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.     

Plasminogen activator inhibitor‐1 inhibits factor VIIa bound to tissue factor

Summary. Background and objective: A growing body of experimental evidence supports broad inhibitory and regulatory activity of plasminogen activator inhibitor 1 (PAI‐1). The present study was designed to investigate whether PAI‐1 inhibits factor (F) VIIa complexed with tissue factor (TF), a well‐known procoagulant risk factor. Methods and results: The ability of PAI‐1 to inhibit FVIIa‐TF activity was evaluated in both clotting and factor X (FX) activation assays. PAI‐1 and its complex with vitronectin inhibit: (i) clotting activity of FVIIa‐TF (PAI‐1_IC50, 817 and 125 nm , respectively); (ii) FVIIa‐TF‐mediated FX activation (PAI‐1_IC50, 260 and 50 nm , respectively); and (iii) FVIIa bound to TF expressed on the surface of stimulated endothelial cells (PAI‐1_IC50, 260 and 120 nm , respectively). The association rate constant (k_a) for PAI‐1 inhibition of FVIIa‐TF was determined using a chromogenic assay. K_a for PAI‐1 inhibition of FVIIa bound to relipidated TF is 3.3‐fold higher than that for FVIIa bound to soluble TF (k_a = 0.09 ± 0.01 and 0.027 ± 0.03 μm ^?1 min^?1, respectively). Vitronectin increases k_a for both soluble and relipidated TF by 3.5‐ and 30‐fold, respectively (to 0.094 ± 0.020 and 2.7 ± 0.2 μm ^?1 min^?1). However, only a 3.5‐ to 5.0‐fold increase in the acylated FVIIa was observed on SDS PAGE in the presence of vitronectin for both relipidated and soluble TF, indicating fast formation of PAI‐1/vitronectin/FVIIa/relipidated TF non‐covalent complex. Conclusions: Our results demonstrate potential anticoagulant activity of PAI‐1 in the presence of vitronectin, which could contribute to regulation of hemostasis under pathological conditions such as severe sepsis, acute lung injury and pleural injury, where PAI‐1 and TF are overexpressed.     

Binding of factor VIIa to the endothelial cell protein C receptor reduces its coagulant activity

BACKGROUND: Endothelial cell protein C receptor (EPCR) binds protein C through its gamma-carboxyglutamic acid (Gla) domain and enhances its thrombin-thrombomodulin complex-dependent activation. So far, only protein C/activated protein C has been shown to interact with EPCR. Factor VII (FVII), the coagulation trigger upon tissue factor (TF) interaction, is a serine protease whose Gla domain is highly homologous to the Gla domain of protein C. OBJECTIVES: To characterize the binding of FVII/FVIIa to EPCR and its functional consequences. METHODS AND RESULTS: We demonstrated by surface plasmon resonance (SPR) that FVII/FVIIa binds to EPCR through its Gla domain. At therapeutic concentrations, FVIIa reduced the activation of protein C by 40%. Soluble EPCR (sEPCR) was also able to prolong dose-dependently the clotting time induced by the FVIIa-TF complex. SPR and amidolytic experiments showed that FVIIa is able to interact simultaneously with TF and EPCR, thus ruling out the possibility that the effect of EPCR on clotting time was due to the inhibition of the binding between FVIIa and TF. sEPCR inhibited dose-dependently the activation of FX by the FVIIa-TF complex. Notably, blocking the binding site of EPCR on the endothelial surface increased the generation of FXa 2-fold. CONCLUSIONS: EPCR binds to FVII/FVIIa and inhibits the procoagulant activity of the FVIIa-TF complex.     

