Thrombocytosis in cancer patients: when more is not better-in fact, the opposite.

Introduction
Platelets are the smallest morphological elements of blood without a nucleus, formed as a result of fragmentation of the cytoplasm of megakaryocytes (MKs). Platelets survive in the bloodstream for 7–10 days and in a healthy person, their number is around 150–350*103/μL of blood. They are rich in granules containing bioactive molecules and express surface receptors that mediate adhesion and signaling [1, 2]. Platelets have a fundamental role in the blood clotting process and are responsible not only for hemostasis and vascular integrity, but also participate in inflammatory processes, wound healing and in immunological defense mechanisms against microorganisms [3]. In the context of cancer, many studies have also pointed to the paradoxical role of platelets in being actively involved in cancer progression. This occurs through a variety of mechanisms, including the stabilization of newly formed blood vessels and the stimulation of blood vessel formation in the vicinity of tumors and newly developing metastatic processes immunomodulating the tumor environment, protecting circulating tumor cells (CTCs) from immune surveillance, promoting interactions of tumor cells with stromal components, creating premetastatic niches, releasing growth factors that directly stimulate tumor cell proliferation and survival, and promoting tumor cell adhesion and extravasation [4]. Elevated platelet counts are frequently observed in cancer patients, and this is associated with poorer prognosis. These interactions underscore the role of platelets as active accomplices in the progression of various types of cancers rather than being merely passive bystanders in the process [5–18]. Given their pro-tumorigenic effects, antiplatelet therapies are emerging as potential strategies for cancer treatment. Drugs such as aspirin and P2Y12 inhibitors, traditionally used to prevent cardiovascular events, have demonstrated potential for reducing metastasis in preclinical studies and some clinical trials [19–26]. In addition, new agents targeting platelet growth factors or adhesion molecules are being investigated. However, balancing the benefits of antiplatelet therapy with the risk of bleeding remains a major challenge. Future studies need to focus on identifying biomarkers to stratify patients who would most benefit from antiplatelet therapy and exploring combination therapies with other anticancer drugs. Understanding the precise mechanisms of platelet involvement in cancer would pave the way for more effective and personalized therapeutic strategies. Because thrombocytosis appears to be something of a double-edged sword, platelet-targeted therapies hold promise for improving outcomes for cancer patients.

The structure of blood platelets
In their resting state, platelets typically present as discoid structures measuring approximately 2 μm across, 0.5 μm thick, and encompassing a volume of 7 µm3. Internally, platelets feature a complex ultrastructure. This includes an open canalicular system (OCS) formed by infoldings of the surface membrane, and a dense tubular system (DTS) composed of a closed channel network derived from the endoplasmic reticulum. Further structural components include a spectrin-containing membrane skeleton, a cytoskeleton based on actin filaments and a marginal band of microtubules. Additionally, platelets house various organelles, notably alpha-granules, dense granules, peroxisomes, lysosomes, and mitochondria [27, 28]. The dense tubular system contains a number of substances and enzymes essential for cell function, including arachidonic acid-rich phospholipids, calcium ions, phospholipase A2, cyclooxygenase, thromboxane Ca2+ synthetase, and ATPase [1]. The platelet cytoplasm contains numerous granules in which active substances are stored and released during platelet activation [29]. Additionally, the membrane is equipped with transport systems such as sodium pumps and adenosine triphosphate pumps, which control the intracellular ionic environment of platelets, as well as numerous integrin proteins, whose ends are located on both sides of the membrane and are responsible for transmitting signals through it, thus determining the contact between platelets and their external environment [30]. The plasma membrane and membrane proteins constitute a large part of the platelet cell mass in relation to other cells. Platelets express receptors belonging to sixteen distinct classes, with several of these classes also encompassing subtypes. Many of these receptors are also present on other cells, while some are expressed only on platelets [31, 32]. Activation is a prerequisite for the surface expression of selected platelet receptors. Receptors requiring platelet pre-activation are typically those sequestered within the internal alpha-granules or dense granules. Following cellular activation by agonists such as thrombin or ADP, the membranes of these storage granules fuse with the plasma membrane (a process known as exocytosis), resulting in the rapid translocation and surface expression of the specific adhesion molecules. Prominent examples of molecules whose expression is strictly dependent on this activation and fusion process include P-Selectin (CD62P) and the CD40 ligand (CD40L or CD154). In contrast to the receptors that must be translocated to the surface, the most abundant platelet receptor, integrin glycoprotein IIb/IIIa (GPIIb/IIIa), is already constitutively present on the plasma membrane in its resting state. The key distinction here is that activation does not cause the surface expression of integrin, but rather triggers a critical conformational change-a molecular switch-that transforms the receptor from an inactive, low-affinity binding state into an active, high-affinity state. This change is mandatory for the receptor to successfully bind its primary ligands, such as fibrinogen or von Willebrand factor (vWF), which ultimately drives platelet-to-platelet aggregation [33]. This extensive variety of receptors underpins the capacity of platelets to generate discriminate cellular outcomes in response to different physiological settings and pathological events. The constant regulation of platelet responses, often via positive or negative feedback loops, is crucial for hemostasis.

Megakaryopoiesis and the regulation of platelet-forming pathways
Hematopoiesis is a multifaceted biological process where hematopoietic stem cells (HSCs) differentiate, giving rise to all mature blood cell types, including platelets. Megakaryopoiesis is the structured progression from HSCs, through progenitor cells, to mature megakaryocytes (MKs), which ultimately produce platelets via repeated endomitosis and cytoplasmic remodeling (Fig. 1). These processes of differentiation, maturation, and fragmentation are strictly controlled by bone marrow endothelial cells, matrix glycosaminoglycans, hematopoietic growth factors (such as SCF and SDF-1), hormones, chemokines, cytokines, and a complex array of transcription factors and microRNAs (miRNAs) [34].

MK maturation and platelet biogenesis involve four main phases [35]:Proliferation of hematopoietic stem cells.

Differentiation of progenitor cells belonging to the common myeloid and granulocytic lineage.

Development of MK and erythrocyte progenitor cells and multi-stage maturation of MKs.

Formation of proplatelets from mature MKs and subsequent platelet release.

