Scientific Report 2007

Molecular and Experimental Medicine

Division of Blood Cell and Vascular Biology

Platelets in Hemostasis, Thrombosis, and Host Defense Mechanisms

Z.M. Ruggeri, F. Almus-Jacobs, R, Habermann, Y. Kamikubo, P. Marchese, R. McClintock, J. Orje, G.M. Podda, J. Roberts, B. Savage, A. Zampolli

Mammalian platelets, small anucleated cells with a diameter of 1–2 μm, are released from bone marrow megakaryocytes into circulating blood. Platelets play a key role in controlling bleeding from wounds but, in the context of degenerative vascular disease, may cause arterial thrombosis and life-threatening conditions such as myocardial infraction and stroke. In other vertebrates, the functions typical of mammalian platelets are carried out by thrombocytes, "white cells" similar in morphology to large lymphocytes, that have a diameter of 10–20 μm. The evolutionary reasons for the transition from thrombocytes to platelets are not known. Thrombocytes not only are involved in controlling bleeding but also have other activities typical of leukocytes, such as phagocytosis and production of inflammatory mediators and cytokines. Mammalian platelets have retained many activities relevant to host defense mechanisms, and their role in inflammation is the topic of continued research efforts.

During the past 20 years, my colleagues and I in the Division of Blood Cell and Vascular Biology have focused on understanding key mechanisms that explain the initiation and regulation of platelet aggregation and clotting in response to vascular injury. Our results have contributed to major advances in understanding the origin of bleeding disorders and the pathogenesis of arterial and venous thrombosis, which together are the leading cause of disease and death in developed countries.

Currently, in collaboration with W. Ruf, Department of Immunology, we are defining the mechanisms responsible for the regulated integration of platelet adhesion and aggregation that occur in activation of the coagulation system and fibrin deposition during thrombus formation. The clots that occlude coronary and cerebral arteries are the result of a fundamental defense process—arresting hemorrhage—that has gone astray for loss of regulation at many potential checkpoints; these clots essentially reflect excessive function of both platelets and coagulation. Our goal is to understand all the interactions that occur in flowing blood exposed to an altered vascular surface that lead to the unregulated deposition of platelet and fibrin clots.

In other studies, we are collaborating with L.G. Guidotti, Department of Molecular and Experimental Medicine, to develop his initial hypothesis that platelets play a key role in immune-mediated processes. Using original mouse models of acute viral hepatitis that he had developed with F.V. Chisari, Department of Molecular and Experimental Medicine, Dr. Guidotti found that platelet depletion reduces the accumulation of virus-specific cytotoxic T lymphocytes (CTLs) in the liver and, consequently, organ damage. This animal model mimics what happens in humans affected by viral hepatitis, when T lymphocytes that recognize viral antigens expressed on liver cells become responsible for the process that replaces functioning liver with scar tissue.

In a first study, we found that transfusion of normal but not activation-blocked platelets to platelet-depleted mice restored accumulation of T lymphocytes and severity of disease. Because it was apparent that platelets are not required for the normal antigen recognition and killing functions of CTLs, these findings suggested that platelets play an essential role in directing lymphocytes to the sites of viral accumulation and inflammation. We verified the validity of this hypothesis with additional studies that have confirmed a more general function of platelets in viral clearance.

We found that in mice infected with different isolates of lymphocytic choriomeningitis virus, a mild hemorrhagic anemia develops, which becomes severe and eventually lethal in animals depleted of platelets or lacking integrin β₃. Lethal hemorrhagic anemia is mediated by virus-induced IFN-α/β that causes platelet dysfunction, mucocutaneous blood loss, and suppression of erythropoiesis. In addition to the life-threatening hemorrhagic anemia, platelet-depleted mice do not mount an efficient CTL response and clear the virus. Transfusion of functional platelets into these animals reduces hemorrhage, prevents death, and restores CTL-induced viral clearance in a manner partially dependent on CD40 ligand. These results indicate that upon activation, platelets expressing integrin β₃ and CD40 ligand are required to protect the host against the induction of a lethal hemorrhagic diathesis dependent on IFN-α/β and to clear lymphocytic choriomeningitis virus infection through CTLs.

Our specific interest in this project stems from the observation that platelet activation is required to direct T lymphocytes to their target sites. This requirement implies that platelets must be able to recognize specifically the occurrence of virus-induced pathogenic processes, presumably through markers of inflammation directly or indirectly presented onto vascular surfaces, and in turn signal the location to T lymphocytes for arrest and extravasation. Once these mechanisms are defined in detail, it may be possible to use drugs that modulate platelet function to influence the course of viral infection and immunopathogenic processes mediated by T lymphocytes.

Publications

Bergmeier, W., Piffath, C.L., Goerge, T., Cifuni, S.M., Ruggeri, Z.M., Ware, J., Wagner, D.D. The role of platelet adhesion receptor GPIbα far exceeds that of its main ligand von Willebrand factor in arterial thrombosis. Proc. Natl. Acad. Sci. U. S. A. 103:16900, 2006.

Donadelli, R., Banterla, F., Galbusera, M., Capoferri, C., Bucchioni, S., Gastoldi, S., Nosari, S., Monteferrante, G., Ruggeri, Z.M., Bresin, E., Scheiflinger, F., Rossi, E., Martinez, C., Coppo, R., Remuzzi, G., Noris, M.; the International Registry of Recurrent and Familial HUS/TTP. In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura. Thromb Haemost. 96:454, 2006.

Kasirer-Friede, A., Moran, B., Nagrampa-Orje, J., Swanson, K., Ruggeri, Z.M., Schraven, B., Neel, B.G., Koretzky, G., Shattil, S.J. ADAP is required for normal α_IIbβ₃ activation by VWF/GP Ib-IX-V and other agonists. Blood 109:1018, 2007.

Kisucka J., Butterfield, C.E., Duda, D.G., Eichenberger, S.C., Saffaripour, S., Ware, J., Ruggeri, Z.M., Jain, R.K., Folkman, J., Wagner, D.D. Platelets and platelet adhesion support angiogenesis while preventing excessive hemorrhage. Proc. Natl. Acad. Sci. U. S. A. 103:855, 2006.

Konstantinides, S., Ware, J., Marchese, P., Almus-Jacobs, F., Loskutoff, D., Ruggeri, Z.M. Distinct antithrombotic consequences of platelet glycoprotein Ibα and VI deficiency in a mouse model of arterial thrombosis. J. Thromb. Haemost. 4:2014, 2006.

Reininger, A.J., Heijnen, H.F.G., Schumann, H., Specht, H.M., Schramm, W., Ruggeri, Z.M. Mechanism of platelet adhesion to von Willebrand factor and microparticle formation under high shear stress. Blood 107:3537, 2006.

Ruggeri, Z.M. Platelet interactions with vessel wall components during thrombogenesis. Blood Cells Mol. Dis. 36:145, 2006.

Ruggeri, Z.M., Orje, J.N., Habermann, R., Federici, A.B., Reininger, A.J. Activation-independent platelet adhesion and aggregation under elevated shear stress. Blood 108:1903, 2006.

Sadler, J.E, Budde, U., Eikenboom, J.C., et al. Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J. Thromb. Haemost. 4:2103, 2006.

Savage, B., Ruggeri, Z.M. Platelet thrombus formation in flowing blood. In: Platelets, 2nd ed. Michelson, A.D. (Ed.). Academic Press, San Diego, 2006, p. 359.