Activity and regulation of factor VIIa analogs with increased potency at the endothelial cell surface

BACKGROUND: Variants of recombinant factor VIIa (rFVIIa) with increased intrinsic activity have been developed to improve efficacy in the treatment of bleeding disorders in the future. The increased potency of FVIIa variants was demonstrated in limited in vitro and in vivo studies. However, further characterization of FVIIa variants is needed to evaluate their potential clinical use. METHODS: In the present study, we investigated the interactions of two FVIIa variants, FVIIa(Q) and FVIIa(DVQ), with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT), and vascular endothelium. TF-FVIIa activity or its inhibition was measured directly in an amidolytic activity assay or for its ability to activate factor X. RESULTS: Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type (WT) FVIIa in the absence of tissue factor (TF). In the presence of TF, TFPI, TFPI-Xa, and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the WT FVIIa failed to generate significant amounts of FXa on unperturbed endothelial cells, FVIIa variants, particularly FVIIa(DVQ), generated a substantial amount of FXa on unperturbed endothelium. Annexin V fully attenuated the FVIIa-mediated activation of FX on unperturbed endothelial cells. On stimulated human umbilical vein endothelial cells, FVIIa and FVIIa variants activated FX at similar rates, and annexin V blocked the activation only partly. AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. Interestingly, despite significant differences observed in FXa generation on unperturbed endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions. CONCLUSION: Overall, the present data suggest that although FVIIa variants generate substantial amounts of FXa, they do not generate excessive thrombin on the surface of endothelium.     

A GPI-anchored co-receptor for tissue factor pathway inhibitor controls its intracellular trafficking and cell surface expression

BACKGROUND: Tissue factor pathway inhibitor (TFPI) lacks a membrane attachment signal but it remains associated with the endothelial surface via its association with an, as yet, unidentified glycosyl phosphatidylinositol (GPI)-anchored co-receptor. OBJECTIVES/METHODS: Cellular trafficking of TFPI within aerolysin-resistant ECV304 and EA.hy926 cells, which do not express GPI-anchored proteins on their surface, was compared with their wild-type counterparts. RESULTS AND CONCLUSIONS: Although aerolysin-resistant cells produce normal amounts of TFPI mRNA, TFPI is not expressed on the cell surface and total cellular TFPI is greatly decreased compared with wild-type cells. Additionally, normal, not increased, amounts of TFPI are secreted into conditioned media indicating that TFPI is degraded within the aerolysin-resistant cells. Confocal microscopy and studies using metabolic inhibitors demonstrate that aerolysin-resistant cells produce TFPI and transport it into the Golgi with subsequent degradation in lysosomes. The experimental results provide no evidence that cell surface TFPI originates from secreted TFPI that binds back to a GPI-anchored protein. Instead, the data suggest that TFPI tightly, but reversibly, binds to a GPI anchored co-receptor in the ER/Golgi. The co-receptor then acts as a molecular chaperone for TFPI by trafficking it to the cell surface of wild-type cells or to lysosomes of aerolysin-resistant cells. TFPI that escapes co-receptor binding is secreted through the same pathway in both wild-type and aerolysin-resistant cells. The data provide a framework for understanding how TFPI is expressed on endothelium.     

TF-FⅦa复合物影响人卵巢癌细胞尿激酶型纤溶酶原激活物受体mRNA表达的研究

目的克隆人组织因子(TF),研究组织因子-活化凝血因子Ⅶ复合物(TF-FⅦa)对人卵巢癌细胞内尿激酶型纤溶酶原激活物(u-PA)及其受体(u-PAR)mRNA表达的影响,探讨该复合物在肿瘤浸润、转移中的作用机制.方法采用分子克隆技术构建人TF真核表达载体pcDNA3-TFcDNA;以脂质体介导转染人卵巢癌细胞系A2780,筛选稳定表达的转染细胞A2780-TF.以FⅦa分别刺激A2780细胞和A2780-TF细胞,采用RT-PCR方法检测细胞内u-PA及u-PAR mRNA水平变化.结果①构建产物经基因测序证实为pcDNA3-TFcDNA重组体;②转染细胞A2780-TF内TF-mRNA水平显著增高转染细胞为3.91±0.28,未转染细胞为0.97±0.23(P<0.01);转染细胞表面TF表达显著增高转染细胞为(48.56±9.53)%,未转染细胞为(2.73±1.15)%(P<0.01);③FⅦa刺激对A2780细胞内u-PA、u-PAR mRNA水平均无显著影响;④FⅦa呈浓度依赖性诱导A2780-TF细胞内u-PAR mRMA水平增高,而不影响u-PA mRNA水平;FⅦa刺激A2780-TF细胞内u-PAR mRNA水平增高的作用具有一定的时相性;⑤抗TF单抗可阻断FⅦa诱导A2780-TF细胞内u-PAR mRNA转录的作用.结论 TF通过与FⅦa形成复合物而上调人卵巢癌细胞内u-PAR mRNA的表达,可能藉此增强肿瘤侵袭及转移作用.     

