The rediscovery and isolation of TFPI

Summary.  Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type proteinase inhibitor that produces factor (F)Xa-dependent feedback inhibition of the factor VIIa/tissue factor (FVIIa/TF) catalytic complex that is responsible for the initiation of coagulation. Since 1985, when Rapaport and colleagues reported that the lipoprotein fraction of plasma contained a FXa-dependent inhibitor of FVIIa/TF, myriad articles have established its biochemical structure, its mechanism of action, and its physiological importance. This brief personal account reviews historical studies that established the existence of the inhibitor and the events that led to its initial isolation.     

A novel anticoagulant activity assay of tissue factor pathway inhibitor I (TFPI).

Tissue factor (TF) pathway inhibitor I (TFPI) is the physiological inhibitor of TF-induced blood coagulation. Circulating blood contains full-length TFPI and TFPI truncated at the C-terminal end. Previous studies have shown that full-length TFPI exerts a stronger anticoagulant effect on diluted prothrombin time (DPT) than truncated TFPI, and it has been suggested that full-length TFPI is biologically more important in vivo. The objective of this study was to develop and validate an assay of TFPI anticoagulant activity. TFPI anticoagulant activity was assayed using a modified DPT assay. Plasmas were incubated in the absence and the presence of TFPI-blocking antibodies. Results were expressed as a ratio with the clotting time in the presence of anti-TFPI as the denominator. The ratio was normalized against a ratio obtained with a reference plasma. The assay was compared with assays of TFPI free antigen, total antigen, and bound TFPI, and TFPI chromogenic substrate activity. We performed all tests in 436 healthy individuals. The normalized TFPI anticoagulant ratio was strongly associated with TFPI free antigen (r = 0.73) but was weakly associated with TFPI chromogenic substrate activity (r = 0.46), TFPI total antigen (r = 0.48), and bound TFPI (r = 0.30). TFPI chromogenic substrate activity was strongly associated with TFPI total antigen (r = 0.73). We have developed a novel assay of TFPI anticoagulant activity in plasma, which may be considered a functional assay of full-length TFPI. Further studies are needed to establish the role of TFPI anticoagulant activity for thrombotic disorders.     

Low tissue factor pathway inhibitor (TFPI) together with low antithrombin allows sufficient thrombin generation in neonates

Summary.  Neonates have an excellent hemostasis despite, in comparison to adults, markedly decreased and delayed ability to generate thrombin. Only 30–50% of peak adult thrombin activity can be produced in neonatal plasma by means of conventional in vitro assays. We show that in contrast to conventional activation, activation with small amounts of lipidated tissue factor (<10 pmol L⁻¹) results in shorter clotting times and faster activated factor X- and thrombin generation in neonates compared with adults due to the concomitant action of low tissue factor pathway inhibitor and antithrombin. The concentrations of both inhibitors in cord plasma are approximately 50% of the respective adult values. After addition of 2.5 pmol L⁻¹ lipidated tissue factor, cord plasma clotted ∼90 s earlier than adult plasma and the amount of free thrombin generated was ∼90% of adult value (291 ± 14 vs. 329 ± 16 nmol L⁻¹ min⁻¹, P  < 0.01). Our results might help to explain the clinically observed excellent hemostasis of neonates despite low levels of procoagulant factors.     

Elevated TFPI levels in female hyperhomocysteinemic patients

OBJECTIVES: Hyperhomocysteinemia is a risk factor for cardiovascular diseases, while tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation. We investigated the in vivo influence of elevated homocysteine concentrations on TFPI levels. METHODS: Total TFPI levels of 82 hyperhomocysteinemic patients were determined. RESULTS: The patients' TFPI levels were significantly elevated compared to blood donors, with a more pronounced effect in females than in males. CONCLUSIONS: We have shown a gender-dependent TFPI elevation in hyperhomocysteinemic patients.     

TF及TFPI在急性白血病患者血浆中检测的临床意义

目的：检测急性白血病（AL）患者化疗前后血浆中组织因子（TF）及组织因子途径抑制物（TFPI）的含量变化,探讨血浆TF及TFPI在AL病情进展、疗效观察、预后判断中的意义,为AL患者发生凝血功能异常提供临床诊断和治疗依据。方法：选择2009年7月—2010年2月间住院的各型初诊AL患者48例,正常对照组20例,采集空腹外周肘静脉血,应用酶联免疫吸附法（ELISA）检测AL患者血浆中TF及TFPI的含量,同时检测其他的临床相关指标,比较血浆TF及TFPI含量在AL患者化疗前后的变化。结果：初诊的AL患者经治疗缓解后血浆TF和TFPI含量均较治疗前明显下降,差异有统计学意义;急性早幼粒细胞白血病（APL）患者化疗前血浆TF和TFPI含量均明显高于其他类型AL,APL患者化疗前后血浆TF和TFPI含量均有明显差异;合并DIC组的AL患者血浆TF和TFPI含量均明显高于不合并DIC组的AL,且化疗前后血浆TF和TFPI含量有显著差异。结论：初诊的AL患者血浆中TF和TFPI的含量均明显增高,而治疗缓解后降低,提示血浆中TF和TFPI含量变化与疾病的发生、进展及临床疗效有一定关系;血浆中TF和TFPI含量的升高与DIC的发生密切相关,在一定程度上对DIC的疗效判断具有预示作用。     

