Paradigm of biased PAR1 (protease-activated receptor-1) activation and inhibition in endothelial cells dissected by phosphoproteomics

Thrombin is the key serine protease of the coagulation cascade and mediates cellular responses by activation of PARs (protease-activated receptors). The predominant thrombin receptor is PAR1, and in endothelial cells (ECs), thrombin dynamically regulates a plethora of phosphorylation events. However, it has remained unclear whether thrombin signaling is exclusively mediated through PAR1. Furthermore, mechanistic insight into activation and inhibition of PAR1-mediated EC signaling is lacking. In addition, signaling networks of biased PAR1 activation after differential cleavage of the PAR1 N terminus have remained an unresolved issue. Here, we used a quantitative phosphoproteomics approach to show that classical and peptide activation of PAR1 induce highly similar signaling, that low thrombin concentrations initiate only limited phosphoregulation, and that the PAR1 inhibitors vorapaxar and parmodulin-2 demonstrate distinct antagonistic properties. Subsequent analysis of the thrombin-regulated phosphosites in the presence of PAR1 inhibitors revealed that biased activation of PAR1 is not solely linked to a specific G-protein downstream of PAR1. In addition, we showed that only the canonical thrombin PAR1 tethered ligand induces extensive early phosphoregulation in ECs. Our study provides detailed insight in the signaling mechanisms downstream of PAR1. Our data demonstrate that thrombin-induced EC phosphoregulation is mediated exclusively through PAR1, that thrombin and thrombin-tethered ligand peptide induce similar phosphoregulation, and that only canonical PAR1 cleavage by thrombin generates a tethered ligand that potently induces early signaling. Furthermore, platelet PAR1 inhibitors directly affect EC signaling, indicating that it will be a challenge to design a PAR1 antagonist that will target only those pathways responsible for tissue pathology

ICAM-1 clustering on endothelial cells recruits VCAM-1

In the initial stages of transendothelial migration, leukocytes use the endothelial integrin ligands ICAM-1 and VCAM-1 for strong adhesion. Upon adhesion of the leukocyte to endothelial ICAM-1, ICAM-1 is clustered and recruited to the adhered leukocyte, promoting strong adhesion. In this study, we provide evidence for the colocalization of VCAM-1 at sites of ICAM-1 clustering. Anti-ICAM-1 antibody-coated beads were used to selectively cluster and recruit ICAM-1 on primary human endothelial cells. In time, co-localization of ICAM-1 and VCAM-1 around the adherent beads was observed. Biochemical pull-down assays showed that ICAM-1 clustering induced its association to VCAM-1, suggesting a physical link between these two adhesion molecules. The association was partly dependent on lipid rafts as well as on F-actin and promoted adhesion. These data show that VCAM-1 can be recruited, in an integrin-independent fashion, to clustered ICAM-1 which may serve to promote ICAM-1-mediated leukocyte adhesion

The composition of the mesenchymal stromal cell compartment in human bone marrow changes during development and aging

Life-long hematopoiesis depends on the support of mesenchymal stromal cells within the bone marrow. Therefore, changes in the hematopoietic compartment that occur during development and aging probably correlate with variation in the composition of the stromal cell microenvironment. Mesenchymal stromal cells are a heterogeneous cell population and various subtypes may have different functions. In accordance with others, we show that CD271 and CD146 define distinct colony-forming-unit-fibroblast containing mesenchymal stromal cell subpopulations. In addition, analysis of 86 bone marrow samples revealed that the distribution of CD271brightCD146− and CD271brightCD146+ subsets correlates with donor age. The main subset in adults was CD271brightCD146−, whereas the CD271brightCD146+ population was dominant in pediatric and fetal bone marrow. A third subpopulation of CD271−CD146+ cells contained colony-forming-unit-fibroblasts in fetal samples only. These changes in composition of the mesenchymal stromal cell compartment during development and aging suggest a dynamic system, in which these subpopulations may have different functions