MKs originate from HSCs [36, 37]. HSCs proceed through Multipotent Progenitors (MPPs), which are crucial for lineage commitment, lose their multipotency to become unipotent precursor cells, progressing through the common myeloid progenitor (CMP) lineage to the bipotent megakaryocyte/erythroid progenitor (MEP), which proliferates and undergoes endomitosis to become a MK [2]. Direct MEP formation from HSCs has also been described [38]. A defining characteristic of MKs is their large size (50–100 µm) and a single, multilobed, polyploid nucleus. Key transcription factors regulating MK differentiation and polyploidization include RUNX1, GATA-1, FLI1, SCL, SRF c-Myb [34]. These are, in turn, regulated by miRNAs, short noncoding RNA molecules that modulate gene expression [39]. The developmental trajectory of megakaryopoiesis is precisely mapped by the sequential expression of Cluster of Differentiation (CD) surface markers, which serve as crucial tools for identifying and isolating cells across the differentiation hierarchy [1]. This progression from uncommitted HSCs to mature megakaryocytes is fundamentally characterized by a reciprocal shift in marker expression. Initially, progenitors are defined by the expression of general hematopoietic markers such as CD34 (a glycoprotein indicative of immaturity) and CD38 (an indicator of transitional commitment), both of which are progressively lost or downregulated as the path towards the cell’s ultimate role is increasingly defined. Upon commitment to the megakaryocytic lineage, the cell initiates expression of the lineage-specific adhesion molecules CD41 (Integrin αIIb) and CD61 (Integrin β3). These markers form the GPIIb/IIIa complex, the earliest definitive megakaryocytic phenotype, and their expression remains high through maturation and on circulating platelets. The final stage of differentiation and terminal maturation is marked by the later acquisition and high expression of CD42b (Glycoprotein Ibα), which is integral to the vWF receptor complex necessary for platelet adhesion, thus signaling the cell's readiness for proplatelet formation.
The primary cytokine driving MK proliferation and differentiation is thrombopoietin (TPO) [40]. All progenitor cells leading to MKs (HSC, CMP, MEP) express the TPO receptor, c-mpl [41]. Regulation functions as a feedback loop: a decrease in platelet count leads to an increase in circulating TPO, stimulating bone marrow stem cells. TPO signaling triggers the internalization of the c-Mpl–TPO complex and activates multiple pathways, including JAK2, STAT3/STAT5, MAPK/ERK, and PI3K/AKT [42–45]. TPO specifically promotes JAK2 phosphorylation, which activates STAT3 and STAT5 transcription factors [44]. These cascades drive the activation of megakaryocyte-specific transcription factors and regulate gene expression vital for MK development. Importantly, TPO-independent pathways exist for megakaryopoiesis [46], with alternative effectors like ICF1 [47] and CCL5 [48]. The Notch signaling pathway is also a key regulator, with its pharmacological inhibition promoting human megakaryopoiesis in vitro [49].
Following signaling activation, the MK progressively grows, synthesizes platelet proteins, loses its proliferative capacity, and develops a polyploid nucleus through endomitosis. Endomitosis is a modified mitosis where DNA replication occurs multiple times without cytoplasmic division, resulting in DNA content ranging from 8 to 128N. This polyploidy intensifies MK metabolism, multiplies gene expression, and increases the synthesis of proteins, membranes, and organelles necessary for platelet production [50].
The differentiation phase includes the formation of proplatelets [51]. Initially, the MK's cell membrane invaginates, forming an extensive network of demarcation membranes (DMS) [52]. Proplatelet formation involves the organized fragmentation of the mature MK cytoplasm, where platelet receptors (like integrins) are exposed, and granules (lysosomes, dense granules, and α-granules containing proteins like vWF and platelet factor 4 (PF4) are formed. The process starts with the creation of long extensions from a single site on the MK [40]. These extensions, which resemble strings of beads [53], are stretched into the blood vessel lumen, where blood shear forces facilitate their fragmentation. During this phase, cell nuclei are extruded and phagocytosed, and the cytoskeleton is fully reorganized by actin depolymerization and tubulin polymerization [54]. Microtubule bundles, consisting of alfa and beta tubulin dimers and dynein, are the main system driving proplatelet extension [55]. Ultimately, proplatelets undergo fragmentation after further actin-mediated bending and branching, releasing several thousand platelets from the replicating ends [56].
Approximately 1 × 1011platelets are produced daily, a rate that can increase up to 20-fold during periods of high demand [57]. Congenital platelet disorders stem from quantitative or qualitative defects in MK differentiation, with over 30 associated genes identified, including seven key transcription factors like RUNX1, GATA1, and FLI1 [57, 58]. The sequential activation of these factors explains why different mutations lead to distinct clinical manifestations [59]. Current research views megakaryopoiesis as a dynamic and adaptable process, suggesting the existence of HSCs strongly biased towards MK and platelet production [60].
As mentioned previously, the classical route involves transit through sequential multipotential progenitor stages (MPP, CMP, MEP). In contrast, the direct pathway facilitates rapid megakaryocyte generation, increasing its activity during conditions of physiological stress, including aging, inflammation, regeneration following progenitor depletion, and induction by the DNA damage response. The demonstration of the existence of a second, direct pathway of megakaryocyte differentiation was made possible by studies using: single cell transplantation, single-cell RNA sequencing, in vivo barcoding in mice and in vitro culture in humans. This direct differentiation mechanism yields functionally distinct megakaryocytes that release hyper-reactive platelets, suggesting its critical role in emergency thrombopoiesis and hemostasis under acute conditions. The HSC compartment exhibits significant functional heterogeneity with respect to lineage commitment and self-renewal. Single-cell transplantation studies have identified a subset of primitive HSCs, often marked by vWF + expression, that are platelet-restricted (P-HSCs) and possess high long-term reconstitution potential. These P-HSCs establish a direct megakaryocyte differentiation pathway that bypasses the conventional MPP intermediates. In contrast, vWF-HSCs primarily contribute to the stepwise pathway, differentiating into multi-lineage progenitors and MPPs. This conserved dual organization dictates that megakaryopoiesis proceeds via two molecularly and functionally distinct routes: a rapid direct route from vWF + P-HSCs and a classical route from multipotential vWF-HSCs. The direct MK pathway functions as an emergency thrombopoiesis mechanism, preferentially responding to progenitor or MK depletion (e.g., after myeloablation or malaria infection) and leading to increased platelet recovery kinetics and pro-thrombotic activity in aged mice. This pathway also generates functionally distinct MK populations, favoring the production of niche-supporting MKs with high ploidy over the lower-ploidy immune MKs favored by the stepwise pathway, though the ultimate MK phenotype is influenced by the microenvironment. Myeloproliferative neoplasms (MPNs) are associated with the expansion and preferential activation of the direct megakaryocyte pathway. This enhanced engagement of the direct pathway may contribute to both the pathogenesis of the disease and the elevated risk of thrombotic complications through the generation of hyper-reactive platelets. The two megakaryocyte differentiation pathways are differentially regulated by inflammatory signals and aging; specifically, Interferon (IFN-I) signaling and associated Interferon-Stimulated Genes (ISGs) are enriched in platelet-biased HSCs and promote the direct pathway, while lipopolysaccharide (LPS) predominantly stimulates the stepwise pathway to generate immune megakaryocytes. Consistent with the elevated thrombosis risk in the elderly, the direct pathway's contribution to megakaryopoiesis increases significantly with age, rising from an estimated 50% in young mice to 80% in aged mice, a shift potentially driven by chronic inflammatory signaling associated with aging [61, 62].

