Karin Grunebaum Cancer Research Foundation Fellow Profile

Elisabeth Battinelli Boston, MA

Instructor of Medicine, Harvard Medical School

Fellow Profile

Current Site Of Practice: Brigham and Women's Hospital
Hospital Affiliation: Brigham and Women's
Focus of Research: Platelets and Angiogenesis
Fellowship Year: 2012 – 2013
Attended: Harvard Medical School


Nitric oxide induces apoptosis in megakaryocytic cell lines

Cytokines that stimulate inducible nitric oxide (NO) synthase can suppress the growth and differentiation of normal human bone marrow cells, including megakaryocytes. Since NO promotes apoptosis in other cell systems, we chose to study the determinants of apoptosis in megakaryocytic cells. We show that both exogenous and endogenous sources of NO can induce apoptosis in megakaryocytoid cell lines. The megakaryocyte growth factor thrombopoietin suppresses NO-induced apoptosis, whereas treatment with peroxynitrite, a cytotoxic product formed when NO reacts with superoxide, promotes apoptosis. Superoxide inhibitors suppress NO-induced apoptosis, and pretreatment with megakaryocyte growth and maturation factors attenuates NO-induced apoptosis. These data show that NO modulates megakaryocyte apoptosis and suggest that this process may occur in the cytokine-rich marrow milieu to regulate megakaryocyte turnover.

Blood. 2000 Jun 1;95(11):3451-9

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.

Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14458-63

Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles.

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 megakaryocyte-derived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P(-), LAMP-1(-) and expressed full-length 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 microparticles

Blood. 2009 Jan 29;113(5):1112-21. Epub 2008 Sep 18

Norrie disease gene: characterization of deletions and possible function

Positional cloning experiments have resulted recently in the isolation of a candidate gene for Norrie disease (pseudoglioma; NDP), a severe X-linked neurodevelopmental disorder. Here we report the isolation and analysis of human genomic DNA clones encompassing the NDP gene. The gene spans 28 kb and consists of 3 exons, the first of which is entirely contained within the 5' untranslated region. Detailed analysis of genomic deletions in Norrie patients shows that they are heterogeneous, both in size and in position. By PCR analysis, we found that expression of the NDP gene was not confined to the eye or to the brain. An extensive DNA and protein sequence comparison between the human NDP gene and related genes from the database revealed homology with cysteine-rich protein-binding domains of immediate--early genes implicated in the regulation of cell proliferation. We propose that NDP is a molecule related in function to these genes and may be involved in a pathway that regulates neural cell differentiation and proliferation.

Genomics. 1993 May;16(2):533-

A mutation in the Norrie disease gene (NDP) associated with X-linked familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a hereditary disorder characterized by an abnormality of the peripheral retina. Both autosomal dominant (adFEVR) and X-linked (XLFEVR) forms have been described, but the biochemical defect(s) underlying the symptoms are unknown. Molecular analysis of the Norrie gene locus (NDP) in a four generation FEVR family (shown previously to exhibit linkage to the X-chromosome markers DXS228 and MAOA (Xp11.4-p11.3)) reveals a missense mutation in the highly conserved region of the NDP gene, which caused a neutral amino acid substitution (Leu124Phe), was detected in all of the affected males, but not in the unaffected family members, nor in normal controls. The observations suggest that phenotypes of both XLFEVR and Norrie disease can result from mutations in the same gene.

Nat Genet. 1993 Oct;5(2):180-3

Characterization of a mutation within the NDP gene in a family with a manifesting female carrier.

No abstract available

Hum Mol Genet. 1993 Oct;2(10):1727-9.

Characterization and mapping of the mouse NDP (Norrie disease) locus (Ndp).

Norrie disease is a severe X-linked recessive neurological disorder characterized by congenital blindness with progressive loss of hearing. Over half of Norrie patients also manifest different degrees of mental retardation. The gene for Norrie disease (NDP) has recently been cloned and characterized. With the human NDP cDNA, mouse genomic phage libraries were screened for the homolog of the gene. Comparison between mouse and human genomic DNA blots hybridized with the NDP cDNA, as well as analysis of phage clones, shows that the mouse NDP gene is 29 kb in size (28 kb for the human gene). The organization in the two species is very similar. Both have three exons with similar-sized introns and identical exon-intron boundaries between exon 2 and 3. The mouse open reading frame is 393 bp and, like the human coding sequence, is encoded in exons 2 and 3. The absence of six nucleotides in the second mouse exon results in the encoded protein being two amino acids smaller than its human counterpart. The overall homology between the human and mouse NDP protein is 95% and is particularly high (99%) in exon 3, consistent with the apparent functional importance of this region. Analysis of transcription initiation sites suggests the presence of multiple start sites associated with expression of the mouse NDP gene. Pedigree analysis of an interspecific mouse backcross localizes the mouse NDP gene close to Maoa in the conserved segment, which runs from CYBB to PFC in both human and mouse.

Mamm Genome. 1996 Feb;7(2):93-7

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

Circ Res. 1999 Jun 25;84(12):1416-21

Cancer and thrombosis

The risk of venous thromboembolic events (VTE) in patients with cancer is well established. Malignancy screening in patients who present with their first episode of VTE is recommended only if the history or physical findings are suggestive of an underlying problem, however. Thrombotic events remain a significant cause of death in cancer patients and their treatment remains a major challenge in the management of cancer. Low-molecular-weight heparins are safe, effective options for treatment and prophylaxis and may prolong survival in this patient population. It remains to be seen, however, if this treatment will influence cancer outcomes.

Curr Hematol Rep. 2005 Sep;4(5):378-84

Delivering new insight into the biology of megakaryopoiesis and thrombopoiesis

The 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 FINDINGS:In 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.SUMMARY:This 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.

Curr Opin Hematol. 2007 Sep;14(5):419-26.

Selective sorting of alpha-granule proteins

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.

J Thromb Haemost. 2009 Jul;7 Suppl 1:173-6.

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.

Hematol Oncol Clin North Am. 2011 Apr;25(2):323-33

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.

Blood. 2011 Aug 4;118(5):1359-69. Epub 2011 Jun 16

Reversal of new oral anticoagulants

No abstract available

Circulation. 2011 Oct 4;124(14):1508-10

Venous thromboembolism overview

This article gives a general overview of venous thromboembolism (VTE). Pathophysiology, presentation, diagnosis, and initial management of VTE are briefly reviewed. More difficult management problems are reviewed in greater depth, including duration of anticoagulation, treatment of superficial venous thrombosis, and controversies surrounding bridging therapy, with a brief review of currently available new oral anticoagulants.

Hematol Oncol Clin North Am. 2012 Apr;26(2):345-67, ix.