Mechanisms of factor VIIa‐catalyzed activation of factor VIII

Summary. Background: Factor (F)VIIa, complexed with tissue factor (TF), is a primary trigger of blood coagulation, and has extremely restricted substrate specificity. The complex catalyzes limited proteolysis of FVIII, but these mechanisms are poorly understood. Objectives: In the present study, we investigated the precise mechanisms of FVIIa/TF‐catalyzed FVIII activation. Results: FVIII activity increased ～4‐fold within 30 s in the presence of FVIIa/TF, and then decreased to initial levels within 20 min. FVIIa (0.1 nm ), at concentrations present physiologically in plasma, activated FVIII in the presence of TF, and this activation was more rapid than that induced by thrombin. The heavy chain (HCh) of FVIII was proteolyzed at Arg⁷⁴⁰ and Arg³⁷² more rapidly by FVIIa/TF than by thrombin, consistent with the enhanced activation of FVIII. Cleavage at Arg³³⁶ was evident at ～1 min, whilst little cleavage of the light chain (LCh) was observed. Cleavage of the HCh by FVIIa/TF was governed by the presence of the LCh. FVIII bound to Glu‐Gly‐Arg‐active‐site‐modified FVIIa (K_d, ～0.8 nm ) with a higher affinity for the HCh than for the LCh (K_d, 5.9 and 18.9 nm ). Binding to the A2 domain was particularly evident. Von Willebrand factor (VWF) modestly inhibited FVIIa/TF‐catalyzed FVIII activation, in keeping with the concept that VWF could moderate FVIIa/TF‐mediated reactions. Conclusions: The results demonstrated that this activation mechanism was distinct from those mediated by thrombin, and indicated that FVIIa/TF functions through a ‘priming’ mechanism for the activation of FVIII in the initiation phase of coagulation.     

Recombinant nematode anticoagulant protein c2, an inhibitor of tissue factor/factor VIIa, attenuates coagulation and the interleukin-10 response in human endotoxemia

Summary.  The tissue factor–factor (F)VIIa complex (TF/FVIIa) is responsible for the initiation of blood coagulation under both physiological and pathological conditions. Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent inhibitor of TF/FVIIa, mechanistically distinct from tissue factor pathway inhibitor. The first aim of this study was to elucidate the pharmacokinetics and pharmacodynamics of a single intravenous (i.v.) dose of rNAPc2. The second aim was to study its effect on endotoxin-induced coagulation and inflammation. Initially, rNAPc2 was administered to healthy volunteers in three different doses. There were no safety concerns and the pharmacokinetics were consistent with previous studies, in which rNAPc2 was administered subcutaneously. rNAPc2 elicited a dose-dependent reduction of the endogenous thrombin potential and a selective prolongation of prothrombin time. Subsequently, the effect on endotoxin-induced coagulation and inflammation was studied. The administration of rNAPc2 completely blocked the endotoxin-induced thrombin generation, as measured by plasma prothrombin fragment F1+2. The endotoxin-induced effect on fibrinolytic parameters such as plasmin–antiplasmin complexes and plasminogen activator inhibitor type 1 was not affected by rNAPc2. The administration of rNAPc2 attenuated the endotoxin-induced rise in interleukin (IL)-10, without affecting the rise in other cytokines. In conclusion, rNAPc2 is a potent inhibitor of TF/FVIIa, which was well tolerated and could safely be used intravenously in this Phase I study in healthy male volunteers. A single i.v. dose rNAPc2 completely blocked endotoxin-induced thrombin generation without affecting the fibrinolytic response. In addition, rNAPc2 attenuated the endotoxin-induced rise in IL-10, without affecting the rises in other cytokines.     

Evolutionary conservation of the allosteric activation of factor VIIa by tissue factor in lamprey

Essentials

• Tissue factor (TF) enhances factor VIIa (FVIIa) activity through structural and dynamic changes.
• We analyzed conservation of TF‐activated FVIIa allosteric networks in extant vertebrate lamprey.
• Lamprey Tf/FVIIa molecular dynamics show conserved Tf‐induced structural/dynamic FVIIa changes.
• Lamprey Tf activation of FVIIa allosteric networks follows molecular pathways similar to human.

Summary

Background

Previous studies have provided insight into the molecular basis of human tissue factor (TF) activation of activated factor VII (FVIIa). TF‐induced allosteric networks of FVIIa activation have been rationalized through analysis of the dynamic changes and residue connectivities in the human soluble TF (sTF)/FVIIa complex structure during molecular dynamics (MD) simulation. Evolutionary conservation of the molecular mechanisms for TF‐induced allosteric FVIIa activation between humans and extant vertebrate jawless fish (lampreys), where blood coagulation emerged more than 500 million years ago, is unknown and of considerable interest.

Objective

To model the sTf/FVIIa complex from cloned Petromyzon marinus lamprey sequences, and with comparisons to human sTF/FVlla investigate conservation of allosteric mechanisms of FVIIa activity enhancement by soluble TF using MD simulations.

Methods

Full‐length cDNAs of lamprey tf and f7 were cloned and characterized. Comparative models of lamprey sTf/FVIIa complex and free FVIIa were determined based on constructed human sTF/FVIIa complex and free FVIIa models, used in full‐atomic MD simulations, and characterized using dynamic network analysis approaches.

