Douglas B Cines

University of Pennsylvania, Filadelfia, Pennsylvania, United States

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Publications (290)2340.79 Total impact

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    ABSTRACT: Platelet-factor 4 (PF4) has been recently shown to inhibit infection by a broad range of HIV-1 isolates in vitro. We found that the inhibitory effects of PF4 are limited to a defined concentration range where PF4 exists largely in a monomeric state. Under these conditions, PF4 bound the HIV-1 envelope protein and inhibited HIV-1 attachment to the cell surface. However, as concentrations increased to the point where PF4 exists largely in tetrameric or higher-order forms, viral infection in vitro was enhanced. Enhancement could be inhibited by mutations in PF4 that shift the oligomeric equilibrium towards the monomeric state, or by using soluble glycosaminoglycans (GAGs) to which tetrameric PF4 avidly binds. We conclude that at physiologically relevant concentrations, oligomeric PF4 enhances infection by HIV-1 by interacting with the viral envelope protein as well as cell surface GAGs, enhancing virus attachment to the cell surface. This effect was not specific to HIV-1, as enhancement was seen with some but not all other viruses tested. The biphasic effects of PF4 on HIV-1 infection suggest that native PF4 will not be a useful antiviral agent, and that PF4 could contribute to the hematologic abnormalities commonly seen in HIV-infected individuals by enhancing virus infection in the bone marrow.
    No preview · Article · Feb 2016 · AIDS research and human retroviruses
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    Full-text · Dataset · Jan 2016
  • C. H. Villa · V. R. Muzykantov · D. B. Cines
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    ABSTRACT: Red blood cells (RBCs) participate in haemostasis and thrombosis through their biochemical properties, biomechanical and rheological properties, intercellular interactions, assembly of coagulation factors, release of procoagulant signaling compounds, and composition of the fibrin clot. Targeting anti-thrombotic and anti-inflammatory drugs to RBCs provides a new approach to mitigate thrombosis and other intravascular pathologies.
    No preview · Article · Jan 2016 · ISBT Science Series
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    ABSTRACT: The use of fibrinolytic agents to prevent new thrombus formation is limited by an increased risk of bleeding due to lysis of hemostatic clots that prevent hemorrhage in damaged blood vessels. We sought to develop an agent that provides thromboprophylaxis without carrying a significant risk of causing systemic fibrinolysis or disrupting hemostatic clots. We previously showed that platelet (PLT) α granule-delivered urokinase plasminogen activator (uPA) is highly effective in preventing thrombosis, while being associated with little systemic fibrinolysis or bleeding. Here, we generated a chimeric prodrug composed of a single-chain version of the variable region of an anti-αIIbβ3 mAb fused to a thrombin-activatable, low-molecular-weight pro-uPA (PLT/uPA-T). PLT/uPA-T recognizes human αIIbβ3 on both quiescent and activated platelets and is enzymatically activated specifically by thrombin. We found that this prodrug binds tightly to human platelets even after gel filtration, has a prolonged half-life in mice transgenic for human αIIb compared with that of uPA-T, and prevents clot formation in a microfluidic system. Importantly, in two murine injury models, PLT/uPA-T did not lyse preexisting clots, even when administration was delayed by as little as 10 minutes, while it concurrently prevented the development of nascent thrombi. Thus, PLT/uPA-T represents the prototype of a platelet-targeted thromboprophylactic agent that selectively targets nascent over preexisting thrombi.
    Full-text · Article · Dec 2015 · The Journal of clinical investigation
