Recent studies in patients with ovarian cancer suggest that tumor growth may be accelerated following cessation of antiangiogenesis therapy; however, the underlying mechanisms are not well understood. In this study, we aimed to compare the effects of therapy withdrawal to those of continuous treatment with various antiangiogenic agents. Cessation of therapy with pazopanib, bevacizumab, and the human and murine anti-VEGF antibody B20 was associated with substantial tumor growth in mouse models of ovarian cancer. Increased tumor growth was accompanied by tumor hypoxia, increased tumor angiogenesis, and vascular leakage. Moreover, we found hypoxia-induced ADP production and platelet infiltration into tumors after withdrawal of antiangiogenic therapy, and lowering platelet counts markedly inhibited tumor rebound after withdrawal of antiangiogenic therapy. Focal adhesion kinase (FAK) in platelets regulated their migration into the tumor microenvironment, and FAK-deficient platelets completely prevented the rebound tumor growth. Additionally, combined therapy with a FAK inhibitor and the antiangiogenic agents pazopanib and bevacizumab reduced tumor growth and inhibited negative effects following withdrawal of antiangiogenic therapy. In summary, these results suggest that FAK may be a unique target in situations in which antiangiogenic agents are withdrawn, and dual targeting of FAK and VEGF could have therapeutic implications for ovarian cancer management.
Ovarian cancer patients have a high risk of developing venous thrombosis. The membrane lipid bilayer of platelets and platelet-derived microparticles (PMP) provides a platform for assembly of coagulation proteins and generation of blood clots.
We compared the lipid composition of platelets and PMPs in patients with ovarian cancer to those in healthy subjects. We used shotgun lipidomics to quantify 12 classes and 177 species of lipids.
We found a significant change in 2 classes of lipids in platelets and PMPs isolated from ovarian cancer patients: higher phosphatidylinositol and lower lyso-phosphatidylcholine. The level of 28 species of lipids was also significantly altered in the direction of an increase in the pro-coagulant and a reduction in the anticoagulant lipids. We found that cancer platelets expressed less lipid phosphate phosphatase 1 (LPP1), a key enzyme in phospholipid biosynthesis pathways, than normal platelets. The reduction in LPP1 might contribute to the changes in the lipid profile of cancer platelets.
Our results support a procoagulant lipid profile of platelets in ovarian cancer patients that can play a role in the increased risk of venous thrombosis in these patients.
As far as we are aware, our study is the first study on platelet lipidomics in ovarian cancer. The importance of our findings for the future studies are: 1) a similar change in lipid profile of platelets and PMP may be responsible for hypercoagulability in other cancers, and 2) plasma level of high-risk lipids for venous thrombosis may be useful biomarkers.
•Lipid composition of platelet and PMP is altered in ovarian cancer.•The change in lipid composition of platelet and PMP is in a procoagulant direction.•LPP1 enzyme is reduced in cancer platelets.
Platelet; Lipidomics; Venous thrombosis; Hypercoagulability; Ovarian cancer; Lipid phosphate phosphatase 1
We hypothesized that platelet levels during therapy could serve as a biomarker for response to therapy and that manipulation of platelet levels could impact responsiveness to chemotherapy.
The medical records of patients with recurrent or progressive ovarian cancer were retrospectively queried for changes in platelet and CA-125 levels during primary therapy. In vitro co-culture experiments and in vivo orthotopic models of human ovarian cancer in mice were used to test the effect of modulating platelet levels on tumor growth and responsiveness to docetaxel.
Thrombocytosis at the diagnosis of ovarian cancer correlated with decreased interval to progression (p = 0.05) and median overall survival (p = 0.007). Mean platelet levels corrected during primary therapy and rose at recurrence. Contrary to treatment-responsive patients, in a cohort of patients refractory to primary therapy, platelet levels did not normalize during therapy. In A2780, HeyA8, and SKOV3-ip1 ovarian cancer cell lines, platelet co-culture protected against apoptosis (p < 0.05). In orthotopic models of human ovarian cancer, platelet depletion resulted in 70% reduced mean tumor weight (p < 0.05). Compared to mice treated with docetaxel, mice treated with both docetaxel and platelet-depleting antibody had a 62% decrease in mean tumor weight (p = 0.04). Platelet transfusion increased mean aggregate tumor weight 2.4-fold (p < 0.05), blocked the effect of docetaxel on tumor growth (p = 0.55) and decreased tumor cell apoptosis. Pre-transfusion aspirinization of the platelets blocked the growth-promoting effects of transfusion.
