Platelets, in addition to their function in hemostasis, play an important role in wound healing and tumor growth. Because platelets contain angiogenesis stimulators and inhibitors, the mechanisms by which platelets regulate angiogenesis remain unclear. As platelets adhere to activated endothelium, their action can enhance or inhibit local angiogenesis. We therefore suspected a higher organization of angiogenesis regulators in platelets. Using double immunofluorescence and immunoelectron microscopy, we show that pro-and antiangiogenic proteins are separated in distinct subpopulations of ␣-granules in platelets and megakaryocytes. Double immunofluorescence labeling of vascular endothelial growth factor (VEGF) (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor), or for thrombospondin-1 and basic fibroblast growth factor, confirms the segregation of stimulators and inhibitors into separate and distinct ␣-granules. These observations motivated the hypothesis that distinct populations of ␣-granules could undergo selective release. The treatment of human platelets with a selective PAR4 agonist (AYPGKF-NH 2 ) resulted in release of endostatin-containing granules, but not VEGF-containing granules, whereas the selective PAR1 agonist (TFLLR-NH 2 ) liberated VEGF, but not endostatin-containing granules. In conclusion, the separate packaging of angiogenesis regulators into pharmacologically and morphologically distinct populations of ␣-granules in megakaryocytes and platelets may provide a mechanism by which platelets can locally stimulate or inhibit angiogenesis. IntroductionAngiogenesis, the process of new vessel development, plays an essential role in embryogenesis, but postnatal angiogenesis is limited to sites of abnormal vascular surface. An activated vascular endothelium can be induced by tissue injury or wound healing, by hormonal cycling such as in pregnancy and ovulation, or by tumor-induced vessel growth. In all of these circumstances, platelets act as the initial responder to vascular change and provide a flexible delivery system for angiogenesis-related molecules. [1][2][3][4] The process of postnatal angiogenesis is regulated by a continuous interplay of stimulators and inhibitors of angiogenesis, and their imbalance contributes to numerous inflammatory, malignant, ischemic, and immune disorders. 5 There is a revived interest in the overlap between angiogenesis and platelets 6 because several clinical trials have now shown that anticoagulation can improve cancer survival 7,8 beyond the benefit derived from the treatment of deep vein thrombosis alone.It is known that platelets stimulate endothelial cells in culture and can promote the assembly of capillary-like structures in vitro. 9,10 Platelets may modulate angiogenesis by releasing promoters such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), and matrix metalloproteinases (MMPs). 1,6,[11][12][13][14][15][16][17][18] The repertoire o...

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Clinical trials with antiangiogenic agents have not been able to validate plasma or serum levels of angiogenesis regulators as reliable markers of cancer presence or therapeutic response. We recently reported that platelets contain numerous proteins that regulate angiogenesis. We now show that accumulation of angiogenesis regulators in platelets of animals bearing malignant tumors exceeds significantly their concentration in plasma or serum, as well as their levels in platelets from non-tumor-bearing animals. This process is selective, as platelets do not take up a proportional amount of other plasma proteins (eg, albumin), even though these may be present at higher concentrations. We also find that VEGFenriched Matrigel pellets implanted subcutaneously into mice or the minute quantities of VEGF secreted by microscopic subcutaneous tumors (0.5-1 mm 3 ) result in an elevation of VEGF levels in platelets, without any changes in its plasma levels. The profile of other angiogenesis regulatory proteins (eg, platelet-derived growth factor, basic fibroblast growth factor) sequestered by platelets also reflects the presence of tumors in vivo before they can be macroscopically evident. The ability of platelets to selectively take up angiogenesis regulators in cancerbearing hosts may have implications for the diagnosis and management of many angiogenesis-related diseases and provide a guide for antiangiogenic therapies. IntroductionPlatelets play a major role in hemostasis, as well as in tissue repair, maintenance of endothelium, and vascular tone. They may also facilitate delivery of angiogenesis regulators and other growth factors to sites of pathologic angiogenesis. 1,2 Correlative studies suggest that increasing platelet counts may be linked to tumor progression. 3,4 We and others have reported previously that platelets contain several proteins that regulate angiogenesis. [5][6][7][8] We have now discovered that the platelet concentrations of angiogenesis regulatory proteins, although relatively constant and stable under physiologic conditions, are modified by and reflect the presence of a tumor. In the presence of microscopic (Ͻ 1.0 mm) tumors in a mouse, circulating platelets sequester increased concentrations of angiogenesis regulatory proteins, without a corresponding elevation in their plasma levels. The uptake of angiogenesis regulatory proteins is selective, as platelets do not take up other plasma proteins. For example, although albumin is present in plasma at much higher concentrations than, for example, vascular endothelial growth factor (VEGF), albumin levels in platelets do not differ in the presence or absence of tumors.In this study, we used a high-throughput surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry (SELDIToF MS), which permitted a rapid analysis of a large number of samples in a highly efficient and reproducible manner. 9,10 In this open-ended proteomic comparison of platelets from tumor-bearing and non-tumor-bearing animals, the majority of identified differentiall...

