Functional cyclophilin D moderates platelet adhesion, but enhances the lytic resistance of fibrin

In the course of thrombosis, platelets are exposed to a variety of activating stimuli classified as ‘strong’ (e.g. thrombin and collagen) or ‘mild’ (e.g. ADP). In response, activated platelets adhere to injured vasculature, aggregate, and stabilise the three-dimensional fibrin scaffold of the expanding thrombus. Since ‘strong’ stimuli also induce opening of the mitochondrial permeability transition pore (MPTP) in platelets, the MPTP-enhancer Cyclophilin D (CypD) has been suggested as a critical pharmacological target to influence thrombosis. However, it is poorly understood what role CypD plays in the platelet response to ‘mild’ stimuli which act independently of MPTP. Furthermore, it is unknown how CypD influences platelet-driven clot stabilisation against enzymatic breakdown (fibrinolysis). Here we show that treatment of human platelets with Cyclosporine A (a cyclophilin-inhibitor) boosts ADP-induced adhesion and aggregation, while genetic ablation of CypD in murine platelets enhances adhesion but not aggregation. We also report that platelets lacking CypD preserve their integrity in a fibrin environment, and lose their ability to render clots resistant against fibrinolysis. Our results indicate that CypD has opposing haemostatic roles depending on the stimulus and stage of platelet activation, warranting a careful design of any antithrombotic strategy targeting CypD

Microfluidic hemophilia models using blood from healthy donors

Abstract Background Microfluidic clotting assays permit drug action studies for hemophilia therapeutics under flow. However, limited availability of patient samples and Inter‐donor variability limit the application of such assays, especially with many patients on prophylaxis. Objective To develop approaches to phenocopy hemophilia using modified healthy blood in microfluidic assays. Methods Corn trypsin inhibitor (4 µg/mL)‐treated healthy blood was dosed with either anti–factor VIII (FVIII; hemophilia A model) or a recombinant factor IX (FIX) missense variant (FIX‐V181T; hemophilia B model). Treated blood was perfused at 100 s−1 wall shear rate over collagen/tissue factor (TF) or collagen/factor XIa (FXIa). Results Anti‐FVIII partially blocked fibrin production on collagen/TF, but completely blocked fibrin production on collagen/FXIa, a phenotype reversed with 1 µmol/L bispecific antibody (emicizumab), which binds FIXa and factor X. As expected, emicizumab had no significant effect on healthy blood (no anti‐FVIII present) perfused over collagen/FXIa. The efficacy of emicizumab in anti‐FVIII‐treated healthy blood phenocopied the action of emicizumab in the blood of a patient with hemophilia A perfused over collagen/FXIa. Interestingly, a patient‐derived FVIII‐neutralizing antibody reduced fibrin production when added to healthy blood perfused over collagen/FXIa. For low TF surfaces, reFIX‐V181T (50 µg/mL) fully blocked platelet and fibrin deposition, a phenotype fully reversed with anti‐TFPI. Conclusion Two new microfluidic hemophilia A and B models demonstrate the potency of anti‐TF pathway inhibitor, emicizumab, and a patient‐derived inhibitory antibody. Using collagen/FXIa‐coated surfaces resulted in reliable and highly sensitive hemophilia models

SPARC Reanalysis Intercomparison Project (S-RIP) Final Report

The S-RIP activity focuses predominantly on reanalyses, although some chapters include diagnostics from operational analyses when appropriate. Many of the chapters focus primarily on newer reanalysis systems that assimilate upper-air measurements and produce data at relatively high resolution (i.e., ERA-Interim, JRA55, MERRA, MERRA-2, and CFSR). The ERA5 reanalysis, which was released during the latter stages of the activity, is not fully evaluated but is included in some intercomparisons. Selected long-term reanalyses that assimilate only surface meteorological observations (e.g., NOAA-CIRES 20CR, ERA-20C, and CERA-20C) are also evaluated where appropriate. Some chapters include comparisons with older reanalyses (NCEP-NCAR R1, NCEP-DOE R2, ERA-40, and JRA-25/JCDAS), because these products have been extensively used in the past and are still being used for some studies, and because such comparisons can provide insight into the potential shortcomings of past research results. Other chapters only include a subset of these reanalysis data sets, since some reanalyses have already been shown to perform poorly for certain diagnostics or do not extend high enough (e.g., pressures less than 10hPa) in the atmosphere. At the beginning of each chapter an explanation is given as to why specific reanalysis data sets were included or excluded. The minimum intercomparison period is 1980-2010. This period starts with the availability of MERRA-2 shortly after the advent of high-frequency remotely sensed data in late 1978 and ends with the transition between CFSR and CFSv2. Some chapters also consider the pre-satellite era before 1979 and/or include results for more recent years. Some chapters use shorter intercomparison periods for some diagnostics due to limitations in the observational record available for comparison and/or computational resources

Integrated microarray and multiplex cytokine analyses of Kaposi's Sarcoma Associated Herpesvirus viral FLICE Inhibitory Protein K13 affected genes and cytokines in human blood vascular endothelial cells

<p>Abstract</p> <p>Background</p> <p>Kaposi's sarcoma (KS) associated herpesvirus (KSHV) is the etiological agent of KS, a neoplasm characterized by proliferating spindle cells, extensive neoangiogenesis and a prominent inflammatory infiltrate. Infection of blood vascular endothelial cells with KSHV in vitro results in their spindle cell transformation, which is accompanied by increased expression of inflammatory chemokines and cytokines, and acquisition of lymphatic endothelial markers. Mimicking the effect of viral infection, ectopic expression of KSHV-encoded latent protein vFLIP K13 is sufficient to induce spindle transformation of vascular endothelial cells. However, the effect of K13 expression on global gene expression and induction of lymphatic endothelial markers in vascular endothelial cells has not been studied.</p> <p>Methods</p> <p>We used gene array analysis to determine change in global gene expression induced by K13 in human vascular endothelial cells (HUVECs). Results of microarray analysis were validated by quantitative RT-PCR, immunoblotting and a multiplex cytokine array.</p> <p>Results</p> <p>K13 affected the expression of several genes whose expression is known to be modulated by KSHV infection, including genes involved in immune and inflammatory responses, anti-apoptosis, stress response, and angiogenesis. The NF-κB pathway was the major signaling pathway affected by K13 expression, and genetic and pharmacological inhibitors of this pathway effectively blocked K13-induced transcriptional activation of the promoter of CXCL10, one of the chemokines whose expression was highly upregulated by K13. However, K13, failed to induce expression of lymphatic markers in blood vascular endothelial cells.</p> <p>Conclusion</p> <p>While K13 may account for change in the expression of a majority of genes observed following KSHV infection, it is not sufficient for inducing lymphatic reprogramming of blood vascular endothelial cells.</p