Platelet-driven effects of chemotherapy response may explain clinical observations.
Platelets; docetaxel; ovarian cancer; aspirin
Detection of donor-specific anti-HLA antibodies (DSA) has been associated with graft rejection in all forms of transplantation. The mechanism by which DSA increase the risk of graft failure remains unclear. We hypothesized that complement-binding DSA are associated with engraftment failure in hematopoietic stem-cell transplantation and analyzed 122 haploidentical transplant recipients tested prospectively for DSA. Retrospective C1q testing was done on 22 allosensitized recipients. Twenty-two of 122 patients (18%) had DSA, 19 of which were females (86%). Seven patients with DSA (32%) rejected the graft. Median DSA level at transplant for patients who failed to engraft was 10,055 MFI versus 2,065 MFI for those who engrafted (p=0.007). Nine patients with DSA were C1q positive in the initial samples with median DSA level 15,279 MFI (range 1,554-28,615), compared with 7 C1q negative patients with median DSA level 2,471 MFI (665-12,254) (p=0.016). Of 9 patients, who tested positive for C1q in the initial samples, 5 patients remained C1q positive at time of transplant [all with high DSA levels (median 15,279, range 6,487-22,944)] and experienced engraftment failure, while 4 patients became C1q negative pre-transplant and all engrafted the donor cells (p=0.008). In conclusion, patients with high DSA levels (> 5,000 MFI) and complement-binding antibodies (C1q positive) appear to be at much higher risk of primary graft failure. C1q should be assessed in patients with DSA prior to hematopoietic stem-cell transplantation. Reduction of DSA to non-complement binding levels might prevent engraftment failure in hematopoietic stem cell transplantation.
Donor-specific anti-HLA antibodies; complement-binding DSA; C1q; graft rejection; hematopoietic stem cell transplantation; desensitization; buffy coat
Growing understanding of the role of thrombocytosis, high platelet turnover, and the presence of activated platelets in the circulation in cancer progression and metastasis has brought megakaryocytes into focus. Platelet biology is essential to hemostasis, vascular integrity, angiogenesis, inflammation, innate immunity, wound healing, and cancer biology. However, before megakaryocyte/platelet-directed therapies can be considered for clinical use, understanding of the mechanism and biology of paraneoplastic thrombocytosis in malignancy is required. Here, we provide an overview of the clinical implications, biological significance, and mechanisms of paraneoplastic thrombocytosis in the context of ovarian cancer.
The complement system has been shown to regulate T cell activation and alloimmune responses in graft-versus-host disease (GVHD). Mice deficient in the central component of complement system C3 have significantly lower GVHD-related mortality/morbidity and C3 modulates Th1/Th17 polarization in mouse GVHD. To investigate whether anti-complement therapy has any impact on human T cell activation, a drug candidate Compstatin was used to inhibit C3 activation in this study. We found the frequency of IFN-γ (Th1), IL-4 (Th2), IL-17 (Th17), IL-2 and TNF-α producing cells were significantly reduced among activated CD4+ cells in the presence of Compstatin. Compstatin treatment decreased the proliferation of both CD4+ and CD8+ T cells upon TCR stimulation. However, Compstatin does not affect the production of IL-2 and TNF-α in activated CD8+ T cells, and the differentiation of CD8+ T cells into distinct memory and effector subsets remained intact. Furthermore, we examined complement deposition in the skin and lip biopsy samples of patients diagnosed with cutaneous GVHD. C3 deposition was detected in the squamous epithelium and dermis, blood vessels and damaged sweat glands, and associated with gland damage and regeneration. We conclude that C3 mediates Th1/Th17 polarization in human T cell activation and skin GVHD in patients.
