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Vascular Endothelial Cells Produce Coagulation Factors That Control Their Growth via Joint Protease-Activated Receptor and C5a Receptor 1 (CD88) Signaling

2022; Elsevier BV; Volume: 192; Issue: 2 Linguagem: Inglês

10.1016/j.ajpath.2021.09.011

1525-2191

Devin Cao, Michael G. Strainic, Daniel Counihan, Shiva Sridar, Fengqi An, Wasim Hussain, Alvin H. Schmaier, Marvin T. Nieman, M. Edward Medof,

Apelin-related biomedical research

As per the classical view of the coagulation system, it functions solely in plasma to maintain hemostasis. An experimental approach modeling vascular reconstitution was used to show that vascular endothelial cells (ECs) endogenously synthesize coagulation factors during angiogenesis. Intracellular thrombin generated from this synthesis promotes the mitotic function of vascular endothelial cell growth factor A (VEGF-A). The thrombin concurrently cleaves C5a from EC-synthesized complement component C5 and unmasks the tethered ligand for EC-expressed protease-activated receptor 4 (PAR4). The two ligands jointly trigger EC C5a receptor-1 (C5ar1) and PAR4 signaling, which together promote VEGF receptor 2 growth signaling. C5ar1 is functionally associated with PAR4, enabling C5a or thrombin to elicit Gαi and/or Gαq signaling. EC coagulation factor and EC complement component synthesis concurrently down-regulate with contact inhibition. The connection of these processes with VEGF receptor 2 signaling provides new insights into mechanisms underlying angiogenesis. Knowledge of endogenous coagulation factor/complement component synthesis and joint PAR4/C5ar1 signaling could be applied to other cell types. As per the classical view of the coagulation system, it functions solely in plasma to maintain hemostasis. An experimental approach modeling vascular reconstitution was used to show that vascular endothelial cells (ECs) endogenously synthesize coagulation factors during angiogenesis. Intracellular thrombin generated from this synthesis promotes the mitotic function of vascular endothelial cell growth factor A (VEGF-A). The thrombin concurrently cleaves C5a from EC-synthesized complement component C5 and unmasks the tethered ligand for EC-expressed protease-activated receptor 4 (PAR4). The two ligands jointly trigger EC C5a receptor-1 (C5ar1) and PAR4 signaling, which together promote VEGF receptor 2 growth signaling. C5ar1 is functionally associated with PAR4, enabling C5a or thrombin to elicit Gαi and/or Gαq signaling. EC coagulation factor and EC complement component synthesis concurrently down-regulate with contact inhibition. The connection of these processes with VEGF receptor 2 signaling provides new insights into mechanisms underlying angiogenesis. Knowledge of endogenous coagulation factor/complement component synthesis and joint PAR4/C5ar1 signaling could be applied to other cell types. The complement and coagulation systems have been envisioned as residing exclusively in the plasma, deriving solely from the liver, and functioning independently of each other in innate immunity and in hemostasis, respectively. Because of this, although effects of complement activation fragments on the coagulation cascade and vice versa have been reported, they have been attributed to cross talk and regarded as being modulatory rather than integral to one pathway or the other.1Leibundgut G. Lee J.H. Strauss B.H. Segev A. Tsimikas S. Acute and long-term effect of percutaneous coronary intervention on serially-measured oxidative, inflammatory, and coagulation biomarkers in patients with stable angina.J Thromb Thrombolysis. 