Novel insights into the regulation of coagulation by factor V isoforms,tissue factor pathway inhibitorα, and protein S

Factor V (FV) is a regulator of both pro‐ and anticoagulant pathways. It circulates as a single‐chain procofactor, which is activated by thrombin or FXa to FVa that serves as cofactor for FXa in prothrombin activation. The cofactor function of FVa is regulated by activated protein C (APC) and protein S. FV can also function as an anticoagulant APC cofactor in the inhibition of FVIIIa in the membrane‐bound tenase complex (FIXa/FVIIIa). In recent years, it has become clear that FV also functions in multiple ways in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. Of particular importance is a FV splice variant (FV‐Short) that serves as a carrier and cofactor to TFPIα in the inhibition of FXa. FV‐Short is generated through alternative splicing of exon 13 that encodes the large activation B domain. A highly negatively charged binding site for TFPIα is exposed in the C‐terminus of the FV‐Short B domain, which binds the positively charged C‐terminus of TFPIα, thus keeping TFPIα in circulation. The binding of TFPIα to FV‐Short is also instrumental in localizing the inhibitor to the surface of negatively charged phospholipids, where TFPIα inhibits FXa in process that is stimulated by protein S. Plasma FV activation intermediates and partially proteolyzed platelet FV similarly bind TFPIα with high affinity and regulate formation of prothrombinase. The novel insights gained into the interaction between FV isoforms, TFPIα, and protein S have opened a new avenue for research about the mechanisms of coagulation regulation and also for future development of therapeutics aimed at modulating coagulation.     

妇科恶性肿瘤患者血浆TFPI的检测及分析

目的研究血浆组织因子途径抑制物(TFPI)抗原含量及活性对妇科恶性肿瘤患者的临床意义。方法应用双抗体夹心酶联免疫吸附法及发色底物法对 4 9例妇科恶性肿瘤患者血浆TFPI抗原含量及活性进行测定。结果妇科恶性肿瘤患者血浆TFPI抗原含量及活性明显增高 ,且晚期患者较早期患者血浆TFPI抗原含量及活性明显增高。结论检测妇科恶性肿瘤患者血浆TFPI抗原含量及活性有助于诊断及监测病情     

创伤弧菌脓毒症大鼠肝组织因子和组织因子途径抑制物的基因表达及抗生素的影响

目的 研究创伤弧菌(VV)脓毒症大鼠肝组织的组织因子(TF)和组织因子途径抑制物(TFPI)基因表达及抗生素头孢哌酮钠联用乳酸左旋氧氟沙星对其的影响.方法 110只雄性SD大鼠随机(随机数字法)分为正常对照组(NC组,10只)、创伤弧菌脓毒症组(VV组,分5个哑组,每亚组10只)、创伤弧菌脓毒症药物干预组(AA组,分5个亚组,每亚组10只).构建创伤弧菌脓毒症模型及药物干预模型.RT-PCR检测大鼠肝组织TF mRNA和TFPI mRNA的基因表达水平.应用SPSS 12.0进行t检验、方差分析等处理.结果 与NC组相比,VV组染菌后2 h,6 h,9 h,12 h,16 h TF mRNA表达均明显升高(P<0.05),其中染菌6 h表达最高;从组染菌后9 h,12 h的TF mRNA仍明显高于NC组(P<0.05),后逐渐减少.VV组与AA组的肝组织TFPI mRNA与NC组相比,均无明显改变(P>0.05).与相同时间点的VV组相比,AA组16 h TF mRNA明显降低(P相似文献   

Atorvastatin or transgenic expression of TFPI inhibits coagulation initiated by anti‐nonGal IgG binding to porcine aortic endothelial cells