Complement; GVHD; Compstatin; Th1; Th17
Intracranial hemorrhage (ICH) is a common complication in acute myeloid leukemia (AML) patients with an incidence rate of 6.3% (1). Bleeding disorders related to disseminated intravascular coagulation (DIC) are common complications in AML cases (2). Recombinant activated Factor VII [rFVIIa (NovoSeven®)] is approved for the treatment of bleeding complications with FVIII or FIX inhibitors in patients with congenital FVII deficiency. Use of rFVIIa for the treatment of acute hemorrhage in patients without hemophilia has been successful (3, 4). Herein, we describe the successful use of rFVIIa in a patient with acute ICH in the setting of newly diagnosed AML.
recombinant activated factor VII; acute myeloid leukemia; intracranial hemorrhage
Most clinical studies of heparin-induced thrombocytopenia have not included cancer patients who have high risk of thromboembolism, frequent exposure to heparin, and many potential causes of thrombocytopenia other than heparin-induced thrombocytopenia. To estimate the incidence and prevalence of heparin-induced thrombocytopenia in cancer patients, we identified cases based on diagnostic codes, anti-heparin antibody testing, and clinical characteristics (4T score) at a comprehensive cancer center between 1 October 2008 and 31 December 2011. We estimated that the prevalence of heparin-induced thrombocytopenia to be 0.02% among all cancer patients and 0.24% among cancer patients exposed to heparin. The annual incidence of heparin-induced thrombocytopenia was 0.57 cases per 1000 cancer patients exposed to heparin. Of the 40 cancer patients with the International Classification of Diseases (Ninth Revision; ICD-9) code for heparin-induced thrombocytopenia, positive anti-heparin antibody, and 4T score ≥4, 5 (12.5%) died of related thromboembolic or hemorrhagic complications. In a multivariate logistic regression model, male gender was a significant (p = 0.035) factor, and non-hematological malignancy was a significant (p = 0.017) factor associated with anti-heparin antibody positivity. Future studies may further examine the risk factors associated with heparin-induced thrombocytopenia in larger cohorts.
Heparin-induced thrombocytopenia; cancer; incidence; prevalence
We describe a role for the complement system in enhancing cancer growth. Cancer cells secrete complement proteins that stimulate tumor growth upon activation. Complement promotes tumor growth via a direct autocrine effect that is partially independent of tumor-infiltrating cytotoxic T cells. Activated C5aR and C3aR signal through the PI3K/AKT pathway in cancer cells, and silencing the PI3K or AKT gene in cancer cells eliminates the progrowth effects of C5aR and C3aR stimulation. In patients with ovarian or lung cancer, higher tumoral C3 or C5aR mRNA levels were associated with decreased overall survival. These data identify a role for tumor-derived complement proteins in promoting tumor growth, and they therefore have substantial clinical and therapeutic implications.
Complement factor H (fH) is a plasma protein that regulates activation of the alternative pathway, and mutations in fH are associated with a rare form of thrombotic microangiopathy (TMA), known as atypical hemolytic uremic syndrome (aHUS). A more common TMA is thrombotic thrombocytopenic purpura, which is caused by the lack of normal ADAMTS-13-mediated cleavage of von Willebrand factor (VWF). We investigated whether fH interacts with VWF and affects cleavage of VWF. We found that factor H binds to VWF in plasma, to plasma-purified VWF, and to recombinant A1 and A2 domains of VWF as detected by co-immunoprecipitation (co-IP) and surface plasmon resonance assays. Factor H enhanced ADAMTS-13-mediated cleavage of recombinant VWF-A2 as determined by quantifying the cleavage products using Western-blotting, enhanced cleavage of a commercially available fragment of VWF-A2 (FRETS-VWF73) as determined by fluorometric assay, and enhanced cleavage of ultralarge (UL) VWF under flow conditions as determined by cleavage of VWF-platelet strings attached to histamine stimulated endothelial cells. Using recombinant full-length and truncated fH molecules, we found that the presence of the C-terminal half of fH molecule is important for binding to VWF-A2 and for enhancing cleavage of the A2 domain by ADAMTS-13. We conclude that factor H binds to VWF and may modulate cleavage of VWF by ADAMTS-13.
Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is mediated by the activation of recipient dendritic cells (DCs) and subsequent proliferation of donor T cells. Recently complement system has been shown to modulate adaptive immunity through the interaction between the complement system and lymphocytes. Complement proteins participate in the activation of DCs, antigen presentation to T cells, and proliferation of T cells. Our studies with a murine model of bone marrow transplantation (BMT) demonstrate that complement system regulates alloimmune responses in GVHD. Mice deficient in the central component of the complement system (C3−/−) had significantly lower GVHD-related mortality and morbidity compared to the wild type (WT) recipient mice. The number of donor-derived T cells including IFNγ+, IL17+ and IL17+IFNγ+ subsets was decreased in secondary lymphoid organs of C3−/− recipients. Furthermore, there was a reduction of recipient CD8α+CD11c+ in lymphoid organs. We conclude C3 regulates Th1/17 differentiation in BMT, and define a novel function of the complement system in GVHD.
