We have used confocal and widefield microscopy to image thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and of a brightfield channel. Vascular injury is induced with a laser focused through the microscope optics. We observed platelet deposition, tissue factor accumulation and fibrin generation after laser-induced endothelial injury in a single developing thrombus. The initiation of blood coagulation in vivo entailed the initial accumulation of tissue factor on the upstream and thrombus-vessel wall interface of the developing thrombus. Subsequently tissue factor was associated with the interior of the thrombus. Tissue factor was biologically active, and was associated with fibrin generation within the thrombus.

Using a laser-induced endothelial injury model, we examined thrombus formation in the microcirculation of wild-type and genetically altered mice by real-time in vivo microscopy to analyze this complex physiologic process in a system that includes the vessel wall, the presence of flowing blood, and the absence of anticoagulants. We observe P-selectin expression, tissue factor accumulation, and fibrin generation after platelet localization in the developing thrombus in arterioles of wild-type mice. However, mice lacking P-selectin glycoprotein ligand 1 (PSGL-1) or P-selectin, or wild-type mice infused with blocking P-selectin antibodies, developed platelet thrombi containing minimal tissue factor and fibrin. To explore the delivery of tissue factor into a developing thrombus, we identified monocyte-derived microparticles in human platelet–poor plasma that express tissue factor, PSGL-1, and CD14. Fluorescently labeled mouse microparticles infused into a recipient mouse localized within the developing thrombus, indicating that one pathway for the initiation of blood coagulation in vivo involves the accumulation of tissue factor– and PSGL-1–containing microparticles in the platelet thrombus expressing P-selectin. These monocyte-derived microparticles bind to activated platelets in an interaction mediated by platelet P-selectin and microparticle PSGL-1. We propose that PSGL-1 plays a role in blood coagulation in addition to its known role in leukocyte trafficking.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laserinduced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation. IntroductionPlatelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kDa membrane glycoprotein that is expressed on a range of blood cells including platelets, monocytes, neutrophils, B lymphocytes, some T lymphocyte subsets, and also on vascular endothelial cells. [1][2][3][4] This member of the immunoglobulin superfamily has been reported to be associated with a wide range of functions, depending on the cell of interest. These include transendothelial migration of leukocytes, 5-7 integrin regulation, [8][9][10][11][12][13][14][15][16] modulation of T-and Blymphocyte antigen receptor signaling, 17,18 B-lymphocyte development, 19 vasculogenesis, 20 apoptosis, 21,22 and protection against endotoxic shock. 23 Several lines of investigation have recently determined that PECAM-1 is involved in the negative regulation of platelet function in vitro. The activation of PECAM-1 prior to the stimulation of platelets results in the inhibition of platelet aggregation and the inhibition of activatory signaling mechanisms. 24,25 Of particular note, therefore, are the observations that mouse platelets deficient in PECAM-1 are hyperresponsive to stimulation with collagen and demonstrate enhanced aggregation, secretion, and adhesion to this agonist. 26 Platelets from PECAM-1-deficient mice have also been shown to form larger thrombi in vitro under physiologic flow conditions. 25 PECAM-1 participates in homophilic ligand-binding interactions [27][28][29] ; indeed, such interactions between PECAM-1 molecules on the same cell and between cells are believed to underlie most of its identified functions. Additional potential ligand-binding interactions have been reported, such as with integrin ␣ v ␤ 3 ...

Summary. We have developed novel instrumentation using confocal and widefield microscopy to image and analyze thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and a brightfield channel, and supports laser-induced injury through the microscope optics. Although this imaging facility requires interface of multiple hardware components, the primary challenge in vascular imaging is careful experimental design and interpretation. This system has been used to localize tissue factor during thrombus formation, to observe defects in thrombus assembly in genetically altered mice, to study the kinetics of platelet activation and P-selectin expression following vascular injury, to analyze leukocyte rolling on arterial thrombi, to generate three-dimensional models of thrombi, and to analyze the effect of antithrombotic agents in vivo.

The adhesion molecule von Willebrand factor (vWF) activates platelets upon binding 2 surface receptors, glycoprotein (GP) Ib-V-IX and integrin IIbβ3. We have used 2 approaches to selectively activate GP Ib using either the snake venom lectin alboaggregin-A or mutant recombinant forms of vWF (▵A1-vWF and RGGS-vWF) with selective binding properties to its 2 receptors. We show that activation of GP Ib induces platelet aggregation, secretion of 5-hydroxy tryptamine (5-HT), and an increase in cytosolic calcium. Syk becomes tyrosine phosphorylated and activated downstream of GP Ib, and associates with several tyrosine-phosphorylated proteins including the Fc receptor γ-chain through interaction with Syk SH2 domains. GP Ib physically associates with the γ-chain in GST-Syk-SH2 precipitates from platelets stimulated through GP Ib, and 2 Src family kinases, Lyn and Fyn, also associate with this signaling complex. In addition, GP Ib stimulation couples to tyrosine phosphorylation of phospholipase Cγ2. The Src family-specific inhibitor PP1 dose-dependently inhibits phosphorylation of Syk, its association with tyrosine-phosphorylated γ-chain, phosphorylation of PLCγ2, platelet aggregation, and 5-HT release. The results indicate that, upon activation, GP Ib is physically associated with FcR γ-chain and members of the Src family kinases, leading to phosphorylation of the γ-chain, recruitment, and activation of Syk. Phosphorylation of PLCγ2 also lies downstream of Src kinase activation and may critically couple early signaling events to functional platelet responses.

