Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity.

The effects of bovine activated protein C (APC) on the fibrinolytic activity of cultured bovine aortic endothelial cells were investigated. Confluent monolayers were incubated with purified APC under various conditions and changes in total fibrinolytic activity and in the level of plasminogen activator and plasminogen activator inhibitor (antiactivator) were monitored. The addition of APC to the cells in the absence of other blood or plasma components led to a rapid, dose-dependent increase of fibrinolytic activity both in the media and in cellular extracts. For example, 3.4 micrograms of APC per ml resulted in a 15-fold increase of fibrinolytic activity in the medium within 1 hour. The enhanced fibrinolytic activity reflected increases in both the urokinase-related and tissue-type plasminogen activators produced by these cells. Interestingly, treatment of cells with APC also caused a rapid, dose-dependent decrease in antiactivator activity. Diisopropyl fluorophosphate-inactivated APC did not decrease antiactivator or increase plasminogen activator. Although a small but significant direct (i.e., cell-independent) effect of APC on both fibrinolytic activity and antiactivator activity could be demonstrated, the major portion of these changes appeared to be cell-mediated. These observations indicate that the fibrinolytic potential of cultured endothelial cells is increased by APC and that the enzyme active site is essential for this change. Moreover, the results suggest that one of the primary mechanisms for this stimulation of endothelial cell fibrinolytic activity involves an APC-mediated decrease in antiactivator.     

Stimulation of endothelial cell proliferation by rat granulosa cell-conditioned medium

The ability of granulosa cells to produce mitogenic factors for vascular endothelial cells, factors which could potentially mediate angiogenesis in the ovary, was examined. Granulosa cells were obtained from preovulatory follicles of immature rats 48 h after priming with PMSG (20 IU). The cells (1 X 10(6)/well) were cultured in 3 ml serum-free medium 199 (M199) at 37 C without further treatment or in the presence of LH (100 ng/ml) or FSH (20 ng/ml). Since oxygen tension has been shown to regulate the production of angiogenic factors by other cell types, the cultures were carried out with either a high (20%) or a low (2%) oxygen concentration in the culture chamber. After 48 h, the medium was collected, filtered (0.2 micron), and frozen until tested for mitogenic effects on sparsely plated fetal bovine aortic endothelial cells. A 1:1 mixture of granulosa cell-conditioned M199 with fresh M199 plus 1% dialyzed fetal bovine serum resulted in 7- to 8-fold increases in endothelial cell numbers over the 4-day test period compared to controls (fresh M199 + 1% dialyzed fetal bovine serum only). Neither gonadotropin treatment nor the oxygen concentration during the conditioning period influenced the proliferation-stimulating activity of the medium. Medium conditioned by granulosa cells in 2% oxygen, however, did have an additional effect on endothelial cell morphology; the cells were more elongated and aligned than those treated with medium conditioned by granulosa cells in 20% oxygen, which showed a typical cobblestone morphology. Preliminary characterization studies indicate that both high (greater than 30,000) and low (less than 10,000) mol wt mitogenic factors are present. The mitogenic activity is heat resistant but partially destroyed by trypsin. The morphology-altering activity is confined to high mol wt fractions (greater than 30,000). These studies demonstrate that granulosa cells from preovulatory follicles release one or more factors in vitro which are mitogenic for endothelial cells. Furthermore, conditioned medium from granulosa cells cultured in low oxygen induces morphological changes in endothelial cells which suggest an increased propensity for migration.     

Activated protein C induces the release of microparticle-associated endothelial protein C receptor