Results

Allosteric paths of correlated motion from Tf contact points in lamprey sTf/FVIIa to the FVIIa active site were determined and quantified, and were found to encompass residue–residue interactions along significantly similar paths compared with human.

Conclusions

Despite low conservation of residues between lamprey and human proteins, 30% TF and 39% FVII, the structural and protein dynamic effects of TF activation of FVIIa appear conserved and, moreover, present in extant vertebrate proteins from 500 million years ago when TF/FVIIa‐initiated extrinsic pathway blood coagulation emerged.

     

Tissue factor enhances protease-activated receptor-2-mediated factor VIIa cell proliferative properties

In addition to its hemostatic functions, factor (F)VIIa exhibits cell proliferative properties as seen in angiogenesis and tumor growth. A role for tissue factor (TF) and protease-activated receptors (PAR)-1 and -2 in cell proliferation remain to be clarified. We tested the hypothesis that FVIIa induces cell proliferation by a mechanism involving TF and PAR-2. Human recombinant FVIIa induced cell proliferation of human BOSC23 cells transfected with plasmid containing human TF DNA sequence. Because DNA primase 1 (PRIM1) plays an essential role in cell proliferation, we used the cloned PRIM1 promoter upstream of the reporter gene chloramphenicol acetyl transferase (CAT) to elucidate the mode of action of FVIIa. FVIIa evoked a dose-dependent increase in cell proliferation and PRIM1 induction, which were markedly potentiated (4-5-fold) by the presence of TF and abrogated by TF antisense oligonucleotide. PRIM1 induction by FVIIa was also abolished by PAR-2 but not by PAR-1 antisense. In contrast, thrombin induced a small increase in CAT activity which was unaffected by TF, but was prevented only by PAR-1 antisense as well as the thrombin inhibitor hirudin. Proliferative properties of FVIIa were associated with a TF-dependent increase in intracellular calcium and were mediated by a concordant phosphorylation of p44/42 MAP kinase. In conclusion, data reveal that FVIIa induces PRIM1 and ensuing cellular proliferation via a TF- and of the PARs entirely PAR-2-dependent pathway, in distinction to that of thrombin which is PAR-1-dependent and TF-independent. We speculate that FVIIa-TF-PAR-2 inhibitors may be effective in suppressing cell proliferation.     

Characterization of a tissue factor/factor VIIa-dependent model of thrombosis in hypercholesterolemic rabbits

Summary.  Tissue factor (TF) expressed in arterial atherosclerotic plaque plays a key role in activating the extrinsic coagulation pathway and triggering acute coronary syndromes. In this study, we developed and characterized a TF–factor (F)VIIa-mediated thrombosis model in rabbits. Balloon catheter-induced endothelial denudation in the femoral artery and a 4-week high cholesterol diet produced a localized atherosclerotic plaque at the injured site. High levels of TF mRNA and TF protein antigen (152 ± 25 vs. 49 ± 12 pg mg⁻¹ protein in normal vessels) were detected in these atherosclerotic plaques. Plasma FVII coagulant activity (FVII:C) was significantly increased in the hypercholesterolemic rabbits (36 ± 1 s) compared with the normal rabbits (44 ± 1 s, P  < 0.0001). Plaque rupture was induced by balloon angioplasty, which resulted in thrombus formation in the injured vessel segment after a brief period of stasis. FVIIai, a specific TF–FVIIa inhibitor, was administered intravenously to rabbits before plaque rupture at 0.3 and 1.0 mg kg⁻¹. FVIIai dose-dependently reduced thrombus mass (14.7 ± 2.5 and 5.9 ± 2.2 mg, respectively, vs. 21.6 ± 1.9 mg in the control group). PD198961, a novel factor Xa inhibitor, and argatroban, a thrombin inhibitor, also dose-dependently inhibited thrombosis. These results indicate that thrombus formation in this model is initiated by the activation of TF–FVIIa pathway, which is attributed to TF expression in the atherosclerotic plaque and enhanced plasma FVII coagulant activity. This model may be useful for evaluating in vivo efficacy of new antithrombotic drugs, particularly TF–FVIIa inhibitors.     

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.     