Summary. Background: Intravascular thrombosis remains a barrier to successful xenotransplantation. Tissue factor (TF) expression on porcine aortic endothelial cells (PAECs), which results from their activation by xenoreactive antibodies (Abs) to Galα1,3Gal (Gal) and subsequent complement activation, plays an important role. Objectives: The present study aimed to clarify the role of Abs directed against nonGal antigens in the activation of PAECs to express functional TF and to investigate selected methods of inhibiting TF activity. Methods: PAECs from wild‐type (WT), α1,3‐galactosyltransferase gene‐knockout (GT‐KO) pigs, or pigs transgenic for CD46 or tissue factor pathway inhibitor (TFPI), were incubated with naïve baboon serum (BS) or sensitized BS (with high anti‐nonGal Ab levels). TF activity of PAECs was assessed. Results: Only fresh, but not heat‐inactivated (HI), naïve BS activated WT PAECs to express functional TF. Similarly, PAECs from CD46 pigs were resistant to activation by naïve BS, but not to activation by fresh or HI sensitized BS. HI sensitized BS also activated GT‐KO PAECs to induce TF activity. TF expression on PAECs induced by anti‐nonGal Abs was inhibited if serum was pretreated with (i) an anti‐IgG Fab Ab or (ii) atorvastatin, or (iii) when PAECs were transgenic for TFPI. Conclusions: Anti‐nonGal IgG Abs activated PAECs to induce TF activity through a complement‐independent pathway. This implies that GT‐KO pigs expressing a complement‐regulatory protein may be insufficient to prevent the activation of PAECs. Genetic modification with an ‘anticoagulant’ gene (e.g. TFPI) or a therapeutic approach (e.g. atorvastatin) will be required to prevent coagulation dysregulation after pig‐to‐primate organ transplantation.     

Exenatide enhances cognitive performance and upregulates neurotrophic factor gene expression levels in diabetic mice

Exenatide is a potent and selective agonist for the GLP‐1 (glucagon‐like peptide‐1) receptor. Recent studies are focused on the effects of GLP‐1 analogues on hippocampal neurogenesis, cognition, learning and memory functions. The aim of this study was to assess the effects of chronic exenatide treatment (0.1 μg/kg, s.c, twice daily for 2 weeks) on spatial memory functions by using the modified elevated plus maze (mEPM) test and emotional memory functions by using the passive avoidance (PA) test in streptozotocin/nicotinamide (STZ‐NA)‐induced diabetic mice. As the genes involved in neurite remodelling are among the primary targets of regulation, the effects of diabetes and chronic administration of exenatide on brain‐derived neurotrophic factor (BDNF) and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) messenger ribonucleic acid (mRNA) levels in the hippocampus of mice were also determined using quantitative real‐time polymerase chain reaction (RT‐PCR). This study revealed that in the mEPM and PA tests, type‐2 diabetes‐induced mice exhibited significant impairment of learning and memory which were ameliorated by GLP‐1 receptor agonist exenatide. Quantitative RT‐PCR revealed that CREB and BDNF gene expression levels were downregulated in diabetic mice, and these alterations were increased by exenatide treatment. Since, exenatide improves cognitive ability in STZ/NA‐induced diabetic mice and activates molecular mechanisms of memory storage in response to a learning experience, it may be a candidate for alleviation of mood and cognitive disorder.     

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.     

Heme binds to factor VIII and inhibits its interaction with activated factor IX

Summary. Background: Heme is a redox active macrocyclic compound that is released upon tissue damage or hemorrhages. The extracellular release of large amounts of heme saturates scavenging heme‐binding proteins. Free heme has been proposed to affect coagulation and has been co‐purified with the factor VIII (FVIII)‐von Willebrand factor (VWF) complex. The sites from which heme is released upon injury overlap with the sites to which FVIII is targeted for performing its hemostatic functions. Objectives: To investigate the interaction of heme with FVIII and the consequence for the procoagulant activity of FVIII in vitro. Methods and results: Heme bound to several sites on FVIII with high apparent affinity. Heme‐binding inhibited FVIII procoagulant activity in a dose‐dependent manner. FVIII inactivation in the presence of saturating amounts of heme implicated a reduced interaction of FVIII with activated FIX, as shown by ELISA, surface plasmon resonance and fluorescence quenching. Heme‐mediated inactivation of FVIII was prevented by VWF, but not by human serum albumin, a heme‐binding protein known for its protective activity in hemolytic conditions. Conclusions: Our data identify FVIII as a novel heme‐binding protein. Occupation of high affinity heme‐binding sites on FVIII at low concentrations of free heme did not inactivate FVIII. Conversely, large molar excesses of heme over FVIII, which correspond to conditions of extensive heme release, inhibited FVIII activity in vitro. It remains to be demonstrated whether, under such conditions, heme‐mediated modulation of the activity of FVIII plays some role in the regulation of coagulation.