组织型纤溶酶原激活剂（t—PA）工程细胞系遗传特性及成瘤性分析

组织型纤溶酶原激活剂(t-PA)因其在防止血栓形成中起重要作用而受到人们的重视。但由于t-PA在血液中半衰期很短,作为溶栓药,一时难于推广。为了延长半衰期、增强其特异活性,本组构建了t-PA突变体并在CHO-dhfr~-细胞中获得了高效表达。我们在细胞培养基中加入秋水仙素,通过低张、固定、染色,进行染色体分析,结果表明,t-PA工程细胞系染色体条数为20条,畸变类型有异着丝粒。四倍体、裂隙、断片,畸变率为15％,属于正常范围。同时我们对该细胞系进行成瘤性试验,选用4周龄裸鼠作为试验鼠,以Hela细胞为阳性对照,CHO-dhfr~-细胞为阴性对照,试验表明:t-PA工程细胞及表达产物对裸鼠均无成瘤性。     

组织型纤溶酶原激活剂工程细胞系遗传特性及成瘤性分析

构建了突变体t-PA,使其在CHO-dhfr细胞中表达。获得t-PA工程细胞系。对该细胞系进行了遗传特性分析,在培养基中加入秋水仙素,通过低张、固定处理并将细胞滴片、染色,进行染色体分析,结果表明,该工程细胞系染色体条数为20条,畸变类型有异着丝粒、四倍体、裂隙、断片、微小体,畸变率为15％,属于正常范围。同时进行成瘤性检测,选用4周龄裸鼠作为试验鼠,以人宫颈癌细胞(Hela细胞)为阳性对照,CHO-dhfr细胞为阴性对照,试验表明,t-PA工程细胞及表达产物对裸鼠均无成瘤性。     

组织型纤溶酶原激活剂突变体细胞t—PA基因拷贝数测定及分析

组织型纤溶酶原激活剂（t—PA）溶栓特异性高,但半衰期短。本组构建了增强纤维蛋白亲合力和延长半衰期的t—PA突变体表达细胞株。测定工程细胞株基因拷贝数是细胞生物学特性鉴定的一个方面。我们采用狭缝杂交法和SouthernBlot法对该细胞株t—PA基因进行了拷贝数测定,结果为30—60个拷贝。     

人t-PA溶栓突变体的研究进展

人t-PA在机体循环中的纤溶系统中起重要作用,是一种内源性溶血栓因子,t-PA蛋白分子可直接用于溶栓治疗,但天然的t-PA分子在体内半衰期短,极最被清除,因而限制其广泛应用,根据它的结构特点而改造的一系列t-PA变体分子将成为新一代溶栓药物,在溶栓治疗中广泛应用。     

Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats.

In order to assess which part of the tissue-type plasminogen activator (t-PA) molecule should be (genetically) modified to obtain more-slowly-clearing mutants, two-chain t-PA and its isolated heavy and light chains were radiolabelled and injected into rats. The vast majority of t-PA and the heavy chain disappeared from the blood circulation with half-lives of 2.3 and 1.0 min respectively. The clearance of the light chain was biphasic, owing to complex-formation with plasma proteinase inhibitors. The disappearance of di-isopropylphospho-light chain, which has a blocked active site, was nearly monophasic, with a half-life of 5.7 min. Organ distribution studies showed that hepatic clearance constituted the major pathway in all cases. These results strongly suggest that t-PA is recognized by the liver primarily through the heavy chain.     

