Factor Xa inhibition by immobilized recombinant tissue factor pathway inhibitor

The recombinant form of the endogenous anticoagulant, tissue factor pathway inhibitor (rTFPI), is a potent inhibitor of Factor Xa (FXa) and the tissue factor–factor VIIa (TF:VIIa) complex. Surface-immobilized rTFPI reduces the thrombogenicity and intimal hyperplasia associated with synthetic vascular grafts in animal models and specifically reduces fibrin deposition on collagen-impregnated Dacron grafts from native blood in an in vitro flow model. The FXa inhibitory capacity of rTFPI in the bulk phase has been demonstrated in static systems and immobilized rTFPI reduces fibrin deposition in whole blood in vitro and animal studies; however, the specific mode of this anticoagulation has not been studied. Therefore, a comparison was made between the FXa binding capacity of two forms of immobilized rTFPI, i.e., passively adsorbed and covalently bound. The rTFPI-coated surfaces were evaluated using a parallel-plate flow reactor and comparing the amount of FXa exiting the flow chamber after exposure to an rTFPI-coated versus an uncoated plate. The results demonstrate that adsorbed rTFPI exhibits increased binding capacity (1.5–3.6 times) the expected stoichiometry via interactions with the C-terminus, whereas covalently-bound rTFPI interacts with FXa in a 1:1 stoichiometry. Thus, the results imply that specific FXa inhibition is a key component of the anticoagulant effect of rTFPI-coated surfaces and that passive adsorption of rTFPI to glass surfaces produces a more effective coating than covalent binding of rTFPI.     

Factor Xa inhibition by immobilized recombinant tissue factor pathway inhibitor

The recombinant form of the endogenous anticoagulant, tissue factor pathway inhibitor (rTFPI), is a potent inhibitor of Factor Xa (FXa) and the tissue factor-factor VIIa (TF:VIIa) complex. Surface-immobilized rTFPI reduces the thrombogenicity and intimal hyperplasia associated with synthetic vascular grafts in animal models and specifically reduces fibrin deposition on collagen-impregnated Dacron grafts from native blood in an in vitro flow model. The FXa inhibitory capacity of rTFPI in the bulk phase has been demonstrated in static systems and immobilized rTFPI reduces fibrin deposition in whole blood in vitro and animal studies; however, the specific mode of this anticoagulation has not been studied. Therefore, a comparison was made between the FXa binding capacity of two forms of immobilized rTFPI, i.e., passively adsorbed and covalently bound. The rTFPI-coated surfaces were evaluated using a parallel-plate flow reactor and comparing the amount of FXa exiting the flow chamber after exposure to an rTFPI-coated versus an uncoated plate. The results demonstrate that adsorbed rTFPI exhibits increased binding capacity (1.5-3.6 times) the expected stoichiometry via interactions with the C-terminus, whereas covalently-bound rTFPI interacts with FXa in a 1:1 stoichiometry. Thus, the results imply that specific FXa inhibition is a key component of the anticoagulant effect of rTFPI-coated surfaces and that passive adsorption of rTFPI to glass surfaces produces a more effective coating than covalent binding of rTFPI.     

Tissue factor pathway inhibitor: structure, biology and involvement in disease

Tissue factor (TF)-initiated coagulation plays a significant role in the pathophysiology of many diseases, including cancer and inflammation. Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor, which modulates initiations of coagulation induced by TF. In a factor (F) Xa-dependent feedback system, TFPI binds directly and inhibits the TF-FVII/FVIIa complex. Normally, TFPI exists in plasma both as a full-length molecule and as variably carboxy-terminal truncated forms. TFPI also circulates in complex with plasma lipoproteins. The levels and the dual inhibitor effect of TFPI on FXa and TF-FVII/FVIIa complex offers insight into the mechanisms of various pathological conditions triggered by TF. The use of selective pharmacological inhibitors has become an indispensable tool in experimental haemostasis and thrombosis research. In vivo administration of recombinant TFPI (rTFPI) in an experimental animal model prevents thrombosis (and re-thrombosis after thrombolysis), reduces mortality from E. coli-induced-septic shock, prevents fibrin deposition on subendothelial human matrix and protects against disseminated intravascular coagulation (DIC). Thus, TFPI may play an important role in modulating TF-induced thrombogenesis and it may also provide a unique therapeutic approach for prophylaxis and/or treatment of various diseases. In this review, we consider structural and biochemical aspects of the TFPI molecule and detail its inhibitory mechanisms and therapeutic implications in various disease conditions.     