Functions of platelets in physiology
The primary function of platelets is hemostasis, a process that inhibits bleeding at sites of vascular injury. Following vascular injury, platelets rapidly adhere to exposed subendothelial matrix proteins such as collagen. This adhesion is mediated by glycoprotein receptors on the platelet surface, particularly glycoprotein Ib (GPIb), which binds to collagen-anchored vWF. This initial adhesion triggers platelet activation, a process characterized by shape change and release of granular contents, including ADP, thromboxane A2 (TXA2) and serotonin. These secreted substances further enlist and activate additional platelets, enhancing the aggregation process through the binding of fibrinogen to GPIIb/IIIa receptors on adjacent platelets, ultimately leading to the formation of a hemostatic plug [63]. Although their role in hemostasis is well understood, it has become increasingly evident that the functions of platelets go far beyond hemostasis itself [64]. Recent studies have emphasized their involvement in immune responses and inflammation, positioning platelets as active participants in the body's defense mechanisms. Platelets can interact with leukocytes, such as neutrophils and monocytes, via P-selectin and other adhesion molecules to form platelet leukocyte aggregates that facilitate the recruitment of leukocytes to sites of inflammation [4, 5, 65, 66]. Additionally, platelets release various cytokines and chemokines, such as PF4 and interleukin-1β (IL-1β), that modulate the inflammatory response and contribute to the engagement and activation of immune cells [3, 4]. In addition, platelets play a key function in tissue repair and wound healing. They are a rich source of growth factors, including platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF). These factors promote the proliferation and migration of fibroblasts, endothelial cells and smooth muscle cells, processes essential for tissue regeneration and angiogenesis [67]. However, in pathological conditions, these same growth factors can contribute to fibrosis, leading to excessive tissue scarring and impaired function [68]. Abnormal platelet function or number also plays a role in diseases other than cancer (as described later in this paper). For example, thrombocytopenia is associated with immune thrombocytopenic purpura (ITP) and some bone marrow disorders, whilst an increased platelet count is related to a greater risk of thrombosis and is often observed in myeloproliferative diseases. Platelet dysfunction, whether congenital, as in Glanzmann's thrombasthenia, or acquired, for example as a result of the use of antiplatelet drugs such as aspirin, can also disturb the balance of hemostasis leading to bleeding [63].

Platelet functions in cancer
Platelet and leukocyte aggregation occurs through the interaction of multiple adhesion factors and signaling pathways. The initial step occurs on the leukocyte and is mediated by the binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1). Additionally, integrins such as glycoprotein GPIIb/IIIa or αIIbβ3, GPIb, ICAM-2, CD40L on platelets, and Mac-1 (αMβ2) on leukocytes, are crucial for platelet–leukocyte aggregates (PLA) adhesion and stability. These interactions lead to the creation of heterotypic cell aggregates that can circulate in the bloodstream or localize to specific sites such as tumor tissues [69–71]. Cancer cells can directly interact with platelets, facilitating metastatic spread and promoting the formation of PLA. Moreover, they can activate platelets by secreting thrombin. Cancer cells express and present tissue factor on their surface, which serves to activate platelets. Activated platelets release growth factors, chemokines, and cytokines that promote cancer cell proliferation, survival and angiogenesis [4]. The formation of cancer cell-platelet-leukocyte complexes increases the extravasation of cancer cells from the bloodstream and invasion of distant tissues, which is a pivotal part of the metastatic cascade. Platelet derived microparticles (PMPs) also contain these bioactive molecules, which further enhance the pro-tumor environment [72]. Leukocytes, especially neutrophils and monocytes, play an integral role in the immune response against tumors. However, in the presence of PLA, their role may change to support tumor progression. Neutrophils in PLA can release neutrophil extracellular traps (NETs), which capture CTCs and facilitate their adhesion to distant sites, promoting metastasis. On the other hand, monocytes can differentiate into tumor associated macrophages (TAMs) under the influence of PDGF, creating an immunosuppressive and proangiogenic tumor microenvironment [4].
PLA contributes to tumor growth by increasing the local availability of growth factors such as VEGF and PDGF. These factors stimulate angiogenesis, which is essential for tumor growth and nutrient delivery [4, 64]. The role of PLA in the metastatic cascade is multifaceted. During intravasation, platelets can surround tumor cells, forming a protective barrier that shields them from detection and destruction by natural killer (NK) cells. In the bloodstream, PLA facilitates the retention of CTCs at distant sites through interactions with endothelial cells and NETs. After extravasation, the release of platelet derived factors promotes the formation and growth of metastatic colonies [4, 73] Fig. 2.

Thrombocytosis in cancer and its consequences
The process of promoting tumor growth and distant metastasis is a fundamental aspect of cancer progression that is facilitated by platelets [74]. Metastasis is a complex and orchestrated process involving multiple cellular and molecular interactions. Platelets play a key role in facilitating the different steps of the metastatic cascade, from tumor cell invasion to extravasation and colonization of distant organs.

