Elisabeth M. Battinelli

Release of angiogenesis regulatory proteins from platelet alpha granules: modulation of physiologic and pathologic angiogenesis.

An association between platelets, angiogenesis, and cancer has long been recognized, but the mechanisms linking them remains unclear. Platelets regulate new blood vessel growth through numerous stimulators and inhibitors of angiogenesis by several pathways, including differential exocytosis of angiogenesis regulators. Herein, we investigated the differential release of angiogenesis stimulators and inhibitors from platelets. Activation of human platelets with adenosine diphosphate (ADP) stimulated the release of VEGF, but not endostatin whereas, thromboxane A(2) (TXA(2)) released endostatin but not VEGF. Platelet releasates generated by activation with ADP promoted migration and formation of capillary structures by human umbilical vein endothelial cells (HUV-EC-Cs) in in vitro angiogenesis models. Conversely, TXA(2)-stimulated platelet releasate inhibited migration and formation of capillary structures. Because tumor growth beyond 1-2 mm(3) is angiogenesis-dependent, we hypothesized that cancer cells preferentially stimulate platelets to secrete their pro-angiogenic payload. In support of this, the breast cancer cell line MCF-7 stimulated secretion of VEGF and a pro-angiogenic releasate from platelets. Furthermore, the antiplatelet agent aspirin inhibited platelet-mediated angiogenesis after exposure to ADP or MCF-7 cells providing a potential mechanism for how aspirin may impact malignancy. Manipulation of differentially mediated release of angiogenic factors from platelets may provide a new modality for cancer treatment.

Deficient Platelet-Derived Nitric Oxide and Enhanced Hemostasis in Mice Lacking the NOSIII Gene

Endothelial nitric oxide synthase (eNOS) has been identified in human platelets. Although platelet-derived nitric oxide (NO) has been shown to inhibit platelet recruitment in vitro, its role in the regulation of the hemostatic response in vivo has not been characterized. To define the role of platelet-derived NO in vivo, we studied mice that lacked a functional eNOS gene (NOSIII). Surface P-selectin expression in platelets from eNOS-deficient mice was not significantly altered; however, bleeding times were markedly decreased in eNOS-deficient versus wild-type mice (77.2+/-3 versus 133.4+/-3 seconds, P<0.00005). To determine the contribution of endothelium- versus platelet-derived NO to the bleeding time, isolated platelets from either eNOS-deficient or wild-type mice were transfused into a thrombocytopenic eNOS-deficient mouse and the bleeding time was measured. The bleeding times in mice transfused with eNOS-deficient platelets were significantly decreased compared with mice transfused with wild-type platelets (Deltableeding time, -24.6+/-9.1 and -3.4+/-5.3 seconds, respectively; P<0.04). Platelet recruitment was studied by measuring serotonin release from a second recruitable population of platelets that were added to stimulated platelets at the peak of NO production. There was 40.3+/-3.7% and 52. 0+/-2.1% serotonin release for platelets added to wild-type or eNOS-deficient platelets, respectively (P<0.05). In summary, mice that lacked eNOS had markedly decreased bleeding times even after endothelial NO production was controlled. These data suggest that the lack of platelet-derived NO alters in vivo hemostatic response by increasing platelet recruitment. Thus, these data support a role for platelet-derived NO production in the regulation of hemostasis.

Selective sorting of alpha-granule proteins.

Summary. One of the main functions of blood platelets is to secrete a variety of substances that can modify a developing thrombus, regulate the growth of the vasculature, promote wound repair, and contribute to cell‐adhesive events. A majority of this vast array of secreted proteins are stored in alpha‐granules. Until recently, it was assumed that platelets contained one homogeneous population of alpha‐granules that undergo complete de‐granulation during platelet activation. This review focuses on the mechanisms of alpha‐granule biogenesis and secretion, with a particular emphasis on recent findings that clearly demonstrate that platelets contain distinct subpopulations of alpha‐granules that undergo differential release during activation. We consider the implications of this new paradigm of platelet secretion, discuss mechanisms of alpha‐granule biogenesis, and review the molecular basis of transport and delivery of alpha‐granules to assembling platelets.

Delivering new insight into the biology of megakaryopoiesis and thrombopoiesis.

Purpose of reviewThe aim of this review is to explore the state of the art knowledge on the cell biological and molecular pathways that regulate megakaryopoiesis and lead to platelet production. Recent findingsIn the last 2 years there has been considerable progress in the elucidation of molecular mechanisms of megakaryocyte development and platelet biogenesis, driven by the application of modern molecular biology approaches to these specialized and unique cells. Studies have for the first time visualized endomitotic spindle dynamics, characterized the maturation of the demarcation membrane system, and delineated the mechanics of organelle transport and microtubule assembly in living megakaryocytes. The role of specific molecules in platelet production has been elucidated in greater detail by combining molecular studies with genetically engineered mice as well as embryonic cell culture systems. SummaryThis review integrates the latest studies of megakaryocyte development into the molecular pathways that regulate megakaryopoiesis and thrombopoiesis. Decoding the pathways of megakaryopoiesis and platelet production should help revolutionize the management of thrombocytopenia and other platelet disorders.