血管内皮生长因子反义寡核苷酸对人白血病细胞血管内皮生长因子表达的影响

目的　探讨血管内皮生长因子 (VEGF)反义寡脱氧核苷酸 (AS ODN)对人白血病细胞系VEGF表达的影响。方法　将VEGFAS ODN与K5 6 2和HL 6 0细胞共孵育 ,采用RT PCR技术、免疫组化方法以及ELISA法观察VEGFAS ODN对人白血病细胞系VEGFmRNA和蛋白水平的影响。应用MTT法观察VEGFAS ODN作用后的白血病细胞培养上清对人脐静脉血管内皮细胞 (ECV30 4 )增殖的影响。结果　K5 6 2细胞和HL 6 0细胞经VEGFAS ODN(2 .5～ 15 .0 μmol L)作用 2 4h后 ,VEGFmRNA的表达量呈剂量依赖性减少 ,而其表达量不受VEGF错义ODN的影响 ;当加入 5 .0 μmol LVEGFAS ODN作用 2 4h后 ,细胞内及其培养上清液中VEGF蛋白水平显著减少 ,其培养上清对ECV30 4细胞的促增殖作用减弱 ,而错义ODN处理组白血病细胞VEGF蛋白水平及其作用未见明显改变。结论　VEGFAS ODN在体外能够抑制人白血病细胞VEGF的表达。     

Monocyte adhesion and transmigration induce tissue factor expression: role of the mitogen-activated protein kinases

The expression of tissue factor (TF) by monocytes that have transmigrated across the endothelium to sites of extravascular inflammation acts both to focus and amplify the inflammatory response. Because clustering of the integrins responsible for endothelial adhesion and transmigration induces tyrosine phosphorylation and activation of the mitogen-activated protein (MAP) kinases, we postulated that transmigration might lead to monocyte activation and TF production. Monocytes were migrated across TNFalpha-primed ECV304 cells grown on fibronectin-coated Transwell chambers in response to FMLP (10(-8) M). After transmigration, monocytes showed a time-dependent increase in surface TF expression and biological procoagulant activity. TF expression was dependent on monocyte adhesion to ECV304 cells. Specifically, TF was not induced by FMLP treatment of suspended monocytes, by migration across fibronectin alone, or by soluble factors induced during migration, whereas monocyte-ECV304 adhesion was sufficient to stimulate TF. Antibodies against CD29 (beta1 integrin), but not against CD18 (beta2 integrin) or CD31 (PECAM-1), inhibited TF expression. Monocyte adhesion to ECV304 cells induced tyrosine phosphorylation of cellular proteins and specifically of the ERK and p38 MAP kinases. Tyrosine kinase inhibition with genistein (10 microg/mL) blocked transmigration, whereas selective ERK inhibition with PD98059 (50 microM) or p38 inhibition with SB203580 (20 microM) did not. However, both ERK and p38 inhibition dose dependently abolished TF expression. These studies suggest that an extravascular focus of infection or inflammation can promote both intravascular thrombosis and extravascular fibrin deposition during the process of adhesion and transmigration across the endothelial barrier. The selective inhibition of the mitogen-activated protein kinases may offer a novel therapeutic means of modulating this inflammatory sequence.     

