Likui Yang

Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells

Ever-increasing evidence in the literature suggests that the antiinflammatory and cytoprotective properties of activated protein C (APC) are mediated through its endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on endothelial cells. However, recent results monitoring the cleavage rate of PAR-1 on human umbilical vein endothelial cells, transfected with an alkaline phosphatase–PAR-1 fusion reporter construct, have indicated that the catalytic activity of thrombin toward PAR-1 is several orders of magnitude higher than that of APC. Because thrombin is required for generation of APC, and because it also functions in the proinflammatory pathways through the activation of PAR-1, it has been difficult to understand how APC can elicit protective cellular responses through the activation of PAR-1 when thrombin is present. In this study we provide a plausible answer to this question by demonstrating that the critical receptors required for both protein C activation (thrombomodulin and EPCR) and APC cellular signaling (EPCR and PAR-1) pathways are colocalized in the membrane lipid rafts in endothelial cells. We further show that the APC cleavage of PAR-1 on cells transfected with a PAR-1 cleavage reporter construct is not sensitive to the cofactor function of EPCR. Thus, the colocalization of EPCR and PAR-1 in lipid rafts is a key requirement for the cellular signaling activity of APC. Thrombomodulin colocalization with these receptors on the same membrane microdomain can also recruit thrombin to activate the EPCR-bound protein C, thereby eliciting PAR-1 signaling events that are involved in the APC protective pathways.

Engineering a Disulfide Bond to Stabilize the Calcium-binding Loop of Activated Protein C Eliminates Its Anticoagulant but Not Its Protective Signaling Properties

In addition to an anticoagulant activity, activated protein C (APC) also exhibits anti-inflammatory and cytoprotective properties. These properties may contribute to the beneficial effect of APC in treating severe sepsis patients. A higher incidence of bleeding because of its anticoagulant function has been found to be a major drawback of APC as an effective anti-inflammatory drug. In this study, we have prepared a protein C variant in which an engineered disulfide bond between two β-sheets stabilized the functionally critical Ca2+-binding 70-80 loop of the molecule. The 70-80 loop of this mutant no longer bound Ca2+, and the activation of the mutant by thrombin was enhanced 60-80-fold independently of thrombomodulin. The anticoagulant activity of the activated protein C mutant was nearly eliminated as determined by a plasma-based clotting assay. However, the endothelial protein C receptor- and protease-activated receptor-1-dependent protective signaling properties of the mutant were minimally altered as determined by staurosporine-induced endothelial cell apoptosis, thrombin-induced endothelial cell permeability, and tumor necrosis-α-mediated neutrophil adhesion and migration assays. These results suggest that the mutant lost its ability to interact with the procoagulant cofactors but not with the protective signaling molecules; thus this mutant provides an important tool for in vivo studies to examine the role of anticoagulant versus anti-inflammatory function of activated protein C.

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.

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.

Identification of a specific exosite on activated protein C for interaction with protease-activated receptor 1.

Activated protein C (APC) is a vitamin K-dependent plasma serine protease which down-regulates the clotting cascade by inactivating procoagulant factors Va and VIIIa by limited proteolysis. In addition to its anticoagulant effect, APC also exhibits cytoprotective and antiinflammatory activity through the endothelial protein C receptor-dependent cleavage of protease activated receptor 1 (PAR-1) on endothelial cells. Recent mutagenesis data have indicated that the basic residues of two surface loops including those on 39 and the Ca2+-binding 70–80 loops constitute interactive sites for both factors Va and VIIIa, thereby mediating the interaction of APC specifically with these procoagulant cofactors. The basic residues of both loops have been discovered to be dispensable for the interaction of APC with PAR-1. It is not known if a similar exosite-dependent interaction contributes to the specificity of APC recognition of PAR-1 on endothelial cells. In this study, we have identified two acidic residues on helix-162 (Glu-167 and Glu-170) on the protease domain of APC which are required for the protease interaction with PAR-1, but not for its interaction with the procoagulant cofactors. Thus, the substitution of either Glu-167 or Glu-170 with Ala eliminated the cytoprotective signaling properties of APC without affecting its anticoagulant activity. These mutants provide useful tools for initiating in vivo studies to understand the extent to which the anticoagulant versus antiinflammatory activity of APC contributes to its beneficial effect in treating severe sepsis.

Thrombomodulin allosterically modulates the activity of the anticoagulant thrombin.

Exosite 1 of thrombin consists of a cluster of basic residues (Arg-35, Lys-36, Arg-67, Lys-70, Arg-73, Arg-75, and Arg-77 in chymotrypsinogen numbering) that play key roles in the function of thrombin. Structural data suggest that the side chain of Arg-35 projects toward the active site pocket of thrombin, but all other residues are poised to interact with thrombomodulin (TM). To study the role of these residues in TM-mediated protein C (PC) activation by thrombin, a charge reversal mutagenesis approach was used to replace these residues with a Glu in separate constructs. The catalytic properties of the mutants toward PC were analyzed in both the absence and presence of TM and Ca2+. It was discovered that, with the exception of the Arg-67 and Lys-70 mutants, all other mutants activated PC with similar maximum rate constants in the presence of a saturating concentration of TM and Ca2+, although their affinity for interaction with TM was markedly impaired. The catalytic properties of the Arg-35 mutant were changed so that PC activation by the mutant no longer required Ca2+ in the presence of TM, but, instead, it was accelerated by EDTA. Moreover, the activity of this mutant toward PC was improved ≈25-fold independent of TM. These results suggest that Arg-35 is responsible for the Ca2+ dependence of PC activation by the thrombin–TM complex and that a function for TM in the activation complex is the allosteric alleviation of the inhibitory interaction of Arg-35 with the substrate.

