Prohibitins are involved in protease‐activated receptor 1‐mediated platelet aggregation

Summary. Background: Prohibitins (PHBs), comprising the two homologous members PHB1 and PHB2, are ubiquitously expressed and highly conserved. The membrane PHBs have been reported to be involved in typhoid fever, obesity, and cancer metastasis. Proteomic studies have revealed the presence of PHBs in human platelets, but the roles of PHBs during platelet aggregation are unknown.Objectives: To investigate the role of PHBs in platelet aggregation. Methods and results: PHB1 and PHB2 were detected on the surfaces of human platelets by flow cytometry and confocal microscopy. The PHBs were distributed in lipid rafts, as determined by sucrose density centrifugation. In addition, the PHBs were associated with protease‐activated receptor 1 (PAR1), as determined by Bm‐TFF2 (a PAR1 agonist)‐affinity chromatography, coimmunoprecipitation, and confocal microscopy. The platelet aggregation, α_IIbβ₃ activation, granular secretion and calcium mobilization stimulated by low concentrations of thrombin (0.05 U mL^?1) or PAR1‐activating peptide (PAR1‐AP) (20 μm ) were reduced or abolished as a result of the blockade of PHBs by anti‐PHB antibodies or their Fab fragments; however, the same results were not obtained with induction by high concentrations of thrombin (0.6 U mL^?1) or protease‐activated receptor 4‐activating peptide (300 μm ). The calcium mobilization in MEG‐01 megakaryocytes stimulated by PAR1‐AP was significantly suppressed by PHB depletion with RNA interference against PHB1 and PHB2. Conclusions: PHBs are localized on the human platelet membrane and are involved in PAR1‐mediated platelet aggregation. Until recently, PHBs were unknown as regulators of PAR1 signaling, and they may be effective targets for antiplatelet therapy.     

Homeostatic effects of coagulation protease‐dependent signaling and protease activated receptors

A homeostatic function of the coagulation system in regard to hemostasis is well established. Homeostasis of blood coagulation depends partially on protease activated receptor (PAR)‐signaling. Beyond coagulation proteases, numerous other soluble and cell‐bound proteases convey cellular effects via PAR signaling. As we learn more about the mechanisms underlying cell‐, tissue‐, and context‐specific PAR signaling, we concurrently gain new insights into physiological and pathophysiological functions of PARs. In this regard, regulation of cell and tissue homeostasis by PAR signaling is an evolving scheme. Akin to the control of blood clotting per se (the fibrin–platelet interaction) coagulation proteases coordinately regulate cell‐ and tissue‐specific functions. This review summarizes recent insights into homeostatic regulation through PAR signaling, focusing on blood coagulation proteases. Considering the common use of drugs altering coagulation protease activity through either broad or targeted inhibitory activities, and the advent of PAR modulating drugs, an in‐depth understanding of the mechanisms through which coagulation proteases and PAR signaling regulate not only hemostasis, but also cell and tissue homeostasis is required.     

Statins prevent tissue factor induction by protease‐activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro

To cite this article: Banfi C, Brioschi M, Lento S, Pirillo A, Galli S, Cosentino S, Tremoli E, Mussoni L. Statins prevent tissue factor induction by protease‐activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro. J Thromb Haemost 2011; 9 : 1608–19. Summary. Background: Protease‐activated receptors (PARs) are G‐protein‐coupled receptors that function in hemostasis and thrombosis, as well as in the inflammatory and proliferative responses triggered by tissue injury. We have previously shown that PAR1 or PAR2 occupancy by specific PAR‐agonist peptides (PAR‐APs) induces tissue factor (TF) expression in human umbilical vein endothelial cells (HUVECs), where TF regulation by PAR1 (but not by PAR2) requires intact endothelial caveolin‐enriched membrane microdomains in which PAR1 and caveolin‐1 associate. Objectives: The aim of this study was to determine the effects of cholesterol‐lowering agents (statins) and cholesterol‐loading lipoprotein on PAR1‐AP‐mediated and PAR2‐AP‐mediated TF induction in HUVECs. Results: Statins completely prevented TF induction by PAR‐APs in an isoprenoid‐independent manner, induced the delocalization of PAR1 from caveolin‐enriched membrane microdomains without affecting PAR1 mRNA, and decreased PAR2 mRNA and protein levels. Statins also prevented PAR‐AP‐mediated extracellular signal‐related kinase 1/2 activation, which is crucial for TF induction. The redistribution of PAR1 is accompanied by the relocation of the membrane microdomain‐associated G‐protein α, caveolin‐1, and Src, which we previously showed to play a key role in signal transduction and TF induction. Conversely, cholesterol loading potently amplified PAR1‐AP‐induced TF, probably as a result of the increased abundance of PAR1 and the Src and G‐protein α signaling molecules in the caveolin‐1‐enriched fraction, without affecting PAR1 mRNA. Conclusions: As PARs have important functions in hemostasis, cancer, thrombosis, and inflammatory processes, our findings that statins prevent TF induction by PAR‐APs altering the membrane localization of PAR1 and the expression of PAR2 suggest that they may provide health benefits other than reducing atherosclerosis.     

