Intravascular release and urinary excretion of tissue factor pathway inhibitor during heparin treatment

Tissue-factor-pathway inhibitor is the principal regulator of tissue factor-induced coagulation. Heparin treatment mobilizes TFPI into the circulation and contributes to the anticoagulant effects of heparins. Previous studies have demonstrated a selective depletion of intravascular TFPI by unfractionated heparin (UFH) but not by low-molecular-weight heparin (LMWH). In this study we sought to investigate the time- and dose-dependent relationships between release of TFPI and lipoprotein lipase (LPL) in respons to UFH and LMWH and to investigate whether the selective depletion of TFPI by UFH but not by LMWH is related to differential urinary excretion of TFPI. Eight healthy males participated in an open crossover study in which participants were assigned to receive (1) continuous infusion of unfractionated heparin (UFH, 450 IU/kg/24 hr); (2) subcutaneous dalteparin, 100 IU/kg given twice at a 12-hr interval; (3) subcutaneous dalteparin, 200 IU/kg given once; or (4) saline-solution infusion. Similar dose-dependent mobilization of TFPI and lipoprotein lipase (LPL), another glucosaminoglycan (GAG)-anchored protein of the endothelial membrane, was observed after both subcutaneous and intravenous administration of heparins. However, UFH induced a more efficient release of both TFPI and LPL into plasma than did LMWH at equivalent anti-Xa levels, indicating molecular-weight dependence of the release reactions. However, LPL reached peak levels faster and was more rapidly cleared from the circulation than was TFPI, regardless of the treatment modality. Only trace amounts of TFPI were detected in the urine in a native form (38 kD). UFH and LMWH treatment reduced renal clearance of TFPI compared with the control regimen. Our findings suggest that displacement of TFPI from the endothelial-surface GAG is the main mechanism for TFPI release during heparin treatment in vivo and that differential urinary excretion of TFPI is not the explanation for selective depletion of TFPI during UFH treatment.     

Very low density lipoproteins stimulate surfactant lipid synthesis in vitro.

Surfactant synthesis is critically dependent on the availability of fatty acids. One fatty acid source may be circulating triglycerides that are transported in VLDL, and hydrolyzed to free fatty acids by lipoprotein lipase (LPL). To evaluate this hypothesis, we incubated immortalized or primary rat alveolar pre-type II epithelial cells with VLDL. The cells were observed to surface bind, internalize, and degrade VLDL, a process that was induced by exogenous LPL. LPL induction of lipoprotein uptake significantly increased the rates of choline incorporation into phosphatidylcholine (PC) and disaturated PC, and these effects were associated with a three-fold increase in the activity of the rate-regulatory enzyme for PC synthesis, cytidylyltransferase. Compared with native LPL, a fusion protein of glutathione S-transferase with the catalytically inactive carboxy-terminal domain of LPL did not activate CT despite inducing VLDL uptake. A variant of the fusion protein of glutathione S-transferase with the catalytically inactive carboxy-terminal domain of LPL that partially blocked LPL-induced catabolism of VLDL via LDL receptors also partially blocked the induction of surfactant synthesis by VLDL. Taken together, these observations suggest that both the lipolytic actions of LPL and LPL-induced VLDL catabolism via lipoprotein receptors might play an integral role in providing the fatty acid substrates used in surfactant phospholipid synthesis.     

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.     

Comparison of cell-surface TFPIα and β

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is mainly produced by endothelial cells and alternative mRNA splicing generates two forms, TFPIalpha and TFPIbeta. A portion of expressed TFPI remains associated with the cell surface through both direct (TFPIbeta) and indirect (TFPIalpha) glycosylphosphatidyl-inositol (GPT)-mediated anchorage. OBJECTIVE: Compare the structure and properties of TFPIalpha and TFPIbeta. METHODS: TFPIalpha and TFPIbeta, with protein molecular masses of 36 and 28 kDa, respectively, migrate similarly (46 kDa) on SDS-PAGE. Experiments using specific glycosidases were carried out to determine the different glycosylation pattern of the two forms. ECV304 cells, a cell line with some endothelial properties, were stimulated with IL-lbeta, LPS, and TNFalpha for up to 24 hrs and mRNA levels and protein synthesis were determined. Stable clones of ECV304 cells that express reduced levels of TFPIalpha, TFPIbeta or both were produced using a plasmid-based small-interfering RNA technique. Surface TFPI activity was determined by a two-stage chromogenic assay based on the ability of each form to inhibit FXa activation by FVIIa on cells with comparable amount of tissue factor (TF). RESULTS AND CONCLUSIONS: The deglycosylation studies show that the difference in molecular masses is due to a greater degree of sialylation in O-linked carbohydrate in TFPIbeta. The mRNA and protein levels of neither form of TFPI were affected by stimulation of cells with inflammatory stimuli. Although TFPIalpha comprises 80% of the surface-TFPI, TFPIbeta was responsible for the bulk of the cellular FVIIa/TF inhibitory activity, suggesting a potential alternative role for cell surface TFPIalpha.     