Activated protein C (APC) treatment is now used for patients with severe sepsis. We investigated its effect in vitro on primary, physiologically relevant cells and demonstrate a novel mechanism of endothelial protein C receptor (EPCR) release that is not inhibited by metalloproteinase inhibitors. Exposure of human umbilical vein endothelial cells or monocytes to APC (6.25-100 nM) results in the release of EPCR-containing microparticles, as demonstrated by confocal microscopy and characterized through flow cytometry, enzyme-linked immunosorbent assay quantitation of isolated microparticles, and Western blotting. The phenomenon is time- and concentration-dependent and requires the APC active site, EPCR, and protease activated receptor 1 (PAR1) on endothelial cells. Neither protein C nor boiled or D-Phe-Pro-Arg-chloromethylketone-blocked APC can induce microparticle formation and antibody blockade of EPCR or PAR1 cleavage and activation abrogates this APC action. Coincubation with hirudin does not alter the APC effect. The released microparticle bound is full-length EPCR (49 kDa) and APC retains factor V-inactivating activity. Although tumor necrosis factor-alpha (10 ng/mL) can also induce microparticle-associated EPCR release to a similar extent as APC (100 nM), it is only APC-induced microparticles that contain bound APC. This novel observation could provide new insights into the consequences of APC therapy in the septic patient.     

Activated protein C inhibits platelet prothrombin-converting activity

Bovine platelets that have been activated by thrombin facilitate the conversion of prothrombin to thrombin in the presence of calcium ions and factor Xa. Activated protein C, a vitamin-K-dependent plasma protein, inhibits this platelet prothrombin-converting activity. The inhibition is time dependent and is not reversed by increasing concentrations of factor Xa. However, factor Xa is able to protect the platelet prothrombin-converting activity from inactivation by activated protein C. The activated protein C causes a parallel loss of factor Xa receptor sites and platelet prothrombin-converting activity. Activated protein C may contribute to the regulation of clotting through inactivation of the platelet prothrombin-converting activity.     

C-reactive protein decreases endothelial nitric oxide synthase activity via uncoupling

C-reactive protein (CRP), a cardiovascular risk marker, induces endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) expression and bioactivity in human aortic endothelial cells (HAECs). In this study, we examined the mechanisms by which CRP decreases eNOS activity in HAECs. To this end, we explored different strategies such as availability of tetrahydrobiopterin (BH4)-a critical cofactor for eNOS, superoxide (O₂⁻) production resulting in uncoupling of eNOS and phosphorylation/dephosphorylation of eNOS. CRP treatment significantly decreased levels of BH4 thereby promoting eNOS uncoupling. Pretreatment with sepiapterin, a BH4 precursor, prevented CRP-mediated effects on BH₄ levels, superoxide production as well as eNOS activity. The gene expression and enzymatic activity of GTPCH1, the first enzyme in the de novo biosynthesis of BH₄, were significantly inhibited by CRP. Importantly, GTPCH1 is known to be regulated by cAMP-mediated pathway. In the present study, CRP-mediated inhibition of GTPCH1 activity was reversed by pretreatment with cAMP analogues. Furthermore, CRP-induced O₂⁻ production was reversed by pharmacologic inhibition and siRNAs to p47 phox and p22 phox. Additionally, CRP treatment significantly decreased the eNOS dimer: monomer ratio confirming CRP-mediated eNOS uncoupling. The pretreatment of cells with NO synthase inhibitor (N-nitro-l-arginine methyl ester [l-NAME]) also prevented CRP-mediated O₂⁻ production further strengthening CRP-mediated eNOS uncoupling. Additionally, CRP decreased eNOS phosphorylation at Ser1177 as well as increased phosphorylation at Thr495. CRP appears to mediate these effects through the Fcγ receptors, CD32 and CD64. To conclude, CRP uncouples eNOS resulting in increased superoxide production, decreased NO production and altered eNOS phosphorylation.     

C-reactive protein decreases endothelial nitric oxide synthase activity via uncoupling