Structure-function analysis with tissue-type plasminogen activator. Effect of deletion of NH2-terminal domains on its biochemical and biological properties

Tissue-type plasminogen activator (t-PA) is a mosaic protein containing several distinct structural domains attached to the serine protease catalytic unit present at its COOH terminus. To investigate structure-function relationships in t-PA, we deleted the NH2-terminal domains, finger and epidermal growth factor, by genetic engineering. The genes for the parent and mutant t-PA were expressed in a bovine papilloma virus-dependent mammalian cell system. The secreted proteins were purified to homogeneity. The mutant protein was processed to the expected size of about 60 kDa compared to approximately 68 kDa for the parent t-PA, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography. While the mutant t-PA had amidolytic activity comparable to native t-PA, it did not bind appreciably to fibrin. Consequently, fibrin-dependent enzymic activity, i.e. plasminogen activation in the presence of soluble fibrin and fibrinolysis were lower than with native recombinant t-PA. The effect of deletion of NH2-terminal domains on the plasma half-life (t1/2) was investigated by injecting native and mutant t-PA into mice. While the majority of the t-PA disappeared initially with a t1/2 of about 2 min, mutant t-PA cleared at a much slower rate with t1/2 of about 50 min. These findings suggest that the NH2-terminal domains of t-PA not only determine its specificity for binding to fibrin but also mediate its clearance from plasma in vivo. Furthermore, the catalytic unit in t-PA seems to function autonomously.     

Identification of the mechanism responsible for the increased fibrin specificity of TNK-tissue plasminogen activator relative to tissue plasminogen activator

TNK-tissue plasminogen activator (TNK-t-PA), a bioengineered variant of tissue-type plasminogen activator (t-PA), has a longer half-life than t-PA because the glycosylation site at amino acid 117 (N117Q, abbreviated N) has been shifted to amino acid 103 (T103N, abbreviated T) and is resistant to inactivation by plasminogen activator inhibitor 1 because of a tetra-alanine substitution in the protease domain (K296A/H297A/R298A/R299A, abbreviated K). TNK-t-PA is more fibrin-specific than t-PA for reasons that are poorly understood. Previously, we demonstrated that the fibrin specificity of t-PA is compromised because t-PA binds to (DD)E, the major degradation product of cross-linked fibrin, with an affinity similar to that for fibrin. To investigate the enhanced fibrin specificity of TNK-t-PA, we compared the kinetics of plasminogen activation for t-PA, TNK-, T-, K-, TK-, and NK-t-PA in the presence of fibrin, (DD)E or fibrinogen. Although the activators have similar catalytic efficiencies in the presence of fibrin, the catalytic efficiency of TNK-t-PA is 15-fold lower than that for t-PA in the presence of (DD)E or fibrinogen. The T and K mutations combine to produce this reduction via distinct mechanisms because T-containing variants have a higher K(M), whereas K-containing variants have a lower k(cat) than t-PA. These results are supported by data indicating that T-containing variants bind (DD)E and fibrinogen with lower affinities than t-PA, whereas the K and N mutations have no effect on binding. Reduced efficiency of plasminogen activation in the presence of (DD)E and fibrinogen but equivalent efficiency in the presence of fibrin explain why TNK-t-PA is more fibrin-specific than t-PA.     

Characterization of the interaction in vivo of tissue-type plasminogen activator with liver cells

The interaction in vivo of 125I-labeled tissue-type plasminogen activator (t-PA) with the rat liver and the various liver cell types was characterized. Intravenously injected 125I-t-PA was rapidly cleared from the plasma (t1/2 = 1 min), and 80% of the injected dose associated with the liver. After uptake, t-PA was rapidly degraded in the lysosomes. The interaction of 125I-t-PA with the liver could be inhibited by preinjection of the rats with ovalbumin or unlabeled t-PA. The intrahepatic recognition site(s) for t-PA were determined by subfractionation of the liver in parenchymal, endothelial, and Kupffer cells. It can be calculated that parenchymal cells are responsible for 54.5% of the interaction of t-PA with the liver, endothelial cells for 39.5%, and Kupffer cells for only 6%. The association of t-PA with parenchymal cells was not mediated by a carbohydrate-specific receptor and could only be inhibited by an excess of unlabeled t-PA, indicating involvement of a specific t-PA recognition site. The association of t-PA with endothelial cells could be inhibited 80% by the mannose-terminated glycoprotein ovalbumin, suggesting that the mannose receptor plays a major role in the recognition of t-PA by endothelial liver cells. An excess of unlabeled t-PA inhibited the association of 125I-t-PA to endothelial liver cells 95%, indicating that an additional specific t-PA recognition site may be responsible for 15% of the high affinity interaction of t-PA with this liver cell type. It is concluded that the uptake of t-PA by the liver is mainly mediated by two recognition systems: a specific t-PA site on parenchymal cells and the mannose receptor on endothelial liver cells. It is suggested that for the development of strategies to prolong the half-life of t-PA in the blood, the presence of both types of recognition systems has to be taken into account.     