Evaluation of the roles of lipoprotein lipase and hepatic lipase in lipoprotein metabolism: in vivo and in vitro studies in man

Abstract. The roles of lipoprotein lipase (LPL) and hepatic lipase in very low density lipoprotein (VLDL) and VLDL remnant metabolism were investigated by (1) in vivo studies where the kinetics of VLDL-apo B removal were measured in patients with non-functioning lipoprotein lipase systems, and (2) in vitro studies where the relative capacities of hepatic lipase and LPL to hydrolyse the triglyceride (TG) of different lipoprotein substrates was measured. The results indicated that VLDL-apo B removal was not impaired in patients with non-functional LPL, nor was there any apparent abnormality in the conversion of VLDL-apo B to intermediate- (IDL) and low (LDL) density lipoprotein-apo B. Post-heparin plasma hepatic lipase activity against VLDL was normal in these subjects. Purified normal hepatic lipase had a similar K_m for VLDL-TG hydrolysis (1.57 mmol/l) to that of LPL (1.49 mmol/l). However, at equal lipoprotein TG concentration, hepatic lipase had increasing activity with lipoproteins of decreasing particle size, in the order chylomicrons ≪ VLDL of Sf 100–400 < VLDL of Sf 60–100 < VLDL of Sf 20–60 < IDL. The mean contribution of hepatic lipase to VLDL-TG hydrolysis by post-heparin plasma was 35% in normal controls, but the contribution to IDL-TG hydrolysis was significantly higher (mean = 58%). It is concluded that hepatic lipase plays a significant role in VLDL and, especially, IDL metabolism, at least in patients with non-functioning lipoprotein lipase.     

Antiflammin-2 prevents HL-60 adhesion to endothelial cells and prostanoid production induced by lipopolysaccharides

We studied the effect of antiflammin-2 (AF-2) on adhesion molecule expression by HL-60 cells and endothelial (ECV304) cells stimulated by lipopolysaccharides (LPSs), and on leukocyte-endothelial cell interaction in an in vitro coculture system. The action of AF-2 on prostanoid production in these experimental conditions was also tested. LPS increased the adhesion molecule expression, such as lymphocyte function-associated antigen-1 and membrane attack complex-1 on HL-60 cells and E-selectin and intercellular adhesion molecule-1 on ECV304 cells. The LPS-stimulated adhesion molecule expression on HL-60/ECV304 coculture system was higher than on HL-60 or ECV304 cultures. LPS also induced HL-60 adhesion to ECV304 monolayer and thromboxane B(2) and prostaglandin E(2) (PGE(2)) production in HL-60 culture and PGE(2) in ECV304 culture. Prostanoid production by HL-60/ECV304 cocultures was higher than by simple cultures. AF-2 inhibited the enhancement of adhesion molecule expression induced by LPSs, especially E-selectin. Thus, AF-2 significantly reduced the HL-60 adhesion to endothelial cells stimulated by LPSs. AF-2 also inhibited prostanoid synthesis by ECV304 cells or HL-60/ECV304 coculture challenged by LPSs. In conclusion, AF-2 reduced HL-60 adhesion to endothelial cells, suggesting that it reduces inflammation by blocking leukocyte trafficking and the subsequent eicosanoid production.     

Lipoprotein lipase increases low density lipoprotein retention by subendothelial cell matrix.