C-reactive protein (CRP), a cardiovascular risk marker, induces endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) expression and bioactivity in human aortic endothelial cells (HAECs). In this study, we examined the mechanisms by which CRP decreases eNOS activity in HAECs. To this end, we explored different strategies such as availability of tetrahydrobiopterin (BH4)-a critical cofactor for eNOS, superoxide (O(2)(-)) production resulting in uncoupling of eNOS and phosphorylation/dephosphorylation of eNOS. CRP treatment significantly decreased levels of BH4 thereby promoting eNOS uncoupling. Pretreatment with sepiapterin, a BH4 precursor, prevented CRP-mediated effects on BH(4) levels, superoxide production as well as eNOS activity. The gene expression and enzymatic activity of GTPCH1, the first enzyme in the de novo biosynthesis of BH(4), were significantly inhibited by CRP. Importantly, GTPCH1 is known to be regulated by cAMP-mediated pathway. In the present study, CRP-mediated inhibition of GTPCH1 activity was reversed by pretreatment with cAMP analogues. Furthermore, CRP-induced O(2)(-) production was reversed by pharmacologic inhibition and siRNAs to p47 phox and p22 phox. Additionally, CRP treatment significantly decreased the eNOS dimer: monomer ratio confirming CRP-mediated eNOS uncoupling. The pretreatment of cells with NO synthase inhibitor (N-nitro-l-arginine methyl ester [l-NAME]) also prevented CRP-mediated O(2)(-) production further strengthening CRP-mediated eNOS uncoupling. Additionally, CRP decreased eNOS phosphorylation at Ser1177 as well as increased phosphorylation at Thr495. CRP appears to mediate these effects through the Fcgamma receptors, CD32 and CD64. To conclude, CRP uncouples eNOS resulting in increased superoxide production, decreased NO production and altered eNOS phosphorylation.     

Activated protein C inhibits high mobility group box 1 signaling in endothelial cells

A pathogenic role for high-mobility group box 1 (HMGB1) protein has been postulated in severe sepsis. Activated protein C (APC) is the only drug approved by the Food and Drug Administration for severe sepsis; however, its effect on HMGB1 signaling has never been investigated. Here, we monitored the effect of APC on the lipopolysaccharide-mediated release of HMGB1 and the HMGB1-mediated modulation of proinflammatory responses in HUVECs. APC potently inhibited the release of HMGB1 and down-regulated the adhesion of the monocytic cell line, THP-1, to HMGB1-activated endothelial cells. HMGB1 up-regulated proinflammatory responses by interacting with 3 pathogen-related pattern recognition receptors: TLR2 and TLR4 and the receptor for advanced glycation end products. APC not only inhibited HMGB1 release but also down-regulated the cell surface expression of all 3 HMGB1 receptors in endothelial cells. The protective effects of APC were mediated through endothelial cell protein C receptor (EPCR) and protease-activated receptor 1 (PAR-1). Interestingly, a thrombin derivative containing the Gla-domain of APC recapitulated all protective effects of APC with a 20- to 50-fold higher efficacy. These results suggest that the EPCR- and PAR-1-dependent protective effects of APC in severe sepsis may partially be mediated through the inhibition of HMGB1 signaling and that the chimeric thrombin mutant has potential therapeutic utility for severe sepsis.     

Activated protein C up-regulates procoagulant tissue factor activity on endothelial cells by shedding the TFPI Kunitz 1 domain

Thrombin and activated protein C (APC) signaling can mediate opposite biologic responses in endothelial cells. Given that thrombin induces procoagulant tissue factor (TF), we examined how TF activity is affected by APC. Exogenous or endogenously generated APC led to increased TF-dependent factor Xa activity. Induction required APC's proteolytic activity and binding to endothelial cell protein C receptor but not protease activated receptors. APC did not affect total TF antigen expression or the availability of anionic phospholipids on the apical cell membrane. Western blotting and cell surface immunoassays demonstrated that APC sheds the Kunitz 1 domain from tissue factor pathway inhibitor (TFPI). A TFPI Lys86Ala mutation between the Kunitz 1 and 2 domains eliminated both cleavage and the enhanced TF activity in response to APC in overexpression studies, indicating that APC up-regulates TF activity by endothelial cell protein C receptor-dependent shedding of the Kunitz 1 domain from membrane-associated TFPI. Our results demonstrate an unexpected procoagulant role of the protein C pathway that may have important implications for the regulation of TF- and TFPI-dependent biologic responses and for fine tuning of the hemostatic balance in the vascular system.     