半衰期延长获得PAI－1抗性的t—PA突变体的构建、表达及特性分析

用基因重级及定位突变技术成功地构建了ｔ－ＰＡ的Ｋ１区缺失突变体ｔ－ＰＡｄｅｌＫ１、ＰＡＩ－１结合位点缺失突变体ｔ－ＰＡｄｅｌ（２９６－３０２）及两的组合突变全ｔ－ＰＡｄｅｌ（Ｋ１,２９６－３０２）,并在ＣＯＳ－７细胞中实现三的暂时性表达,在ＣＨＯ细胞中实现了ｔ－ＰＡｄｅｌ（Ｋ１,２９６－３０２）的稳定性表达。对表达产物的生物学特性分析表明,ｔ－ＰＡｄｅｌ（２９６－３０２）及ｔ－ＰＡｄｅｌ（Ｋ１     

Structure and function of human tissue-type plasminogen activator (t-PA)

Full-length tissue-type plasminogen activator (t-PA) cDNA served to construct deletion mutants within the N-terminal "heavy" (H)-chain of the t-PA molecule. The H-chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins ("finger," "epidermal growth factor," "kringles"). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon-intron junctions of the chromosomal t-PA gene. Recombinant t-PA deletion mutant proteins were obtained after transient expression in mouse Ltk- cells, transfected with SV40-pBR322-derived t-PA cDNA plasmids. It is demonstrated that the serine protease moiety of t-PA and its substrate specificity for plasminogen is entirely contained within the C-terminal "light" (L)-chain of the protein. The presence of cDNA, encoding the t-PA signal peptide preceding the remaining portion of t-PA, suffices to achieve secretion of (mutant) t-PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t-PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H-chain, kringle K1, and the epidermal growth-factor-like domain, do not contribute to this property of t-PA. These findings correlate well with the fibrin-binding properties of the rt-PA deletion-mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t-PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L-chain of t-PA. Deleting specific segments of t-PA H-chain cDNA and subsequent transient expression in mouse Ltk- cells of t-PA deletion-mutant proteins did not affect the formation of a stable complex between mutant t-PA and PAI.     

Spontaneous fibrinolysis in whole human plasma. Identification of tissue activator-related protein as the major plasminogen activator causing spontaneous activity in vitro

Spontaneous fibrinolysis of plasma clots was studied by following the lysis of the clots formed in 125I-fibrinogen-supplemented citrated plasma. Lysis of the clots invariably follows sigmoidal kinetics with S50 (the time required for 50% clot lysis) ranging from 3.5 to 4.7 days in 8 samples of pooled blood bank plasma and in the majority of apparently healthy donor plasmas. The spontaneous lysis of factor XII-deficient and prekallikrein-deficient plasmas was found to be similar to that of normal plasma. Addition of ellagic acid or antibodies against kallikrein or urokinase to normal pooled plasma did not alter significantly its rate of spontaneous lysis. On the other hand the addition of antibody against tissue activator (t-PA) inhibited over 80% of the spontaneous fibrinolysis in a 7-day incubation period at 37 degrees C, and the clot visually persisted for more than a month. Therefore, the factor XII-dependent components and prourokinase/urokinase system do not contribute significantly in whole plasma fibrinolysis in vitro, while the t-PA-related protein appears to be the major plasminogen activator responsible for initiating spontaneous fibrinolysis in whole plasma. Exogenous addition of increasing amounts of purified HeLa cell t-PA to normal pooled plasma in the ng/ml range cause progressively faster clot lysis. By extrapolation, normal pooled plasma is found to contain endogenous tissue activator in an amount functionally equivalent to 2 ng/ml of purified 60-kDa t-PA. The molecular nature of the t-PA-related proteins in plasma was studied by zymographic and immunological methods. The major t-PA-related protein in plasma was found to have a molecular mass of 100 kDa as determined by zymography. By incubating purified HeLa 60-kDa t-PA with a t-PA-depleted plasma, the 100-kDa component can be generated in plasma, suggesting that the latter is formed as a result of the binding of 60-kDa t-PA to a binding protein in plasma.     