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    ABSTRACT: Platelet-driven blood clot contraction (retraction) is thought to promote wound closure and secure hemostasis while preventing vascular occlusion. Notwithstanding its importance, clot contraction remains a poorly understood process, partially due to the lack of methodology to quantify its dynamics and requirements. We used a novel automated optical analyzer to continuously track in vitro changes in the size of contracting clots in whole blood and in variously reconstituted samples. Kinetics of contraction was complemented with dynamic rheometry to characterize the viscoelasticity of contracting clots. This combined approach enabled investigation of the coordinated mechanistic impact of platelets, including non-muscle myosin II, red blood cells, fibrin(ogen), factor XIIIa, and thrombin on the kinetics and mechanics of the contraction process. Clot contraction is comprised of three sequential phases, each characterized by a distinct rate constant. Thrombin, Ca(2+), the integrin αIIbβ3, myosin IIa, factor XIIIa-crosslinking, and platelet count all promote one or more phases of the clot contraction process. In contrast, RBCs impair contraction and reduce elasticity, while increasing the overall contractile stress generated by the platelet-fibrin meshwork. A better understanding of the mechanisms by which blood cells, fibrin(ogen), and platelet-fibrin interactions modulate clot contraction may generate novel approaches to reveal and to manage thrombosis and hemostatic disorders.
    Full-text · Article · Nov 2015 · Blood
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    ABSTRACT: Approximately one-half of patients who develop clinical atherosclerosis have normal or only modest elevations in plasma lipids, indicating additional mechanisms contribute to pathogenesis. In view of increasing evidence that inflammation contributes to atherogenesis, we studied the effect of human neutrophil α-defensins (α-defs) on low density lipoprotein (LDL) trafficking, metabolism, vascular deposition and atherogenesis using transgenic mice expressing human α-defs in their polymorphonuclear leukocytes (Def+/+). Def+/+ mice develop α-def/LDL complexes that accelerate the clearance of LDL from the circulation accompanied by enhanced vascular deposition and retention of LDL, induction of endothelial cathepsins, increased endothelial permeability to LDL and the development of lipid streaks in the aortic roots when fed a regular diet and at normal plasma levels of LDL. Transplantation of bone marrow from Def+/+ to WT mice increased LDL clearance, increased vascular permeability and increased vascular deposition of LDL, while transplantation of WT bone marrow to Def+/+ mice prevented these outcomes. The same outcome was obtained by treating Def+/+ mice with colchicine to inhibit release of α-defs. These studies identify a potential new link between inflammation and the development of atherosclerosis.
    Preview · Article · Oct 2015 · Journal of Biological Chemistry
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    ABSTRACT: Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via FcγRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcγRIIA, which activates spleen tyrosine kinase (Syk) and leads to the generation of tissue factor and thrombin. The combination of direct platelet activation by HIT immune complexes through FcγRIIA and transactivation by monocyte-derived thrombin markedly increases annexin and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcγRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk.
    No preview · Article · Oct 2015 · Blood
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    ABSTRACT: Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by immune complexes containing platelet factor 4 (PF4), antibodies to PF4 and heparin or cellular glycosaminoglycans (GAGs). Here we solve the crystal structures of the: (1) PF4 tetramer/fondaparinux complex, (2) PF4 tetramer/KKO-Fab complex (a murine monoclonal HIT-like antibody) and (3) PF4 monomer/RTO-Fab complex (a non-HIT anti-PF4 monoclonal antibody). Fondaparinux binds to the 'closed' end of the PF4 tetramer and stabilizes its conformation. This interaction in turn stabilizes the epitope for KKO on the 'open' end of the tetramer. Fondaparinux and KKO thereby collaborate to 'stabilize' the ternary pathogenic immune complex. Binding of RTO to PF4 monomers prevents PF4 tetramerization and inhibits KKO and human HIT IgG-induced platelet activation and platelet aggregation in vitro, and thrombus progression in vivo. The atomic structures provide a basis to develop new diagnostics and non-anticoagulant therapeutics for HIT.
    Preview · Article · Sep 2015 · Nature Communications

