Platelet-derived CD154: ultrastructural localization and clinical correlation in organ transplantation.

CD154 is an immunostimulatory ligand for CD40 that markedly influences alloimmunity. Its presence in platelets suggests that its release and subsequent immune effects are driven by trauma and thus could be relevant following organ transplantation. However, the release of platelet derived CD154 and its consequences have not been investigated in a clinical transplant setting. To better characterize the relationship between platelet activation and CD154 release, we investigated CD154 release by platelets obtained from normal individuals, and patients with two genetic defects that influence platelet granule development. Using these unique patient populations and immune-electron microscopy, we confirmed that CD154 was an alpha granule and not a cell surface protein, and thereafter optimized the methods for its in vivo measurement in humans. We then investigated plasma CD154 levels in kidney and liver transplant recipients and found no evidence that CD154 levels fluctuated systemically as a result of kidney or liver transplant procedures. Paradoxically, we found that kidney transplant patients had significantly lower systemic CD154 levels during episodes of rejection. These data suggest that the immune effects of CD154 are likely mediated through local and not systemic mechanisms, and discourage the use of CD154 as a peripheral biomarker in organ transplantation.

The α-granule proteome: novel proteins in normal and ghost granules in gray platelet syndrome.

BACKGROUND: Deficiencies in granule-bound substances in platelets cause congenital bleeding disorders known as storage pool deficiencies. For disorders such as gray platelet syndrome (GPS), in which thrombocytopenia, enlarged platelets and a paucity of α-granules are observed, only the clinical and histologic states have been defined. OBJECTIVES: In order to understand the molecular defect in GPS, the α-granule fraction protein composition from a normal individual was compared with that of a GPS patient by mass spectrometry (MS). METHODS: Platelet organelles were separated by sucrose gradient ultracentrifugation. Proteins from sedimented fractions were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis, reduced, alkylated, and digested with trypsin. Peptides were analyzed by liquid chromatography-tandem MS. Mascot was used for peptide/protein identification and to determine peptide false-positive rates. MassSieve was used to generate and compare parsimonious lists of proteins. RESULTS: As compared with control, the normalized peptide hits (NPHs) from soluble, biosynthetic α-granule proteins were markedly decreased or undetected in GPS platelets, whereas the NPHs from soluble, endocytosed α-granule proteins were only moderately affected. The NPHs from membrane-bound α-granule proteins were similar in normal platelets and GPS platelets, although P-selectin and Glut3 were slightly decreased, consistent with immunoelectron microscopy findings in resting platelets. We also identified proteins not previously known to be decreased in GPS, including latent transforming growth factor-ß-binding protein 1(LTBP1), a component of the transforming growth factor-ß (TGF-ß) complex. CONCLUSIONS: Our results support the existence of 'ghost granules' in GPS, point to the basic defect in GPS as failure to incorporate endogenously synthesized megakaryocytic proteins into α-granules, and identify specific new proteins as α-granule inhabitants.

Proteomic analysis of platelet alpha-granules using mass spectrometry.

BACKGROUND: Platelets have three major types of secretory organelles: lysosomes, dense granules, and alpha-granules. alpha-Granules contain several adhesive proteins involved in hemostasis, as well as glycoproteins involved in inflammation, wound healing, and cell-matrix interactions. This article represents the first effort to define the platelet alpha-granule proteome using mass spectrometry (MS). METHODS: We prepared a subcellular fraction enriched in intact alpha-granules from human platelets using sucrose gradient ultracentrifugation. alpha-Granule proteins were separated and identified using sodium dodecylsulfate polyacrylamide gel electrophoresis and liquid chromatography-tandem MS. RESULTS: In the sucrose fraction enriched in alpha-granules, we identified 284 non-redundant proteins, 44 of which appear to be new alpha-granule proteins, on the basis of a literature review. Immunoelectron microscopy confirmed the presence of Scamp2, APLP2, ESAM and LAMA5 in platelet alpha-granules for the first time. We identified 65% of the same proteins that were detected in the platelet releasate (J. A. Coppinger et al. [Blood 2004;103: 2096-104]) as well as additional soluble and membrane proteins. Our method provides a suitable tool for analyzing the granule proteome of patients with storage pool deficiencies.

Motor patterns in the stomatogastric ganglion of the lobster Panulirus argus.

1. Acitivity patterns arising from the thirty cells of the stomatogastric ganglion of Panulirus argus are described for both a semi-intact preparation and an isolated one. 2. The thirty or so cells can be divided so far into two functional groupings: the gastric mill group, with at least ten motor elements, and the pyloric group with at least fourteen. There is some, but not extensive, interaction between groups. 3. The main gastric mill activity is arranged in two sets of elements, each of which is composed of reciprocating elements innervating antagonistic muscles. Thus alternation in activity between the single LC and the two LG neurones results in alternate closing and opening of the lateral teeth; alternation between the four GM and single CP units results in alternate protraction and retraction of the medial tooth. 4. The two sets are phased to each other in such a way that they cause gastric mill teeth to operate effectively to masticate food. 5. The main pyloric activity is arranged in a three-part cycle with each of three sets of units active in sequence. Activity in two PD and one AB unit is followed by bursts in IC and LP units followed in turn by activity in up to seven PY units. Activity in a single VD neurone is locked to this cycle in a more complex pattern.