Increased risk of thromboembolism
Thrombosis is often one of the first symptoms of malignancy. Approximately one third of all initial venous thromboembolic events can be related to a developing cancer [75]. Malignant neoplasms are linked to a higher occurrence of venous thromboembolism (VTE) (4% to 20%) and arterial thrombosis (2% to 5%) [76–80]. VTE is the second leading cause of death in cancer patients. The risk of thromboembolic complications in cancer patients is 4 to 6 times higher than in those without cancer. Clinical manifestations of VTE include deep vein thrombosis, pulmonary embolism, nonbacterial thrombotic endocarditis, superficial thrombophlebitis, catheter-related thrombosis, and veno-occlusive liver disease [81–83]. Thrombocytosis is recognized as a significant independent risk factor for the development of VTE. Numerous investigations have shown that patients with malignant tumors have a significantly higher risk of developing VTE [84]. Furthermore, patients with elevated platelet counts prior to cancer development have been found to have a higher risk of VTE compared to those with lower platelet counts [77]. Thrombocytosis has been incorporated into risk stratification models such as the Khorana score, which is commonly used to predict VTE risk in outpatient cancer patients. A platelet count > 350 × 10*9/l as a variable underscores its importance as a predictor of thrombotic risk. The risk of thromboembolism in cancer patients is influenced by multiple factors, including thrombocytosis, tumor location, cancer stage, histopathological tumor type, age, gender, body weight, leukocyte count, surgical treatment, radiotherapy, chemotherapy, hormone therapy, the presence of central venous catheters, immobilization, pregnancy, and prothrombotic mutations [85, 86]. Furthermore, the occurrence of VTE is a significant unfavorable prognostic factor in patients with malignant tumors in whom the overall survival time is significantly shorter compared to those without such complications [85, 87–90]. Additionally, the risk of death due to pulmonary embolism in these patients is significantly higher compared to patients without diagnosed cancer [91].
Interestingly, the incidence of VTE varies across cancer types. Cancers can be divided into 3 main groups based on their risk of VTE: high risk (pancreatic, ovarian, gastric, CNS, hematological and gynecological cancers), intermediate risk (colorectal and lung cancers), and low risk (breast and prostate cancers) [92–94]. This indicates the possibility of cancer type-specific mechanisms contributing to the development of VTE. Chemotherapy is a major contributing risk factor for VTE due to several interrelated mechanisms. Endothelial injury, which exposes procoagulant surfaces and activates the coagulation cascade is one contributory factor. Chemotherapy can also induce release of proinflammatory cytokines and upregulate TF expression on tumor and endothelial cells, enhancing thrombin generation. Certain agents, like platinum-based regimens, are particularly thrombogenic, leading to a significantly higher incidence of VTE in cancer patients undergoing chemotherapy compared to those not exposed to it [82, 95]. The most significant clinical link between chemotherapy and thrombosis was observed in breast cancer patients undergoing chemotherapy [82, 95–98]. In a study by the Eastern Cooperative Oncology Group, VTE occurred significantly more frequently in breast cancer patients receiving chemotherapy and hormone therapy compared to controls [99]. High rates of VTE have also been reported after chemotherapy for other cancers [100, 101]. The risk of VTE is raised six-fold and that of VTE recurrence two-fold in oncological patients undergoing chemotherapy and the estimated annual incidence of VTE in these patients is 10.9% [102–104]. Surgery can increase the risk of postoperative VTE by around two-fold in patients with cancer compared to those without cancer and is related to a three- to four-fold increased likelihood of developing pulmonary embolism after surgery [103, 104]. A study assessing the impact of neurosurgery on thrombosis risk in glioma patients found that those who underwent surgery had a 70% higher likelihood of developing VTE compared to unoperated patients [105]. However, not all studies have found an increased risk of VTE associated with surgery in patients with cancer. An analysis of data from patients with breast, colon or ovarian cancer suggests that major surgery may actually lower the occurrence of VTE. For these three cancer types, surgery was associated with a reduced risk of developing VTE, even after adjusting for factors such as age, race, sex, cancer stage, and the presence of chronic comorbidities. Specifically, patients with ovarian cancer who had undergone gynecologic surgery had a 30% lower risk of VTE, those who had breast surgery demonstrated a 40% reduction in risk, and colorectal cancer patients who received surgical treatment had a 60% lower risk of developing VTE compared to those who did not undergo major surgery [98]. Additional risk factors for thrombosis related to cancer include central venous catheters, prolonged immobilization, use of oral contraceptives, trauma, history of venous thrombosis, hormone therapy, pregnancy, advanced age, prothrombotic mutations like factor V Leiden and prothrombin 20210 A, increased levels of D-dimer, C-reactive protein, and soluble P-selectin, a body mass index of 35 kg/m2 or higher, the presence of antiphospholipid antibodies and biomarkers, and leukocyte counts exceeding 11 × 103/μL [97, 106–108].

Proliferation and angiogenesis
Platelets synthesize various cytokines and growth factors, including PDGF and TGF-β, which stimulate tumor cell proliferation and promote their survival and angiogenesis. Once activated, platelets release these growth factors into the tumor microenvironment, where they interact with cognate receptors on tumor cells to promote cell cycle progression and inhibit apoptosis [109, 110]. The process of angiogenesis plays a key role in tumor growth and metastasis. The platelets and the microparticles they release contribute to angiogenesis by promoting the growth of new vessels and stabilizing existing ones [109, 111, 112]. This occurs through the release of proangiogenic factors, including VEGF, basic fibroblast growth factor (bFGF), PDGF, and epidermal growth factor (EGF), which promote endothelial cell proliferation, migration and enhanced permeability [109, 110]. Studies involving patients with lung, breast, colon and kidney cancer have found a correlation between platelet count and VEGF levels [113–115]. High platelet counts, which are associated with high plasma VEGF levels, have been associated with shorter overall survival (OS) in these patients [116, 117]. In turn, glycoprotein VI present on the surface of platelets has a stabilizing effect on the integrity of tumor vessels [118].