Coagulation under flow: the influence of flow-mediated transport on the initiation and inhibition of coagulation

A mathematical model of intravascular coagulation is presented; it encompasses the biochemistry of the tissue factor pathway, platelet activation and deposition on the subendothelium, and flow- and diffusion-mediated transport of coagulation proteins and platelets. Simulation experiments carried out with the model indicate the predominant role played by the physical processes of platelet deposition and flow-mediated removal of enzymes in inhibiting coagulation in the vicinity of vascular injury. Sufficiently rapid production of factors IXa and Xa by the TF:VIIa complex can overcome this inhibition and lead to formation of significant amounts of the tenase complex on the surface of activated platelets and, as a consequence, to substantial thrombin production. Chemical inhibitors are seen to play almost no (TFPI) or little (AT-III and APC) role in determining whether substantial thrombin production will occur. The role of APC is limited by the necessity for diffusion of thrombin from the site of injury to nearby endothelial cells to form the thrombomodulin-thrombin complex and for diffusion in the reverse direction of the APC made by this complex. TFPI plays an insignificant part in inhibiting the TF:VIIa complex under the conditions studied whether its action involves sequential binding of TFPI to Xa and then TFPI:Xa to TF:VIIa, or direct binding of TFPI to Xa already bound to the TF:VIIa complex.     

Computational Modeling of Factor Xa Inhibition by Immobilized Tissue Factor Pathway Inhibitor

Coating surfaces of implanted devices with anticoagulants can reduce thrombosis and studies using a recombinant form of endogenous tissue factor pathway inhibitor (rTFPI) are promising. The anticoagulant function of immobilized rTFPI is thought to occur primarily by its inhibition of plasma clotting factor Xa (FXa); however the kinetics of this reaction at a surface are as yet unknown. To better understand the surface inhibition reaction under flow conditions, a theoretical model was developed delineating the roles of mass transport and reaction kinetics for an in vitro parallel plate device used in prior experimental studies [Hall et al., J. Biomech. Eng. 120:484–490, 1998]. As a first approximation, the kinetics of inhibition of FXa by rTFPI reported for static, homogeneous systems was considered. The unsteady convection–diffusion equation was solved for different wall-shear rates and inlet concentrations of FXa using the computational fluid dynamics software CFD-ACE (ESI Software Group). The results show that the heterogeneous inhibition reaction is diffusion controlled prior to saturation of the rTFPI. The experimental results compare favorably with the model at the lower shear rates (100–400 s⁻¹). At higher shear rates (>400 s⁻¹) the theoretical results follow the same trend as the experimental results but show a greater inhibition of FXa, implying an effect of flow or shear on the inhibition reaction.     

组织因子途径抑制因子及其医学应用前景

外源凝血途径起始于组织因子 / F a活性复合物的形成。组织因子途径抑制因子是主要的凝血抑制因子之一 ,通过和 TF/ F a以及 FXa形成四聚体抑制这一过程。研究表明 ,组织因子途径抑制在诸如动脉粥样硬化 ,血管再狭窄 ,弥漫性血管内凝血等疾病可能有潜在的治疗功能     

人组织因子途径抑制因子重组蛋白表达菌株的建立

组织因子途径抑制因子是机体凝血过程的主要抑制因子，在血栓形成性疾病的防治中具有广阔的应用前景。本研究采用pGEX—2T为表达载体、大肠杆菌细胞为表达宿主进行了人组织因子途径抑制因子重组蛋白的制备。制备的重组蛋白产量较高，纯化过程简单，且具有良好的抑制组织因子的功能。     

In vitro flow evaluation of recombinant tissue factor pathway inhibitor immobilized on collagen impregnated Dacron

Recombinant tissue factor pathway inhibitor (rTFPI) was immobilized on collagen impregnated (CI) knitted Dacron surfaces and its resistance to fibrin deposition evaluated following exposure to nonanticoagulated whole blood. Recombinant TFPI readily adsorbed to the CI Dacron surface and maintained its inhibitory activity. Under static conditions, rTFPI treated CI Dacron showed little fibrin deposition when compared with untreated surfaces. Treated samples exposed to flowing native blood at wall shear rates of 100 or 200 sec(-1) also demonstrated reduced fibrin deposition (up to 56%) compared with untreated samples. To assess the relative roles of the contact and tissue factor pathways in fibrin formation on artificial grafts, flow studies were performed with whole blood containing corn trypsin inhibitor, a potent inhibitor of FXIIa and contact activation. Corn trypsin inhibitor reduced fibrin deposition on untreated CI Dacron by 40%. Immobilized rTFPI alone, or corn trypsin inhibitor in combination with immobilized rTFPI, reduced fibrin deposition by 58% and 61%, respectively. These data suggest that immobilized rTFPI slows fibrin deposition on the vascular graft material by inhibiting both the contact pathway and blood borne tissue factor procoagulant activity arising from either the alternatively spliced form of tissue factor or from tissue factor containing microparticles.     