Lipoprotein lipase (LPL), the rate-limiting enzyme for hydrolysis of plasma lipoprotein triglycerides, is a normal constituent of the arterial wall. We explored whether LPL affects (a) lipoprotein transport across bovine aortic endothelial cells or (b) lipoprotein binding to subendothelial cell matrix (retention). When bovine milk LPL was added to endothelial cell monolayers before addition of 125I-labeled LDL, LDL transport across the monolayers was unchanged; but, at all concentrations of LDL tested (1-100 micrograms), LDL retention by the monolayers increased more than fourfold. 125I-labeled LDL binding to extracellular matrix increased when LPL was added directly to the matrix or was added to the basolateral side of the endothelial cell monolayers. Increased LDL binding required the presence of LPL and was not associated with LDL aggregation. LPL also increased VLDL, but not HDL, retention. Monoclonal anti-LPL IgG decreased both VLDL and LDL retention in the presence of LPL. Lipoprotein transport across the monolayers increased during hydrolysis of VLDL triglyceride (TG). In the presence of LPL and VLDL, VLDL transport across the monolayers increased 18% and LDL transport increased 37%. High molar concentrations of oleic acid to bovine serum albumin (3:1) in the medium increased VLDL transport approximately 30%. LDL transport increased 42% when oleic acid was added to the media. Therefore, LPL primarily increased retention of LDL and VLDL. A less remarkable increase in lipoprotein transport was found during hydrolysis of TG-containing lipoproteins. We hypothesize that LPL-mediated VLDL and LDL retention within the arterial wall potentiates conversion of these lipoproteins to more atherogenic forms.     

Tissue factor pathway inhibitor-γ is an active alternatively spliced form of tissue factor pathway inhibitor present in mice but not in humans

Summary.  Background:  Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor procoagulant activity produced as two alternatively spliced isoforms, TFPIα and TFPIβ, which differ in domain structure and mechanism for cell surface association. 3' Rapid amplification of cDNA ends was used to search for new TFPI isoforms. TFPIγ, a new alternatively spliced form of TFPI, was identified and characterized. Methods:  The tissue expression, cell surface association and anticoagulant activity of TFPIγ were characterized and compared to those of TFPIα and TFPIβ through studies of mouse and human tissues and expression of recombinant proteins in Chinese hamster ovary (CHO) cells. Results:  TFPIγ is produced by alternative splicing using the same 5'-splice donor site as TFPIβ and a 3'-splice acceptor site 276 nucleotides beyond the stop codon of TFPIβ in exon 8. The resulting protein has the first two Kunitz domains connected to an 18 amino acid C-terminal region specific to TFPIγ. TFPIγ mRNA is differentially produced in mouse tissues but is not encoded within the human TFPI gene. When expressed in CHO cells, TFPIγ is secreted into conditioned media and effectively inhibits tissue factor procoagulant activity. Conclusions:  TFPIγ is a third alternatively spliced form of TFPI that is widely expressed in mouse tissues but not made by human tissues. It contains the first two Kunitz domains and is a secreted, rather than a cell surface-associated, protein. It is a functional anticoagulant and may partially explain the resistance of mice to coagulopathy in tissue factor-mediated models of disease.     

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.     

Apolipoprotein B metabolism in subjects with deficiency of apolipoproteins CIII and AI. Evidence that apolipoprotein CIII inhibits catabolism of triglyceride-rich lipoproteins by lipoprotein lipase in vivo.

Previous data suggest that apolipoprotein (apo) CIII may inhibit both triglyceride hydrolysis by lipoprotein lipase (LPL) and apo E-mediated uptake of triglyceride-rich lipoproteins by the liver. We studied apo B metabolism in very low density (VLDL), intermediate density (IDL), and low density lipoproteins (LDL) in two sisters with apo CIII-apo AI deficiency. The subjects had reduced levels of VLDL triglyceride, normal LDL cholesterol, and near absence of high density lipoprotein (HDL) cholesterol. Compartmental analysis of the kinetics of apo B metabolism after injection of 125I-VLDL and 131I-LDL revealed fractional catabolic rates (FCR) for VLDL apo B that were six to seven times faster than normal. Simultaneous injection of [3H]glycerol demonstrated rapid catabolism of VLDL triglyceride. VLDL apo B was rapidly and efficiently converted to IDL and LDL. The FCR for LDL apo B was normal. In vitro experiments indicated that, although sera from the apo CIII-apo-AI deficient patients were able to normally activate purified LPL, increasing volumes of these sera did not result in the progressive inhibition of LPL activity demonstrable with normal sera. Addition of purified apo CIII to the deficient sera resulted in 20-50% reductions in maximal LPL activity compared with levels of activity attained with the same volumes of the native, deficient sera. These in vitro studies, together with the in vivo results, indicate that in normal subjects apo CIII can inhibit the catabolism of triglyceride-rich lipoproteins by lipoprotein lipase.     

