Samuel M. Silver, MD, PhD, FACP, University of Michigan, Ann Arbor, MI

Eric F. Grabowski, MD, ScD, Harvard Medical School – Massachusetts General Hospital, Boston, MA

Nigel S. Key, MD, University of North Carolina at Chapel Hill, Chapel Hill, NC

Joel Moake, MD, Baylor College of Medicine, Texas Medical Center, Houston, TX
Introduction of the Recipient of the Mary Rodes Gibson Memorial Award in Hemostasis and Thrombosis (a trainee with the highest scoring abstract at the ASH annual meeting submitted in the field of hemostasis and thrombosis at the ASH annual meeting)

Benjamin T. Kile, PhD, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
An Intrinsic Cell Death Program Determines Platelet Life Span

Barring their consumption in hemostatic processes, platelets exhibit a circulating lifespan in humans of ~10 days. We have recently shown that the mechanism underlying this finite existence is an intrinsic cell death program. Platelets require the Bcl-2 family protein, Bcl-x_L, for survival. Bcl-x_L functions to restrain the activity of the pro-death regulators, Bak and Bax. Genetic deletion of the gene encoding Bcl-x_L or pharmacological inhibition of the protein with the BH3 mimetic ABT-737 cause thrombocytopenia via dose-dependent reductions in platelet lifespan. Conversely, genetic deletion of Bak and Bax extends lifespan and renders platelets entirely refractory to the effects of ABT-737. Thus, the balance between pro-survival Bcl-x_L and pro-death Bak and Bax maintains platelet viability; initiation of apoptosis circumscribes an individual platelet's existence. Attention is now focused on two major questions. The first of these is how the death program is regulated. Our current model is that the Bcl-x_L:Bak pathway functions as a "molecular clock." In this scenario, the amount of Bcl-x_L a platelet inherits determines its lifespan. In the absence of significant new protein synthesis, platelets are unable to counter an inexorable decline in Bcl-x_L relative to Bak. Eventually, a threshold is reached, upon which pro-apoptotic Bak is freed and platelet apoptosis is induced. This is supported by evidence that Bcl-x_L has a shorter half life than Bak and that ABT-737 preferentially targets older platelets (presumably containing less Bcl-x_L). Current efforts are directed at testing the veracity of the model, and examining the possible existence of signals promoting platelet survival. The second major question is whether the Bcl-x_L:Bak pathway is required for aspects of platelet function other than death. One possibility is that it operates as a discrete switch, dormant until triggered at the end of a platelet's useful life, and playing no other functional role. Alternatively, the pathway may be activated during hemostasis. Supporting this latter proposition are the plethora of platelet functional responses that resemble apoptosis. We are now untangling the role of Bak/Bax in these responses and evaluating the potential utility of Bak/Bax inhibition as a means to extend platelet life span and viability during storage prior to transfusion.

Bone-marrow-derived cells contribute to physiological and pathological vascular remodeling throughout ontogenesis and adult life. During tissue regeneration and tumor growth, hemangiogenic cells of hematopoietic and endothelial lineage are mobilized from the bone marrow and home to neo-angiogenic niches in the periphery, acting in synergy with mature hematopoietic cells, growth factors, cytokines, and chemokines. Plasma elevation of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), stromal-derived factor (SDF)-1α, and stem cell factor (Kit-ligand), induce mobilization of VEGF receptor-2 (VEGFR2)⁺ endothelial progenitor cells (EPCs) and VEGFR1⁺ CXCR4⁺ hematopoietic progenitor cells (HPCs). At the neo-angiogenic niche, VEGFR1⁺CXCR4⁺ cells, or hemangiocytes, stabilize newly forming vessels, promoting the incorporation of VEGFR2-expressing EPCs. While VEGFR1⁺CXCR4⁺ cells provide peri-vascular support, platelets, as the primary storage and delivery vehicles for pro- and anti-angiogenic growth factors, including VEGF-A, angiostatin and thrombospondin (TSP), are also key contributors to new vessel growth. Site-specific deployment of stromal-derived factor (SDF)-1α by platelets may orchestrate the migration patterns of CXCR4⁺ hemangiogenic cells, directing the local angiogenic stimulus within an organ. Key factors, such as matrix metalloproteinase-9 (MMP-9), enable the mobilization of hemangiogenic cells from the bone marrow and mediate the elaboration of SDF-1α from platelets. Moreover, current evidence implies that VEGFR1⁺CXCR4⁺ hemangiocytes and platelets contribute instructively and structurally to the vascularization of tumors and ischemic tissue recovery. These insights have profound clinical implications; inhibition of VEGFR1, CXCR4, and platelet-deployed SDF-1α may be an effective strategy to block the growth of hemangiogenic-dependent tumors, while the introduction of VEGFR1⁺ CXCR4⁺ hemangiocytes may accelerate revascularization and tissue regeneration.