Effect of site-directed PEGylation of trichosanthin on its biological activity, immunogenicity, and pharmacokinetics

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) with multiple biological and pharmacological activities. It has been approved effective in the clinical treatment of AIDS and tumor, but its strong immunogenicity and short plasma half-life have limited the clinical administration. To reduce the immunogenicity and prolong the plasma half-life of this compound, three TCS muteins (M(1), M(2) and M(3)) and two PEGylated TCS muteins (PM(1) and PM(2)) were constructed by site-directed mutagenesis and PEGylation, respectively. Compared with the unmodified TCS, both PEGylated TCS showed a 3- to 4-fold decrease in immunogenicity, a 0.5- to 0.8-fold decrease in non-specific toxicity, and a 4.5- to 6-fold increase in plasma half-life. But there is a problem of activity reduction. The increased circulating half-life in vivo may compensate for the reduced activity. Together with the other benefits of PEGylation such as reduced immunogenicity and toxicity, it is worthwhile to further explore the potential application of the PEGylated TCS as a better therapeutic agent for AIDS and tumor.     

Plasma levels of t-PA and PAI-1 correlate with the formation of experimental post-surgical peritoneal adhesions

This study has evaluated whether systemic changes of plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) parallel the adhesions development and whether they could be used as predictors of adhesion risk. This has been studied in an animal model of post-surgical peritoneal adhesion by monitoring for 10 days the plasma and tissue levels of t-PA and PAI-1. The results showed that both tissular and plasmatic levels of t-PA were decreased in concomitance with the development of peritoneal adhesions. In contrast, PAI-1 was found increased into the tissue and into the plasma samples of the rats taken at 5 and 10 days time points. Inflammatory mediators such as ICAM-1, VCAM-1, and IL-6 within the peritoneal lavage fluid also correlated with the adhesion formation process. In conclusion, post-surgical peritoneal adhesions provide alterations of local inflammatory components and local and systemic fibrinolytic components, possibly with PAI-1 quenching t-PA. This may have potential for the identification of high-risk patients.     

Inactivation of tissue plasminogen activator in plasma. Demonstration of a complex with a new rapid inhibitor

A new specific and sensitive method for determination of tissue plasminogen activator (t-PA) in plasma samples has been used to demonstrate the presence of a fast inhibitor to t-PA in plasma. By adding [35S]Met internally labeled t-PA (Mr approximately 70,000) to plasma, we were able to demonstrate the rapid formation of a stable complex with an apparent molecular weight of about 115,000 as estimated by gel filtration. The complex was partially purified by immunoadsorbtion chromatography on insolubilized antibodies against porcine t-PA, and a molecular weight of about 120,000 was found by dodecyl sulfate-polyacrylamide gel electrophoresis. From the apparent molecular weight of the complex (120,000) and the molecular weight of t-PA (70,000), a molecular weight of about 50,000 would be expected for the inhibitor. However, gel filtration of inhibitor-rich plasma resulted in the appearance of a symmetrical peak of t-PA inhibitory activity with an apparent molecular weight of about 205,000. The reason for this discrepancy is not known, but several different models are possible.     