  • No preview · Article · Aug 2015 · Cancer Research
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    ABSTRACT: A safety analysis of pooled data from clinical studies of romiplostim, a thrombopoietin (TPO) receptor agonist, in which patients with immune thrombocytopaenia (ITP) received romiplostim, placebo, or medical standard of care (SOC) Rodeghiero et al. (Eur J Haematol 91:423-436, 2013), has been updated. Included are data from 14 trials spanning 2002-2011; placebo- and SOC-arm data are pooled. Most patients (n = 1059) were female (61 %) and Caucasian (85 %); 38 % had undergone splenectomy; 23 were children. Mean (SD) baseline platelet count was 20.6 (16.5) × 10(9)/L. Mean (SD) weekly dose of romiplostim was 4.2 (2.8) µg/kg; total exposure was 1520 patient-years. Overall, 921 patients received romiplostim only, 65 received placebo/SOC only, and 73 received placebo/SOC followed by romiplostim. Rates of haemorrhage (romiplostim, 205/100 patient-years; placebo/SOC, 263/100), thrombosis (both, 5.5/100 patient-years), haematological malignancy/myelodysplastic syndrome (romiplostim, 0.5/100 patient-years; placebo/SOC, 2.7/100), and non-haematological tumours (romiplostim, 2.2/100 patient-years; placebo/SOC, 3.6/100) were comparable among groups. Bone marrow reticulin was reported in 17 patients and collagen in one patient receiving romiplostim; one patient receiving placebo/SOC had reticulin reported. Three patients developed neutralizing antibodies to romiplostim, but not to endogenous TPO. This integrated analysis of the safety profile of romiplostim in patients with ITP is consistent with previously reported studies; no new safety concerns emerged.
    No preview · Article · Jul 2015 · International journal of hematology
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    ABSTRACT: For several decades, researchers have used erythrocytes for drug delivery of a wide variety of therapeutics in order to improve their pharmacokinetics, biodistribution, controlled release and pharmacodynamics. Approaches include encapsulation of drugs within erythrocytes, as well as coupling of drugs onto the red cell surface. This review focuses on the latter approach, and examines the delivery of red blood cell (RBC)-surface-bound anti-inflammatory, anti-thrombotic and anti-microbial agents, as well as RBC carriage of nanoparticles. Herein, we discuss the progress that has been made in surface loading approaches, and address in depth the issues relevant to surface loading of RBC, including intrinsic features of erythrocyte membranes, immune considerations, potential surface targets and techniques for the production of affinity ligands.
    No preview · Article · Jul 2015 · Therapeutic delivery
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    ABSTRACT: Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies to a self-antigen, platelet factor (4) and heparin. The reasons why antibodies form to PF4/heparin, but not to PF4 bound to other cellular glycosaminoglycans are poorly understood. To investigate differences in cellular responses to cell-bound PF4 and PF4/heparin complexes, we studied the internalization of each by peripheral blood-derived monocytes, dendritic cells and neutrophils. Using unlabeled, fluorescently-labeled antigen and/or labeled monoclonal antibody to PF4/heparin complexes (KKO), we show that PF4/heparin complexes are taken up by monocytes in a heparin-dependent manner and are internalized by human monocytes and dendritic cells, but not by neutrophils. Complexes of PF4/low-molecular weight heparin and complexes composed of heparin and murine PF4, protamine, or lysozyme are internalized similarly, suggesting a common endocytic pathway. Uptake of complexes is mediated by macropinocytosis, as shown by inhibition using cytochalasin D and amiloride. Internalized complexes are transported intact to late endosomes, as indicated by co-staining of vesicles with KKO and lysosomal associated membrane protein-2 (LAMP-2). Lastly, we show cellular uptake is accompanied by expression of MHCII and CD83 co-stimulatory molecules. Taken together, these studies establish a distinct role for heparin in enhancing antigen uptake and activation of the initial steps in the cellular immune response to PF4-containing complexes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    No preview · Article · May 2015 · Journal of Thrombosis and Haemostasis
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    ABSTRACT: ADAMTS13 metalloprotease cleaves von Willebrand factor (VWF), thereby inhibiting platelet aggregation and arterial thrombosis. An inability to cleave ultra large VWF resulting from hereditary or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma exchange is the most effective initial therapy for TTP to date. Here, we report characterization of transgenic mice expressing recombinant human ADAMTS13 (rADAMTS13) in platelets and its efficacy in inhibiting arterial thrombosis and preventing hereditary and acquired antibody-mediated TTP in murine models. Western blotting and fluorescent resonance energy transfer (FRETS) assay detect full-length rADAMTS13 protein and its proteolytic activity, respectively, in transgenic (Adamts13(-/-)Plt(A13)), but not in wild type (WT) and Adamts13(-/-) (KO) platelets. The expressed rADAMTS13 is released upon stimulation with thrombin and collagen, but less with 2MesADP. Platelet-delivered rADAMTS13 is able to inhibit arterial thrombosis after vascular injury and prevent the onset and progression of Shigatoxin-2 or recombinant murine VWF (rmVWF)-induced TTP syndrome in mice despite lack of plasma ADAMTS13 activity resulting from the ADAMTS13 gene deletion or the antibody-mediated inhibition of plasma ADAMTS13 activity. These findings provide a proof-of-concept that platelet-delivered ADAMTS13 may be explored as a novel treatment for arterial thrombotic disorders including hereditary and acquired TTP in the presence of anti-ADAMTS13 autoantibodies. Copyright © 2015 American Society of Hematology.
    No preview · Article · Mar 2015 · Blood
