Substitution of stable isotopes in Chlorella

Replacement of biologically important isotopes in the alga Chlorella by corresponding heavier stable isotopes produces increasingly greater deviations from the normal cell size and changes the quality and distribution of certain cellular components. The usefulness of isotopically altered organisms increases interest in the study of such permuted organisms

Thrombus Formation: Direct Real‐Time Observation and Digital Analysis of Thrombus Assembly in a Living Mouse by Confocal and Widefield Intravital Microscopy

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

Discovery of 1,3-Diaminobenzenes as Selective Inhibitors of Platelet Activation at the PAR1 Receptor

A high-throughput screen of the NIH-MLSMR compound collection, along with a series of secondary assays to identify potential targets of hit compounds, previously identified a 1,3-diaminobenzene scaffold that targets protease-activated receptor 1 (PAR1). We now report additional structure–activity relationship (SAR) studies that delineate the requirements for activity at PAR1 and identify plasma-stable analogues with nanomolar inhibition of PAR1-mediated platelet activation. Compound 4 was declared as a probe (ML161) with the NIH Molecular Libraries Program. This compound inhibited platelet aggregation induced by a PAR1 peptide agonist or by thrombin but not by several other platelet agonists. Initial studies suggest that ML161 is an allosteric inhibitor of PAR1. These findings may be important for the discovery of antithrombotics with an improved safety profile

Parmodulins Inhibit Thrombus Formation Without Inducing Endothelial Injury Caused by Vorapaxar

Protease-activated receptor-1 (PAR1) couples the coagulation cascade to platelet activation during myocardial infarction and to endothelial inflammation during sepsis. This receptor demonstrates marked signaling bias. Its activation by thrombin stimulates prothrombotic and proinflammatory signaling, whereas its activation by activated protein C (APC) stimulates cytoprotective and antiinflammatory signaling. A challenge in developing PAR1-targeted therapies is to inhibit detrimental signaling while sparing beneficial pathways. We now characterize a novel class of structurally unrelated small-molecule PAR1 antagonists, termed parmodulins, and compare the activity of these compounds to previously characterized compounds that act at the PAR1 ligand–binding site. We find that parmodulins target the cytoplasmic face of PAR1 without modifying the ligand-binding site, blocking signaling through Gαq but not Gα13 in vitro and thrombus formation in vivo. In endothelium, parmodulins inhibit prothrombotic and proinflammatory signaling without blocking APC-mediated pathways or inducing endothelial injury. In contrast, orthosteric PAR1 antagonists such as vorapaxar inhibit all signaling downstream of PAR1. Furthermore, exposure of endothelial cells to nanomolar concentrations of vorapaxar induces endothelial cell barrier dysfunction and apoptosis. These studies demonstrate how functionally selective antagonism can be achieved by targeting the cytoplasmic face of a G-protein–coupled receptor to selectively block pathologic signaling while preserving cytoprotective pathways

T granules in human platelets function in TLR9 organization and signaling

Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. In this paper, we show that the TLR9 transcript is specifically up-regulated during pro-PLT production and is distributed to a novel electron-dense tubular system-related compartment we have named the T granule. TLR9 colocalizes with protein disulfide isomerase and is associated with either VAMP 7 or VAMP 8, which regulates its distribution in PLTs on contact activation (spreading). Preincubation of PLTs with type IV collagen specifically increased TLR9 and CD62P surface expression and augmented oligodeoxynucleotide (ODN) sequestration and PLT clumping upon addition of bacterial/viral ODNs. Collectively, this paper (a) tracks TLR9 to a new intracellular compartment in PLTs and (b) describes a novel mechanism of TLR9 organization and signaling in human PLTs

Climacteric Lowers Plasma Levels of Platelet-Derived Microparticles: A Pilot Study in Pre-versus Postmenopausal Women

Background: Climacteric increases the risk of thrombotic events by alteration of plasmatic coagulation. Up to now, less is known about changes in platelet-(PMP) and endothelial cell-derived microparticles (EMP). Methods: In this prospective study, plasma levels of microparticles (MP) were compared in 21 premenopausal and 19 postmenopausal women. Results: No altered numbers of total MP or EMP were measured within the study groups. However, the plasma values of CD61-exposing MP from platelets/megakaryocytes were higher in premenopausal women (5,364 x 10(6)/l, range 4,384-17,167) as compared to postmenopausal women (3,808 x 10(6)/l, range 2,009-8,850; p = 0.020). This differentiation was also significant for the subgroup of premenopausal women without hormonal contraceptives (5,364 x 10(6)/l, range 4,223-15,916; p = 0.047; n = 15). Furthermore, in premenopausal women, higher plasma levels of PMP exposing CD62P were also present as compared to postmenopausal women (288 x 10(6)/l, range 139-462, vs. 121 x 10(6)/l, range 74-284; p = 0.024). This difference was also true for CD63+ PMP levels (281 x 10(6)/l, range 182-551, vs. 137 x 10(6)/l, range 64-432; p = 0.015). Conclusion: Climacteric lowers the level of PMP but has no impact on the number of EMP in women. These data suggest that PMP and EMP do not play a significant role in enhancing the risk of thrombotic events in healthy, postmenopausal women. Copyright (C) 2012 S. Karger AG, Base

Analysis of the potential of cancer cell lines to release tissue factor-containing microvesicles: correlation with tissue factor and PAR2 expression

BackgroundDespite the association of cancer-derived circulating tissue factor (TF)-containing microvesicles and hypercoagulable state, correlations with the incidence of thrombosis remain unclear.MethodsIn this study the upregulation of TF release upon activation of various cancer cell lines, and the correlation with TF and PAR2 expression and/or activity was examined. Microvesicle release was induced by PAR2 activation in seventeen cell lines and released microvesicle density, microvesicle-associated TF activity, and phoshpatidylserine-mediated activity were measured. The time-course for TF release was monitored over 90 min in each cell line. In addition, TF mRNA expression, cellular TF protein and cell-surface TF activities were quantified. Moreover, the relative expression of PAR2 mRNA and cellular protein were analysed. Any correlations between the above parameters were examined by determining the Pearson’s correlation coefficients.ResultsTF release as microvesicles peaked between 30–60 min post-activation in the majority of cell lines tested. The magnitude of the maximal TF release positively correlated with TF mRNA (c = 0.717; p