The tick‐derived inhibitor Ixolaris prevents tissue factor signaling on tumor cells

Summary Background: Tissue factor (TF) is frequently overexpressed in cancer cells and correlated with more aggressive tumor phenotypes and poor prognosis. In addition to promoting coagulation‐dependent metastasis and cancer‐associated thrombosis, tumor cell‐expressed TF mediates direct cell signaling involving the protease‐activated receptor (PAR) 2. Ixolaris is a tick‐derived inhibitor of the TF–factor (F)VIIa–Xa coagulation initiation complex which blocks primary tumor growth and angiogenesis in glioblastoma and melanoma models. Methods: In this study we address the anti‐tumor effects of Ixolaris in TF–VIIa–PAR2 signaling‐dependent breast cancer models, a xenograft model of highly aggressive human MDA‐MB‐231mfp cells and a syngeneic model of PAR2‐deficient and replete PyMT mouse mammary carcinoma cells. Results: Ixolaris potently inhibited the procoagulant activity of human MDA‐MB‐231mfp or murine PyMT breast cancer cells. Ixolaris blocked signaling by the ternary TF–FVIIa–FXa complex, and, surprisingly, at higher concentrations also the binary TF–FVIIa complex on MDA‐MB‐231 cells. We show that Ixolaris interacts with certain residues in the human VIIa protease domain that are involved in PAR2 cleavage. In contrast to human VIIa, Ixolaris was a poor inhibitor of murine TF–FVIIa signaling and did not attenuate PAR2‐dependent tumor growth in a syngeneic mouse model of breast cancer progression. Conclusion: These data show that Ixolaris inhibits PAR2 cleavage specifically by human TF signaling complexes and suggest that Ixolaris may block tumor growth of human cell models with ectopic FVIIa expression through inhibition of direct TF–FVIIa–PAR2 signaling as well as its anticoagulant activity.     

Tissue factor around dermal vessels has bound factor VII in the absence of injury

BACKGROUND: 'Idling' or ongoing low-level activity of the tissue factor (TF) pathway is a postulated mechanism by which the coagulation process can become active without a lag period at sites of injury. OBJECTIVE: To determine whether TF around cutaneous vessels has bound factor VIIa in the absence of injury, and thus could participate in the idling process. METHODS: Immunostaining of mouse skin with antibodies against a 15-residue peptide from the sequence of mouse TF, and against the whole extracellular portion of TF. RESULTS: The whole TF antibody recognized TF in squamous epithelium and around vessels in the dermis. By contrast, the monospecific antibody only recognized TF in the squamous epithelium, but not around vessels. We also found that biotinylated, active site-inhibited FVIIa (FVIIai) bound to tissue sections in the same areas in which TF was recognized by the monospecific antibody (squamous epithelium), but did not bind around vessels. Molecular modeling revealed that FVIIa and FX binding to TF masked a significant part of the surface of the target peptide. CONCLUSIONS: In the aggregate, these data are most consistent with the interpretation that TF in perivascular sites has bound FVIIa, even in the absence of any injury. The presence of endogenously bound FVIIa prevents the subsequent binding of the monospecific antibody or exogenous FVIIai to perivascular TF.     

Platelet binding and activity of a factor VIIa variant with enhanced tissue factor independent activity

Summary. Background and objectives: Platelet binding and activity play important roles in the efficacy of factor VIIa (FVIIa) as a bypassing agent for hemophilia treatment. An analog of FVIIa with increased tissue factor (TF)‐independent activity, NN1731, has been produced by introducing three amino acid changes in the protease domain. NN1731 has a conformation similar to TF‐bound FVIIa, even in the absence of TF. This results in much greater intrinsic proteolytic activity, but similar activity in the presence of TF. Objectives: We hypothesized that these changes would not alter binding to platelets or phospholipid, a characteristic thought to be localized to the Gla domain. The goal of the current work was to compare platelet binding and activity of NN1731 and wild‐type FVIIa. Methods/Results: FVIIa and NN1731 bound identically to phospholipid vesicles as assessed by both activity assays and electrophoretic quasielastic light scattering techniques. However, NN1731 bound to a greater number of sites on activated platelets than FVIIa, as assessed by flow cytometry. Removal of the Gla domain abolished binding of both FVIIa and NN1731. Inhibition of the active site did not reduce NN1731 binding to the level of FVIIa. When corrected for the amount of protein bound, NN1731 had greater activity than FVIIa on platelet surfaces. Conclusions: While the Gla domain is essential for FVIIa binding to platelets, changes in the protease domain in NN1731 enhanced platelet binding as well as proteolytic activity. Features in addition to lipid composition appear to contribute to binding of rFVIIa and, especially, NN1731 to platelets.     

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.     