  • Adam Cuker · Eline T Luning Prak · Douglas B Cines
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    ABSTRACT: Primary immune thrombocytopenia (ITP) in adults often assumes a chronic course that requires persistent monitoring and treatment. Medical therapy has traditionally been viewed as a means of temporarily raising the platelet count with little or no potential to induce long-term platelet responses off treatment. However, several recent studies have tested the hypothesis that intensive medical therapy administered early in the disease course may ameliorate or even cure ITP. In this review, we propose a biological rationale for medical intervention that simultaneously targets the innate and adaptive immune responses administered early in the course of disease. We also critically examine data on long-term outcomes after single-agent and multi-agent medical therapy. Intensive regimens that target inflammation and adaptive immunity (e.g., combination high-dose dexamethasone and rituximab) appear to improve response rates at 6 to 12 months compared with standard first-line therapy (e.g., prednisone, high-dose dexamethasone alone) in newly diagnosed patients. Controlled trials with extended follow-up are needed to determine whether these intensive regimens induce more cures compared with standard treatment or merely delay relapse at the expense of potentially greater toxicity. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.
    No preview · Article · Mar 2015 · Seminars in Thrombosis and Hemostasis
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    ABSTRACT: Persistent intracranial hemorrhage (ICH) is a major cause of death and disability after traumatic brain injury (TBI) for which no medical treatment is available. Delayed bleeding is often ascribed to consumptive coagulopathy initiated by exposed brain tissue factor. We examined an alternative hypothesis, namely that marked release of tissue type plasminogen activator (tPA) followed by delayed synthesis and release of urokinase plasminogen activator (uPA) from injured brain leads to post-traumatic bleeding by causing premature clot lysis. Using a murine model of severe TBI, we found ICH is reduced in tPA(-/-) and uPA(-/-) mice but increased in PAI(-/-) mice compared to wild-type (WT) mice. tPA(-/-), but not uPA(-/-), mice developed a systemic coagulopathy post-TBI. Tranexamic acid inhibited ICH expansion in uPA(-/-), but not in tPA(-/-) mice. Catalytically inactive tPA-S(481)A inhibited plasminogen activation by tPA and uPA, attenuated ICH, lowered plasma D-dimers, lessened thrombocytopenia and improved neurological outcome in WT, tPA(-/-) and uPA(-/-) mice. ICH expansion was also inhibited by tPA-S(481)A in WT mice anticoagulated with warfarin. These data demonstrate that protracted endogenous fibrinolysis induced by TBI is primarily responsible for persistent ICH and post-TBI coagulopathy in this model and offer a novel approach to interrupt bleeding. Copyright © 2015 American Society of Hematology.
    Preview · Article · Feb 2015 · Blood
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    ABSTRACT: Background: Existing approaches for measuring hemostasis parameters require multiple platforms, can take hours to provide results, and generally require 1-25 mL of sample. We developed a diagnostic platform that allows comprehensive assessment of hemostatic parameters on a single instrument and provides results within 15 min using 0.04 mL of blood with minimal sample handling. Methods: T2 magnetic resonance (T2MR) was used to directly measure integrated reactions in whole blood samples by resolving multiple water relaxation times from distinct sample microenvironments. Clotting, clot contraction, and fibrinolysis stimulated by thrombin or tissue plasminogen activator, respectively, were measured. T2MR signals of clotting samples were compared with images produced by scanning electron microscopy and with standard reference methods for the following parameters: hematocrit, prothrombin time, clot strength, and platelet activity. Results: Application of T2MR methodology revealed conditions under which a unique T2MR signature appeared that corresponded with the formation of polyhedral erythrocytes, the dynamics and morphology of which are dependent on thrombin, fibrinogen, hematocrit, and platelet levels. We also showed that the T2MR platform can be used for precise and accurate measurements of hematocrit (%CV, 4.8%, R(2) = 0.95), clotting time (%CV, 3.5%, R(2) = 0.94), clot strength (R(2) = 0.95), and platelet function (93% agreement with light transmission aggregometry). Conclusions: This proof-of-concept study demonstrates that T2MR has the potential to provide rapid and sensitive identification of patients at risk for thrombosis or bleeding and to identify new biomarkers and therapeutic targets with a single, simple-to-employ analytic approach that may be suitable for routine use in both research and diverse clinical settings.
    Preview · Article · Jun 2014 · Clinical Chemistry
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    Full-text · Article · Jun 2014 · American Journal of Hematology
  • Douglas B Cines · Adam Cuker · John W Semple
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    ABSTRACT: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by antibody-mediated platelet destruction. The platelet, as an accessible target, has made ITP an attractive disorder in the study of autoimmunity. However, the pathogenesis of ITP has proven complex with diverse pre-existing challenges to the immune system in the form of infection, genetic predisposition, underlying autoimmune repertoire, inhibition of platelet production, perturbations of cell mediated affector and effector pathways, sequestered harbors within lymphoid organs, and responsiveness to intervention. This chapter surveys key new insights into the pathogenesis of ITP and attempts to integrate them into a model that may serve as a template for future investigation.
    No preview · Article · Mar 2014 · La Presse Médicale