The relationship of TFPI, Lp(a), and oxidized LDL antibody levels in patients with coronary artery disease

OBJECTIVES: The aim of the present study was to determine and correlate tissue factor pathway inhibitor (TFPI), lipoprotein (a) (Lp(a)), oxidized low-density lipoprotein (LDL) antibody (oLAB), and thiobarbituric acid reactive substances (TBARS; as a marker of lipid peroxidation) levels in patients with coronary artery disease (CAD) and in a control group. DESIGN AND METHODS: Peripheral blood samples from patients with coronary heart disease were provided by the Department of Cardiology. Serum oLAB, Lp(a), plasma total TFPI, and plasma-free TFPI levels were determined by ELISA. Serum TBARS levels were determined by a spectrophotometric method using thiobarbituric acid. RESULTS: The CAD and the control group were matched for age and sex. Serum Lp(a), oLAB, and plasma total TFPI levels in patients with coronary heart disease were found to be significantly higher than in the control group (P < 0.001). But there was no difference in plasma-free TFPI levels between patients with CAD and the control group (P > 0.05). In patients with single (P < 0.05), double, and triple vessel (P < 0.01) disease, the mean serum Lp(a) levels were significantly higher than in the control group. On the other hand, in patients with single vessel disease (P < 0.05), double vessel disease (P < 0.05), and triple vessel disease (P < 0.001), plasma total TFPI levels were found to be significantly higher than in the control group. We also found a significant positive correlation (r = 0.28, P < 0.05) between serum Lp(a) and plasma total TFPI levels in CAD. In the patient group, TBARS, total cholesterol, triglyceride (TRG), and LDL cholesterol levels were found to be significantly higher than those in the control group. In addition, high-density lipoprotein (HDL) cholesterol levels were found to be significantly lower than the control group. CONCLUSIONS: These results suggest that elevated plasma levels of total TFPI, Lp(a), and oLAB may be useful diagnostic and monitoring markers in patients with CAD.     

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.     

Temporal expression of alternatively spliced forms of tissue factor pathway inhibitor in mice

Summary.  Background: Mouse tissue factor pathway inhibitor (TFPI) is produced in three alternatively spliced isoforms that differ in domain structure and mechanism for cell surface binding. Tissue expression of TFPI isoforms in mice was characterized as an initial step for identification of their physiological functions. Methods and Results: Sequence homology demonstrates that TFPIα existed over 430 Ma while TFPIβ and TFPIγ evolved more recently. In situ hybridization studies of heart and lung did not reveal any cells exclusively expressing a single isoform. Although our previous studies have demonstrated that TFPIα mRNA is more prevalent than TFPIβ or TFPIγ mRNA in mouse tissues, western blot studies demonstrated that TFPIβ is the primary protein isoform produced in adult tissues, while TFPIα is expressed during embryonic development and in placenta. Consistent with TFPIβ as the primary isoform produced within adult vascular beds, the TFPI isoform in mouse plasma migrates like TFPIβ in SDS-PAGE and mice have a much smaller heparin-releasable pool of plasma TFPIα than humans. Conclusions: The data demonstrate that alternatively spliced isoforms of TFPI are temporally expressed in mouse tissues at the level of protein production. TFPIα and TFPIβ are produced in embryonic tissues and in placenta while adult tissues produce almost exclusively TFPIβ.     

抗VEGF抗体介导靶向超声造影剂的体外实验研究

目的以抗血管内皮因子（VEGF）抗体为配体研制能与血管内皮细胞特异性结合的靶向脂质体超声造影剂并检测其体外寻靶能力。 方法以静电吸附法将抗VEGF抗体连接到脂质体造影剂微泡的表面；体外培养ECV304人血管内皮细胞，用免疫荧光法检测靶向造影剂与其体外结合能力，以普通造影剂为对照组。 结果所制备的靶向超声造影剂与普通微泡无显著差异；免疫荧光实验结果显示靶向造影剂能在体外与血管内皮细胞特异性结合。 结论携抗VEGF抗体的脂质体靶向造影剂能通过静电吸附法成功制备，且在体外能与血管内皮细胞特异性结合。     