Activated protein C protects vascular endothelial cells from apoptosis in malaria and in sepsis

Objective In malaria and sepsis, apoptotic endothelial damage is preventable in vitro by antioxidants and protease inhibitors. Activated protein C, which has anti‐apoptotic effects, improves survival in sepsis. Therefore, we studied whether activated protein C prevents endothelial cell apoptosis, induced by serum from patients with malaria or sepsis. Methods Endothelial cells were incubated with patient sera (Plasmodium falciparum malaria, Escherichia coli sepsis, Staphylococcus aureus sepsis) or culture supernatants of the respective organisms, with or without neutrophils. Activated protein C was used to reduce endothelial cell apoptosis in vitro. The proportion of apoptotic endothelial cells was determined by TUNEL staining. Results The apoptosis‐inducing effect of patient sera or culture supernatants (P. falciparum, E. coli, S. aureus) on endothelial cells was augmented by neutrophils and reduced by activated protein C in the presence of neutrophils. Pre‐incubating either endothelial cells or neutrophils with activated protein C also reduced the endothelial cell apoptosis rate. The pro‐apoptotic effect of P. falciparum supernatant was reduced by pan‐caspase inhibitor and caspase 8 inhibitor, but not by caspase 9 inhibitor. The pro‐apoptotic effect of E. coli and S. aureus supernatants was also reduced by caspase 9 inhibitor. Conclusions Activated protein C protects vascular endothelial cells from apoptosis triggered by patient sera or culture supernatants in combination with neutrophils. It seems to act both on neutrophils and on endothelial cells. Activated protein C blocks caspase‐8‐dependent apoptosis, which accounts for endothelial damage in sepsis and malaria. Therefore, activated protein C might offer clinical benefit not only in sepsis but also in malaria.     

Activated protein C action in inflammation

Activated protein C (APC) is a natural anticoagulant that plays an important role in coagulation homeostasis by inactivating the procoagulation factor Va and VIIIa. In addition to its anticoagulation functions, APC also has cytoprotective effects such as anti‐inflammatory, anti‐apoptotic, and endothelial barrier protection. Recently, a recombinant form of human APC (rhAPC or drotrecogin alfa activated; known commercially as ‘Xigris’) was approved by the US Federal Drug Administration for treatment of severe sepsis associated with a high risk of mortality. Sepsis, also known as systemic inflammatory response syndrome (SIRS) resulting from infection, is a serious medical condition in critical care patients. In sepsis, hyperactive and dysregulated inflammatory responses lead to secretion of pro‐ and anti‐inflammatory cytokines, activation and migration of leucocytes, activation of coagulation, inhibition of fibrinolysis, and increased apoptosis. Although initial hypotheses focused on antithrombotic and profibrinolytic functions of APC in sepsis, other agents with more potent anticoagulation functions were not effective in treating severe sepsis. Furthermore, APC therapy is also associated with the risk of severe bleeding in treated patients. Therefore, the cytoprotective effects, rather than the anticoagulant effect of APC are postulated to be responsible for the therapeutic benefit of APC in the treatment of severe sepsis.     

Activated protein C variants with normal cytoprotective but reduced anticoagulant activity

Recombinant activated protein C (APC), a well-defined anticoagulant enzyme, reduced mortality in severe sepsis patients in a phase 3 trial. However, 2 potent anticoagulants, antithrombin III and recombinant tissue factor pathway inhibitor, failed to do so, implying the physiologic relevance of APC's less well-defined anti-inflammatory and antiapoptotic activities. Recombinant APC therapy conveys an increased risk of serious bleeding complications due to APC anticoagulant activity. To generate recombinant APC variants with reduced risk of bleeding due to reduced anticoagulant activity, we dissected APC's anticoagulant activity from its cytoprotective activity by site-directed mutagenesis. Using staurosporine-induced endothelial cell apoptosis assays, we show here that Ala mutations (RR229/230AA and KKK191_ 193AAA) in 2 APC surface loops that severely reduce anticoagulant activity result in 2 APC variants that retain normal antiapoptotic activity that requires protease activated receptor-1 and endothelial cell protein C receptor. Thus, it is possible to reduce anticoagulant activity while preserving antiapoptotic activity of recombinant APC variants. We suggest that therapeutic use of such APC variants may reduce serious bleeding risks while providing the beneficial effects of APC acting directly on cells.     