  • No preview · Article · Mar 2014
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    ABSTRACT: Contraction of blood clots is necessary for hemostasis, wound healing and to restore flow past obstructive thrombi, but little has been known about the structure of contracted clots or the role of erythrocytes in contraction. We found that contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral erythrocytes within. The same results were obtained after initiation of clotting with various activators and also with clots from reconstituted human blood and mouse blood. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, were also observed in human arterial thrombi taken from patients. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a tightly packed array. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and help explain how fibrinolysis is greatly retarded as clots contract.
    Full-text · Article · Dec 2013 · Blood

Publication Stats

14k Citations
2,340.79 Total Impact Points

Institutions

  • 1984-2015
    • University of Pennsylvania
      • • Department of Pathology and Laboratory Medicine
      • • Department of Pathology
      • • Division of Hematology/Oncology
      • • Department of Medicine
      Filadelfia, Pennsylvania, United States
  • 2007-2013
    • William Penn University
      Filadelfia, Pennsylvania, United States
    • University of California, Santa Barbara
      • Department of Chemical Engineering
      Santa Barbara, California, United States
    • University of Illinois at Chicago
      Chicago, Illinois, United States
  • 2002-2013
    • University of Toronto
      • • Department of Laboratory Medicine and Pathobiology
      • • Department of Paediatrics
      Toronto, Ontario, Canada
    • University of New Mexico
      Albuquerque, New Mexico, United States
    • University of Maryland, Baltimore
      Baltimore, Maryland, United States
  • 2012
    • Weill Cornell Medical College
      New York, New York, United States
  • 1983-2008
    • Hospital of the University of Pennsylvania
      • • Department of Pathology and Laboratory Medicine
      • • Department of Obstetrics and Gynecology
      • • Division of Hematology/Oncology
      Philadelphia, PA, United States
  • 2006
    • Harvard University
      Cambridge, Massachusetts, United States
    • University of Texas Health Science Center at Tyler
      Tyler, Texas, United States
  • 1998-2006
    • The Children's Hospital of Philadelphia
      • Division of Hematology
      Philadelphia, Pennsylvania, United States
  • 2004
    • University of Pittsburgh
      Pittsburgh, Pennsylvania, United States
    • Justus-Liebig-Universität Gießen
      • Department of Internal Medicine
      Gieben, Hesse, Germany
  • 2002-2003
    • Louisiana State University in Shreveport
      Shreveport, Louisiana, United States
  • 1989-2003
    • The Scripps Research Institute
      • Department of Cell and Molecular Biology
      La Jolla, CA, United States
  • 2001
    • Case Western Reserve University
      • Division of Hematology and Oncology
      Cleveland, OH, United States
  • 2000
    • McGill University
      Montréal, Quebec, Canada
  • 1999
    • Tel Aviv Sourasky Medical Center
      • Hematology
      Tell Afif, Tel Aviv, Israel
  • 1995-1998
    • University of Michigan
      • Division of Hematology and Oncology
      Ann Arbor, Michigan, United States
  • 1997
    • University of California, Los Angeles
      Los Angeles, California, United States
    • Tel Aviv University
      • Sackler Faculty of Medicine
      Tell Afif, Tel Aviv, Israel
  • 1988-1994
    • Temple University
      • Department of Medicine
      Filadelfia, Pennsylvania, United States
  • 1991
    • Cornell University
      • Department of Obstetrics and Gynecology
      Итак, New York, United States