Transcriptional program induced by factor VIIa-tissue factor, PAR1 and PAR2 in MDA-MB-231 cells

BACKGROUND: Factor VIIa (FVIIa) binding to tissue factor (TF) induces cell signaling via the protease activity of FVIIa and protease-activated receptor 2 (PAR2). OBJECTIVE: We examined how the gene-expression profile induced by FVIIa corresponds to the profiles induced by protease-activated receptor 1 (PAR1) or PAR2 agonists using MDA-MB-231 breast carcinoma cells that constitutively express TF, PAR1 and PAR2. RESULTS AND CONCLUSIONS: Out of 8500 genes, FVIIa stimulation induced differential regulation of 39 genes most of which were not previously recognized as FVIIa regulated. All genes regulated by FVIIa were similarly regulated by a PAR2 agonist peptide confirming FVIIa signaling via PAR2. An appreciable fraction of the PAR2-regulated genes was also regulated by a PAR1 agonist peptide suggesting extensive redundancy between FVIIa/PAR2 signaling and thrombin/PAR1 signaling. The FVIIa regulated genes encode cytokines, chemokines and growth factors, and the gene repertoire induced by FVIIa in MDA-MB-231 cells is consistent with a role for TF-FVIIa signaling in regulation of a wound healing type of response. Interestingly, a number of genes regulated exclusively by FVIIa/PAR2-mediated cell signaling in MDA-MB-231 cells were regulated by thrombin and a PAR1 agonist, but not by FVIIa, in the TF-expressing glioblastoma U373 cell line.     

组织因子/活化因子Ⅶ复合物影响人卵巢癌细胞侵袭及转移能力的研究

目的　研究组织因子 /活化因子Ⅶ (TF/FⅦa)复合物对人卵巢癌细胞体外侵袭能力及体内转移能力的影响。方法　①采用分子克隆及基因转染技术构建高效表达TF的人卵巢癌细胞系A2 780 /TF ;②采用Boyden小室法计数FⅦa刺激后A2 780、A2 780 /TF穿透Matrigel迁移到PVPF膜背面的细胞数 ;③建立人卵巢癌细胞转移的实验动物模型 ,研究TF对人卵巢癌细胞在裸鼠体内形成转移灶的能力。结果　①经分子克隆技术构建得到pcDNA3 TFcDNA重组体 ;②稳定转染的A2 780 /TF细胞内TFmRNA水平显著增高 :转染细胞为 3.99± 0 .15 ,未转染细胞为 0 .97± 0 .2 3(P <0 .0 1) ;转染细胞表面TF表达也显著增高 :转染细胞中约 (48.5 6± 9.5 3) %细胞表面有TF抗原 ,而未转染细胞中仅 (2 .73± 1.15 ) % (P <0 .0 1) ;③A2 780 /TF细胞穿膜细胞数为 15 7.3± 19.2 ,经FⅦa刺激后穿膜细胞数显著增多 ,为 4 4 7.7± 39.4 (P <0 .0 1) ;与FⅦa和抗TF单抗共孵育的A2 780 /TF ,穿膜细胞数接近刺激前的基础水平 ;④种植A2 780细胞的裸鼠中 2 2 .2 %可见肺部转移灶 ,种植A2 780 /TF细胞的裸鼠中 88.9%发生肺转移 (P <0 .0 1)。结论　①将所构建的TF真核表达载体转入人卵巢癌细胞系 ,获得稳定、高效表达TF的人卵巢癌细胞系A2 780 /TF ,为研究TF     

Pharmacological interruption of acute thrombus formation with minimal hemorrhagic complications by a small molecule tissue factor/factor VIIa inhibitor: comparison to factor Xa and thrombin inhibition in a nonhuman primate thrombosis model

This study was designed to evaluate the antithrombotic efficacy and bleeding propensity of a selective, small-molecule inhibitor of tissue factor/factor VIIa (TF/VIIa) in comparison to small-molecule, selective inhibitors of factor Xa and thrombin in a nonhuman primate model of thrombosis. Acute, spontaneous thrombus formation was induced by electrolytic injury to the intimal surface of a femoral blood vessel, which results in thrombus propagation at the injured site. The TF/FVIIa inhibitor 3-amino-5-[1-[2-([4-[amino(imino)methyl]benzyl]amino)-2-oxoethyl]-3-chloro-5-(isopropylamino)-6-oxo-1,6-dihydropyrazin-2-yl]benzoic acid dihydrochloride (PHA-927F) was fully effective in prevention of thrombosis-induced vessel occlusion at a dose of 400 microg/kg/min, i.v., in the arterial vasculature (femoral artery). Neither the effective dose nor multiples up to 4.4-fold the effective arterial plasma concentration elicited any significant effect on bleeding time or blood loss from either the bleeding time site or the surgical (femoral isolation) site. Small-molecule inhibitors of factor Xa or thrombin were effective arterial antithrombotic agents; however, in contrast to the TF/FVIIa inhibitor, they both elicited substantial increases in bleeding propensity at the effective dose and at multiples of the effective plasma concentration. These data indicate that TF/VIIa inhibition effectively prevented arterial thrombosis with less impact on bleeding parameters than equivalent doses of factor Xa and thrombin inhibitors.     