Protection against shear stress and immunological supervision
Platelets interact dynamically with CTCs via ADP, TXA2, MMPs, and TF, which stimulate platelet aggregation to form heterotypic aggregates that protect tumor cells from immune surveillance and shear forces during transport in the bloodstream [119]. Platelet-tumor cell communication increases CTC attachment to endothelial cells in the systemic microvasculature, promoting their extravasation into the surrounding tissues. Surface molecules such as P-selectin, GP Ib-IX-V, GP IIb-IIIa, GP V, GP VI and C-type lectin-like receptor 2 (CLEC-2) on activated platelets enable them to adhere to tumor cells, forming a protective barrier layer for NK cells and T cells, preventing the elimination of CTCs [104, 120–122]. It is worthy of note, that the expression of CLEC-2 and its activator, podoplanin, has been identified in numerous cancers, such as colon, lung and bladder cancer, and thereby represents a promising therapeutic target [123, 124]. Furthermore, studies suggest that CLEC-1B, another platelet surface receptor, may serve as a prognostic biomarker and potentially regulate the immune response in hepatocellular carcinoma [125]. In addition, tumor dissemination is facilitated by tumor cell-induced platelet aggregation. Integrins in conjunction with fibrinogen or vWF mediate this aggregation by binding to GP IIb/IIIa and integrins on platelets [126, 127]. Activation of the endothelial P2Y2 receptor via P-selectin further promotes the accumulation of tumor cells in the capillary wall, facilitating their migration into the subendothelial matrix of a distant organ [128, 129].

CTC extravasation
Epithelial-to-mesenchymal transition (EMT) has a crucial role in cancer progression, during which tumor cells acquire a mesenchymal phenotype that enhances their capacity to infiltrate surrounding tissues and metastasize to distant sites [130]. This transition plays a key role in the generation of CTCs, which serve as a link between primary tumors and metastatic colonies. Platelet-derived TGF-β has been found to play a key role in this process by acting synergistically with thrombin-activated platelet-tumor cell complexes to activate the TGF β/Smad and NF-κB pathways in tumor cells. As a result, tumor cells undergo a phenotypic shift toward invasiveness and increased metastatic potential. Inhibiting NF-κB signaling in tumor cells or suppressing TGF-β1 expression represent potential therapeutic strategies to impede this deleterious transition and limit metastasis [131].

CTC masking
Moreover, platelets and tumor cells participate in the exchange of certain membrane proteins, which leads to tumor cells effectively masking themselves and making their precise recognition difficult [132]. Additionally, platelets secrete factors that modulate the local tissue environment and prepare specific niches, or places where CTCs can nest. Establishing metastatic colonies in distant organs requires the creation of a favorable microenvironment that nourishes the initial tumor cells and promotes their survival and growth [133, 134]. This occurs by preconditioning the metastatic microenvironment through the creation of extracellular matrix (ECM) and granulocyte recruitment [135], neovascularization, and the establishment of an immunosuppressive environment around developing metastases, thereby facilitating immune evasion. In a study focusing on a lung cancer model, knockdown of the platelet ADP receptor (P2Y12) resulted in reduced lung levels of fibronectin, a key component of the ECM. Fibronectin levels are elevated in the connective tissue of premetastatic organs and form an integral component of the acellular matrix within the metastatic niche. Consequently, the absence of a platelet-mediated increase in fibronectin led to a reduced rate of metastasis in this model [136].

Immunomodulation
PDGF can modulate the activity of immune cells, including T cells, natural killer cells, and myeloid-derived suppressor cells (MDSC), shaping the immune landscape and influencing tumor progression [4]. They can pass on their MHC class I proteins to tumor cells, making it difficult for NK cells to recognize CTCs. Equally, TGF-β released by platelets downregulates the NKG2D receptor on NK cells [137]. Moreover, TGF-β-1 contained in released platelet-derived microparticles stimulates the expression of Foxp3 in CD4 + cells and the increase of microRNA 183. MicroRNA 183 induces a decrease in the concentration of DNAX activating protein 12 kDa, which leads to the dysfunction of NK cell surface receptors such as NKG2D, NKp30, DNAM-1 [138, 139]. The increase in Foxp3 expression induces the transition of CD4 + cells into regulatory T lymphocytes capable of killing activated T lymphocytes [140].
Moreover, platelets enhance the expression of PD-L1 on the surface of cancer cells, thereby facilitating tumor immune evasion. This effect is mediated through VEGF and PDGF which up-regulate PD-L1 expression at both the transcriptional and protein levels. Importantly, the use of anti-platelet agents, such as eptifibatide, has been found to effectively inhibit this platelet-driven increase in PD-L1 and restore T-cell activation. These findings highlight a novel mechanism by which platelets contribute to tumor progression and suggest that targeting platelet activity may represent a promising adjuvant strategy to improve the efficacy of immune checkpoint inhibitors in cancer therapy [141].
A further study has provided evidence that platelets contribute to immune evasion by delivering PD-L1 to cancer cells, even in tumors originally lacking PD-L1 expression. Platelet-derived PD-L1 supports tumor growth and suppresses T-cell-mediated cytotoxicity, thereby offering an explanation for the clinical benefit of PD-1/PD-L1 inhibitors observed in patients with PD-L1–negative tumors. These findings emphasize the significance of non-cancer and non-immune sources of PD-L1 within the tumor microenvironment and highlight the role of platelets as key modulators of immune resistance. The results also suggest that combining PD-L1 checkpoint blockade with antiplatelet therapy may represent a promising therapeutic strategy for patients with PD-L1–negative cancers [142].
A recent study has identified a critical mechanism by which platelets promote tumor progression and metastasis through the immunosuppressive actions of TXA2. By engaging the ARHGEF1-dependent pathway in T cells, TXA2 suppresses T cell receptor signaling, proliferation, and effector functions, thereby weakening immune control over disseminating cancer cells. The findings demonstrate that pharmacological inhibition of COX-1—using aspirin or selective COX-1 inhibitors—as well as platelet-specific deletion of COX-1 restores T cell activity, enhances immune-mediated rejection of metastases, and significantly reduces metastatic burden. These results provide mechanistic insight into how platelet-derived TXA2 fosters cancer progression and highlight the therapeutic potential of COX-1 inhibition to limit metastasis and improve long-term outcomes in cancer patients [143].