Vascular cell attachment and procoagulant activity on metal alloys

The attachment and growth of vascular smooth muscle cells on biomaterials used as components of devices implanted in the vascular space may influence the biocompatibility of such materials. The nature of the materials may affect the attachment and/or the activation of these cells' procoagulant responses. Therefore, the main objective of this study was to measure the strength of adhesion of these vascular cells to potential biomaterials (titanium, zirconium alloys, and stainless steel) by exposing them to a range of shear stresses (50-300 dyn cm^-2) in a parallel plate flow chamber. The procoagulant responses of the cells were evaluated by measuring the tissue factor (TF) activity promoted by the different materials under flow conditions. The materials supported distinctly different levels of initial cell adhesion in static culture. However, the fraction of adherent cells did not decline significantly with incrementally increasing shear stress within the range tested. TF expression, as measured by factor Xa (FXa) production, was material-dependent. For example, cells cultured on Ti1313 exhibited more FXa production (13.2 nM 10^-5 cells) than Ti1313(DH) (8.5 nM 10^-5 cells) or stainless steel (2 nM 10^-5 cells). Thus, our studies indicate that the level of adhesion, strength of attachment and the expression of procoagulant activity of adherent vascular cells depend strongly on the nature of the underlying biomaterial.     

肾上腺髓质素对oxLDL诱导的HUVECs组织因子及其抑制物表达的干预作用

目的：探讨肾上腺髓质素（AM）对氧化低密度脂蛋白（oxLDL）影响脐静脉内皮细胞（HUVECs）组织因子（TF）及其抑制物（TFPI）表达的干预作用，并探讨可能的信号转导途径。 方法： 进行人HUVECs原代培养，分别采用发色底物法和RT-PCR技术检测TF及TFPI蛋白活性及mRNA水平的变化，并给予8-溴-环腺苷酸（cAMP拮抗剂）、PD098059（MAPKK抑制剂）及H7（PKC抑制剂）观察AM影响TFPI表达的信号途径。 结果： AM呈剂量依赖性地抑制oxLDL诱导的HUVECs TF蛋白活性和mRNA表达的升高；AM可增加HUVECs TFPI的表达，并能减轻oxLDL所致的TFPI蛋白活性和mRNA水平的降低；AM通过cAMP和MAPK途径影响TFPI的变化。 结论： AM可以减低oxLDL对HUVECs TF和TFPI表达的影响，进而改善动脉粥样硬化的凝血状态，延缓其进程。     

Simulation of a mathematical model of the role of the TFPI in the extrinsic pathway of coagulation

Blood coagulation is a complex physiological process. With the development of biochemistry and molecular biology, especially the discovery of the tissue factor pathway inhibitor (TFPI) and further research, the researches of tissue factor (TF) and TFPI have been the hot topics now. Based on this result, we provide a new concept of blood coagulation system and provide a dynamic model to describe the role of TFPI of this pathway. Through stability analysis of the equilibrium point and numerical simulations, we obtain some important conclusions. All these results may have great significance for lesions of cardiac vessels and cerebral vessels, haemophilia, DIC, thrombosis formation and malignancy.     

Tissue factor-dependent coagulation is preferentially up-regulated within arterial branching areas in a baboon model of Escherichia coli sepsis

Endothelium plays a critical role in the pathobiology of sepsis by integrating systemic host responses and local rheological stimuli. We studied the differential expression and activation of tissue factor (TF)-dependent coagulation on linear versus branched arterial segments in a baboon sepsis model. Animals were injected intravenously with lethal doses of Escherichia coli or saline and sacrificed after 2 to 8 hours. Whole-mount arterial segments were stained for TF, TF-pathway inhibitor (TFPI), factor VII (FVII), and markers for endothelial cells (ECs), leukocytes, and platelets, followed by confocal microscopy and image analysis. In septic animals, TF localized preferentially at branches, EC surface, leukocytes, and platelet aggregates and accumulated in large amounts in the subendothelial space. FVII strongly co-localized with TF on ECs and leukocytes but less so with subendothelial TF. TFPI co-localized with TF and FVII on endothelium and leukocytes but not in the subendothelial space. Focal TF increases correlated with fibrin deposition and increased endothelial permeability to plasma proteins. Biochemical analysis confirmed that aortas of septic baboons expressed more TF mRNA and protein than controls. Branched segments contained higher TF protein levels and coagulant activity than equivalent linear areas. These data suggest that site-dependent endothelial heterogeneity and rheological factors contribute to focal procoagulant responses to E. coli.     

