The redistribution of ,8-thromboglobulin (#TG), platelet Factor 4 (PF4), and fibrinogen from the alpha granules of the platelet after stimulation with thrombin was studied by morphologic and immunocytochemical techniques. The use of tannic acid stain and quick-freeze techniques revealed several thrombin-induced morphologic changes. First, the normally discoid platelet became rounder in form, with filopodia, and the granules clustered in its center. The granules then fused with one another and with elements of the surface-connected canalicular system (SCCS) to form large vacuoles in the center of the cell and near the periphery. Neither these vacuoles nor the alpha granules appeared to fuse with the plasma membrane, but the vacuoles were connected to the extracellular space by wide necks, presumably formed by enlargement of the narrow necks connecting the SCCS to the surface of the unstimulated cell. The presence of fibrinogen, #TG, and PF4 in corresponding large intracellular vacuoles and along the platelet plasma membrane after thrombin stimulation was demonstrated by immunocytochemical techniques in saponin-permeabilized and nonpermeabilized platelets. Immunocytochemical labeling of the three proteins on frozen thin sections of thrombin-stimulated platelets confirmed these findings and showed that all three proteins reached the plasma membrane by the same pathway. We conclude that thrombin stimulation of platelets causes at least some of the fibrinogen, #TG, and PF4 stored in their alpha granules to be redistributed to their plasma membranes by way of surface-connected vacuoles formed by fusion of the alpha granules with elements of the SCCS. Platelets are stimulated by a variety of agents, including thrombin, to change in shape, to release several ofthe proteins stored in their granules, and to form aggregates. In vivo, these aggregates may form a hemostatic plug. The secretion of the contents of alpha granules

Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.

Hereditary macrothrombocytopenia and prolonged bleeding times are associated with the recessive mouse pigment dilution gene gunmetal (gm). Other platelet abnormalities include a mild storage pool deficiency and abnormal expression of two low-molecular-weight guanosine triphosphate binding proteins. These studies were designed to further elucidate the cause of the macrothrombocytopenia. The life span of gunmetal mouse platelets was not significantly different from normal. However, rates of platelet synthesis, measured by sulfate incorporation, were decreased to 25% of normal values. Bone marrow transplantation of normal marrow cells corrected the thrombocytopenia. Furthermore, direct morphologic analysis of mature mutant marrow megakaryocytes by transmission electron microscopy showed reductions in the normal cytoplasmic demarcation membrane system, areas of abnormal membrane complexes, and an increased incidence of emperipolesis. Mutant platelets were relatively more heterogeneous in size and contained unusual elongated and striated inclusions. Mutant megakaryocyte numbers were increased threefold to fivefold over normal numbers in marrow and spleen. Thus, the efficiency of platelet production from gunmetal megakaryocytes is reduced by an order of magnitude. Mutant marrow had a greater proportion of 32N and a smaller proportion of 8N megakaryocytes. Collectively, the results indicate that the gunmetal gene acts intrinsically in megakaryocytes and that an abnormality in this gene causes significant qualitative and quantitative effects on platelet production.

Murine megakaryocyte (MK) colonies in soft-agar cultures were immunocytochemically stained with platelet antiserum and an immuno-alkaline phosphatase procedure. Subsequently, cytochemical staining for acetylcholinesterase was used to confirm the specificity of the immunolabelling technique. The correlation of numbers of megakaryocyte colonies enumerated by independent observers was excellent. A comparable platelet antiserum directed against human platelet epitopes was utilized to identify human MK colonies in soft-agar cultures of human bone marrow. Using this method, we determined that the frequency of detectable human MK colonies in our agar culture system was maximal between days 10 and 12. The immunocytochemical staining technique we have developed for identification of MK colonies in soft-agar cultures yielded good cellular morphology and produced an intensely specific label against a clear background; it therefore facilitated accurate enumeration of MK colonies. This non-fluorescent method avoids dependence upon a non-permanent marker, and allows the simultaneous enumeration of positive and negative colonies.

Mononuclear phagocytes secrete over 50 different proteins that are regulated during differentiation and that are under the influence of various materials and factors in their extracellular milieu as part of the inflammatory response. The complex nature of the regulation of the expression of these molecules is displayed by apolipoprotein E (ApoE). ApoE mRNA first appears as monocytes differentiate into macrophages, and this expression is paralleled by the secretion of ApoE by the cells. In mature macrophages ApoE synthesis and secretion are decreased by activation of macrophages with endotoxin and interferon-gamma. Although these macrophages contain abundant translatable ApoE mRNA, little ApoE is synthesized, suggesting that this decrease occurs largely at the translational level. ApoE is also controlled at the level of secretion. ApoE is concentrated in the Golgi complex of macrophages and is also found in endoplasmic reticulum, secretion vesicles and coated vesicles. When macrophages come in contact with immune complexes the intracellular ApoE compartment degranulates rapidly. Therefore, ApoE is regulated at the levels of secretion, translation and transcription.

The distribution of β-thromboglobulin, platelet factor 4, and fibrinogen in unstimulated platelets was investigated by several immunocytochemical techniques. All three substances were found to be localized in the majority of platelet alpha granules either by immunoperoxidase methods on saponin-treated platelets or by colloidal gold immunoconjugates on frozen thin sections. The optimal conditions for preparing and fixing platelets for immunocytochemistry were also determined. Platelets obtained from blood dripped directly into fixative or anticoagulated blood were compared systematically with respect to shape. Temperature was found to be the most important variable. Immediately fixed platelets were generally disc-shaped, regardless of the temperature of the fixative. Reducing the temperature of blood (stored with anticoagulant) before fixation resulted in more swollen and fewer disc-shaped platelets. However, if the blood was mixed with an anticoagulant and maintained at 37° C for 1 h before fixation, the same number of disc-shaped platelets were present as in samples from blood fixed immediately. The intracellular localization of β-thromboglobulin, platelet factor 4, and fibrinogen was consistent regardless of platelet preparatory procedure, but several technical problems were encountered with respect to plasma membrane labelling when control experiments were analysed. Immediately fixed, non-permeabilized platelet plasma membranes were always labelled, no matter which control substances or immunoperoxidase markers were used. However, when platelets were washed by centrifugation, the plasma membranes were negative. Exposure to saponin markedly diminished labelling of the plasma membranes. Optimal techniques for the immunocytochemical demonstration of these alpha granule proteins in platelets are presented in this report.

HE COMPLEX regulation of megakaryocytopoiesis T has been extensively re~iewed.'.~ Although mega- karyocyte development is a continuous process, it has been useful to consider early and late stages. The early stage comprises the transition of stem cells to committed mega- karyocyte precursors and is measured by megakaryocyte colony number and size in culture. In vitro, this stage is stimulated by one or more megakaryocyte colony-stimulat- ing factors (Meg-CSF) that have been identified in the urine and plasma of patients with aplastic anemia,536 amega- karyocytic thrombocytopenia,' and thrombocytopenia fol- lowing chemotherapy.8 The late stage comprises the matura- tion of megakaryocyte precursors into platelet producing cells and may be assessed by megakaryocyte number, size, ploidy or acetylcholinesterase activity (in certain species) or by measuring isotope incorporation into newly forming platelets.' Megakaryocyte maturation and platelet produc-