Chemotherapy resistance
Both experimental studies and clinical observations indicate the participation of platelets in the protection of tumor cells against the action of anticancer drugs. In ovarian or colon adenocarcinoma cells treated with 5-fluorouracil, platelets have been shown to repair damaged DNA [144]. Equally, in advanced gastric cancer, platelet aggregation has been found to increase resistance to chemotherapy [145] and the resistance of pancreatic cancer to gemcitabine is the result of ADP and ATP released from platelets [146]. Platelets were also found to protect non-small cell lung cancer cells against the action of cisplatin [147].

Lymphangiogenesis
Finally, platelets also appear to contribute to the establishment of the tumor lymphatic network, as the a-granules contain VEGF-C, the most important regulator of lymphangiogenesis [148]. Furthermore, an association has been found between podoplanin, expressed in various types of tumors, and tumor cell-induced platelet aggregation (TCIPA) [149]. Although it has not yet been confirmed that platelets direct lymphatic vessel formation in the tumor microenvironment, there is at least one study in esophageal cancer that has shown a link between platelet numbers, in the circulation as well as in the tumor microenvironment, and lymphangiogenesis [150].

Use of platelet count as a prognostic factor
Thrombocytosis may be one of the first symptoms of an ongoing cancer process and should not be underestimated. It could be considered as a potential marker for the presence of certain types of cancer (colon, lung, ovarian or stomach cancer) [151, 152]. Platelet counts are elevated in lung cancer regardless of the type [153]. For example, a platelet count at the high end of the normal range (325–400 × 10E9/l) in people aged 60 plus may serve as a potential indicator of an increased risk for cancer development [154]. Thrombocytosis is an unfavorable prognostic factor in many common cancers. It is associated with an increased risk of disease recurrence, metastasis and cancer-related mortality. It has been linked to worse oncological outcomes in esophageal cancer [13, 14], gastric cancer [15–18], pancreatic cancer, colon cancer, early [5] and advanced [6] breast cancer, ovarian cancer [7, 8], genitourinary cancers [9–11], and lung cancer [12]. Thrombocytosis correlates with advanced disease, larger tumor size, and metastatic proliferation in different types of cancer. An elevated platelet count at diagnosis is an independent prognostic factor for poor prognosis, with higher platelet counts indicating poorer clinical outcome and decreased overall survival. Thrombocytosis can affect treatment response and therapeutic outcomes in cancer patients, affecting both conventional cytotoxic therapies and new targeted drugs. High platelet counts are associated with decreased sensitivity to chemotherapy and radiotherapy, potentially contributing to treatment resistance and disease relapse.

Esophageal cancer
In a study of nearly 300 patients with esophageal squamous cell carcinoma, thrombocytosis, defined as a platelet count greater than a mean of 293 × 10*9/L, was detected in 21% of patients. Thrombocytosis emerged as a significant independent prognostic factor for overall survival. This association was particularly evident in patients with disease stage III and IV, but not in those with disease stage I and II. On multivariate analysis it was revealed that thrombocytosis, together with higher T stage, tumor size, and lymph node involvement, was associated with poorer survival outcomes [13].

Gastric cancer
In a study including 1593 patients with gastric adenocarcinoma, thrombocytosis (defined as a platelet count above 400 × 10*9/L) was present in 6.4% of participants [15]. All patients underwent gastrectomy with negative margins and extensive D2 lymph node dissection. Thrombocytosis was linked with higher T stage, lymph node involvement, and poorer survival. However, the multivariate analysis did not support the expectation that thrombocytosis should be of significant prognostic value for long-term survival, while T stage and lymph node involvement continued to be statistically significant predictors of long-term survival in these patients. Thrombocytosis on the other hand, was strongly associated with recurrence, especially for distant metastases (by blood), but not with locoregional recurrence or peritoneal seeding [15]. In another study of 369 patients with gastric cancer, thrombocytosis occurred in 11.4% and was associated with poorer survival [16]. The 1-year and 3-year survival of patients with thrombocytosis was 72.9% and 23.4% accordingly and in those without thrombocytosis, 85.7% and 52.4%. It was observed that the extent of tumor invasion was positively correlated with lymph node involvement [16]. In a smaller series of 98 patients who underwent surgery for gastric cancer, preoperative thrombocytosis had been present in 21% and was significantly linked to poorer overall survival [17]. Among these patients, the 5-year survival rate for those with thrombocytosis was 9.5% and for those without thrombocytosis 31.2%. Interestingly, expression of the proangiogenic enzyme thymidine phosphorylase/PDGF was related to thrombocytosis and both were found to be stand-alone predictors of survival in multivariate analysis [123]. Finally, a study of 181 patients with gastric cancer, evaluating platelet count and serum VEGF levels as prognostic factors, revealed no correlation with either overall or progression-free survival. However, the VEGF to platelet ratio was significantly related to progression-free survival in multivariate analysis [18]. This may be attributed to the pathophysiological role of activated platelet derived VEGF in driving the neoplastic process.

Pancreatic cancer
In a similar vein, preoperative thrombocytosis was evaluated with regard to its prognostic significance in patients undergoing surgical resection for pancreatic adenocarcinoma. The results, confirmed by multivariate regression analysis, demonstrated a significant association between thrombocytosis and decreased levels of overall survival [155]. Furthermore, in a separate cohort of patients with operable pancreatic cancer, thrombocytosis was associated with poorer disease-free survival. The median progression-free survival was significantly shorter in the group of patients with thrombocytosis compared to the group with normal platelet counts: 4.9 months vs. 46.5 months, respectively. Interestingly, the prognosis was even better in the subgroup of patients who maintained normal platelet counts after surgery [156]. In contrast to these results, a study including pancreatic, duodenal, and biliary tract cancer showed that lower platelet counts adversely affected both overall and disease-free survival. Lower preoperative platelet counts were associated with positive surgical margins, which may potentially be an explanation for the unfavorable prognostic association. Another possible clarification for this discrepancy in comparison to previous studies could be the use of a lower threshold for defining a high platelet count, which was set at 300× 10*9/L [157].