2016; 41: 569-580Google Scholar For example, although the immediate response to trauma includes both proteolysis of fibrinogen to form fibrin and complement activation, the two processes have been regarded as being unconnected and serving unrelated purposes. Both coagulation and complement system products exert effects on vascular endothelial cells (ECs). Thrombin is connected to EC tissue factor production and procoagulant surface protein expression during coagulation.2Arachiche A. Mumaw M.M. de la Fuente M. Nieman M.T. Protease-activated receptor 1 (PAR1) and PAR4 heterodimers are required for PAR1-enhanced cleavage of PAR4 by alpha-thrombin.J Biol Chem. 2013; 288: 32553-32562Google Scholar, 3Lin H. Liu A.P. Smith T.H. Trejo J. Cofactoring and dimerization of proteinase-activated receptors.Pharmacol Rev. 2013; 65: 1198-1213Google Scholar, 4Nakanishi-Matsui M. Zheng Y.W. Sulciner D.J. Weiss E.J. Ludeman M.J. Coughlin S.R. PAR3 is a cofactor for PAR4 activation by thrombin.Nature. 2000; 404: 609-613Google Scholar, 5Rezaie A.R. Protease-activated receptor signalling by coagulation proteases in endothelial cells.Thromb Haemostasis. 2014; 112: 876-882Google Scholar Complement activation fragments C3a and C5a are linked to EC integrin expression and proinflammatory cytokine production during inflammation.5Rezaie A.R. Protease-activated receptor signalling by coagulation proteases in endothelial cells.Thromb Haemostasis. 2014; 112: 876-882Google Scholar Thrombin confers its modulatory effects on ECs via protease-activated receptors (PARs). Its cleavage of the N-termini of these G-protein–coupled receptors (GPCRs) unmasks their tethered ligands, thereby inducing their signaling. Four PAR family members (PAR1 to PAR4) exist, and PAR1 and PAR4, which frequently function cooperatively, are expressed on ECs.2Arachiche A. Mumaw M.M. de la Fuente M. Nieman M.T. Protease-activated receptor 1 (PAR1) and PAR4 heterodimers are required for PAR1-enhanced cleavage of PAR4 by alpha-thrombin.J Biol Chem. 2013; 288: 32553-32562Google Scholar,3Lin H. Liu A.P. Smith T.H. Trejo J. Cofactoring and dimerization of proteinase-activated receptors.Pharmacol Rev. 2013; 65: 1198-1213Google Scholar,6Duvernay M.T. Temple K.J. Maeng J.G. Blobaum A.L. Stauffer S.R. Lindsley C.W. Hamm H.E. Contributions of protease-activated receptors PAR1 and PAR4 to thrombin-induced GPIIbIIIa activation in human platelets.Mol Pharmacol. 2017; 91: 39-47Google Scholar,7Zhao P. Metcalf M. Bunnett N.W. Biased signaling of protease-activated receptors.Front Endocrinol. 2014; 5: 67Google Scholar In addition to their expression on ECs, PARs are widely expressed on other cell types and implicated in many cell functions.5Rezaie A.R. Protease-activated receptor signalling by coagulation proteases in endothelial cells.Thromb Haemostasis. 2014; 112: 876-882Google Scholar Although C5a receptor-1 (C5ar1) was long thought to be strictly limited to myeloid phagocytic cells,8Jagels M.A. Travis J. Potempa J. Pike R. Hugli T.E. Proteolytic inactivation of the leukocyte C5a receptor by proteinases derived from Porphyromonas gingivalis.Infect Immunity. 1996; 64: 1984-1991Google Scholar, 9Proctor L.M. Arumugam T.V. Shiels I. Reid R.C. Fairlie D.P. Taylor S.M. Comparative anti-inflammatory activities of antagonists to C3a and C5a receptors in a rat model of intestinal ischaemia/reperfusion injury.Br J Pharmacol. 2004; 142: 756-764Google Scholar, 10Gerard C. Gerard N.P. C5A anaphylatoxin and its seven transmembrane-segment receptor.Annu Rev Immunol. 1994; 12: 775-808Google Scholar it now is known to function in adaptive immune cells11Paiano J. Harland M. Strainic M.G. Nedrud J. Hussain W. Medof M.E. Follicular B2 cell activation and class switch recombination depend on autocrine C3ar1/C5ar1 signaling in B2 cells.J Immunol. 