组织因子途径抑制因子对生物材料表面血小板粘附的影响

组织因子途径抑制因子（tissue factor pathway inhibitor,TFPI）是组织因子凝血途径的主要抑制因子，具有抑制组织因子、凝血因子VIIa、和Xa的功能。我们以前的研究显示TFPI在体外可以明显延长被修饰材料表面的凝血时间，在体内显著减少材料表面的血栓形成。本文观察了TFPI包被对聚乙烯和聚氯乙烯材料表面血小板粘附的影响。结果显示，通过TFPI处理后，上述两种材料表面的血小板粘附数目较对照组明显减少，提示TFPI可通过抑制血小板在材料表面的粘附起到改善生物材料血液相容性的作用。     

重组组织因子途径抑制物对大鼠肾系膜细胞凋亡及Fas和bcl-2表达的影响

目的 观察重组组织因子途径抑制物(rTFPI)对大鼠肾脏系膜细胞(MsC)凋亡的影响,并探讨其可能相关的凋亡机制.方法 免疫组织化学(ABC)法检测人肾穿组织中TFPI表达;免疫荧光法检测培养正常大鼠MsC中TFPI表达;将rTFPI及其不同结构域蛋白分别作用于大鼠MsC,Hoechst 33258染色观察MsC细胞核形态变化;流式细胞仪定量检测MsC细胞凋亡率;DNA片段凝胶电泳分析MsC凋亡情况;Western blot检测凋亡相关激酶半胱氨酸蛋白酶3(caspase-3)、bel-2以及Fas蛋白的表达.结果 系膜增生型肾小球肾炎肾小球内TFPI表达高于轻微病变者;正常大鼠MsC中有TFPI表达;rTFPI及其C末端能诱导大鼠MsC发生凋亡,并具有剂量和时间依赖性劝口入rTFPI后,MsC的凋亡率分别为对照组的2.1、3.0及4.9倍(P<0.05).活性形式的caspase-3和Fas蛋白表达明显高于对照组,bcl-2表达无明显变化.结论 rTFPI能诱导正常大鼠MsC发生凋亡,并且其主要功能区位于C末端,死亡受体Fas/FasL信号通路可能参与了rTFPI诱导MsC凋亡的过程.     

TFPI inhibits lectin pathway of complement activation by direct interaction with MASP‐2

The lectin pathway (LP) of complement has a protective function against invading pathogens. Recent studies have also shown that the LP plays an important role in ischemia/reperfusion (I/R)‐injury. MBL‐associated serine protease (MASP)‐2 appears to be crucial in this process. The serpin C1‐inhibitor is the major inhibitor of MASP‐2. In addition, aprotinin, a Kunitz‐type inhibitor, was shown to inhibit MASP‐2 activity in vitro. In this study we investigated whether the Kunitz‐type inhibitor tissue factor pathway inhibitor (TFPI) is also able to inhibit MASP‐2. Ex vivo LP was induced and detected by C4‐deposition on mannan‐coated plates. The MASP‐2 activity was measured in a fluid‐phase chromogenic assay. rTFPI in the absence or presence of specific monoclonal antibodies was used to investigate which TFPI‐domains contribute to MASP‐2 inhibition. Here, we identify TFPI as a novel selective inhibitor of MASP‐2, without affecting MASP‐1 or the classical pathway proteases C1s and C1r. Kunitz‐2 domain of TFPI is required for the inhibition of MASP‐2. Considering the role of MASP‐2 in complement‐mediated I/R‐injury, the inhibition of this protease by TFPI could be an interesting therapeutic approach to limit the tissue damage in conditions such as cerebral stroke, myocardial infarction or solid organ transplantation.     

Suppression of the tissue factor-dependent coagulation cascade: a contributing factor for the development of intratumoral hemorrhage in glioblastoma

To clarify factors that may contribute to the development of intratumoral hemorrhage, we analyzed the expression of tissue factor (TF), an initiator of the extrinsic coagulation pathway, and of tissue factor pathway inhibitor (TFPI) in glioblastomas with or without massive intratumoral hematoma. Among 196 glioma cases reviewed, there were 13 with macroscopic intratumoral hemorrhage. We focused on the glioblastomas and used immunoblot- and immunohistochemical methods to compare the expression of TF and TFPI in 9 glioblastomas with macroscopic hematoma and 30 glioblastomas without macroscopic hemorrhage. Although TF was expressed in most glioblastomas irrespective of the presence or absence of macroscopic hemorrhage, the staining patterns differed significantly: TF-positive glioma cells were diffusely present in the non-hemorrhage group; in the group with hemorrhage, positive cells, primarily macrophages, were scattered throughout the tissue examined. The expression of TFPI was significantly higher in the group with than in the group without hemorrhage. Our results suggest that local suppression of the TF-dependent coagulation cascade is a contributing factor that permits the occurrence of intratumoral hemorrhage.     

