• Anticoagulants inhibit release of angiogenic proteins from platelets.Platelets are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. Because of the propensity for clotting, patients with malignancy are often anticoagulated with heparin products, which paradoxically offer a survival benefit by an unknown mechanism. We hypothesized that antithrombotic agents alter the release of angiogenesis regulatory proteins from platelets. Our data revealed that platelets exposed to heparins released significantly decreased vascular endothelial growth factor (VEGF) in response to adenosine 59-diphosphate or tumor cells (MCF-7 cells) and exhibited a decreased angiogenic potential. The releasate from these platelets contained decreased proangiogenic proteins. The novel anticoagulant fondaparinux (Xa inhibitor) demonstrated a similar impact on the platelet angiogenic potential. Because these anticoagulants decrease thrombin generation, we hypothesized that they disrupt signaling through the platelet protease-activated receptor 1 (PAR1) receptor. Addition of PAR1 antagonists to platelets decreased VEGF release and angiogenic potential. Exposure to a PAR1 agonist in the presence of anticoagulants rescued the angiogenic potential. In vivo studies demonstrated that platelets from anticoagulated patients had decreased VEGF release and angiogenic potential. Our data suggest that the mechanism by which antithrombotic agents increase survival and decrease metastasis in cancer patients is through attenuation of platelet angiogenic potential. (Blood. 2014;123(1):101-112) IntroductionCancer cells are surrounded by and interact with a complex milieu consisting of but not limited to endothelial cells, mast cells, macrophages, stromal cells, and lymphocytes. In fact, tumor cells live in intimate symbiosis with the rest of the body and appear to hijack normal physiological processes to aid their progression and growth. The recognition that tumor growth and metastasis is not exclusively an independent process for tumor cells suggests that disrupting the tumor microenvironment might provide a novel treatment modality for malignancy.Although platelets are best known for their role in hemostasis and thrombosis, a substantial amount of data supports the idea that platelets play important roles in tumorigenesis, contributing to inflammation, angiogenesis, and metastatic dissemination of tumor cells.1 Platelet count can be a prognostic factor in cancer, patients presenting with thrombocytosis having a poor survival in a variety of cancers including breast cancer.2-5 Conversely, the presence of thrombocytopenia is associated with a survival benefit and decreased metastasis.6-9 For tumors to grow beyond 1 to 2 mm 3 , they must establish their own blood supply through angiogenesis, and there is substantial evidence that angiogenesis is regulated by platelets. [10][11][12] In malignancy, platelets are the major serum source of many potent proangiogenic proteins, including more than 80% of circulating vascul...

Key Points• CCL5 increases MK ploidy and subsequent proplatelet formation in a CCR5-dependent manner.• CCL5 may act to increase platelet counts during physiological stress.In times of physiological stress, platelet count can transiently rise. What initiates this reactive thrombocytosis is poorly understood. Intriguingly, we found that treating megakaryocytes (MKs) with the releasate from activated platelets increased proplatelet production by 47%. Platelets store inflammatory cytokines, including the chemokine ligand 5 (CCL5, RANTES); after TRAP activation, platelets release over 25 ng/mL CCL5. We hypothesized that CCL5 could regulate platelet production by binding to its receptor, CCR5, on MKs. Maraviroc (CCR5 antagonist) or CCL5 immunodepletion diminished 95% and 70% of the effect of platelet releasate, respectively, suggesting CCL5 derived from platelets is sufficient to drive increased platelet production through MK CCR5. MKs cultured with recombinant CCL5 increased proplatelet production by 50% and had significantly higher ploidy. Pretreating the MK cultures with maraviroc prior to exposure to CCL5 reversed the augmented proplatelet formation and ploidy, suggesting that CCL5 increases MK ploidy and proplatelet formation in a CCR5-dependent manner. Interrogation of the Akt signaling pathway suggested that CCL5/CCR5 may influence proplatelet production by suppressing apoptosis. In an in vivo murine acute colitis model, platelet count significantly correlated with inflammation whereas maraviroc treatment abolished this correlation. We propose that CCL5 signaling through CCR5 may increase platelet counts during physiological stress. (Blood. 2016;127(7):921-926) IntroductionCirculating blood platelets are specialized cells that function to minimize bleeding and blood vessel injury. As such, platelets play a critical role in both normal and disease physiology. Large progenitor cells in the bone marrow called megakaryocytes (MKs) release platelets by extending long processes, designated proplatelets, into sinusoidal blood vessels.1 Despite the importance of platelets in thrombosis and hemostasis, the mechanism by which MKs complete differentiation and release platelets is poorly understood. Specifically, very little is known about what triggers mature, resting MKs to form proplatelets. Platelet counts rise transiently in the setting of physiological stress, such as myocardial infarction, infection, inflammation, and malignancy. [2][3][4] What initiates this upregulation is not well understood and has largely been attributed to an inflammatory response and increased cytokine release. [5][6][7] One cytokine that is highly expressed in inflammatory states is CCL5 (RANTES).8 CCL5, which is abundant in human platelets, signals predominantly through CCR5, a 7-transmembrane G-protein-coupled receptor that mediates diverse signaling cascades. 9 Methods Platelet purification and activationBlood collection was performed with institutional review board/institutional animal care and use committee approval and in accordance wi...