Hepatocellular cancer
Platelets appear to have a multifaceted relationship with liver cancer. On the one hand, thrombocytopenia is a key predictor of hepatocellular cancer (HCC) recurrence following liver resection in patients with cirrhosis [158]. Conversely, thrombopoietin, a cytokine necessary for platelet production, is synthesized in the liver and can lead to thrombocytosis if the tumor cells mimic normal liver cells and produce the cytokine or if the tumor cells stimulate its production by the liver [159, 160]. It is worth noting that extreme thrombocytosis has been observed in both hepatocellular carcinoma and hepatoblastoma, a liver tumor in children and adolescents. Patients with hepatoma multiforme have been shown to have significantly increased levels of thrombopoietin compared to controls, though only minimally increased levels of IL-6 indicates that thrombopoietin most likely acts downstream of IL-6 in the pathway triggering platelet production. IL-6 can stimulate hepatocytes to increase TPO production via IL-6 receptor–STAT3 signaling. In liver tumors, like hepatoma multiforme, TPO secretion by the tumor itself can be markedly elevated, even when IL-6 is only mildly increased. This indicates that TPO acts downstream of IL-6 in the pathway or can be directly produced by tumor cells, thus triggering platelet production. [161]. Moreover, patients with HCC and thrombocytosis tend to have larger tumors and better liver function compared to patients with normal platelet counts [162]. A comprehensive study of 1,154 patients found that the incidence of thrombocytosis in HCC was 2.7%. Furthermore, platelet counts and thrombopoietin levels were found to correlate with treatment efficacy, such that platelet counts decreased after tumor resection and increased with recurrence. Thrombocytosis was significantly linked to younger patient age, greater malignancy progression, emergence of portal vein thrombosis due to tumor involvement, and a shorter median survival of less than 5 months compared with more than 12 months in patients without thrombocytosis [163].

Colorectal cancer
Similar results were found for colorectal cancer, where thrombocytosis, defined as a platelet count greater than 400 × 10*9/L, was identified as a prognostic factor in a comprehensive study of 1513 patients who underwent surgery. It was observed that patients with thrombocytosis had significantly shorter overall survival compared to those with a platelet count within the normal range. Furthermore, although the rate of locoregional recurrence were not significantly different, patients with thrombocytosis had a higher rate of overall recurrence and of recurrence with distant metastases, the risk persisting for 5 years after surgery [164]. Equally, in a retrospective analysis of 150 patients who had undergone surgery for colon cancer, those with preoperative thrombocytosis had a 5-year survival rate of 13.3% compared to 56.3% in those with normal preoperative platelet counts. Thrombocytosis, lymph node involvement, grade, and occurrence of neural invasion were significantly associated with poorer survival outcomes [165]. Additionally, studies focusing on patients with rectal cancer have shown that preoperative thrombocytosis is associated with a decreased response to chemoradiotherapy and an increased risk of local recurrence [166]. This pattern was replicated in a subsequent study of 198 patients with lymph node metastases, where thrombocytosis (defined as platelet count greater than 400 × 10*9/L) was independently related to significantly worse survival outcomes, along with specific parameters associated with the tumor—its grade, depth and lymphatic invasion [167]. In addition, there is an ongoing search for biomarkers to assess the risk of VTE in cancer patients. A potential candidate is PF4, which is a marker of platelet activation. In a study of patients with pancreatic cancer it was shown that increased levels of PF4 were associated with an almost three-fold higher risk of VTE in these patients [168] Table 1.

Management of thrombocytosis in cancer

Antiplatelet therapy
Targeting platelet-tumor interactions represents a promising avenue for therapeutic intervention in cancer treatment. Strategies aimed at inhibiting platelet activation, aggregation, and secretion of pro-tumor factors have demonstrated efficacy in preclinical.
models and early-phase clinical trials, emphasizing the therapeutic potential of antiplatelet agents in cancer treatment. Antiplatelet therapy, consisting mainly of aspirin and P2Y12 receptor inhibitors, has attracted considerable interest as an adjunct to cancer treatment. By attenuating platelet activation and modulating platelet-tumor cell interactions, antiplatelet drugs can exert antitumor effects and improve clinical outcomes when used in combination with standard anticancer therapies. Aspirin is a widely used antiplatelet drug, which exerts pleiotropic effects on platelet function, inflammation and tumor biology by irreversibly inhibiting cyclooxygenase (COX) enzymes. P2Y12 inhibitors, such as clopidogrel and ticagrelor, inhibit ADP-mediated platelet activation and have shown potential as adjunctive therapies in cancer patients. Most clinical trials have focused on evaluating aspirin for its potential as an anticancer agent. A 2012 meta-analysis of 5 randomized controlled trials (RCTs) found that aspirin significantly reduced the risk of metastasis and cancer-related mortality in patients with adenocarcinoma, regardless of the organ of tumor origin [19]. Similarly, another meta-analysis of 23 RCTs with 82,868 participants concluded that the daily use of aspirin was related to a lower incidence of cancer deaths [172]. However, some RCTs failed to establish such correlations [20, 21]. The somewhat unexpected results of the ASPREE study demonstrated an increased risk of serious gastrointestinal bleeding and an increase in deaths and late-stage cancers in older people on aspirin prophylaxis. The authors of the study suggested that aspirin-induced immunosuppression may lead to decreased control of tumor growth and metastasis, and their results argue against recommending aspirin for cancer prevention in patients aged 70 years and older [21]. There are currently many clinical trials underway to assess the effect of aspirin on cancer outcomes. Of note, the ADD-ASPIRIN trial is actively enrolling patients who have undergone prior treatment for early-stage breast, stomach, esophageal, prostate or colon cancer. The study is designed to determine whether five years of aspirin prophylaxis after initial cancer treatment can delay or prevent cancer recurrence [22, 173]. Recent studies have explored the hypothesis that the anticancer effects of aspirin are due to its inhibition of premetastatic niche formation. In a mouse model of experimental lung metastasis, TXA2, a prostanoid product of COX-1, was identified as responsible for this prometastatic effect. Inhibition of the COX-1/TXA2 pathway in platelets led to diminished aggregation on tumor cells, decreased endothelial activation and tumor cell adhesion, as well as impaired recruitment of monocytes/macrophages that promote metastasis, thereby inhibiting the establishment of premetastatic niches [23]. Previous studies have suggested that other platelet activation pathways may also contribute to the formation of the intravascular metastatic niche. For example, clopidogrel, a P2Y12 receptor antagonist, and eptifibatide, an αIIbβ3 integrin inhibitor, have been associated with a reduction in metastasis in an experimental model [24, 25]. However, these findings were not confirmed in a more recent study [23]. Additional research is required to gain a better understanding of the role of distinct platelet activation pathways at various stages of metastatic development, including EMT and extravasation. Such efforts hold promise for identifying antiplatelet agents to combat micrometastatic niche formation and novel therapeutic targets [24, 26]. Xanthorrhizol has been studied for its anti-inflammatory and antiplatelet properties that may indirectly influence cancer progression. By reducing inflammation and platelet activation, it could inhibit processes like metastasis, platelet-tumor interactions and angiogenesis. Though direct effects on platelets in cancer require further study, evidence shows that xanthorrhizol may exert its effects by targeting various signaling pathways, kinases, cytokines and transcription factors, promoting apoptosis and cell cycle arrest across multiple cancer types [174–176] Table 2.