Polyphosphate amplifies proinflammatory responses of nuclear proteins through interaction with receptor for advanced glycation end products and P2Y1 purinergic receptor

The extracellular nuclear proteins, histone H4 (H4) and high mobility group box 1 (HMGB1), released by injured cells during the activation of inflammation and coagulation pathways provoke potent inflammatory responses through interaction with pathogen-related pattern recognition receptors (ie, Toll-like receptors [TLRs] and receptor for advanced glycation end products [RAGE]) present on vascular and innate immune cells. Inorganic polyphosphate (polyP) has emerged as a key modulator of coagulation and inflammation. Here, we demonstrate that polyP binds to both H4 and HMGB1 with high affinity, thereby dramatically potentiating their proinflammatory properties in cellular and in vivo models. By using small interfering RNA knockdowns, pharmacologic inhibitors and extracellular domains of the receptors TLR2, TLR4, RAGE, and P2Y1 as competitive inhibitors, we demonstrate that polyP amplifies H4- and HMGB1-mediated inflammatory signaling in human umbilical vein endothelial cells specifically through interaction with the RAGE and P2Y1 receptors, thereby eliciting intracellular Ca(2+) release. Finally, we demonstrate that the natural anticoagulant protease, activated protein C, potently inhibits polyP-mediated proinflammatory effects of both nuclear proteins in cellular and in vivo systems.

Protein Z-dependent Protease Inhibitor Binds to the C-terminal Domain of Protein Z

Protein Z (PZ) is a multidomain vitamin K-dependent plasma protein that functions as a cofactor to promote the inactivation of factor Xa (fXa) by PZ-dependent protease inhibitor (ZPI) by three orders of magnitude. To understand the mechanism by which PZ improves the reactivity of fXa with ZPI, we expressed wild-type PZ, PZ lacking the γ-carboxyglutamic acid domain (GD-PZ), and a chimeric PZ mutant in which both Gla and EGF-like domains of the molecule were substituted with identical domains of fXa. The ZPI binding and the cofactor function of the PZ derivatives were characterized in both binding and kinetic assays. The binding assay indicated that all PZ derivatives interact with ZPI with a similar dissociation constant (KD) of ∼7 nm. However, the apparent KD for the chimeric PZ-mediated ZPI inhibition of fXa was elevated 6-fold on PC/PS vesicles and its capacity to function as a cofactor to accelerate the ZPI inhibition of fXa was also decreased 6-fold. The cofactor activity of GD-PZ was dramatically impaired; however, the deletion mutant exhibited a normal cofactor function in solution. A chimeric activated protein C mutant containing the Gla domain of fXa was susceptible to inhibition by ZPI in the presence of PZ. These results suggest that: (i) the ZPI interactive site of PZ is located within the C-terminal domain of the cofactor and (ii) a specific interaction between the Gla domains of PZ and fXa contributes ∼6-fold to the acceleration of the ZPI inhibition of fXa on phospholipid membranes.

Identification of Factor Xa Residues Critical for Interaction with Protein Z-dependent Protease Inhibitor: Both Active-site and Exosite Interactions Are Required for Inhibition

Protein Z-dependent protease inhibitor (ZPI) is a plasma serpin, which can rapidly inactivate factor Xa (fXa) in the presence of protein Z (PZ), negatively charged phospholipids, and Ca2+. To investigate the mechanism by which ZPI inactivates fXa, we expressed the serpin in mammalian cells and characterized its reactivity with both wild-type and selected mutants of fXa that 1) contained substitutions in the autolysis loop and the heparin binding exosite, 2) lacked the first EGF-like domain (fXa-des-EGF-1), or 3) contained the Gla domain of protein C (fXa/PC-Gla). Inhibition studies in both the presence and absence of PZ revealed that Arg-143, Lys-147, and Arg-154 of the autolysis loop and Lys-96, Lys-169, and Lys-236 of the heparin binding exosite are required for recognition of ZPI, with Arg-143 being essential for the interaction. Similar studies with fXa-des-EGF-1 and fXa/PC-Gla suggested that protein-protein interaction with either the Gla or the EGF-1 domain may not play a dominant role in the PZ-dependent recognition of fXa by the serpin on phospholipid vesicles. Further studies showed that an inactive Ser-195 to Ala mutant of fXa effectively competes with wild-type fXa for binding to the non-serpin inhibitors tissue factor pathway inhibitor and recombinant tick anticoagulant peptide, but does not compete for binding to ZPI. This suggests that the catalytic residue of fXa is required for interaction with ZPI.

Factor X/Xa Elicits Protective Signaling Responses in Endothelial Cells Directly via PAR-2 and Indirectly via Endothelial Protein C Receptor-dependent Recruitment of PAR-1

We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.