The mechanisms of paraneoplastic thrombocytosis in ovarian cancer and the role that platelets play in abetting cancer growth are unclear.
We analyzed clinical data on 619 patients with epithelial ovarian cancer to test associations between platelet counts and disease outcome. Human samples and mouse models of epithelial ovarian cancer were used to explore the underlying mechanisms of paraneoplastic thrombocytosis. The effects of platelets on tumor growth and angiogenesis were ascertained.
Thrombocytosis was significantly associated with advanced disease and shortened survival. Plasma levels of thrombopoietin and interleukin-6 were significantly elevated in patients who had thrombocytosis as compared with those who did not. In mouse models, increased hepatic thrombopoietin synthesis in response to tumor-derived interleukin-6 was an underlying mechanism of paraneoplastic thrombocytosis. Tumor-derived interleukin-6 and hepatic thrombopoietin were also linked to thrombocytosis in patients. Silencing thrombopoietin and interleukin-6 abrogated thrombocytosis in tumor-bearing mice. Anti–interleukin-6 antibody treatment significantly reduced platelet counts in tumor-bearing mice and in patients with epithelial ovarian cancer. In addition, neutralizing interleukin-6 significantly enhanced the therapeutic efficacy of paclitaxel in mouse models of epithelial ovarian cancer. The use of an antiplatelet antibody to halve platelet counts in tumor-bearing mice significantly reduced tumor growth and angiogenesis.
These findings support the existence of a paracrine circuit wherein increased production of thrombopoietic cytokines in tumor and host tissue leads to paraneoplastic thrombocytosis, which fuels tumor growth. We speculate that countering paraneoplastic thrombocytosis either directly or indirectly by targeting these cytokines may have therapeutic potential. (Funded by the National Cancer Institute and others.)
Almost a trillion platelets pass through the pulmonary circulation every minute, yet little is known about how they support pulmonary physiology or contribute to the pathogenesis of lung diseases. When considering this conundrum, three questions jump out: Does platelet production in the lungs occur? Why does severe thrombocytopenia—which undercuts the principal physiological role of platelets to effect hemostasis—not lead to pulmonary hemorrhage? Why does atherothrombosis—which platelets initiate, maintain, and trigger is other critically important arterial beds—not develop in the pulmonary artery? The purpose of this review is to explore these and derivative questions by providing data within a conceptual framework that begins to organize a subject that is largely unassembled.
cell signaling; hemostasis; platelets; thrombosis; vascular biology
Anemia is a common finding among patients with chronic heart failure. Although co-morbidities, such as kidney failure, might contribute to the pathogenesis of anemia, many patients with heart failure do not have any other obvious etiology for their anemia. We investigated whether anemia in heart failure is associated with an elevation in hepcidin concentration.
We used time-of-flight mass spectrometry to measure hepcidin concentration in urine and serum samples of patients with heart failure and in control subjects. We found that the concentration of hepcidin was lower in urine samples of patients with heart failure compared to those of control subjects. Serum hepcidin was also reduced in heart failure but was not significantly lower than that in controls. There were no significant differences between hepcidin levels in patients with heart failure and anemia compared to patients with heart failure and normal hemoglobin. We concluded that hepcidin probably does not play a major role in pathogenesis of anemia in patients with chronic heart failure.
Anemias; Cytokines; Iron
The association between cancer and venous thromboembolism (VTE) has been well documented in the literature. Prevention and treatment of VTE in cancer patients is imperative. Typically, the mainstay regimen for VTE prevention and treatment has been anticoagulation therapy, unless contraindicated. This therapy consists of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), factor Xa inhibitor, or vitamin K antagonist (VKA). Current guidelines recommend LMWH over VKA for the treatment of VTE in cancer patients. Factor-specific anticoagulants have been proven safe and effective, and recently factor Xa inhibitors have emerged as a treatment alternative to heparins and VKA. Currently, three factor Xa inhibitors have been identified: fondaparinux (the only one approved so far by the US Food and Drug Administration), idraparinux (in clinical trials), and idrabiotaparinux (in clinical trials). This paper will examine the role of these agents, focusing on fondaparinux, for the prevention and treatment of VTE in cancer patients.