Chemotherapy
Chemotherapeutic drugs target rapidly dividing cells, which include not only cancer cells, but also normal bone marrow cells responsible for producing blood components such as platelets. Bone marrow MKs, the precursor cells of platelets, are particularly susceptible to the cytotoxic effects of chemotherapy. As a result, many chemotherapy regimens lead to reduced platelet production by the bone marrow. This occurs primarily through their adverse effects on hematopoietic stem and progenitor cells (HSPCs), particularly those involved in the megakaryocyte-platelet lineage. Agents such as carboplatin, busulfan, bis-chloronitrosourea, cisplatin and etoposide have been reported to induce either acute or long-term bone marrow injury, leading to chemotherapy-induced thrombocytopenia. Carboplatin and cisplatin cause increased apoptosis in HSPCs, including MKs precursors, and are associated with elevated DNA fragmentation in bone marrow. Busulfan has been shown to significantly inhibit hematopoietic function without directly inducing HSPCs apoptosis, indicating long-term suppression of hematopoiesis. These agents can impair the self-renewal and proliferation capacity of HSPCs by inducing oxidative stress and senescence, particularly in the case of long-term exposure. Etoposide and cisplatin further contribute to bone marrow suppression by generating reactive oxygen species, activating TGF-β1 signaling, and damaging the bone marrow niche, including stromal and endothelial components. This niche damage disrupts the supportive microenvironment necessary for HSC maintenance and MKs maturation. Moreover, 5-fluorouracil has been shown to reduce the number of functional MKs and damage sinusoidal vessels critical for thrombopoiesis. Thrombocytopenia may vary in severity depending on the specific drug used, dose and duration of treatment. Combining antiplatelet drugs with conventional chemotherapy, targeted drugs, or immunotherapy offers promise for synergistic antitumor effects and improved treatment response. By targeting both procoagulant and protumorigenic platelet functions, combination therapy strategies may enhance therapeutic efficacy and overcome resistance mechanisms in cancer [178–181].

Therapeutic apheresis
Therapeutic platelet apheresis is increasingly recognized as a crucial part of the treatment process for patients with solid tumors. Although traditionally associated with hematologic malignancies [182], emerging evidence suggests that platelet apheresis may provide significant benefits in the context of solid tumors [183]. Clinical reports demonstrate rapid improvement in symptoms and thromboembolic complications after thrombapheresis in cases where other cytoreductive and first-line treatment methods have failed. Thrombapheresis can also be used in patients with extreme increases in thrombocytosis after splenectomy [63]. One of the main clinical applications of platelet apheresis in solid tumors is the management of cancer-related thrombocytosis. By reducing the number of platelets, thrombapheresis may help reduce the risk of venous thrombosis, stroke, and myocardial infarction in patients with solid tumors. This intervention is particularly valuable in emergency situations where rapid platelet reduction is needed to prevent life-threatening complications. Despite its advantages, thrombapheresis is not without its challenges. One major concern is the need for reliable venous access, as the procedure requires the collection and return of blood over an extended period of time. Additionally, thrombapheresis can cause adverse reactions, including hypocalcemia, hypovolemia, and citrate toxicity, which must be carefully monitored and managed during the procedure. New research is exploring the potential of predictive biomarkers to optimize the use of thrombapheresis in cancer treatment [184]. Identifying biomarkers that can predict response to thrombapheresis and its associated risks could enable more personalized and effective treatment strategies. In addition, advances in apheresis technology could improve the efficiency and safety of the procedure, making it more accessible and less burdensome for patients [183].

Summary
Platelets fulfill an essential role in hemostasis, but their functions go far beyond the traditionally recognized tasks related to blood clotting, as they are also critically involved in the pathogenesis of cancer. Platelet activation in response to factors secreted by cancer cells and their exchanges with leukocytes support the development of metastases by protecting CTCs from the immune response and stimulating angiogenesis and proliferation of cancer cells. Platelets can also participate in the formation of pre-metastatic niches, supporting the attachment of malignant cells to the blood vessel lining and their migration to distant tissues. Increased numbers of platelets, which often occur in cancer patients, are associated with a worse prognosis, a higher risk of thrombosis and an increased tendency to metastasis. Antiplatelet therapies, such as the use of aspirin and P2Y12 inhibitors, have shown potential in reducing the risk of metastases and improving the efficacy of cancer treatment in preclinical and clinical studies. However, the effect of low-dose aspirin on cancer growth and metastases may differ in patients of different ages. In the elderly it may be associated with a higher incidence of cancer-related mortality, as observed in the ASPREE trial and its use is related to a higher risk of bleeding. These factors emphasize the need for further research to identify appropriate biomarkers and combination treatment strategies. Understanding the mechanisms involving platelets in oncogenesis may enable the development of personalized therapeutic strategies, reducing the risk of complications and improving patient prognosis. Research of this kind on biomarkers and combination therapies with anticancer drugs may contribute to more effective treatment of cancer patients in the future.