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The online version of this article contains a supplementary appendix. BackgroundThe endothelial protein C receptor plays an important role within the protein C pathway in regulating coagulation and inflammation. Recently, we described that endothelial protein C receptor can be released in vitro in microparticulate form from primary endothelial cells by exogenous activated protein C. Activated protein C bound to this endothelial protein C receptor retains anticoagulant activity and we hypothesize that this microparticulate endothelial protein C receptor-activated protein C complex can also cleave endothelial protease-activated receptor 1 to modulate inflammation and increase cell survival. Our main objective was, therefore, to study the effect that microparticle-associated endothelial protein C receptor-activated protein C has on endothelial function. Design and MethodsMini-arrays were used and probed with cDNA obtained from endothelial cells after treatment with microparticle-associated endothelial protein C receptor-activated protein C and results were confirmed by real time polymerase chain reaction. The functional relevance of changes at gene level were further analyzed by endothelial apoptosis and permeability assays, in the presence and absence of specific blockade of endothelial protein C receptor, protein C and protease-activated receptor 1. ResultsGene profiling of endothelial cells stimulated by 40 nmol/L activated protein C on microparticles showed significant changes in anti-apoptotic and inflammatory pathways. This was accompanied by protease-activated receptor 1-dependent anti-apoptotic and barrier protective effects, the latter of which also involved sphingosine 1-phosphate receptor and vascular endothelial growth factor receptor-2/ kinase insert domain receptor. Protein C blockade reversed these effects showing specificity for activated protein C on microparticles. Furthermore, confocal microscopy and enzyme-linked immunosorbent assay of plasma obtained from septic patients during recombinant activated protein C treatment showed evidence of their presence in vivo. ConclusionsActivated protein C on microparticle-associated endothelial protein C receptor release can induce protease-activated receptor 1-dependent endothelial effects. The mechanisms underlying barrier protection involve sphingosine 1-phosphate receptor and kinase insert domain receptor.Key words: activated protein C, endothelial protein C receptor, microparticles, protease activated receptor, vascular endothelial growth factor receptor-2.Citation: Pérez-Casal M, Downey C, Cutillas-Moreno B, Zuzel M, Fukudome K, and Toh CH. Microparticle-associated endothelial protein C receptor and the induction of cytoprotective and antiinflammatory effects. Haematologica 2009; 94:387-394. doi:10.3324/haematol.13547 ©2009 Ferrata Storti Foundation. This is an open-access paper. Microparticle-associated endothelial protein C receptor and the induction of cytoprotective and anti-inflammatory effects

IntroductionActivated protein C (APC) induces release of microparticles (MP) from primary physiological cells, which are found in patients undergoing treatment with recombinant human APC (rhAPC) for severe sepsis. We hypothesised that APC on these circulating MPs activate endothelial protease-activated receptor 1 (PAR1) to induce anti-apoptotic and anti-inflammatory properties that can improve patient outcome.MethodsThis was an experimental study on clinical samples in an intensive care setting, and included patients with severe sepsis who fulfilled criteria for treatment with rhAPC. The number of CD13+ MPs from the patients were analysed to determine their origin. They were also quantified for endothelial protein C receptor (EPCR) and APC expression. Clinical relevance of these MPs were ascertained by comparing survival between the group receiving rhAPC (n = 25) and a control group of untreated patients (n = 25). MPs were also incubated with endothelial cells to analyse apoptotic gene expression, cytoprotection and anti-inflammatory effects.ResultsrhAPC treatment induced a significant increase in circulating MP-associated EPCR by flow cytometry (P < 0.05) and by quantitative ELISA (P < 0.005). APC expression also showed significant increases (P < 0.05). Numerically, CD13+ MPs were higher in rhAPC-treated survivors versus non-survivors. However, the number of non-survivors was low and this was not significantly different. APC on MPs was demonstrated to induce anti-apoptotic and endothelial barrier effects through the activation of endothelial PAR1.ConclusionsrhAPC treatment in patients with sepsis significantly increases circulating EPCR + MPs. These MPs were noted to express APC, which has specific anti-apoptotic and anti-inflammatory effects, with a non-significant correlative trend towards survival. This suggests that MPs could disseminate APC function and activate endothelial PAR1 at distal vascular sites.