Activated protein C induces endothelial cell proliferation by mitogen-activated protein kinase activation in vitro and angiogenesis in vivo

Activated protein C (APC), a natural anticoagulant, has recently been demonstrated to activate the mitogen-activated protein kinase (MAPK) pathway in endothelial cells in vitro. Because the MAPK pathway is implicated in endothelial cell proliferation, it is possible that APC induces endothelial cell proliferation, thereby causing angiogenesis. We examined this possibility in the present study. APC activated the MAPK pathway, increased DNA synthesis, and induced proliferation in cultured human umbilical vein endothelial cells dependent on its serine protease activity. Antibody against the endothelial protein C receptor (EPCR) inhibited these events. Early activation of the MAPK pathway was inhibited by an antibody against protease-activated receptor-1, whereas neither late and complete activation of the MAPK pathway nor endothelial cell proliferation were inhibited by this antibody. APC activated endothelial nitric oxide synthase (eNOS) via phosphatidylinositol 3-kinase-dependent phosphorylation, followed by activation of protein kinase G, suggesting that APC bound to EPCR might activate the endothelial MAPK pathway by a mechanism similar to that of VEGF. APC induced morphogenetic changes resembling tube-like structures of endothelial cells, whereas DIP-APC did not. When applied topically to the mouse cornea, APC clearly induced angiogenesis in wild-type mice, but not in eNOS knockout mice. These in vitro events induced by APC might at least partly explain the angiogenic activity in vivo. This angiogenic activity of APC might contribute to maintain proper microcirculation in addition to its antithrombotic activity.     

Age-related decreases in lymphocyte protein kinase C activity and translocation are reduced by aerobic fitness

This study investigated the effects of advancing age and long-term aerobic fitness on lymphocyte protein kinase C (PKC) activity and translocation. Lymphocytes were obtained from young (20-36 years old) and older (61-78 years old) healthy men who were either aerobically conditioned or deconditioned. Both baseline PKC activity and the response of this enzyme to the direct PKC stimulating agent, phorbol 12-myristate, 13-acetate (PMA) or to the mitogen, phytohaemagglutinin (PHA), were measured in partially purified extracts of cytosolic and membranous fractions of lymphocytes. Basal PKC activity, PMA-induced redistribution of PKC, and PHA-induced enhancement of PKC activity were reduced among older subjects in both lymphocyte cytosolic and membranous fractions. However, the magnitudes of these reductions were smaller among the older subjects who were aerobically fit. Lymphocyte PKC activity and translocation may be biological markers of aging, and the maintenance of aerobic fitness into later life may serve to slow the rate at which activation of this enzyme declines during senescence.     

系统性红斑狼疮抗活化的蛋白C研究

目的　了解抗活化的蛋白C(APCR)在系统性红斑狼疮 (SLE)患者中的发生情况 ,并进一步探讨SLE患者发生血栓的机制。方法　采用APC KPTT法 ,ELISA法和PTT LA法分别对5 0例SLE患者及 2 0例正常对照 (NC)进行APCR、抗心磷脂抗体 (ACA)和狼疮抗凝物 (LA)检测。结果　SLE患者APCR阳性率 ( 5 8% )明显高于NC组 ( 5 % ) (P <0 0 0 5 ) ,APCR阳性患者中血栓发生率( 2 7 6% )明显高于APCR阴性患者 ( 4 8% ) (P <0 0 1) ,患者LA阳性率 ( 2 2 % )明显高于对照组 ( 0 /10 ) (P <0 0 5 ) ,患者ACA IgG及IgM明显高于对照组 (P <0 0 5 ) ,而IgA与对照组差异不显著 (P >0 0 5 ) ,LA阳性组中的APCR阳性率 ( 90 9% )明显高于LA阴性组 ( 5 3 8% ) (P <0 0 5 ) ,ACA阳性组中的APCR阳性率 ( 64 7% )与ACA阴性组 ( 60 6% )未发现明显相关性 (P >0 0 5 )。结论　APCR在国人SLE患者中有较高的发生率且与LA有明显相关性 ,APCR可能是SLE患者诱发血栓的主要原因之一。