丹参提取物单体对肿瘤坏死因子诱导内皮细胞组织因子表达的干预作用

背景目前认为肿瘤坏死因子-α(TNF-α)可诱导人内皮细胞(HUVECs)组织因子基因的表达,而丹参提取物单体(764-3)对其干预作用如何?其机制有待于进一步探讨.目的研究764-3对TNF-α诱导HUVECs组织因子基因表达的干预作用,及单个内皮细胞内游离钙([Ca2+]1)水平的影响,以探讨764-3对防止心血管血栓栓塞性疾病的可能机制.设计随机对照的实验研究.单位协和医科大学基础医学研究所病理生理学系的实验室.材料实验于1998-05/1999-09在中国医学科学院,中国协和医科大学基础医学研究所病理生理学系实验室完成.脐带取自北京妇产医院.干预进行体外培养ECV304细胞株和人脐静脉内皮细胞,采用限制性内切酶法构建含有人TF基因不同上游调控序列的荧光素酶报告基因质粒,经脂质体法转染内皮细胞,用TNF-α(100 U/mL)及764-3(30 mg/L)处理细胞.主要观察指标测定细胞裂解物中荧光素酶及β半乳糖苷酶的活性.以Fluo-3/AM为荧光指示剂,用激光共聚焦显像系统观测[Ca2+]i的改变.结果在TF基因上游序列-244/+121 bp存在下,TNF-α可使转染内皮细胞荧光素酶表达量较对照组明显增加,764-3使这种增加有所降低(P＜0.05).而-111/+121 bp存在时,TNF-α组较对照组荧光素酶表达量无明显差异,均比-244/+121 bp存在时明显降低,此时,764-3并未引起荧光素酶表达量明显改变.激光扫描共聚焦显像,TNF-α可引起人内皮细胞[Ca2+]i持续缓慢升高,加入764-3后[Ca2+]i迅速大幅度降低,逐渐恢复到基线水平.结论764-3可抑制TNF-α诱导的HUVECs TF基因表达的增强,这种作用依赖于该基因上游序列-244/+121 bp的存在,推测胞内游离钙离子可能参与此抑制作用.     

小干扰RNA下调cyclophilin-D蛋白表达对内皮细胞抗氧化损伤的作用

背景:线粒体基质中的Cyclophilin-D蛋白(CypD)在线粒体损伤过程中起着特殊作用。目前对于CypD蛋白与血管内皮细胞功能的关系,以及CypD蛋白在动脉粥样硬化和血管狭窄发生、发展中的作用仍未明确。目的:观察小干扰RNA基因沉默对内皮细胞CyPD蛋白表达及氧化应激损伤、凋亡的影响。方法:采用含500μmol/LH2O2的PBS液处理ECV304细胞,小干扰RNA沉默ppif基因以建立CyPD蛋白缺陷型细胞凋亡模型,空白对照组以单纯PBS液处理。流式细胞仪检测ECV304细胞凋亡率,电镜观察细胞超微结构。结果与结论:CyPD缺陷模型组细胞凋亡率为(32.51±6.6)%,明显低于500μmol/LH2O2处理组(52.57±5.84)%,P=0.001。电镜观察结果显示,CyPD缺陷型细胞模型组细胞凋亡数量明显较500μmol/LH2O2处理组减少,形态改变也较轻,多为早期凋亡特征。提示下调CyPD蛋白水平可明显减轻血管内皮细胞氧化应激损伤和凋亡。     

Glycosylation of tissue factor is not essential for its transport or functions

See also Morrissey JH. Low‐carb tissue factor? This issue, pp 1508–10.DOI: 10.1111/j.1538‐7836.2011.04332.x . Summary. Background: Glycosylation plays an important role in protein function. The importance of glycosylation for tissue factor (TF) function is unclear. Objective: The aim of the present study is to investigate the importance of TF glycosylation in transport to the cell surface and its coagulant and signaling functions. Methods: Endothelial cells and peripheral blood mononuclear cells (PBMC) were treated with tunicamycin to inhibit N‐linked glycosylation. Site‐specific mutagenesis of one or more potential N‐linked glycosylation sites in TF was used to generate TF mutants lacking glycans. TF expression at the cell surface was determined in binding assays using ¹²⁵I‐FVIIa or ¹²⁵I‐TF mAb and confocal microscopy. TF coagulant activity was measured by factor (F) Xa generation assay, and TF signaling function was assessed by measuring cleavage of protease activated receptor 2 (PAR2) and activation of p44/42 MAPK. Results: Tunicamycin treatment reduced TF activity at the endothelial cell surface; however, this reduction was found to be the result of decreased TF protein production in tunicamycin‐treated cells. Tunicamycin treatment had no significant effect on TF activity or antigen levels in PBMC. No significant differences were observed in TF protein expression and procoagulant activity among cells transfected to express either wild‐type TF or TF mutants. A fully non‐glycosylated TF is shown to bind FVIIa and interact with FX with the same efficiency as that of wild‐type TF. Non‐glycosylated TF is also capable of supporting FVIIa cleavage of PAR2 and PAR2‐dependent p44/42 MAPK activation. Conclusions: Glycosylation is not essential for TF transport and coagulant or signaling functions.     