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surfacn. Lifc Sci 43:68-9. 1988. 2 Lw S. Agc distnbution of erychnxytc population in lalc prcgnancy. G~IICCO~ Obstci lnvcw 3&147-149. IYYO. 3 Lurk S. Changes in age distnbution of erythnxytes dunng pregnancy A longitudinal study Gymcol obstct Invest X141-144. 1993. 80.123-126. 1992 tion. J Lab Clin Med 64.668474.196). 4. ~u r k S. ~a m n D: Life span of crythrocytcs in laic pregnancy Obstet GYIICCOI 5 . Danon D. Marykovsky Y: Dctcrminatm of dcnsiiy distnbuiion of red cell plpula-the Arg 91 Gln mutation. which also results in recessive type 2N VWD. is present in the asymptomatic heterozygous stale in 2% of Dutch individuals 151. Whether the Arg 53 Trp has a similar frequency in Spanish populations is unknown. JM. Alcvy YG. Sadlcr JE: Stmclurc of the gcnc An humun von Willchmnd factor. J B i d Chcm 264:19514. 19H9. Gaucher C. Mcrcicr B, Joricux S. Ou&ir D, Muuricr C ldcntification of Iwo point mutations in the von Willchrund factor sene of three fumilics with chc "Normandy" variunt of von Willchrund diwaw. Br f llacmalol 78:x)6. 1991. Muurier C: von Willcbrrrnd discrcsc mrrlyucruding 1s hacmnphilis A. Thromb Hacmost 67:391, 1992. Eikenboom JCJ. Rcitrmn PII, Pccrlinck KMJ. BriCt E Rcccsnivc inhcrilancc of von Wilkbrund's divcase type 1. Lancet 341:Y82. 19Y3. Mitncuso DJ. TUICY EA. Wc~tficld LA, W o d l NK. Shclton-lnlI*I BB. Sorace Crescentic Glomerulonephrltls During Treatment With Interferona,,

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Activated protein C (APC) administration is now used for treating patients with severe sepsis. We investigated its effect on primary, physiologically relevant cells and demonstrate a novel mechanism of endothelial protein C receptor (EPCR) release from the cell surface. Exposure of human umbilical vein endothelial cells or monocytes to APC (from physiological levels of 0.5 up to 100nM) resulted in the increasing release of EPCR-containing microparticles (EPCR-MP), as demonstrated by confocal microscopy. Further characterisation through flow cytometry showed a concomitant fall in EPCR levels from the cell surface. This release of EPCR could not be inhibited by the metalloproteinase inhibitors 1, 10-phenanthroline or Ro31-9790, unlike soluble EPCR (sEPCR) that is metalloproteinase cleaved at the cell surface following thrombin or pro- inflammatory cytokine stimulation. Western blotting confirmed the molecular weight of EPCR-MP to be identical to the full-length membrane form (49 kD) and different from sEPCR (45 kDa). APC was also bound to EPCR-MP and could be quantified by ELISA using EPCR capture and APC detection by chromogenic substrate, S2366. Using an initial factor Va incubation step followed by a prothrombinase assay, the APC bound to EPCR-MP could significantly reduce thrombin generation. This was abrogated in the presence of excess α1-antitrypsin, an APC inhibitor. By contrast, APC bound to sEPCR could no longer inactivate factor Va. Further characterisation showed the APC induction of EPCR-MP to be time dependent with increasing release over 24 hours, as quantified by ELISA. The phenomenon also required the active site of APC. Neither protein C, heat-inactivated or D-Phe-Pro-Arg-chloromethylketone-blocked APC could induce EPCR-MP formation. Co-incubation with hirudin (6mM) did not alter the APC effect and excluded any role of contaminating thrombin. This novel observation provides new insights into the consequences of APC therapy in the septic patient as well as demonstrating for the first time that there can be 2 circulating forms of EPCR. Unlike sEPCR however, EPCR-MP can facilitate and potentially disseminate the anticoagulant activity of bound APC.

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