2019; 203: 379-388Google Scholar, 12Strainic M.G. Shevach E.M. An F. Lin F. Medof M.E. Absence of signaling into CD4(+) cells via C3aR and C5aR enables autoinductive TGF-beta1 signaling and induction of Foxp3(+) regulatory T cells.Nat Immunol. 2013; 14: 162-171Google Scholar, 13Lalli P.N. Strainic M.G. Yang M. Lin F. Medof M.E. Heeger P.S. Locally produced C5a binds to T cell-expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis.Blood. 2008; 112: 1759-1766Google Scholar, 14Strainic M.G. Liu J. Huang D. An F. Lalli P.N. Muqim N. Shapiro V.S. Dubyak G.R. Heeger P.S. Medof M.E. Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells.Immunity. 2008; 28: 425-435Google Scholar as well as other cell types.15O'Barr S.A. Caguioa J. Gruol D. Perkins G. Ember J.A. Hugli T. Cooper N.R. Neuronal expression of a functional receptor for the C5a complement activation fragment.J Immunol. 2001; 166: 4154-4162Google Scholar Recent studies16Hwang M.S. Strainic M.G. Pohlmann E. Kim H. Pluskota E. Ramirez-Bergeron D.L. Plow E.F. Medof M.E. VEGFR2 survival and mitotic signaling depends on joint activation of associated C3ar1/C5ar1 and IL-6R-gp130.J Cell Sci. 2019; 132: jcs219352Google Scholar have shown that EC C5 synthesis and C5a production are interconnected with vascular endothelial cell growth factor A (VEGF-A) function. Thrombin, however, has been regarded as functioning solely in the context of coagulation in plasma. Thrombin is generated from prothrombin in a complex termed prothrombinase that is produced by factor Va binding to factor Xa and factor II (FII) on the EC surface. Factor Xa generation, leading to the assembly of prothrombinase, is induced by the actions of factor IXa and factor VIIIa or factor VIIa and tissue factor. These processes are tightly regulated by homeostasis. The same is true for assembly of the canonical C5 convertases (C4b2a3b and C3bBbC3b) that gives rise to C5a. A previous study17Huber-Lang M. Sarma J.V. Zetoune F.S. Rittirsch D. Neff T.A. McGuire S.R. Lambris J.D. Warner R.L. Flierl M.A. Hoesel L.M. Gebhard F. Younger J.G. Drouin S.M. Wetsel R.A. Ward P.A. Generation of C5a in the absence of C3: a new complement activation pathway.Nat Med. 2006; 12: 682-687Google Scholar linked plasma C5a generation with thrombin in a mouse model of immune complex–mediated lung injury. Although thrombin cleaved purified complement component C5 to C5a in vitro, the authors proposed that the process constituted a compensatory mechanism for C5 cleavage connected with increased thrombin generation in plasma during disease. A role for this process in a nonhemostatic physiological process was not proposed. C5a was recently linked with physiological VEGF-A growth induction. However, based on prior immune cell activation studies, it was theorized to be generated by the conventional alternative pathway C5 convertase C3bBbC3b. These studies demonstrated that interacting dendritic cell–CD4+ cell partners each generate C5a (and C3a) from complement components that are endogenously synthesized by both cell types,12Strainic M.G. Shevach E.M. An F. Lin F. Medof M.E. Absence of signaling into CD4(+) cells via C3aR and C5aR enables autoinductive TGF-beta1 signaling and induction of Foxp3(+) regulatory T cells.Nat Immunol. 2013; 14: 162-171Google Scholar, 14Strainic M.G. Liu J. Huang D. An F. Lalli P.N. Muqim N. Shapiro V.S. Dubyak G.R. Heeger P.S. Medof M.E. Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells.Immunity. 