Antiphospholipid antibodies (aPLA) have been shown to enhance thrombus formation by increasing the expression of adhesive receptors such as P‐selectin on endothelial cells. The P‐selectin counter‐receptor on leucocytes is P‐selectin glycoprotein ligand‐1 (PSGL‐1). We have previously described a variable number of tandem repeats (VNTR) polymorphism in the mucin‐like region of PSGL‐1, with three alleles: allele A, 16 repeats; allele B, 15 repeats; and allele C, 14 repeats.
We compared the PSGL‐1 VNTR allele and genotype frequencies in 90 patients with antiphospholipid syndrome (APS) with thrombosis, 39 patients with persistent aPLA positivity without thrombosis, and 203 healthy controls.
The frequency of the B allele was significantly higher in patients with APS with thrombosis compared with patients without thrombosis (p = 0.023). When we compared the groups by genotype frequencies, we found a markedly higher frequency of the AB genotype in patients with APS with thrombosis than in aPLA‐positive patients without thrombosis (38.9% vs 10.3%, p = 0.001) or in normal population (38.9% vs 22.2%, p<0.01).
We suggest that the VNTR polymorphism of PSGL‐1 is a significant determinant of thrombotic predisposition in patients with APS. Furthermore, risk appears to correlate best with the combination of alleles inherited rather than with the presence of any particular allele.
antiphospholipid syndrome; P‐selectin; P‐selectin glycoprotein ligand‐1; PSGL‐1 VNTR polymorphism; thrombosis
Background and Objectives
The complement system is a biochemical cascade composed of several plasma proteins that can interact with endothelial cells and blood cells, including platelets. In order to investigate the effect of the complement system on platelets, we studied platelet function in C3-deficient mice that lack complement activity.
Method and Results
Tail-cut bleeding time was prolonged and platelet aggregation in response to protease activated receptor-4 (PAR4) peptide was decreased in C3-deficient mice compared to wild-type littermates. Platelet aggregation in response to other agonists (ADP and collagen) was similar between C3-deficient mice and their normal littermates. Isolated platelets from wild-type mice aggregate less in C3-deficient plasma than in normal plasma and conversely, addition of plasma from wild-type mice or plasma-purified C3 improved aggregation of C3-deficient platelets. We also monitored formation of murine arteriole or venule thrombi in an intravital microscopy thrombosis model. We found that C3-deficient mice had a significantly delayed thrombotic response in arterioles compared to their wild-type littermates. Furthermore, thrombi in C3-deficient mice were less stable and embolized more frequently than those in wild-type mice.
Platelets of C3-deficient mice have subnormal function resulting in a prolonged tail-cut bleeding time and delayed thrombosis after vessel wall injury.
platelet; complement system; thrombosis
The complement system is an important part of innate immunity; however, as with other parts of the immune system, the complement system can become pathologically activated and create or worsen disease. Anticomplement reagents have been studied for several years, but only recently have they emerged as a viable therapeutic tool. Here, we describe the role of the complement system in a wide array of diseases, as well as the use of anticomplement therapy as treatment for these diseases in animal models and in human clinical trials. Specifically, we will discuss the role of anticomplement therapy in paroxysmal nocturnal hemoglobinuria, glomerulonephritis, and heart disease, including coronary artery disease, myocardial infarction, and coronary revascularization procedures such as percutaneous coronary angioplasty and coronary artery bypass graft surgery.
complement; paroxysmal nocturnal hemoglobinuria; glomerulonephritis; myocardial infarction; cardiopulmonary bypass
Inflammation and thrombosis are two responses that are linked through a number of mechanisms, one of them being the complement system. Various proteins of the complement system interact specifically with platelets, which, in turn, activates them and promotes thrombosis. In this paper, we show that the converse is also true: activated platelets can activate the complement system. As assessed by flow cytometry and immunoblotting, C3 deposition increased on the platelet surface upon cell activation with different agonists. Activation of the complement system proceeded to its final stages, which was marked by the increased generation of the anaphylotoxin C3a and the C5b-9 complex. We identified P-selectin as a C3b-binding protein, and confirmed by surface plasmon resonance binding that these two proteins interact specifically with a dissociation constant of 1 μM. Using heterologous cells expressing P-selectin, we found that P-selectin alone is sufficient to activate the complement system, marked by increases in C3b deposition, C3a generation, and C5b-9 formation. In summary, we have found that platelets are capable of activating the complement system, and have identified P-selectin as a receptor for C3b capable of initiating complement activation. These findings point out an additional mechanism by which inflammation may localize to sites of vascular injury and thrombosis.