Induction of platelet formation from megakaryocytoid cells by nitric oxide

Although the growth factors that regulate megakaryocytopoiesis are well known, the molecular determinants of platelet formation from mature megakaryocytes remain poorly understood. Morphological changes in megakaryocytes associated with platelet formation and removal of senescent megakaryocytes are suggestive of an apoptotic process. Previously, we have established that nitric oxide (NO) can induce apoptosis in megakaryocytoid cell lines. To determine whether there is an association between NO-induced apoptosis and platelet production, we exposed Meg-01 cells to S-nitrosoglutathione (GSNO) with or without thrombopoeitin (TPO) pretreatment and used flow cytometry and electron microscopy to assess platelet-sized particle formation. Meg-01 cells treated with TPO alone produced few platelet-sized particles (<3% of total counts), whereas treatment with GSNO alone produced a significant percentage of platelet-sized particles (22 ± 4% of total counts); when combined with TPO pretreatment, however, GSNO led to a marked increase in platelet-sized particle production (48 ± 3% of total counts). Electron microscopy confirmed that Meg-01 cells treated with TPO and GSNO yielded platelet-sized particles with morphological features specific for platelet forms. The platelet-sized particle population appears to be functional, because addition of calcium, fibrinogen, and thrombin receptor-activating peptide led to aggregation. These results demonstrate that NO facilitates platelet production, thereby establishing the essential role of NO in megakaryocyte development and thrombopoiesis.

Reversal of New Oral Anticoagulants

Although the options for therapeutic modalities such as chemotherapy and antibiotics have flourished, anticoagulant management has remained stagnant since the first agents were developed during World War II. Traditional anticoagulants widely used to treat thromboembolic disease include heparin-based drugs and warfarin. These drugs are cumbersome to administer. They have a narrow therapeutic window to achieve adequate anticoagulation without bleeding, and they exhibit interactions with food and other drugs that led to the requirement for frequent monitoring by laboratory testing. Until recently, few other options remained for antithrombotic therapy. Recently, however, there has been an influx of new anticoagulants poised to enter the market, providing more therapeutic options for patients. They promise to be better than current agents because they achieve similar, if not better, efficacy with less bleeding and fewer interactions with food and drugs and without the need for frequent monitoring. Article see p 1573 These new agents work by targeting specific components of the coagulation cascade, such as thrombin itself or its precursor, factor Xa. Dabigatran etexilate is a direct inhibitor of both free and clot-associated thrombin. It is a prodrug that is converted to its active compound, dabigatran, with a peak plasma concentration achieved 1.5 hours after ingestion, and it has a half-life of 14 to 17 hours. Unlike warfarin, it is not metabolized by cytochrome p450, so there are few drug interactions. It does, however, interact with the efflux transporter P-glycoprotein, leading to concern about alterations in efficacy when used with P-glycoprotein inhibitors or inducers such as rifampin, quinidine, ketoconazole, and verapamil. A number of studies have proved its efficacy as an anticoagulant for the prevention and treatment of venous and arterial thromboembolic events, including prevention after total knee or hip arthroplasty, treatment of acute venous thromboembolic events, and prevention of stroke associated with atrial fibrillation. …

The Role of Thrombophilia in Pregnancy

Thrombotic disease is a major cause of peripartum morbidity and mortality worldwide. Development of thrombosis in pregnancy is multifactorial due to the physiologic changes of pregnancy—which induce a relative hypercoagulable state—as well as physical changes leading to increased stasis and also the effects of both the inherited and the acquired thrombophilias. In this review, we discuss the impact of each of these factors on the development of thrombosis as well as the evidence for the impact of pregnancy-associated thrombosis on pregnancy outcome. We then discuss the use of both prophylactic and therapeutic anticoagulation during pregnancy and the puerperium. We review the indications and dosing recommendations for administration of anticoagulation in a context of discussing the evidence including the lack of evidence and formal guidelines in this area. We briefly address the role of the new oral anticoagulants in pregnancy and conclude that significant further research in women with thrombophilias and pregnancy-associated thrombosis may help clarify the management of this condition in the future.

Thrombophilias in Pregnancy

Thrombophilic conditions are associated with an increased risk of venous thromboembolic events (VTE) during pregnancy. Thrombophilic disorders are either acquired, as in antiphospholipid syndrome, or inherited, as in factor V Leiden. Both are associated with VTE but acquired disorders can also increase the risk of arterial events. However, there is controversy as to whether they may adversely affect other pregnancy outcomes including pregnancy loss, placental abruption, severe preeclampsia, and stillbirth. This article discusses the effect of thrombophilias on pregnancy.

CCL5 derived from platelets increases megakaryocyte proplatelet formation

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.

Tamoxifen Directly Inhibits Platelet Angiogenic Potential and Platelet-Mediated Metastasis

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 antitumor 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 proangiogenic regulator vascular endothelial growth factor. 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, chemokine (C-X-C motif) ligand 1, chemokine (C–C motif) ligand 5, epidermal growth factor, chemokine (C-X-C motif) ligand 5, platelet-derived growth factor dimeric isoform BB, whereas antiangiogenic angiopoietin-1 was elevated. Platelets isolated from patients on tamoxifen maintenance therapy were also found to have decreased activation responses, diminished vascular endothelial growth factor release, and lower angiogenic and metastatic potential. Conclusions— We demonstrate that tamoxifen and its metabolite 4-hydroxytamoxifen directly alter 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.