Co-Authors: Isabelle Petit, PhD, Daniel Rafii, MD, PhD, and David Lyden, MD, PhD, New York Hospital-Cornell Medical Center, New York, NY

Hemostasis is the orchestrated interplay of blood components to prevent blood loss in the case of vessel injury. Thrombosis represents the other side of the coin, a misrouted physiological process occurring on a diseased vessel wall. The hemodynamic process of axial migration, which occurs mainly at arterial shear rates, concentrates the bulk of red cells in the center of the blood vessel thereby leaving a red cell-free and platelet-rich boundary layer adjacent to the vessel wall. Platelets moving in that plasma layer constantly scan the endothelial surface for defects, which – when detected – are immediately sealed to close the leak. This process termed primary hemostasis can be divided into three phases. The first step is initial platelet arrest and adhesion: the platelets adhere to a thrombogenic surface, even from fast-flowing blood via the glycoprotein (GP) Ibα receptor, which requires no prior activation and binds to von Willebrand factor (VWF). Other proteins like fibronectin can substitute for VWF to a certain extent. Following outside-in and inside-out signalling, other receptors are activated, i.e., integrin αIIbβ3 (GPIIb/IIIa), integrin α2β1, GP VI, which then engage their respective ligands, namely VWF, fibrinogen, collagen, and other subendothelial matrix proteins. The second step is aggregation: again, flowing platelets first need to arrest via VWF bound to already adherent ones. Then, permanent adhesion is ensured by platelets sticking to each other through fibrinogen molecules bridging integrin αIIbβ3 receptors on adjacent platelets, respectively. When regular hemostasis is sealing a vessel leak, high shear rates reciprocal to the narrowing of the vessel occur. In order to assure sufficient platelet adhesion, even under such unfavorable flow conditions, an additional mechanism can be operative: reversible platelet aggregates form without prior activation mediated only by GPIb-VWF interaction at shear rates above 15,000 to 20,000 s^-1.¹ This mechanism would ensure the arrest of large numbers of platelets, which subsequently could be activated and incorporated into the growing thrombus. The third step is secretion: during activation the content of platelet granules is released by exocytosis, for example, VWF, fibrinogen, P-selectin, thrombospondin, fibronectin, and ADP. The latter additionally augment and propagate formation of a platelet aggregate exposing large amounts of procoagulant membranes, which accelerate clotting several thousand fold. The final hemostatic plug or thrombus develops by subsequent secondary hemostasis, i.e., the activation of the coagulation cascade and the generation of fibrin, which stabilizes the aggregate against shear stress. Platelet derived microparticles, which are also generated by GPIb-VWF interaction under high shear rates of 10,000 s^-1 and above, exhibit an activated and procoagulant surface and shorten clotting time.² Whether tissue factor plays its crucial role in secondary hemostasis in the blood-borne or the tissue-borne version is still under debate.

1. Ruggeri ZM, Orje JN, Habermann R, Federici AB, Reininger AJ. Activation-independent platelet adhesion and aggregation under elevated shear stress. Blood. 2006;108:1903-10. Epub June 13, 2006

2. Reininger AJ, Heijnen HF, Schumann H, Specht HM, Schramm W, Ruggeri ZM. Mechanism of platelet adhesion to von Willebrand factor and microparticle formation under high shear stress. Blood. 2006;107:3537-45. Epub January 31, 2006