Regulation of tissue factor coagulant activity on cell surfaces

Summary. Tissue factor (TF) is a transmembrane glycoprotein and an essential component of the factor VIIa‐TF enzymatic complex that triggers activation of the coagulation cascade. Formation of TF‐FVIIa complexes on cell surfaces not only trigger the coagulation cascade but also transduce cell signaling via activation of protease‐activated receptors. Tissue factor is expressed constitutively on cell surfaces of a variety of extravascular cell types, including fibroblasts and pericytes in and surrounding blood vessel walls and epithelial cells, but is generally absent on cells that come into contact with blood directly. However, TF expression could be induced in some blood cells, such as monocytes and endothelial cells, following an injury or pathological stimuli. Tissue factor is essential for hemostasis, but aberrant expression of TF leads to thrombosis. Therefore, a proper regulation of TF activity is critical for the maintenance of hemostatic balance and health in general. TF‐FVIIa coagulant activity at the cell surface is influenced not only by TF protein expression levels but also independently by a variety of mechanisms, including alterations in membrane phospholipid composition and cholesterol content, thiol‐dependent modifications of TF allosteric disulfide bonds, and other post‐translational modifications of TF. In this article, we critically review the key literature on mechanisms by which TF coagulant activity is regulated at the cell surface in the absence of changes in TF protein levels with specific emphasis on recently published data and provide the authors’ perspective on the subject.     

八肽缩胆囊素对脂多糖诱导血管内皮细胞诱生型一氧化氮合酶表达变化的抑制作用

目的探讨八肽缩胆囊素（CCK-8）对脂多糖（LPS）诱导血管内皮细胞诱生型一氧化氯合酶（iNOS）表达变化的影响。方法培养人脐静脉内皮细胞株ECV-304细胞。用0．01、0．1和1mg／L LPS处理2～24h，用生理盐水、10mol／LCCK-8和0．1mg／L LPS＋10^-8、10^-7、10^-8mol／L CCK-8处理16h；用比色法检测培养液中一氧化氮（NO）含量、细胞NOS活性，免疫细胞化学及蛋白质免疫印迹法检测iNOS蛋白表达。结果与生理盐水处理的对照组比较，LPS诱导培养液NO含量增多、细胞NOS活性增高、iNOS蛋白表达上调；CCK-8剂量依赣性抑制LPS的上述效应。而单独作用对iNOS蛋白表达、NOS活性和NO含量均无明显影响。结论CCK-8可以明显抑制LPS引起ECV-304细胞iNOS蛋白表达上调。减少NO生成。     

Endothelial cell protein C receptor‐mediated redistribution and tissue‐level accumulation of factor VIIa

Summary. Background: Recent studies show that activated factor VII (FVIIa) binds to the endothelial cell protein C receptor (EPCR) on the vascular endothelium; however, the importance of this interaction in hemostasis or pathophysiology is unknown. Objective: The aim of the present study was to investigate the role of the FVIIa interaction with EPCR on the endothelium in mediating FVIIa transport from the circulation to extravascular tissues. Methods: Wild‐type, EPCR‐deficient or ECPR‐over‐expressing mice were injected with human recombinant (r)FVIIa (120 μg kg^?1 body weight) via the tail vein. At varying time intervals after rFVIIa administration, blood and various tissues were collected to measure FVIIa antigen and activity levels. Tissue sections were analyzed by immunohistochemistry for FVIIa and EPCR. Results: The data reveal that, after intravenous (i.v.) injection, rFVIIa rapidly disappears from the blood and associates with the endothelium in an EPCR‐dependent manner. Immunohistochemical analyses revealed that the association of FVIIa with the endothelium was maximal at 30 min and thereafter progressively declined. The FVIIa association with the endothelium was undetectable at time points exceeding 24 h post‐FVIIa administration. The levels of rFVIIa accumulated in tissue correlate with expression levels of EPCR in mice and FVIIa associated with tissues remained functionally active for periods of at least 7 days. Conclusions: The observation that an EPCR‐dependent association of FVIIa with the endothelium is most pronounced soon after rFVIIa administration and subsequently declines temporally, combined with the retention of functionally active FVIIa in tissue homogenates for extended periods, indicates that FVIIa binding to EPCR on the endothelium facilitates the transport of FVIIa from circulation to extravascular tissues where TF resides.     