Elevated plasma tissue factor levels in neonates with umbilical arterial catheter-associated thrombosis

Catheterization of the umbilical artery has been a useful aid in the management of sick neonates for the past few decades. However, it is associated with various complications. Reported studies strongly suggest a significant role of intravascular catheterization in the development of aortic thrombi. Increase in thrombosis of large vessels is believed to be related to mechanical injury in the catheterized vessels, which provide direct exposure of blood to tissue factor (TF), the primary cellular initiator of the extrinsic coagulation pathway. This study was conducted to determine the levels of plasma TF, tissue factor pathway inhibitor (TFPI) and D-dimer (DD) in infants with umbilical arterial catheter (UAC)-associated thrombosis. Quantification of TF was carried out using an in-house sandwich ELISA, whereas TFPI and DD levels were measured with commercial immunoassay kits. Infants with UAC inserted were found to have significantly higher levels of plasma TF (p < 0.001) than baseline levels. However, there were no significantly elevated levels of TFPI or DD. Infants with UAC-associated thrombosis demonstrated a greater increase of TF level (median: 414.5 pg/mL; range: -76.0, 6667.0) than infants without UAC-associated thrombosis (105.0 pg/mL; -976.0, 9480.0; p = 0.009) following UAC insertion. Our findings indicate that quantification and monitoring of TF levels could predict thrombus formation in infants with indwelling UAC. Following umbilical arterial catheterisation, infants with an approximately 3-fold rise in plasma TF levels were most at risk of developing abdominal aorta thrombosis as confirmed by real-time abdominal ultrasonography.     

Bioactive Hydrogel Substrates: Probing Leukocyte Receptor–Ligand Interactions in Parallel Plate Flow Chamber Studies

The binding of activated integrins on the surface of leukocytes facilitates the adhesion of leukocytes to vascular endothelium during inflammation. Interactions between selectins and their ligands mediate rolling, and are believed to play an important role in leukocyte adhesion, though the minimal recognition motif required for physiologic interactions is not known. We have developed a novel system using poly(ethylene glycol) (PEG) hydrogels modified with either integrin-binding peptide sequences or the selectin ligand sialyl Lewis X (SLe^X) within a parallel plate flow chamber to examine the dynamics of leukocyte adhesion to specific ligands. The adhesive peptide sequences arginine–glycine–aspartic acid–serine (RGDS) and leucine–aspartic acid–valine (LDV) as well as sialyl Lewis X were bound to the surface of photopolymerized PEG diacrylate hydrogels. Leukocytes perfused over these gels in a parallel plate flow chamber at physiological shear rates demonstrate both rolling and firm adhesion, depending on the identity and concentration of ligand bound to the hydrogel substrate. This new system provides a unique polymer-based model for the study of interactions between leukocytes and endothelium as well as a platform to develop improved scaffolds for cardiovascular tissue engineering.     

脂多糖诱导血管内皮细胞凝血途径相关因子基因表达变化

目的：观察脂多糖(LPS)对脐静脉血管内皮细胞(HUVECs)表达组织因子(TF)、组织因子抑制物(TFPI)和凝血酶调节蛋白(TM)的影响。方法：应用胰酶消化HUVECs并进行传代培养，用生长良好的第2、3代细胞进行实验。同时应用CCK-8测定细胞在不同浓度的LPS(1-100 mg/L)处理前后细胞活性；应用反转录聚合酶链反应(RT-PCR)法检测细胞内TM、TF和TFPI mRNA水平。结果：浓度为10 mg/L的LPS对细胞活力与对照组相比没有显著差异。浓度为10 mg/L的LPS作用使HUVECs显著上调TF mRNA表达，6-24 h可以使细胞TFPI mRNA表达下调,以后渐恢复正常表达，72 h达到正常对照组水平，同时下调TM mRNA表达。结论：LPS(10 mg/L)对HUVECs的活性不造成直接的影响，可显著上调HUVECs的TF mRNA转录，抑制TFPI mRNA 的转录，而不改变TM mRNA的转录，这可能与LPS在感染过程中诱导血栓形成,血液凝固和DIC发生相关。