The action of high-dose factor VIIa (FVIIa) in a cell-based model of hemostasis

We have developed a cell-based model of hemostasis. This model suggests that the defect in hemophilia is specifically a failure of platelet-surface factor Xa (FXa) generation, leading to a failure of platelet surface thrombin generation. Activation of FX by FVIIa/tissue factor (TF) does not compensate for a lack of FXa activation on the platelet surface by the FVIIIa/FIXa complex. This is because plasma protease inhibitors prevent FXa from moving through the fluid phase from the TF-bearing cell to the platelet surface. We have previously proposed a platelet-dependent mechanism of action for high-dose factor VIIa (FVIIa; Novoseven, Novo Nordisk, Copenhagen, Denmark). Our data suggest that, when present at high levels, FVIIa binds to activated platelets and activates small amounts of FX independent of TF. This platelet-surface FXa can partially restore platelet-surface thrombin generation in hemophilia. Recently, van't Veer and colleagues reported results from an in vitro model in which coagulation reactions were initiated by relipidated TF. The authors concluded that high-dose FVIIa may exert a hemostatic effect in hemophilia by overcoming inhibition of FVIIa/TF activity by zymogen FVII. By contrast, we found that plasma levels of FVII did not slow thrombin generation in a model system initiated with cell-associated TF. This discrepancy highlights the potential differences between the studies of the coagulation reactions assembled on living cells compared to phospholipid vesicles. Our data suggest that in a cellular system high-dose FVIIa acts primarily by enhancing the rate of thrombin generation on platelet surfaces and not by overcoming inhibition by zymogen FVII of TF-dependent activation of FX.     

Factor VIIa-mediated tenase function on activated platelets under flow

BACKGROUND: Tissue factor (TF) and/or active factor (F)VIIa may be stored inside resting platelets. OBJECTIVES: The objective of this study was to examine if platelets, following activation of GPVI, could support tenase and prothrombinase activity without any exogenously added tissue factor. METHODS: Thrombin (IIa) formation on gel-filtered platelets with added factors or the clotting of platelet-free plasma (PFP) or platelet-rich plasma (PRP) supplemented with corn trypsin inhibitor (CTI) (to inhibit factor XIIa) was studied in well plate assays with a fluorogenic thrombin substrate or in flow assays by fibrin visualization. RESULTS: Pretreatment of convulxin (CVX)-stimulated, fibrinogen-adherent, gel-filtered platelets with anti-TF, anti-FVII/VIIa, or 1 nm PPACK [inhibitor of FVIIa, factor XIa and factor (F)IIa] delayed fibrin deposition on platelets perfused with PFP/CTI at 62.5 s(-1). Anti-TF or anti-FVII/VIIa also attenuated thrombin generation in plate assays using recalcified PRP/CTI treated with CVX. Anti-TF or anti-FVII/VIIa (but not inhibited factor IXa) delayed the burst in thrombin production by gel-filtered platelets suspended in prothrombin and CVX by 14 min and 40 min, respectively. Anti-FVII/VIIa completely eliminated thrombin generation on fibrinogen-adherent, gel-filtered platelets pretreated with 10 micro m PPACK and 10 micro m EGR-CK [inhibitor of factor (F)Xa], rinsed, and then supplemented with CVX, prothrombin, and FX. Addition of anionic phospholipid to PFP/CTI or to a mixture of prothrombin, FX, and recVIIa was not sufficient to generate detectable tenase activity. Lastly, isolated, unactivated neutrophils suspended in FX, FII and recVIIa supported a very low level of thrombin generation sensitive to antagonism of P-selectin, CD18, and TF. CONCLUSIONS: Activated platelets supported tenase and prothrombinase activity by elevating the function or level of FVIIa and exposing active FVIIa or FVIIa-cofactor(s), distinct from anionic lipid, that may be, in part, TF.