Molecular basis of protease‐activated receptor 1 signaling diversity

Protease‐activated receptors (PARs) are a family of highly conserved G protein‐coupled receptors (GPCRs) that respond to extracellular proteases via a unique proteolysis‐dependent activation mechanism. Protease‐activated receptor 1 (PAR1) was the first identified member of the receptor family and plays important roles in hemostasis, inflammation and malignancy. The biology underlying PAR1 signaling by its canonical agonist thrombin is well characterized; however, definition of the mechanistic basis of PAR1 signaling by other proteases, including matrix metalloproteases, activated protein C, plasmin, and activated factors VII and X, remains incompletely understood. In this review, we discuss emerging insights into the molecular bases for “biased” PAR1 signaling, including atypical PAR1 proteolysis, PAR1 heterodimer and coreceptor interactions, PAR1 translocation on the membrane surface, and interactions with different G‐proteins and β‐arrestins upon receptor activation. Moreover, we consider how these new insights into PAR1 signaling have acted to spur development of novel PAR1‐targeted therapeutics that act to inhibit, redirect, or fine‐tune PAR1 signaling output to treat cardiovascular and inflammatory disease. Finally, we discuss some of the key unanswered questions relating to PAR1 biology, in particular how differences in PAR1 proteolysis, signaling intermediate coupling, and engagement with coreceptors and GPCRs combine to mediate the diversity of identified PAR1 signaling outputs.     

Protease‐activated receptor‐1 cleaved at R46 mediates cytoprotective effects

Summary. Background: Activated protein C (aPC) mediates powerful cytoprotective effects through the protease‐activated receptor‐1 (PAR1) that translate into reduced harm in mouse injury models. However, it remains elusive how aPC‐activated PAR1 can mediate cytoprotective effects whereas thrombin activation does the opposite. Objectives: We hypothesized that aPC and thrombin might induce distinct active conformations in PAR1 causing opposing effects. Methods: We analyzed antibody binding to, and cleavage and signalling of PAR1 in either endogenously expressing endothelial or overexpressing 293T cells. Results: In thrombin‐cleaved PAR1 neither the tethered ligand nor the hirudin‐like domain were available for anti‐PAR1 ATAP2 and WEDE15 binding unless the tethered ligand was quenched. In contrast, aPC irreversibly prevented ATAP2 binding while not affecting WEDE15 binding. Reporter constructs with selective glutamine substitutions confirmed R41 as the only thrombin cleavage site in PAR1, whereas aPC preferentially cleaved at R46. Similarly, we report distinct cleavage sites on PAR3, K38 for thrombin and R41 for aPC. A soluble peptide corresponding to R46‐cleaved PAR1 enhanced the endothelial barrier function and reduced staurosporine toxicity in endothelial as well as in 293T cells if PAR1 was expressed. Overexpression of PAR1 variants demonstrated that cleavage at R46 but not R41 is required for cytoprotective aPC signaling. Conclusions: We provide a novel concept on how aPC and thrombin mediate distinct effects. We propose that the enzyme‐specific cleavage sites induce specific conformations which mediate divergent downstream effects. This unexpected model of PAR1 signaling might lead to novel therapeutic options for the treatment of inflammatory diseases.     

Tissue factor non‐coagulant signaling – molecular mechanisms and biological consequences with a focus on cell migration and apoptosis

Tissue factor (TF), a transmembrane glycoprotein, is the main initiator of the blood coagulation cascade. TF is also recognized as a true signaling receptor. There is accumulating evidence that the downstream signaling effects of the TF complexes are transduced by several mechanisms, including: activation of protease‐activated receptor (PAR)‐1 and PAR‐2, and the PAR‐dependent pathways, via the TF cytoplasmic domain and by transactivation of receptor tyrosine kinases. Triggering of signaling cascades such as the mitogen‐activated protein kinase and phosphoinositide 3‐kinase/AKT pathways couples TF to a multitude of functions within the cell, such as proliferation, cell migration, and survival. Thus, TF has a Janus face; on the one hand, it has vital life‐maintaining functions, and on the other it has harmful effects, exemplified by inflammation, the acute coronary syndromes, and cancer. TF mediates a broad spectrum of signaling mechanisms. Learning more about these different mechanisms/pathways will lead to new treatment strategies, which can ultimately be personalized.     