2008; 28: 425-435Google Scholar and that the GPCR signaling plays an integral role in shaping the T-cell response. Analogous to the findings in immune cells, the studies of C5a production by ECs showed that autocrine C5ar1 signaling plays a requisite role in vascular endothelial cell growth factor receptor 2 (VEGFR2) mitotic signaling. It promotes VEGFR2 autophosphorylation, downstream growth signaling via the canonical VEGFR2 cascades, and EC transition through the cell cycle. The findings of local complement production in ECs16Hwang M.S. Strainic M.G. Pohlmann E. Kim H. Pluskota E. Ramirez-Bergeron D.L. Plow E.F. Medof M.E. VEGFR2 survival and mitotic signaling depends on joint activation of associated C3ar1/C5ar1 and IL-6R-gp130.J Cell Sci. 2019; 132: jcs219352Google Scholar together with the observation17Huber-Lang M. Sarma J.V. Zetoune F.S. Rittirsch D. Neff T.A. McGuire S.R. Lambris J.D. Warner R.L. Flierl M.A. Hoesel L.M. Gebhard F. Younger J.G. Drouin S.M. Wetsel R.A. Ward P.A. Generation of C5a in the absence of C3: a new complement activation pathway.Nat Med. 2006; 12: 682-687Google Scholar that thrombin can cleave C5a from C5 in vitro prompted the hypothesis that ECs might endogenously produce coagulation factors in addition to complement components, and that EC produced thrombin might participate in EC C5a generation. Therefore, this study investigated whether endogenous thrombin generation, if locally produced within ECs, might be mechanistically linked with endogenous C5a generation in ECs, and whether these linkages might participate in EC proliferation and angiogenesis. The sources of cells and major reagents used in each assay are given with each assay method. All cells and reagents used are listed together in detail in the major resource table (Table 1).Table 1Major ResourcesAnimals (in vivo studies)SpeciesVendor or sourceBackground strainSexMouseJackson Laboratories, Bar Harbor, MEC57BL/6M/FMouseC. Gerard, Children's Hospital, Boston, MAC57BL/6, C5ar1−/−M/FMouseJackson LaboratoriesPAR1−/−M/FMouseMutant Mouse Resource & Research Centers, University of California, Davis, CAPAR3−/−PAR4−/−M/FAnimal breedingParentSpeciesVendor or sourceBackground strainOther informationMMouseJackson LaboratoriesC57BL/6FMouseJackson LaboratoriesC57BL/6MMouseC. GerardBALB/c, C5ar1−/−Crossbred for 12 generationsFMouseC. GerardC57BL/6Crossbred for 12 generationsMMouseC. GerardC57BL/6, C5ar1−/−FMouseC. GerardC57BL/6, C5ar1−/−MMouseMutant Mouse Resource & Research CentersC57BL/6, PAR1−/−FMouseMutant Mouse Resource & Research CentersC57BL/6, PAR1−/−MMouseMutant Mouse Resource & Research CentersC57BL/6, PAR3−/−, PAR4−/−FMouseMutant Mouse Resource & Research CentersC57BL/6, PAR3−/−, PAR4−/−AntibodiesTarget antigenVendor or sourceCatalog no.Working concentrationLot no. (preferred but not required)Human/mouse FIIThermoFisher, Carlsbad, CAPA1-430401 μmol/mLSI2444095Mouse FVBioss, Woburn, MABs-1040R5 μg/mLAD121701Mouse FVIIBiossBs-4846R5 μg/mLYE1022WMouse FXBiossBs-9501R5 μg/mL9B26M7Human/mouse C5aRSanta Cruz Biotechnologies, Dallas, TXSC-53795 PE2 μg/mLC1810Mouse C5aBD Biosciences, Franklin Lakes, NJ5580275 μg/mLHuman C5aR&D Systems, Minneapolis, MNAF20375 μg/mLHuman thrombinThermoFisherHYB 109-04-025 μg/mLUI2843443Mouse/human PAR1BD Biosciences6115225 μg/mL6013888Mouse/human PAR4Abcam, Cambridge, UKAb57875 μg/mLGR267332-5Human/mouse VEGFR2Cell Signalling Technologies, Danvers, MA24790.1 μg/mL18ReagentsReagentVendor or sourceCatalog no.Working concentrationLot no. (preferred but not required)C5a receptor antagonist, W-54011Calbiochem, Torrey Pines, CA23441510 ng/mLVorapaxar (PAR1 antagonist)Axon Medchem, Reston, VA1755Batch no. 2TFLLRN (PAR1 agonist)Tocris Biosciences, Minneapolis, MN14640.01–100 ng/mLAY-NH2 (PAR4 agonist)Tocris Biosciences14870.001–10 ng/mL10ABivalirudin trifluoroacetate saltSigma, St. Louis, MOSML105125 μg/mL064M4731VAS252424 (PI-3Kγ inhibitor)Tocris Biosciences367110 μmol/mL2A/153705SU5416 (VEGFR2 antagonist)Tocris Biosciences303710 μmol/mLHuman plasmaPlasmaSourcePlasma deficient in FIIGeorge King Biomedical, Overland