尼莫地平对氧化低密度脂蛋白诱导人血管内皮细胞凋亡的影响

目的研究尼莫地平对不同浓度氧化低密度脂蛋白诱导的人血管内皮细胞凋亡的影响。方法在人脐静脉内皮细胞株ECV304培养基中加入不同浓度氧化低密度脂蛋白和尼莫地平（1.2μg/ml）,作用6-24 h。透射电镜观察内皮细胞ECV304的凋亡情况;流式细胞术（FCM）检测细胞凋亡率;用激光共聚焦显微镜检测钙离子浓度。结果不同浓度氧化低密度脂蛋白能明显诱导培养的内皮细胞发生凋亡,尼莫地平可显著降低氧化低密度脂蛋白诱导的内皮细胞凋亡率（P〈0.01）。结论尼莫地平对氧化低密度脂蛋白诱导的人血管内皮细胞凋亡具有保护作用。     

Dynamical view of membrane binding and complex formation of human factor VIIa and tissue factor

Summary. Background: The molecular mechanism of enhancement of the enzymatic activity of factor VIIa by tissue factor (TF) is not fully understood, primarily because of the lack of atomic models for the membrane‐bound form of the TF–FVIIa complex. Objectives: To construct the first membrane‐bound model of the TF–FVIIa complex, and to investigate the dynamics of the complex in solution and on the surface of anionic membranes by using large‐scale molecular dynamics (MD) simulations in full atomic detail. Methods: Membrane‐bound models of the TF–FVIIa complex and the individual factors were constructed and subjected to MD simulations, in order to characterize protein–protein and protein–lipid interactions, and to investigate the dynamics of TF and FVIIa. Results: The MD trajectories reveal that isolated FVIIa undergoes large structural fluctuation, primarily due to the hinge motions between its domains, whereas soluble TF (sTF) is structurally stable. Upon complex formation, sTF restricts the motion of FVIIa significantly. The results also show that, in the membrane‐bound form, sTF directly interacts with the lipid headgroups, even in the absence of FVIIa. Conclusion: The first atomic models of membrane‐bound sTF–FVIIa, FVIIa and sTF are presented, revealing that sTF forms direct contacts with the lipids, both in the isolated form and in complex with FVIIa. The main effect of sTF binding to FVIIa is spatial stabilization of the catalytic site of FVIIa, which ensures optimal interaction with the substrate, FX.     

银杏黄酮苷元对ox-LDL诱导的人脐静脉内皮细胞P-选择素、LOX-1表达的影响

目的：探讨银杏黄酮苷元（ginkgetin aglycone,GA）对氧化低密度脂蛋白（oxidized-low density lipoprotein,ox-LDL）诱导的人脐静脉内皮细胞P-选择素和植物血凝素样氧化低密度脂蛋白受体-1（lectin—like oxidized low density lipoprotein receptor-1,LOX-1）表达的影响。方法：培养人脐静脉内皮细胞株ECV304,分为对照组、ox-LDL组、LOX-1拮抗剂聚肌苷酸加ox-LDL混合刺激组（聚肌苷酸组）、不同含量GA加ox-LDL混合刺激组（GA6．25mg／L组、GA12．5mg／L组、GA25mg／L组和GA50mg／L）,通过逆转录聚合酶链式反应检测P-选择素mRNA和LOX-1mRNA表达,用ELISA检测各组培养基上清中P-选择素蛋白含量,辣根过氧化物酶免疫组织化学法检测LOX．1蛋白,并作比较。结果：OX-LDL上调内皮细胞P-选择素和LOX-1表达（P〈0．05）;6．25～50mg／LGA明显抑制OX-LDL诱导的内皮细胞P-选择素mRNA和LOX．1mRNA和蛋白表达（P〈0．05）;聚肌苷酸可部分或完全阻断OX-LDL诱导的内皮细胞P．选择素mRNA、LOX-1mRNA及其蛋白表达（P〈0．05）。结论：GA通过抑制LOX-1表达而降低内皮细胞合成和分泌黏附分子P-选择素,这可能是其抗动脉粥样硬化的机制之一。