The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation

Leukocyte transendothelial migration involves the active participation of the endothelium through the formation of apical membrane protrusions that embrace adherent leukocytes, termed docking structures. Using live-cell imaging, we find that prior to transmigration, endothelial docking structures form around 80% of all neutrophils. Previously we showed that endothelial RhoG and SGEF control leukocyte transmigration. In this study, our data reveal that both full-length Trio and the first DH-PH (TrioD1) domain of Trio, which can activate Rac1 and RhoG, interact with ICAM-1 and are recruited to leukocyte adhesion sites. Moreover, upon clustering of ICAM-1, the Rho-guanine nucleotide exchange factor Trio activates Rac1, prior to activating RhoG, in a filamin-dependent manner. We further show that docking structure formation is initiated by ICAM-1 clustering into ring-like structures, which is followed by apical membrane protrusion. Interestingly, we find that Rac1 is required for ICAM-1 clustering, whereas RhoG controls membrane protrusion formation. Finally, silencing endothelial Trio expression or reducing TrioD1 activity without affecting SGEF impairs both docking structure formation and leukocyte transmigration. We conclude that Trio promotes leukocyte transendothelial migration by inducing endothelial docking structure formation in a filamin-dependent manner through the activation of Rac1 and RhoG

Monitoring storage induced changes in the platelet proteome employing label free quantitative mass spectrometry

Shelf life of platelet concentrates is limited to 5-7 days due to loss of platelet function during storage, commonly referred to as the platelet storage lesion (PSL). To get more insight into the development of the PSL, we used label free quantitative mass spectrometry to identify changes in the platelet proteome during storage. In total 2501 proteins were accurately quantified in 3 biological replicates on at least 1 of the 7 different time-points analyzed. Significant changes in levels of 21 proteins were observed over time. Gene ontology enrichment analysis of these proteins revealed that the majority of this set was involved in platelet degranulation, secretion and regulated exocytosis. Twelve of these proteins have been shown to reside in α-granules. Upon prolonged storage (13-16 days) elevated levels of α-2-macroglobulin, glycogenin and Ig μ chain C region were identified. Taken together this study identifies novel markers for monitoring of the PSL that may potentially also be used for the detection of "young" and "old" platelets in the circulation

Syntaxin 5 determines Weibel-Palade body size and von Willebrand factor secretion by controlling Golgi architecture

Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells. VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPB), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde endoplasmic reticulum-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometry-based approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in endothelial cells using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted endothelial cells exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. In brief, we identified SNARE protein STX5 as a novel regulator of WPB biogenesis

The class I scavenger receptor CD163 promotes internalization of ADAMTS13 by macrophages

Internalization of ADAMTS13 by macrophages may contribute to its clearance from the circulation. Here we investigated endocytic mechanisms that contribute to the uptake of ADAMTS13 by macrophages. Human monocyte-derived macrophages were used to monitor the uptake of fluorescently labeled recombinant ADAMTS13 by flow cytometry. Internalization of ADAMTS13 was blocked upon addition of the cell-permeable dynamin inhibitor dynasore. Partial blocking of ADAMTS13 uptake was observed by using mannan; however, uptake was not affected by an antibody that blocked binding to the macrophage mannose receptor CD206, which suggests that other endocytic receptors contribute to the internalization of ADAMTS13 by macrophages. A pull-down with ADAMTS13 and subsequent mass spectrometric analysis identified the class I scavenger receptor CD163 as a candidate receptor for ADAMTS13. Blocking experiments with monoclonal anti-CD163 antibody EDHu-1 resulted in decreased ADAMTS13 internalization by macrophages. Pronounced inhibition of ADAMTS13 uptake by EDHu-1 was observed in CD163 high-expressing macrophages. In agreement with these findings, CD163-expressing Chinese hamster ovary cells were capable of rapidly internalizing ADAMTS13. Surface plasmon resonance revealed binding of ADAMTS13 to scavenger receptor cysteine-rich domains 1-9 and 1-5 of CD163. Taken together, our data identify CD163 as a major endocytic receptor for ADAMTS13 on macrophages