Park, KSPlasma deficient in FVGeorge King BiomedicalPlasma deficient in FVIIGeorge King BiomedicalPlasma deficient in FXGeorge King BiomedicalsiRNATargetVendor or sourceCatalog no.IDWorking concentration, nmol/LHuman PAR1ThermoFisherAM1670814625930Human PAR4ThermoFisher4392420s19497230Cultured cellsNameVendor or sourceSex (F, M, or unknown)bEnd.3ATCC, Manassas, VAUnknownPrimary aortic endothelial cells; normal, human (HAECs)ATCCUnknownHUV-EC-C (HUVECs)ATCCUnknown293 (HEK-293)ATCCUnknownF, female; M, male; C5ar1, C5a receptor-1; FII, factor II; FV, factor V; FVII, factor VII; FX, factor X; HAEC, human aortic endothelial cell; HUVEC, human umbilical vein endothelial cell; ID, identifier; PAR, protease-activated receptor; VEGFR2, vascular endothelial cell growth factor receptor 2. Open table in a new tab F, female; M, male; C5ar1, C5a receptor-1; FII, factor II; FV, factor V; FVII, factor VII; FX, factor X; HAEC, human aortic endothelial cell; HUVEC, human umbilical vein endothelial cell; ID, identifier; PAR, protease-activated receptor; VEGFR2, vascular endothelial cell growth factor receptor 2. Murine bEnd 3 and NIH-3T3 cell lines were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were maintained in EBM-2 media containing BulletKit (Lonza, Basel, Switzerland) without added vitamin K. Except where indicated, cells were seeded at 2.5 × 104 per well in 24-well plates in complete media, after which they were transferred to media containing 0.5% serum and treated as described in the figure legends. The concentrations of C5a, C5ar1-A,12Strainic M.G. Shevach E.M. An F. Lin F. Medof M.E. Absence of signaling into CD4(+) cells via C3aR and C5aR enables autoinductive TGF-beta1 signaling and induction of Foxp3(+) regulatory T cells.Nat Immunol. 2013; 14: 162-171Google Scholar,14Strainic M.G. Liu J. Huang D. An F. Lalli P.N. Muqim N. Shapiro V.S. Dubyak G.R. Heeger P.S. Medof M.E. Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells.Immunity. 2008; 28: 425-435Google Scholar,16Hwang M.S. Strainic M.G. Pohlmann E. Kim H. Pluskota E. Ramirez-Bergeron D.L. Plow E.F. Medof M.E. VEGFR2 survival and mitotic signaling depends on joint activation of associated C3ar1/C5ar1 and IL-6R-gp130.J Cell Sci. 2019; 132: jcs219352Google Scholar,18Strainic M.G. Liu J. An F. Bailey E. Esposito A. Hamann J. Heeger P.S. Medof M.E. CD55 is essential for CD103(+) dendritic cell tolerogenic responses that protect against autoimmunity.Am J Pathol. 2019; 189: 1386-1401Google Scholar thrombin,19Forbes G.L. Merkulova A. Pinheiro A. Lee J. Zeng P. Abdalian S. Walker A.Y. Wnek G.E. Schmaier A.H. Poly (acrylic acid) (PAA) is a contact system activator with properties to stop hemorrhage.Thromb Res. 2020; 193: 142-145Google Scholar, 20Schmaier A.H. A novel antithrombotic mechanism mediated by the receptors of the kallikrein/kinin and renin-angiotensin systems.Front Med (Lausanne). 2016; 3: 61Google Scholar, 21Stavrou E.X. Fang C. Merkulova A. Alhalabi O. Grobe N. Antoniak S. Mackman N. Schmaier A.H. Reduced thrombosis in Klkb1-/- mice is mediated by increased Mas receptor, prostacyclin, Sirt1, and KLF4 and decreased tissue factor.Blood. 2015; 125: 710-719Google Scholar and PAR1 and PAR4 agonists and antagonists22Gupta S. Konradt C. Corken A. Ware J. Nieswandt B. Di Paola J. Yu M. Wang D. Nieman M.T. Whiteheart S.W. Brass L.F. Hemostasis vs. homeostasis: platelets are essential for preserving vascular barrier function in the absence of injury or inflammation.Proc Natl Acad Sci U S A. 2020; 117: 24316-24325Google Scholar,23Nieman M.T. RAPid signaling in platelets.Blood. 