Activated protein C protects against myocardial ischemic/reperfusion injury through AMP‐activated protein kinase signaling

Summary. Background: Activated protein C (APC) is a vitamin K‐dependent plasma serine protease that down‐regulates clotting and inflammatory pathways. It is known that APC exerts a cardioprotective effect by decreasing apoptosis of cardiomyocytes and inhibiting expression of inflammatory mediators after myocardial ischemia. Objectives: The objective of this study was to understand the mechanism of the APC‐mediated cardioprotection against ischemic injury. Methods: Cardioprotective activities of wild‐type APC and two derivatives, having either dramatically reduced anticoagulant activity or lacking signaling activity, were monitored in an acute ischemia/reperfusion injury model in which the left anterior descending coronary artery (LAD) was occluded. Results: APC reduced the myocardial infarct size by a mechanism that was largely independent of its anticoagulant activity. Thus, the non‐anticoagulant APC‐2Cys mutant, but not the non‐signaling APC‐E170A mutant, attenuated myocardial infarct size by EPCR and PAR‐1‐dependent mechanisms. Further studies revealed that APC acts directly on cardiomyocytes to stimulate the AMP‐activated protein kinase (AMPK) signaling pathway. The activation of AMPK by APC ameliorated the post‐ischemic cardiac dysfunction in isolated perfused mouse hearts. Moreover, both APC and APC‐2Cys inhibited production of TNFα and IL‐6 in vivo by attenuating the ischemia/reperfusion‐induced JNK and NF‐κB signaling pathways. Conclusions: APC exerts a cardioprotective function in ischemic/reperfusion injury through modulation of AMPK, NF‐κB and JNK signaling pathways.     

Activated protein C inhibitor for correction of thrombin generation in hemophilia A blood and plasma1

Summary. Background: Replacement therapy for hemophilic patient treatment is costly, because of the high price of pharmacologic products, and is not affordable for the majority of patients in developing countries. Objective: To generate and evaluate low molecular weight agents that could be useful for hemophilia treatment. Methods: Potential agents were generated by synthesizing specific inhibitors [6‐(Lys‐Lys‐Thr‐[homo]Arg)amino‐2‐(Lys[carbobenzoxy]‐Lys[carbobenzoxy]‐O‐benzyl)naphthalenesulfonamide] (PNASN‐1)] for activated protein C (APC) and tested in plasma and fresh blood from hemophilia A patients. Results: In the activated partial thromboplastin time‐based APC resistance assay, PNASN‐1 partially neutralized the effect of APC. In calibrated automated thrombography, PNASN‐1 neutralized the effect of APC on thrombin generation in normal and congenital factor VIII‐deficient plasma (FVIII:C < 1%). The addition of PNASN‐1 to tissue factor‐triggered (5 pm ) contact pathway‐inhibited fresh blood from 15 hemophilia A patients with various degrees of FVIII deficiency (FVIII:C < 1–51%) increased the maximum level of thrombin generated from 78 to 162 nm , which approached that observed in blood from a healthy individual (201 nm ). PNASN‐1 also caused a 47% increase in clot weight in hemophilia A blood. Conclusions: Specific APC inhibitors compensate to a significant extent for FVIII deficiency, and could be used for hemophilia treatment.     

Lipid raft localization regulates the cleavage specificity of protease activated receptor 1 in endothelial cells

Summary.  Background:  The endothelial protein C receptor (EPCR)-dependent cleavage of protease activated receptor 1 (PAR-1) by either activated protein C (APC) or thrombin in lipid rafts initiates protective signaling responses in endothelial cells. Objectives:  To investigate the mechanism by which APC and thrombin interact with and cleave PAR-1 in lipid rafts. Methods:  We constructed two types of PAR-1 cleavage reporter constructs in which a secreted alkaline phosphatase (ALP) was fused to the extracellular domain of PAR-1. The first construct has a transmembrane domain capable of uniformly anchoring the fusion protein to the membrane surface, while the second construct has the recognition sequence for targeting the fusion protein to lipid rafts/caveolae in transfected cells. Results:  Both APC and the Gla-domainless (GD)-APC cleaved the PAR-1 exodomain with similar efficiency in HUVECs transfected with the first construct. Unlike APC, GD-APC did not cleave PAR-1 in cells transfected with the second construct; however, prior treatment of cells with S195A mutants of either protein C or thrombin led to the GD-APC cleavage of PAR-1 with a comparable or higher catalytic efficiency. The same results were obtained if the cellular signaling properties of APC and GD-APC were monitored in the TNF-α-induced endothelial cell apoptosis and permeability assays. Conclusions:  The lipid raft localization renders the scissile bond of the PAR-1 exodomain unavailable for interaction with coagulation proteases. The binding of either the Gla-domain of protein C to EPCR or exosite-1 of thrombin to the C-terminal hirudin-like sequence of PAR-1 changes the membrane localization and/or the conformation of the PAR-1 exodomain to facilitate its recognition and subsequent cleavage by these proteases.