CD36 (platelet glycoprotein IV) is an 88kd transmembrane protein expressed on platelets, megakaryocytes, phagocytic cells, adipocytes, myocytes, capillary endothelium, and specialized epithelia of gut, breast, and eye. On endothelial cells (EC), CD36 functions as a receptor for thrombospondins, mediating their antiangiogenic activity. On fat, muscle, and gut epithelium, it functions to facilitate fatty acid transport, while on phagocytic cells, it mediates recognition and uptake of modified (oxidized) phospholipids, apoptotic cells, and bacterial products. The function of CD36 on platelets has not been well studied. Recent work from our groups has shown that platelet CD36 binds oxidized LDL and specific oxidized phospholipids, and that this interaction led to platelet activation. Importantly, in vivo studies of murine thrombosis showed that CD36 deficiency mitigated the prothrombotic state observed in hyperlipidemic apoE null animals. Using biochemical and pharmacologic approaches, we have begun to define the signaling pathway by which CD36 influences platelet activation, showing a role for both src kinases and specific MAP kinases. Based on our earlier work showing that CD36 on phagocytic cells and endothelial cells can transmit cellular signals and that CD36 binds apoptotic cells via a specific interaction with phosphatidyl serine (PS) and/or oxidized PS, we hypothesized that platelet CD36 might interact with PS exposed on the surface of microparticles (MP) and thereby modulate platelet activation. We isolated MP from cultured human EC and showed by immunofluorescence microscopy and flow cytometry that they bound to resting platelets. Using anti-CD36 monoclonal antibodies and platelets from CD36 null donors, we showed that this interaction was CD36-dependent. Platelet-MP interactions were blocked by treating MP with annexin V to mask exposed PS. Platelets incubated with MP at a 1:9 ratio showed augmented aggregation and activation in response to low doses of ADP. These data suggest a pathogenic link between platelet CD36 and prothrombotic disorders associated with circulating MP, such as inflammation, diabetes, and cancer. To study the role of CD36 in normal platelet function, we studied platelets from CD36 null mice and NakA- human subjects. Aggregation responses to low doses of agonists were blunted in CD36 null platelets, and both mesenteric and carotid thrombosis times in response to low levels of FeCl injury were prolonged in CD36 null mice. In summary, these data suggest a role for CD36 in modulating platelet function and in contributing to pathologic thrombosis in certain disease states.

Co-Authors: Kan Chen, MD, and Keith McCrae, MD, Case Western Reserve University, Cleveland, OH; Maria Febbraio, PhD, Wei Li, MD, PhD, and Eugene Podrez, MD, PhD, Cleveland Clinic, Cleveland, OH; and Arunima Ghosh, MD, Cleveland State University, Cleveland, OH

Tissue factor (TF) is the major cellular activator of the coagulation protease cascade. Under pathologic conditions, TF is expressed by vascular cells, such as monocytes and endothelial cells. TF expression by neutrophils and platelets is controversial. However, recent studies have shown that complement protein C5a induces TF expression in neutrophils. Moreover, platelets were found to contain stored TF pre-mRNA that after activation of the cells is spliced into mature message and translated. At present, the role of platelet TF in thrombosis is unknown. TF is also present in plasma in the form of microparticles, which are small membrane fragments released from activated or apoptotic cells. The contribution of different cell types to the pool of TF-positive microparticles may vary with different diseases. Nevertheless, TF expression within the vasculature is likely to play a major role in thrombosis associated with a variety of diseases, such as cancer, sickle cell disease, antiphospholipid syndrome, and sepsis. Activation of the clotting cascade generates various proteases, including FVIIa, FXa, and thrombin. Thrombin is required for formation of fibrin and activation of platelets. The proteases are also the target of new antithrombotic drugs. Coagulation proteases activate a family of protease-activated receptors (PARs). Thrombin activation of human platelets is mediated by PAR-1, a high affinity receptor, and PAR-4, a low affinity receptor. PAR-1 antagonists are currently in clinical trials for the treatment of thrombosis. Many diseases are associated with both coagulation and inflammation, suggesting that the two processes may be interconnected. Importantly, inhibition of coagulation reduces inflammation in animal models of sepsis. The activation of PARs by coagulation proteases may provide a link between coagulation and inflammation. In support of this notion, in vitro studies have shown that activation of PAR-1 and PAR-2 on endothelial cells induces the expression of inflammatory mediators, such as IL-6 and IL-8. Ongoing studies are analyzing the role of TF, coagulation proteases, and PARs in inflammation in different animal models. These studies may identify novel targets for anti-inflammatory therapy.