2018; 132: 1864-1865Google Scholar were chosen on the basis of previous studies and the manufacturer's directions. Cell numbers were counted manually. Real-time quantitative PCR was performed as described.12Strainic M.G. Shevach E.M. An F. Lin F. Medof M.E. Absence of signaling into CD4(+) cells via C3aR and C5aR enables autoinductive TGF-beta1 signaling and induction of Foxp3(+) regulatory T cells.Nat Immunol. 2013; 14: 162-171Google Scholar Data are given as fold increases relative to basal levels corrected for actin or glyceraldehyde-3-phosphate dehydrogenase. A total of 100 μL of culture supernatant was incubated at 37°C for 5 minutes with 100 μL of plasma deficient in FII, factor V (FV), factor VII (FVII), or factor X (FX; George King Biomedical, Overland Park, KS). A total of 100 μL of Phospoplastin RL (R2 Diagnostics, South Bend, IN) was then added, and time to coagulation was measured while continuously mixing. Percentage clotting activity corresponds to the dilution of pooled human plasma (George King Biomedical), yielding the same clotting time. Enzyme-linked immunosorbent assays for C5a and flow cytometry for phosphorylated STAT3 were performed as described.12Strainic M.G. Shevach E.M. An F. Lin F. Medof M.E. Absence of signaling into CD4(+) cells via C3aR and C5aR enables autoinductive TGF-beta1 signaling and induction of Foxp3(+) regulatory T cells.Nat Immunol. 2013; 14: 162-171Google Scholar Intracellular staining was performed following permeabilization, as in past studies,24Amthauer R. Kodukula K. Brink L. Udenfriend S. Phosphatidylinositol-glycan (PI-G)-anchored membrane proteins: requirement of ATP and GTP for translation-independent COOH-terminal processing.Proc Natl Acad Sci U S A. 1992; 89: 6124-6128Google Scholar and the intracellular signal was calculated following subtraction of the extracellular signal. The assay was performed using the glycogen synthase kinase (GSK)-3β Activity Assay kit (Sigma-Aldrich, St. Louis, MO; catalog number CS0990), per the manufacturer's protocol. Thoracic aortae were obtained from 8- to 14-week–old mice following perfusion with Ringer’s solution. Following removal of fibroadipose tissue, approximately 1-mm segments were suspended in a 6-mm petri dish containing complete EGM-2 (Lonza) with reduced growth factor Matrigel (Corning, Tewskbury, MA). Aortic ring sprouts were imaged on days 6 to 8, and sprout areas were determined by morphometric analysis using MetaMorph (Molecular Devices, San Jose, CA) and a Leica DMI6000 microscope (Wetzlar, Germany). Reduced growth factor Matrigel (as used in sprouting assays; 500 μL) supplemented with heparin (60 U/mL) and VEGF-A (300 ng/mL) was injected subcutaneously into the lower abdominal flank of anesthetized 8- to 14-week–old wild-type (WT) C57BL/6 mice. Ten days after injection, recovered Matrigel plugs were fixed in formalin, embedded in OCT compound, and sectioned. Sections were stained for different coagulation factors [anti-FII (Aviva Systems Biology Corp., San Diego, CA; ARP41757-P050; lot number QC12431), anti-FV (Bioss, Woburn, MA; bs-1040R; lot number AD121701), anti-FVII (Bioss; bs-4846R; lot number YE1022W), or anti-FX (Bioss; bs-9501R; lot number 9B26M7)] followed by AF546-labeled anti-rabbit. After coagulation protein staining, cells were counterstained with AF488-labeled anti-CD31 (clone 390; BD Biosciences, East Rutherford, NJ), mounted in ProLong Gold anitfade reagent with DAPI (Molecular Probes, Eugene, OR; catalog number P36935), and visualized by confocal microscopy. Images were collected using a 63× (1.4 numerical aperture) oil immersion objective on a Leica HyVolution SP8 inverted confocal microscope. Resulting images were subjected to deconvolution using Hyugens Professional (Hilversum, The Netherlands). For C5ar1 and PAR4 colocalization assays, bEnd.3 cells were grown overnight on ibiTreat 8 chamber μ-Slides (ibidi, Martinsried, Germany). The