The adhesion molecule von Willebrand factor (vWF) activates platelets upon binding 2 surface receptors, glycoprotein (GP) Ib-V-IX and integrin IIbβ3. We have used 2 approaches to selectively activate GP Ib using either the snake venom lectin alboaggregin-A or mutant recombinant forms of vWF (▵A1-vWF and RGGS-vWF) with selective binding properties to its 2 receptors. We show that activation of GP Ib induces platelet aggregation, secretion of 5-hydroxy tryptamine (5-HT), and an increase in cytosolic calcium. Syk becomes tyrosine phosphorylated and activated downstream of GP Ib, and associates with several tyrosine-phosphorylated proteins including the Fc receptor γ-chain through interaction with Syk SH2 domains. GP Ib physically associates with the γ-chain in GST-Syk-SH2 precipitates from platelets stimulated through GP Ib, and 2 Src family kinases, Lyn and Fyn, also associate with this signaling complex. In addition, GP Ib stimulation couples to tyrosine phosphorylation of phospholipase Cγ2. The Src family-specific inhibitor PP1 dose-dependently inhibits phosphorylation of Syk, its association with tyrosine-phosphorylated γ-chain, phosphorylation of PLCγ2, platelet aggregation, and 5-HT release. The results indicate that, upon activation, GP Ib is physically associated with FcR γ-chain and members of the Src family kinases, leading to phosphorylation of the γ-chain, recruitment, and activation of Syk. Phosphorylation of PLCγ2 also lies downstream of Src kinase activation and may critically couple early signaling events to functional platelet responses.

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GABA (y-aminobutyric acid) is the principal inhibitory neurotransmitter of the central nervous system and interacts with both ionotropic (GABAA and GAB&) and metabotropic (GABAB) receptors. A GABAB R1 receptor has recently been cloned. However, when GABAB is transfected into clonal cell lines this protein alone does not reach the cell surface. Coexpression of GABAB R1 with the newly identified GABAB R2 protein does result in successfully trafficking to the cell surface. This fully functional GABAB receptor has properties similar to native receptors.We have used the two-hybrid assay to isolate other potential GABAB R 1-interacting proteins. The DNA sequence encoding the intracellular C-terminal region of GABAB Rl(104 amino acids) was used to screen a rat brain cDNA library for interacting proteins. Approximately 600 0-galactosidase positive yeast colonies were isolated, of which three hundred have so far been analysed. From this two separate novel partial clones have specificity for GABAB RI when tested against a wide range of c-terminal baits including mGluR1-7, GluR1-4 (AMPA), NR1, 2A-C (NMDA) and GluR5-6 (lcainate). Domain analysis of the C-terminus of GABAB R1 reveals that both clones interact within the same region. We are currently investigating the interactions in vitro using biochemical pull-down and overlay assays. In addition we are performing co-localisation studies in transiently transfected HEK 293 cells.Yeast 2-hybrid system and -108 Investigation of the effect of interleukin-1 on articular cartilage by tandem electrospray mass spectrometry.The pro-inflammatory cytokine interleukin-1 @-1) is a potent mediator of articular cartilage degradation, and may be involved in the pathogenesis of rheumatoid arthritis (RA) and osteoarthritis, though the underlying molecular mechanisms are currently unknown. Chondrocytes were isolated from porcine articular cartilage, and cultured for 24 h, prior to incubation with recombinant IL-lf? for a further 18 h. Proteins were separated by 1 and 2D electrophoresis, and stained with Coomassie-blue. Bands were excised, subjected to in gel digestion, and mass analysed and partially sequenced by nano-electrospray mass spectrometry using a Micromass Q-Tof. The resulting peptide mass maps and sequence tags were searched against the SWISS-PROT database. The major secreted protein up-regulated by IL-1 was a 38-kDa heparin binding protein (gp38k), a porcine homologue of the human cartilage glycoprotein HCgp39 (also known as YKL-40), which is associated with tissue remodelling processes, and appears to be elevated in rheumatoid arthritis. In cell lysates the most significantly up-regulated protein was manganese-dependent superoxide dismutase (MnSOD), an enzyme which is known to be responsive to IL-1 treatment. Production of MnSOD could detoxify the superoxide radicals induced in chondrocytes by cytokines, but long term production of MnSOD could result in accumulation of hydrogen peroxide, which is a powerful inhibitor of proteoglycan synthesis, and might contribute to the joint des...