The source and significance of blood-borne tissue factor (TF) are controversial. TF mRNA, protein, and TF-dependent procoagulant activity (PCA) have been detected in human platelets, but direct evidence of TF synthesis is missing. Nonstimulated monocyte-free plate-lets from most patients expressed TF mRNA, which was enhanced or induced in all of them after platelet activation. Immunoprecipitation assays revealed TF protein (mainly of a molecular weight [Mr] of approximately 47 kDa, with other bands of approximately 35 and approximately 60 kDa) in nonstimulated platelet membranes, which also increased after activation. This enhancement was con-comitant with TF translocation to the plasma membrane, as demonstrated by immunofluorescence-confocal micros-copy and biotinylation of membrane proteins. Platelet PCA, assessed by factor Xa (FXa) generation, was induced after activation and was inhibited by 48% and 76% with anti-TF and anti-FVIIa, respectively , but not by intrinsic pathway inhibi-tors. Platelets incorporated [ 35 S]-methi-onine into TF proteins with Mr of approximately 47 kDa, approximately 35 kDa, and approximately 60 kDa, more intensely after activation. Puromycin but not actinomycin D or DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) inhibited TF neosynthesis. Thus, human platelets not only assemble the clotting reactions on their membrane, but also supply their own TF for thrombin generation in a timely and spatially circum-scribed process. These observations simplify, unify, and provide a more coherent formulation of the current cell-based model of hemostasis. (Blood. 2007;109:5242-5250)

Myosin is a ubiquitous eukaryotic contractile protein that generates the force responsible for such diverse cellular movements as muscle contraction and cytokinesis. Although there have been numerous studies of sarcomeric myosin heavy chain (MHC) genes, no molecular clones have been reported that encode mammalian nonmuscle MHC. This study presents the molecular genetic characterization of a human nonmuscle MHC that is expressed in fibroblasts, endothelial cells, and macrophages. Human nonmuscle MHC amino acids are weakly homologous (33%) to sarcomeric MHC but are -72% identical to smooth muscle MHC. In contrast to vertebrate sarcomeric MHCs, which generate diversity through the expression of members of a multigene family, an alternative polyadenylylation site is used in the nonmuscle MHC gene to generate multiple transcripts that encode the same protein.

Immunofluorescence and immunogold electron microscopy were employed to examine the assembly of connexins (Cx) 33, 37, and 43 into testis cell gap junctions in mature and postnatal rats. Cx37 was localized by immunofluorescence to the endothelia of blood vessels in both mature and immature testes and was not further characterized. Only Cx43 assembled into Leydig cell gap junctions, but Cx43 also co-assembled with Cx33 in some Sertoli-Sertoli gap junction plaques within and near Sertoli occluding junctions and on adluminal surfaces Assembly of Sertoli gap junctions appeared to be regulated according to the stage of the seminiferous epithelium since Cx33 (and Cx43) immunoreactivity was strong in Sertoli cells from stages II-VII but weak in stages IX-XIV. During postnatal maturation, assembly of Cx33 into gap junctions was regulated independently of Cx43 assembly. Cx43 was present on Sertoli cells of all tubules from postnatal Day 5 through Day 28. In contrast, Cx33 was not apparent on Sertoli cell surfaces until Day 15 and gradually accumulated in all tubules through Day 28. Between postnatal Days 38 and 43, the immunoreactivities of Cx33 and Cx43 became weak in Sertoli cells containing step 9-14 elongated spermatids. Thus, connexin abundance and gap junction composition in Sertoli cells is regulated during testis maturation and the cycle of the seminiferous epithelium.

Because hepatocytes communicate via gap junctions, it has been proposed that Ca2+waves propagate through this pathway and in the process activate Ca2+-dependent cellular responses. We tested this hypothesis by measuring vasopressin-induced glycogenolysis in short-term cultures of rat hepatocytes. A 15-min vasopressin (10−8 M) stimulation induced a reduction of glycogen content that reached a maximum 1–3 h later. Gap junction blockers, octanol or 18α-glycyrrhetinic acid, reduced the effect by 70%. The glycogenolytic response induced by Ca2+ ionophore 8-bromo-A-21387, which acts on each hepatocyte, was not affected by gap junction blockers. Moreover, the vasopressin-induced glycogenolysis was lower (70%) in dispersed than in reaggregated hepatocytes and in dispersed hepatocytes was not affected by gap junction blockers. In hepatocytes reaggregated in the presence of a synthetic peptide homologous to a domain of the extracellular loop 1 of the main hepatocyte gap junctional protein, vasopressin-induced glycogenolysis and incidence of dye coupling were drastically reduced. Moreover, gap junctional communication was detected between reaggregated cells, suggesting that hepatocytes with different vasopressin receptor densities become coupled to each other. The vasopressin-induced effect was not affected by suramin, ruling out ATP as a paracrine mediator. We propose that gap junctions allow for a coordinated vasopressin-induced glycogenolytic response despite the heterogeneity among hepatocytes.

Summary. Background: Glycoprotein VI (GPVI), 60-65 kDa, is a major collagen receptor on platelet membranes involved in adhesive and signaling responses. Mice lacking GPVI have impaired platelet response to collagen and defective primary adhesion and subsequent thrombus formation. Complete or partial deficiency of GPVI in humans is a rare condition presenting as a mild bleeding disorder. The defect in most of the reported patients is acquired and associated with other diseases. To date, only two patients have been characterized at the molecular level who carry different compound heterozygous mutations in the GP6 gene. Objective: To report four unrelated patients from non-consanguineous families who presented with mucocutaneous bleeding. They had absent platelet aggregation and 14 C-5-HT secretion with collagen, convulxin and collagen-related peptide. Results: Flow cytometry and immunofluorescence-confocal microscopy showed an absence of GPVI in non-permeabilized platelets. All the patients had an adenine insertion in exon 6 (c.711_712insA), changing the reading frame and generating a premature 'stop codon' in site 242 of the protein.The mutation predicts the synthesis of the truncated protein before the trans-membrane domain, corresponding to a band of %49 kDa observed in western blots and in permeabilized platelets by immunofluorescence. Platelet mRNA from all the patients was sequenced and contained the corresponding adenine insertion. Heterozygous relatives had no pathological bleeding, normal response to collagen and convulxin and intermediate membrane expression of GPVI. Conclusions: The identification of four unrelated homozygous patients with an identical defect suggests that inherited GPVI deficiency is more frequent than previously suspected, at least in Chile.