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。     

Disposition of tissue factor pathway inhibitor during cardiopulmonary bypass

BACKGROUND: The tissue factor (TF) factor (F) VIIa complex activates coagulation FIX and FX to initiate coagulation, and also cleaves protease activated receptors (PARs) to initiate inflammatory processes in vascular cells. Tissue factor pathway inhibitor (TFPI) is the only specific inhibitor of the TF-FVIIa complex, regulating both its procoagulant and pro-inflammatory properties. Upon heparin infusion during cardiopulmonary bypass (CPB), a heparin releasable pool of endothelial associated TFPI circulates in plasma. Following protamine neutralization of heparin, the plasma TFPI level decreases, but does not return completely to baseline, suggesting that during CPB a fraction of the plasma TFPI becomes heparin-independent. We have investigated the structural and functional properties of plasma TFPI during CPB to further characterize how TFPI is altered during this procedure. METHODS: We enrolled 17 patients undergoing first-time cardiac surgery involving CPB. Plasma samples were obtained at baseline, 5 min and 1 h after start of CPB (receiving heparin), 10 min after protamine administration (off CPB) and 24 h following surgery. Samples were analyzed for full-length and free (non-lipoprotein bound) TFPI antigen by enzyme-linked immunosorbent assay (ELISA) and for TFPI anticoagulant activity using an amidolytic assay. Western blot analysis was used to identify TFPI species of varying molecular weights in three additional patients. Dunnett's test for post hoc comparisons was used for statistical analysis. RESULTS: The ELISA and Western blot data indicated that an increase in full-length TFPI accounted for most of the heparin releasable TFPI. Following heparin neutralization with protamine, the full-length TFPI antigen returned to baseline levels while the free TFPI antigen and the total plasma TFPI activity remained elevated. This was associated with the appearance of a new 38 kDa form of plasma TFPI identified by Western blot analysis. The 38 kDa form of TFPI did not react with an antibody directed against the C-terminal region of TFPI indicating it has undergone proteolysis within this region. All TFPI measurements returned to baseline 24 h following CPB. CONCLUSIONS: During CPB the full-length form of TFPI is the predominant form in plasma because of its prompt release from the endothelial surface following heparin administration. Upon heparin neutralization with protamine, full-length TFPI redistributes back to the endothelial surface. However, a new 38 kDa TFPI fragment is generated during CPB and remains circulating in plasma, indicating that TFPI undergoes proteolytic degradation during CPB. This degradation may result in a decrease in endothelium-associated TFPI immediately post-CPB, and may contribute to the procoagulant and proinflammatory state that often complicates CPB.     

RT-PCR检测白血病细胞VEGF异构体及其受体基因

目的 建立逆转录-聚合酶链反应(RT-PCR)检测白血病细胞血管内皮生长因子(VEGF)异构体及其受体基因的方法。方法 用TRIZOL试剂提取体外培养的细胞系总RNA,M—MLV逆转录酶生成模板cDNA,PCR扩增VEGF异构体基因以及VEGF受体Flt-1基因,扩增产物用含溴化乙锭的2％琼脂糖凝胶电泳后,直接分析电泳结果。结果 白血病细胞(K562和HL-60)出现3条VEGF条带(516bp、588bp、648bp),分别代表VEGF3种异构体(VEGF121,VEGF145,VEGF165)的基因扩增产物,而人脐静脉血管内皮细胞(ECV304)无VEGF条带出现;此外,2种白血病细胞都可以测出VEGF的Flt-1受体基因,与Flt-1阳性细胞ECV304相似。结论 RT-PCR可用于白血病细胞VEGF多种异构体及其Flt-1受体基因的检测。     

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.     

卡维地洛及TNF-α对内皮细胞释放t-PA和PAI-1的影响

目的　研究卡维地洛及肿瘤坏死因子α(TNF α)对内皮细胞分泌组织纤溶酶原激活物 (t PA)和纤溶酶原激活物抑制剂 1(PAI 1)的影响。方法　培养内皮细胞株 (ECV3 0 4) ,分为TNF α刺激组 ,培养基中TNF α加至终浓度为 5、10、2 5、5 0、10 0ng/ml;卡维地洛干预组 ,培养基中加TNF α (5 0ng/ml)后加入卡维地洛 ,终浓度为 2 0、5 0、10 0、2 0 0nmol/L ,2 4h后测定上清中的组织纤溶酶原激活物 (t PA)和纤溶酶原激活物抑制剂 1(PAI 1)的含量。结果　内皮细胞在TNF α刺激 2 4h后 ,其分泌的PAI 1抗原含量与对照组比有明显升高 ,有显著性差异 (P <0 0 5 ) ,而两组间t PA含量无明显差异 (P >0 0 5 )。而卡维地洛干预组却显著降低PAI 1(P <0 0 5 ) ,对t PA含量无明显作用 (P >0 0 5 )。结论　TNF α对内皮细胞株分泌的PAI 1有显著的升高作用 ,而对t PA无显著影响 ;用卡维地洛干预后 ,PAI 1显著降低 ,t PA含量无明显改变。