slides were washed three times with 1× phosphate-buffered saline and fixed with 2% formaldehyde. Cells were stained with a cocktail containing 2 μg/mL of each of anti–C5ar1-AF488 (Abd Serotec, Oxford, UK) and anti–Par4-AF546 (Abcam, Cambridge, UK) for 1 hour. For Matrigel plug assays, plugs were implanted into the inner thighs of WT mice for 10 days. Plugs were harvested, embedded in OCT compound, and sectioned. Sections were stained with anti-FII, anti-FV, anti-FVII, or anti-FX, followed by AF546-labeled anti-rabbit secondary antibody (Ab). Cells were counterstained with anti-CD31 (green). Nuclei were visualized with DAPI (blue). For coagulation factor staining in confluent versus nonconfluent cells, HUVECs were grown to 70% and 100% confluence, fixed with 2% formaldehyde, and blocked with 5% horse serum in phosphate-buffered saline. Cells were stained with anti-FII followed by AF546-labeled anti-rabbit secondary Ab. All images were collected using a 63× (1.4 numerical aperture) oil immersion objective on a Leica HyVolution SP8 inverted confocal microscope. Resulting images were subjected to deconvolution using Hyugens Professional. Cells were lysed in 1× Cell Lysis Buffer (10×; Cell Signaling, Danvers, MA); catalog number 9803) supplemented with 1 mmol/L phenylmethylsulfonyl fluoride and 1 Complete Mini protease inhibitor tablet (Roche, Boston, MA; catalog number 11836153001) for 10 minutes on ice (1 mmol/L Na orthovanadate was used to inhibit phosphatase activity when lysates were to be assayed for kinase activity). Lysates were then sonicated three times for 2 minutes to break apart nucleic acids, after which the cells were centrifuged for 10 minutes at 12,000 × g. Clean supernatants were transferred to new tubes and incubated with Protein-A/G beads (Santa Cruz Biotechnology, Dallas, TX) to preclear the lysates and prevent nonspecific co-immunoprecipitation. Abs against PAR4, C5ar1, GSK-β, and CD59 were added, and samples were incubated overnight at 4°C. Protein-A/G beads were used to pull down Ab, and immunoprecipitations were assayed by Western blot analysis, enzyme-linked immunosorbent assay, or protein activity via kits, as described. Bioluminescence resonance energy transfer (BRET) analyses were performed as described.25de la Fuente M. Noble D.N. Verma S. Nieman M.T. Mapping human protease-activated receptor 4 (PAR4) homodimer interface to transmembrane helix 4.J Biol Chem. 2012; 287: 10414-10423Google Scholar Briefly, HEK293 cells (1 × 105) were transfected with 0.5 μg of Par4-Luc donor plasmid and increasing amounts (0 to 5 μg) of C5ar1–green fluorescent protein (GFP) acceptor plasmid or C5ar1-Luc donor plasmid and increasing amounts (0 to 5 μg) of C5ar1-GFP or Par4-GFP acceptor plasmids. Emission was detected using a PerkinElmer Life Sciences (Waltham, MA) Victor 3 plate reader equipped with the appropriate BRET2 filter set (410 nm with 80-nm bandpass and 515 nm with 30-nm bandpass; PerkinElmer Life Sciences). Emission at 410 and 515 nm was collected immediately after the addition of 5 μmol/L luciferase substrate (coelenterazine 400a; Biotium Inc., Hayward, CA). BRET signal was calculated by the ratio of emission at 515 nm to emission at 410 nm minus the BRET in the absence of GFP. In BRET studies, specific interactions are detected by a hyperbolic increase in the BRET signal (ratio of emission at 510/410 nm) as the ratio of GFP-receptor/RLuc-receptor increased, whereas nonspecific interactions increased linearly. Data were analyzed with Prism 6 (GraphPad, San Diego, CA) using best model (hyperbolic versus linear) for each experiment. GFP expression was determined by excitation at 495 nm and emission at 515 nm. Luciferase expression was determined by adding 5 μm coelenterazine