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Objective— 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. Approach and Results— 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. Conclusions— 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.

It has been proposed that differences may exist between umbilical cord blood (CB) platelets and adult peripheral blood (APB) platelets, including altered protein levels of the main platelet integrins. We have now compared the protein expression profiles of CB and APB platelets employing a label-free comparative proteomics approach. Aggregation studies showed that CB platelets effectively aggregate in the presence of thromboxane A2 analogue, collagen, and peptide agonists of the proteinase-activated receptors 1 and 4. In agreement with previous studies, higher concentrations of the agonists were required to initiate aggregation in the CB platelets. Mass spectrometry analysis revealed no significant difference in the expression levels of critical platelet receptors like glycoprotein (GP)Ib, GPV, GPIX, and integrin αIIbβ3. This was confirmed using flow cytometry-based approaches. Gene ontology enrichment analysis revealed that elevated proteins in CB platelets were in particular enriched in proteins contributing to mitochondrial energy metabolism processes. The reduced proteins were enriched in proteins involved in, among others, platelet degranulation and activation. In conclusion, this study reveals that the CB and APB platelets are distinct. In particular, changes were observed for proteins that belong to metabolic and energy generation processes and not for the critical adhesive platelet integrins and glycoproteins.

Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2M using U87MG cells. The results show that α2M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091-2095 and 2155-2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis. These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 domain that have been implicated in lipid binding.

ispartof: Management & Techniek vol:2018 issue:18 pages:5-6 status: published

The vascular endothelium provides a unique interaction plane for plasma proteins and leukocytes in inflammation. The pro-inflammatory cytokines Tumor Necrosis Factor α (TNFα) and interleukin 1β (IL-1β) have a profound effect on endothelial cells, which includes increased levels of adhesion molecules and a disrupted barrier function. To assess the endothelial response to these cytokines at the protein level, we evaluated changes in the whole proteome, cell surface proteome and phosphoproteome after 24 h of cytokine treatment. The effects of TNFα and IL-1β on endothelial cells were strikingly similar and included changes in proteins not previously associated with endothelial inflammation. Temporal profiling revealed time-dependent proteomic changes, including a limited number of early responsive proteins such as adhesion receptors ICAM1 and SELE. In addition, this approach uncovered a greater number of late responsive proteins, including proteins related to self-antigen peptide presentation, and a transient increase in ferritin. Peptide-based cell surface proteomics revealed extensive changes at the cell surface, which were in agreement with the whole proteome. In addition, site-specific changes within ITGA5 and ICAM1 were detected. Combined, our integrated proteomic data provide detailed information on endothelial inflammation, emphasize the role of the extracellular matrix therein, and include potential targets for therapeutic intervention. SIGNIFICANCE: Pro-inflammatory cytokines induce the expression of cell adhesion molecules in vascular endothelial cells. These molecules mediate the adhesion and migration of immune cells across the vessel wall, which is a key process to resolve infections in the underlying tissue. Dysregulation of endothelial inflammation can contribute to vascular diseases and the vascular endothelium is therefore an attractive target to control inflammation. Current strategies targeting endothelial adhesion molecules, including PECAM, CD99, ICAM1 and VCAM1 do not completely prevent transmigration. To identify additional therapeutic targets, we mapped the endothelial proteome after pro-inflammatory cytokine treatment. In addition to the whole proteome, we assessed the surface proteome to focus on cell adhesion molecules, and the phosphoproteome to uncover protein activation states. Here, we present an integrated overview of affected processes which further improves our understanding of endothelial inflammation and may eventually aid in therapeutic intervention of imbalanced inflammation.

Summary Factor (F)VIII is an essential cofactor in the enzymatic coagulation cascade. A defect in the gene encoding for FVIII results in the X-linked inherited bleeding disorder hemophilia A. In the circulation, FVIII is protected from proteolytic degradation and premature clearance by forming a complex with its carrier protein von Willebrand factor (VWF). While the complex of FVIII and VWF involves one of the strongest protein-protein interactions in the human body, it has remained unclear when and where FVIII and VWF first interact. The most widely accepted view is that FVIII and VWF are secreted from different cells and form a complex in the circulation, merely by virtue of their high-affinity interaction. During the last decade however, increasing evidence has been presented that suggests that the human body should also comprise a releasable storage pool of both proteins. In this thesis, we have studied the relationship between high-affinity interaction in the circulation and intracellular co-storage of FVIII and VWF. We have taken a new approach towards this issue by creating fluorescently tagged VWF and FVIII proteins that are established as being deficient in high-affinity interaction. We have demonstrated that FVIII is stored in VWF-containing storage organelles in absence of high-affinity interaction and that the extent of FVIII storage is independent of its affinity for VWF. In addition, we studied the therapeutic benefit of intracellular co-expression of FVIII and VWF in terms of potential treatment options for hemophilia A. We have shown that blood outgrowth endothelial cells can produce high levels of functional FVIII over a prolonged period of time. In addition, we have demonstrated that a substantial amount of FVIII can be stored in VWF-containing storage organelles whereas the majority of FVIII is released in a constitutive manner. From our data, we conclude that the structural requirements that determine high-affinity interaction between FVIII and VWF in the circulation differ from those that determine intracellular co-trafficking. Our data provide a molecular explanation for the fact that hemophilia A and von Willebrand’s disease patients that suffer from impaired complex assembly of FVIII and VWF in the circulation show concomitant increase of FVIII and VWF plasma levels following treatment with desmopressin. The mechanism that determines FVIII storage in VWF-containing granules as well as the therapeutic potential of FVIII expression in VWF producing cells now awaits further exploration.