Platelet microparticles are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and platelet microparticle levels. Yet little is known about the processes by which platelet microparticles are generated in vivo. We now characterize microparticles derived directly from megakaryocytes. Video microscopy of live mouse megakaryocytes demonstrated that microparticles form as submicron beads along the lengths of slender, unbranched micropodia. These microparticles are CD41 ؉ , CD42b ؉ , and express surface phosphatidylserine. Megakaryocyte microparticle generation is resistant to inhibition of microtubule assembly, which is critical to platelet formation, and augmented by inhibition of actin polymerization. To determine whether circulating microparticles are derived primarily from activated platelets or megakaryocytes, we identified markers that distinguish between these 2 populations. CD62P and LAMP-1 were found only on mouse microparticles from activated platelets. In contrast, full-length filamin A was found in megakaryocytederived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P ؊ , LAMP-1 ؊ and expressed fulllength filamin A, indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62P ؊ and expressed full-length filamin A. Cultured human megakaryocytes elaborated microparticles that were CD41 ؉ , CD42b ؉ , and express surface phosphatidylserine. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet micropar-

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Standard chemotherapeutic drugs, when modified by the frequency and dose of administration, can target angiogenesis. This approach is referred to as antiangiogenic chemotherapy, low-dose chemotherapy, or metronomic chemotherapy. This study evaluated the feasibility of 6 months of metronomic chemotherapy, its toxicity and tolerability, surrogate markers of activity, and preliminary evidence of activity in children with recurrent or progressive cancer. Twenty consecutive children were enrolled and received continuous oral thalidomide and celecoxib with alternating oral etoposide and cyclophosphamide every 21 days for a planned duration of 6 months using antiangiogenic doses of all four drugs. Surrogate markers including bFGF, VEGF, endostatin, and thrombospondin were also evaluated. Therapy was well tolerated in this heavily pretreated population. Toxicities (predominantly reversible bone marrow suppression) responded to dose modifications. Sixty percent of the patients received less than the prescribed 6 months of therapy due to toxicity (one case of deep vein thrombosis), personal choice (1 patient), or disease progression (10 patients). Forty percent of the patients completed the 6 months of therapy, resulting in prolonged or persistent disease-free status. One quarter of all patients continue to be progression free more than 123 weeks from starting therapy. Sixteen percent of patients showed a radiographic partial response. Only elevated thrombospondin-1 levels appeared to correlate with prolonged response. This oral antiangiogenic chemotherapy regimen was well tolerated in this heavily pretreated pediatric population, which showed prolonged or persistent disease-free status, supporting the continued study of antiangiogenic/metronomic chemotherapy in human clinical trials.

Three-dimensional tumor growth is dependent on the perpetual recruitment of host blood vessels to the tumor site. This recruitment process (mainly via angiogenesis) is thought to be triggered, at least in part, by the very same set of genetic alterations (activated oncogenes, inactivated/lost tumor suppressor genes) as those responsible for other aspects of malignant transformation (e.g., aberrant mitogenesis, resistance to apoptosis). Potent oncogenes are able to deregulate expression of both angiogenesis stimulators and inhibitors in cancer cells. For example, mutant ras expression is associated with increased production of vascular endothelial growth factor (VEGF) and downregulation of thrombospondin-1 (TSP-1). Upregulation of VEGF and angiogenesis can also be induced by constitutive activation of other oncogenic proteins (e.g., EGFR, Raf, MEK, PI3K) acting at various levels on the Ras signaling pathway. The mode and the magnitude of such proangiogenic influences can be significantly modified by cell type (fibroblastic or epithelial origin), epigenetic factors (hypoxia, changes in cell density), and/or presence of additional genetic lesions (e.g., preceding loss of p16 or p53 tumor suppressor genes). Activated oncogenes (e.g., ras, src, HER-2) induce co-expression of angiogenic properties concomitantly with several highly selectable traits (increased mitogenesis, resistance to apoptosis), a circumstance that may accelerate selection of the angiogenic phenotype at the cell population level. On the other hand oncogene-induced reduction in growth requirements may also endow tumor cells with a diminished (albeit not abrogated) dependence on (close) proximity to blood vessels, i.e., with reduced vascular dependence. Thus, oncogenes can impact several interconnected aspects of cellular growth, survival, and angiogenesis. Experimental evidence suggests that, in principle, many of these properties (including angiogenesis) can be simultaneously suppressed (and tumor stasis or regression induced) by effective use of the specific oncogene antagonists and signal transduction inhibitors.

Platelets sequester angiogenesis regulatory proteins which suggests an avenue for developing biomarkers to monitor disease. We describe a comparison of angiogenesis regulatory proteins found in platelets of colorectal cancer patients and normal controls. Platelet and plasma content of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), platelet factor 4 (PF4), thrombospondin-1 (TSP-1) and endostatin in 35 patients with colon cancer were compared with 84 age-matched healthy controls using ELISAs. We standardized the platelet preparation procedure, introduced process controls and normalized the respective protein levels to platelet numbers using an actin ELISA. Statistically significant differences were found in the median levels of VEGF, PF4 and PDGF in platelets of patients with cancer compared to healthy individuals. Platelet concentrations in cancer patients versus controls were: VEGF 1.3 versus 0.6 pg/10(6), PF4 18.5 versus 9.4 ng/10(6), and PDGF 34.1 versus 21.0 pg/10(6). Multivariable logistic regression analysis indicated that PDGF, PF4 and VEGF were independent predictors of colorectal carcinoma and as a set provided statistically significant discrimination (area under the curve = 0.893, P < .0001). No significant differences were detected for bFGF, endostatin, or TSP-1. Reference Change Value analysis determined that the differences seen were not clinically significant. Plasma levels yielded no correlations.