It is now recognized that compounds released from tumor cells can activate platelets, causing the release of platelet-derived factors into the tumor microenvironment. Several of these factors have been shown to directly promote neovascularization and metastasis, yet how the feedback between platelet releasate and the tumor cell affects metastatic phenotype remains largely unstudied. Here, we identify that breast tumor cells secrete high levels of interleukin 8 (IL-8, CXCL8) in response to platelet releasate, which promotes their invasive capacity. Furthermore, we found that platelets activate the Akt pathway in breast tumor cells, and inhibition of this pathway eliminated IL-8 production. We therefore hypothesized inhibiting platelets with aspirin could reverse the prometastatic effects of platelets on tumor cell signaling. Platelets treated with aspirin did not activate the Akt pathway, resulting in reduced IL-8 secretion and impaired tumor cell invasion. Of note, patients with breast cancer receiving aspirin had lower circulating IL-8, and their platelets did not increase tumor cell invasion compared with patients not receiving aspirin. Our data suggest platelets support breast tumor metastasis by inducing tumor cells to secrete IL-8. Our data further support that aspirin acts as an anticancer agent by disrupting the communication between platelets and breast tumor cells.

Objective Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in metastasis. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment of breast cancer. Tamoxifen and its metabolites have been shown to directly impact platelet function suggesting that this drug has additional mechanisms of action. The purpose of this study was to determine whether tamoxifen exerts anti-tumor effects through direct platelet inhibition. Approach and Results This study found that pretreatment with tamoxifen leads to a significant inhibition of platelet activation. Platelets exposed to tamoxifen released significantly lower amounts of pro-angiogenic regulator VEGF. In vitro angiogenesis assays confirmed that tamoxifen pretreatment led to diminished capillary tube formation and decreased endothelial migration. Tamoxifen and its metabolite, 4-Hydroxytamoxifen, also significantly inhibited the ability of platelets to promote metastasis in vitro. Using a membrane based array, we identified several proteins associated with angiogenesis metastasis that were lower in activated releasate from tamoxifen treated platelets including angiogenin, CXCL1, CCL5, EGF, CXCL5 and PDGF-BB while anti-angiogenic angiopoietin-1 was elevated. Platelets isolated from patients on tamoxifen maintenance therapy were also found to have decreased activation responses, diminished VEGF release and lower angiogenic and metastatic potential. Conclusions We demonstrate that tamoxifen and its metabolite 4-Hydroxytamoxifen directly alters platelet function leading to decreased angiogenic and metastatic potential. Furthermore, this study supports the idea of utilizing targeted platelet therapies to inhibit the platelet’s role in angiogenesis and malignancy.