proliferation in vivo. Downregulated to absent TM expression is found in metastatic foci, while forced expression of TM in transgenic mouse models of squamous cell carcinoma lacking TM expression leads to a differentiated epithelial-like phenotype—effect regulated by Snai1 transcription factor [57]. Interference of the TM-PrC system that occurs as a result of lower TM and development of acquired resistance to PrC of cellular type is one of the factors for procoagulant conversion of endothelium.

In response to stimuli from the tumor microenvironment, antagonistic deregulation is observed in endothelial expression of the pairs of vWF/ADAMTS13, TF TFPI, PAI-1/plasmi‐ nogen activators. Cytokine-activated endothelium releases high-molecular complexes of vWF, which are hyper-reactive to platelet aggregation, thrombus formation, and adhesion, whereas expression of ADAMTS13 depolymerase is suppressed [58]. Local procoagulant activity of endothelium is potentiated further by the effects of heparanase, which induces expression of TF and simultaneously dissociates its inhibitor TFPI from the endothelial cell surface [59]. And last but not least, the endothelial cells as a primary source of fibrinolytic activators participate in the induction of hypofibrilinolitic state by defective secretion of plasminogen activators in enhancing the expression of a fibrinolytic inhibitor PAI-1 [60]. Different mechanisms of microvascular dysfunction in combination with activated coagulation are the main pathoge‐ netic factors in the development of thrombotic microangiopathy in malignancies.

#### *2.2.3. Switch to proangiogenic phenotype*

Increased angiogenic activity is due to complex processes in which fully differentiated, nonproliferating endothelial cells acquire invasive, migratory, and proliferative properties. The processes of the angiogenic switch are determined by the increased production of positive regulators of angiogenesis (VEGF, FGF2, IL-8, PIGF, FGF-β, PDGF), which originate from tumor cells—they can be mobilized from the extracellular matrix or are released by stromal cells, recruited in the tumor [61]. At the same time, many of these processes are coupled with regulatory coagulation processes. For example, endothelial growth factor VEGF, secreted by the tumor cells, increases endothelial expression of TF, which causes inverse decrease in the expression of the negative angiogenic regulator—thrombospondin [62]. Synthesis and secretion of another potent proangiogenic cytokine—interleukin-8 (IL-8) from endothelial cells —are increased, and the effect is dose dependent on the levels of fibrin deposits [63]. A similar mechanism is responsible for fibrin-induced expression of the gene for TF from umbilical vascular endothelial cells [64]. Additionally, thrombin and coagulation degradation products mediate haptotaxis of endothelial cells by selective exposure of a set of integrins. Thereby, they orientate the formation of capillaries and vasculogenesis in the direction of the angiogenic stimulus [65, 66].

Angiopoietin-1,2/Tie2 system is a key regulator of physiologic endothelial angiogenic activity, which controls the proliferation and differentiation of endothelial cells during embryonic development. Tie2 is an endothelial-specific receptor tyrosine kinase whose ligands are angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2). Angiopoietin-1 is secreted by perivascular cells and in complex with Tie2 stabilizes the endothelium in quiescent state and potentiates the maturation of blood vessels. Angiopoietin-2 acts as an antagonist of Ang-1/Tie2. It is overexpressed during tumor angiogenesis and is responsible for pro-angiogenic endothelial conversion [67]. Tumor blood vessels express structural and functional abnormalities such as abnormal vascular permeability and increased potential for rapid growth and remodeling due to overexpression of Ang-2 [68]. Experimental data support the interdependence of the processes of procoagulant and pro-angiogenic endothelial conversion. Ang-1 inhibits overex‐ pression of TF in HUVECs in a putative mechanism of PI3/Akt signal activation [69]. Throm‐ bin-induced angiogenesis in an in vivo model of chorion allantoic membrane is accompanied by a double increase in expression of mRNA, encoding VEGF and Ang2 [70].

Endothelium performs a crucial role as a regulatory nexus in the processes of hemostasis and angiogenesis. The complexity of interactions allows on the one hand multiple targeting of several pathological mechanisms involved in angiogenesis and tumor progression and on the other hand mediates expression of side effects associated with the system of hemostasis.