H (Invitrogen, Carlsbad, CA) and reading total light emission without a filter. Each BRET assay was performed in three independent experiments. siRNAs targeting human PAR1 (identifier: 146259) and PAR4 (identifier: s194972) were purchased from ThermoFisher (Carlsbad, CA). RNA interference was performed using Lipofectamine RNAiMAX Reagent (ThermoFisher) following the manufacturer’s recommended protocol. Briefly, cells were treated with 30 nmol/L siRNA and transfection reagent at a 1:2 ratio under reduced serum conditions for 48 hours. Cells were returned to complete media, and knockdown was assessed using flow cytometry at 3 and 7 days. Silencer-Cy3 siRNA (ThermoFisher) was used as a nonspecific control. Power calculations and animal numbers were determined using the information provided by http://statpages.org and the therein linked Russ Lenth's power and sample-size calculator obtainable through http://www.stat.uiowa.edu/∼rlenth/Power/index.html#Download_to_run_locally (both last accessed September 12, 2021). To achieve a true difference between means of 0.5 and a power of 0.2 testing the difference between two means via an unpaired, two-tailed t-test, 10 mice were required in each group and there were 10 experimental groups. Statistical significance for all experimental data was determined by t-test (unpaired, two tailed) performed in Microsoft Excel (Seattle, WA), SigmaPlot (Systat Software, Chicago, IL), or GraphPad Prism 6, with a significance threshold value of P < 0.05. Except where indicated, all experiments were repeated at least two times in separate samples. Data are presented as mean values with SD. To test whether ECs endogenously synthesize coagulation factors and, if so, whether VEGF-A stimulation affects their synthesis, a culture system with noncontact inhibited ECs was used to model EC growth as occurs in blood vessel reconstitution. Initial cell numbers were plated such that cells would remain <70% confluent in the presence of VEGF-A at the longest incubation time. In initial studies, murine bEnd.3 ECs incubated for 1 hour were used in the absence or presence of added VEGF-A. Immediately after harvesting the ECs, the washed cells were assayed for coagulation factor mRNA transcripts. The cells incubated with VEGF-A showed 350% to 500% increased amounts of FV, FX, FVII, and FII (prothrombin) mRNAs adjusted to fold increases over media alone (Figure 1A). Whether the mRNA synthesis translates to protein synthesis by measuring coagulation factors in culture supernatants was assessed next. Supernatants of the cells incubated in media alone shortened the clotting times of plasma samples selectively deficient in FII, FV, FVII, and FX (Figure 1B), whereas those of VEGF-A-treated cells more profoundly shortened the clotting times of each (Figure 1B). Primary aortic endothelial cells (HAECs) were used to document that the findings apply to nontransformed primary cells and human ECs. Incubation of HAECs without or with human VEGF-A showed comparable coagulation factor mRNA production (Figure 1C). These data point to endogenous production of functional FV, FX, FVII, and FII by ECs tonically and augmented production of each in response to VEGF-A stimulation. As a first test of whether the EC coagulation factor synthesis occurs in vivo, FV, FX, FVII, and FII production was examined in thoroughly perfused aortae from WT mice. Real-time quantitative PCR of dispase extracts were used to documente FII, FV, and FX as well as tissue factor mRNA expression (Figure 1D). Because aortae contain other cell types (eg, smooth muscle cells), and aortic ECs in situ do not proliferate (under homeostatic conditions), Matrigel plugs were implanted into the flanks of WT mice, plugs were removed 10 days