Ranaviruses such as frog virus 3 ([FV3]family Iridoviridae) are increasingly prevalent pathogens that infect reptiles, amphibians, and fish worldwide. Whereas studies in the frog Xenopus laevis have revealed the critical involvement of CD8 T-cell and antibody responses in host resistance to FV3, little is known about the role played by innate immunity to infection with this virus. We have investigated the occurrence, composition, activation status, and permissiveness to infection of peritoneal leukocytes (PLs) in Xenopus adults during FV3 infection by microscopy, flow cytometry, and reverse transcription-PCR. The total number of PLs and the relative fraction of activated mononucleated macrophage-like cells significantly increase as early as 1 day postinfection (dpi), followed by NK cells at 3 dpi, before the peak of the T-cell response at 6 dpi. FV3 infection also induces a rapid upregulation of proinflammatory genes including arginase 1, interleukin-1␤, and tumor necrosis factor alpha. Although PLs are susceptible to FV3 infection, as evidenced by apoptotic cells, active FV3 transcription, and the detection of viral particles by electron microscopy, the infection is weaker (fewer infectious particles), more transitory, and involves a smaller fraction (less than 1%) of PLs than the kidney, the main site of infection. However, viral DNA remains detectable in PLs for at least 3 weeks postinfection, past the point of viral clearance observed in the kidneys. This suggests that although PLs are actively involved in anti-FV3 immune responses, some of these cells can be permissive and harbor quiescent, asymptomatic FV3.

Objective-To investigate the presence and role of NF-B proteins in megakaryocytes and platelets. The nuclear factor-B (NF-B) transcription factor family is well known for its role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-B family members, including the regulatory inhibitor-B (I-B) and I-kinase (

Thrombocytopenia is a critical problem that occurs in many hematologic diseases, as well as after cancer therapy and radiation exposure. Platelet transfusion is the most commonly used therapy but has limitations of alloimmunization, availability, and expense. Thus, the development of safe, small, molecules to enhance platelet production would be advantageous for the treatment of thrombocytopenia. Herein, we report that an important lipid mediator and a peroxisome

Hemostasis is dependent upon the successful recruitment and activation of blood platelets to the site of a breach in the vasculature. Platelet activation stimulates the rapid reorganization of the cortical actin cytoskeleton, resulting in the transformation of platelets from biconcave disks to fully spread cells. During this process, platelets extend filopodia and generate lamellipodia, resulting in a dramatic increase in the platelet surface area. Kohler-illuminated Nomarski Differential Interference Contrast microscopy has proved an effective tool to characterize platelet morphological changes in real time, and provides a useful tool to identify genetic and pharmacological regulators of platelet function.

Objective-Factor XI (FXI) promotes hemostasis and thrombosis through enhancement of thrombin generation and has been shown to play a critical role in the formation of occlusive thrombi in arterial injury models. The aim of this study was to investigate the mechanisms governing interactions between FXI and platelets. 2 Inherited FXI deficiency causes a mild bleeding diathesis and is protective against ischemic stroke, 3,4 whereas an elevated FXI plasma level is an independent risk factor for thrombotic diseases such as deep vein thrombosis. 5 Consistent with these observations, FXI plays a critical role in experimental thrombus growth in rabbits, mice, and primates. 6 -10 See accompanying article on page 1409 FXI circulates as a disulfide-linked homodimer in complex with plasma high molecular weight kininogen (HK). FXI shares high sequence homology (58% amino acid identity) with the functionally-distinct plasma protein prekallikrein, which also circulates in complex with HK. 11,12 Although the serine protease domain of each FXI subunit is similar to catalytic domains for other coagulation proteases, the noncatalytic portion contains 4 apple domains (A1 to A4), a feature shared only with prekallikrein. 12,13 The FXI A3 domain has been shown to contain binding sites for FIX and for the platelet receptor glycoprotein Ib-IX-V (GPIb). 14,15 FXI binding has been localized to the leucine-rich repeat (LRR) sequences on the NH 2 -terminal globular domain of GPIb␣, at a site distinct from the anionic thrombin-binding domain of GPIb␣. 16,17 It is unknown whether FXI-platelet binding is solely mediated by GPIb␣ or whether other platelet receptor(s) exist that can support interactions with FXI.GPIb␣ has been shown to form a complex on the platelet surface with apolipoprotein E receptor 2 (ApoER2, LRP8), 18 -20 a member of the low-density lipoprotein (LDL) family of receptors. ApoER2 initiates intracellular signaling through the adaptor protein disabled-1 (Dab-1) in platelets. 21 The extracellular domain of ApoER2 consists of 3 regions: (1) the type A-binding repeats of LDL-binding domains displaying a negatively-charged surface, which are responsible for receptor-ligand interactions; (2) type B repeats, which are homologous to regions in the epidermal growth factor precursor; and (3) the protein stack of O-linked sugar domains that separate the LDL-binding domains from the cellular surface. We have recently shown that platelet and leukocyte ApoER2 mediate interactions with the anticoagulant serine protease, activated protein C. 22,23 Here, we present the first evidence that identifies FXI as a ligand for ApoER2.

No abstract

Factor Va serves as the nonenzymatic protein cofactor for the prothrombinase complex, which converts prothrombin to thrombin in the events leading to formation of a hemostatic plug. Several observations support the concept that platelet-derived factor V/Va is physically and functionally distinct and plays a more important role in thrombin generation at sites of vascular injury as compared to its plasma counterpart. Platelet-derived factor V/Va is generated following endocytosis of the plasma-derived molecule by the platelet precursor cells, megakaryocytes, via a two receptor system consisting of low density lipoprotein (LDL) receptor-related protein-1 (LRP-1) and an unidentified specific "binding site". More recently, it was suggested that a cell surface-expressed β-galactoside binding protein, galectin-8, was involved in factor V endocytosis. Endocytosed factor V is trafficked through the cell and retailored prior to its storage in α-granules. Given the essential role of platelet-derived factor Va in clot formation, understanding the cellular and molecular mechanisms that regulate how platelets acquire this molecule will be important for the treatment of excessive bleeding or clotting.

Plasma- and platelet-derived factor Va are essential for thrombin generation catalyzed by the prothrombinase complex; however, several observations demonstrate that the platelet-derived cofactor, which is formed following megakaryocyte endocytosis and modification of the plasma procofactor, factor V, is more hemostatically relevant. Factor V endocytosis, as a function of megakaryocyte differentiation and proplatelet formation, was assessed by flow cytometry and microscopy in CD34 hematopoietic progenitor cells isolated from human umbilical cord blood and cultured for 12 days in the presence of cytokines to induce ex vivo differentiation into megakaryocytes. Expression of an early marker of megakaryocyte differentiation, CD41, endocytosis of factor V, and the percentage of CD41 cells that endocytosed factor V increased from days 6 to 12 of differentiation. In contrast, statistically significant decreases in expression of the stem cell marker, CD34, and in the percentage of CD34 cells that endocytosed factor V were observed. A statistically significant increase in the expression of CD42b, a late marker of megakaryocyte differentiation, was also observed over time, such that by Day 12, all CD42b cells endocytosed factor V and expressed CD41. This endocytosed factor V was trafficked to proplatelet extensions and was localized in a punctate pattern in the cytoplasm consistent with its storage in α-granules. In conclusion, loss of CD34 and expression of CD42b define cells capable of factor V endocytosis and trafficking to proplatelet extensions during differentiation of megakaryocytes ex vivo from progenitor cells isolated from umbilical cord blood.