Peritoneal macrophages express both P-selectin and PSGL-1

Macrophages, phagocytic cells involved in an early phase of host defense, are known to express the P-selectin ligand, PSGL-1. Heretofore, P-selectin has only been found on platelets and endothelial cells. Here, we demonstrate that peritoneal macrophages isolated by peritoneal lavage of unchallenged mice express P-selectin on the plasma membrane. The peritoneal macrophages synthesize P-selectin, as indicated by metabolic labeling experiments. P-Selectin is constitutively expressed on the extracellular surface of macrophages but is only partially colocalized with PSGL-1. P-Selectin is rapidly translocated from the macrophage plasma membrane to intracellular vesicles and to lysosomes. Peritoneal macrophages assemble into cell strings under flow conditions based upon macrophage–macrophage interactions mediated by P-selectin and PSGL-1. This is the first description of a leukocyte shown to express both P-selectin and PSGL-1

Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse

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

Novel γ-carboxyglutamic acid-containing peptides from the venom of Conus textile

Author Posting. © The Author(s), 2006. This is the author's version of the work. It is posted here by permission of Blackwell for personal use, not for redistribution. The definitive version was published in FEBS Journal 273 (2006): 2779-2788, doi:10.1111/j.1742-4658.2006.05355_1.x.The cone snail is the only invertebrate system in which the vitamin K dependent carboxylase (or γ-carboxylase) and its product γ-carboxyglutamic acid (Gla)1 have been identified. It remains the sole source of structural information of invertebrate γ-carboxylase subtrates. Four novel γ- carboxyglutamic acid (Gla)1 containing peptides were purified from the venom of Conus textile and characterized by biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have 6 Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-L-bromotryptophan. Gla(2)-TxVI/A and Gla(2)- TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids –46 to –27) were nearly identical and highly hydrophobic. The predicted propeptide region (–20 to –1) that contains the γ-carboxylation recognition site (γ-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conus γ-carboxylase and synthetic peptide substrates localized the γ-CRS of Gla(1)-TxVI to the region –14 to –1 of the polypeptide precursor: the Km was reduced from 1.8 mM for Gla (1)-TxVI lacking a propeptide to 24 μM when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km 10-fold.This work was supported by grants K2001-03X-04487-27A and K2001- 03GX-04487-27, 08647, 13147 from the Swedish Medical Research Council, the European Union Cono-Euro-Pain (QLK3-CT-2000-00204), the Swedish Foundation for Strategic Research, the Kock Foundation, the Påhlsson Foundation and the Foundation of University Hospital, Malmö

The BILAG-2004 systems tally-a novel way of representing the BILAG-2004 index scores longitudinally

Objective. This was an exploratory analysis to develop a new way of representing BILAG-2004 system scores longitudinally that would be clinically meaningful and easier to analyse in comparison with multiple categorical variables. Methods. Data from a multicentre longitudinal study of SLE patients (the BILAG-2004 index and therapy collected at every visit) were used. External responsiveness analysis of the index suggested the possibility of using counts of systems with specified transitions in scores as a basis to analyse the system scores. Exploratory analyses with multinomial logistic regression were used to examine the appropriateness of this new method of analysing BILAG-2004 system scores. Receiver operating characteristic (ROC) curve analysis was used to assess the performance of this approach. Results. There were 1414 observations from 347 patients. A novel method was devised based on counts of systems with defined transitions in score (BILAG-2004 systems tally, BST). It has six components (systems with major deterioration, systems with minor deterioration, systems with persistent significant activity, systems with major improvement, systems with minor improvement and systems with persistent minimal or no activity). This was further simplified (simplified BST, sBST) into three components (systems with active/worsening disease, systems with improving disease and systems with persistent minimal or no activity). Both versions had expected associations with change in therapy. ROC curve analyses demonstrated that both versions had similar good performance characteristics (areas under the curve >0.80) in predicting increase in therapy. Conclusion. The BST and sBST provide alternative approaches to representing BILAG-2004 disease activity longitudinally. Further validation of their use is required

Accumulation of Tissue Factor Into Developing Thrombi In Vivo Is Dependent Upon Microparticle P-Selectin Glycoprotein Ligand 1 And Platelet P-Selectin

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

Accumulation of Tissue Factor into Developing Thrombi In Vivo Is Dependent upon Microparticle P-Selectin Glycoprotein Ligand 1 and Platelet P-Selectin

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

PTP-1B is an essential positive regulator of platelet integrin signaling

Outside-in integrin αIIbβ3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein–tyrosine phosphatase (PTP)–1B in this process. In resting platelets, c-Src forms a complex with αIIbβ3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to αIIbβ3 triggers PTP-1B recruitment to the αIIbβ3–c-Src–Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B–deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from αIIbβ3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B–deficient platelets are defective in outside-in αIIbβ3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in αIIbβ3 signaling in platelets