t—PA昆虫细胞表达及特性分析

With the RT-PCR technique, human tissue-type plasminogen activator (t-PA) cDNA was amplified from human melanoma cells. The sequence was proved to be the same as those reported by foreign researchers. The t-PA cDNA containing whole reading frame was inserted into the transfer vector pBacPAK8. The constructed pBac-tPA and the linear BacPAK6 virus DNA were cotransfected with Tn-5B-1 insect cells by lipofectin-mediated transfection method. Eleven pure recombinant viruses were isolated by plaque assay, and one of them, nominated BactPA3, was selected by PCR identification and biological activity comparison. The t-PA activity expressed in serum-containing media reach the highest level at 72 h postinfection. The activity was 3.04 x 10(3) IU/ml, e.g. 1.8 x 10(4) IU/10(6) cells. The highest expression level in serum-free media was almost the same, but needed more time (at 132 h postinfection). SDS-PAGE fibrin autography showed that the molecular weight of the expressed t-PA was about 68 kDa. Its stimulation by fibrinogen, affinity with fibrin and inactivation in plasma were almost the same as the native t-PA purified from human melanoma cell culture. The half-life of t-PA expressed in serum-free media was seven minutes.     

Report of a clinical trial of a Canadian preparation of antihemophilic factor.

A clinical trial of a Canadian preparation of antihemophilic factor has been completed. The mean percentage increase in the factor VIII coagulant activity in the plasma was determined to be 2.1 +/- 0.8 per unit per kilogram dose, which compares very favourably with published data. The mean half-life of antihemophilic factor was found to be 14.7 hours. Side effects and reactions to the product were minimal and the product has been rated as clinically effective. No significant differences between lots were noted in the in vivo response or the half-life.     

The stratagem utilized by the plasminogen activator from the snake Trimeresurus stejnegeri to escape serpins

The plasminogen activator isolated from the venom of the snake Trimeresurus stejnegeri (TSV-PA) triggers plasmin production, along with tissue-type plasminogen activators (t-PA) and urokinase (u-PA). The half-life of TSV-PA in plasma is remarkable. We unveil in this paper two of the molecular mechanisms allowing TSV-PA to escape inhibition by plasma serpins. The first involves a phenylalanine at position 193 (chymotrypsinogen numbering system). Phe(193) distinguishes TSV-PA from nearly all trypsin-like proteinases, having glycine at this position. A mutant of TSV-PA (F193G), in which Phe(193) had been replaced by a glycine, was inactivated by plasminogen activator inhibitor 1 (PAI-1) and alpha(2)-antiplasmin 100-fold more rapidly than the wild-type enzyme. The second mechanism originates from the 37-loop of TSV-PA. Swapping the 37-loop of TSV-PA for either that of t-PA or that of u-PA also increased dramatically the rate of inactivation by PAI-1. Loop swapping and F193G mutations were additive, resulting in a rate of inactivation by PAI-1 that was 4 orders of magnitude higher than for the wild-type enzyme. The potential role of Phe(193) and of the 37-loop in the immunity of TSV-PA toward alpha(1)-antitrypsin and antithrombin is also discussed.     

Increased t-PA yields using ultrafiltration of an inhibitory product from CHO fed-batch culture

Fed-batch operation for the production of t-PA using Chinese Hamster Ovary (CHO) cells was optimized using serial and parallel experimentation. The feed, an isotonic concentrate, was improved to obtain 2- to 2.5-fold increases in integrated viable cell days versus batch. With a low glucose inoculum train, the viability index was further increased up to 4.5-fold. Hydrolysates were substituted for the amino acid portion of the concentrate with no significant change in fed-batch results. The concentrate addition rate was based on a constant 4 pmol/cell.day glucose uptake rate that maintained a relatively constant glucose concentration (approximately 3 mM). Increased viable cell indices did not lead to concomitant increases in t-PA concentrations compared to batch. The fed-batch concentrate and feeding strategy were shown to be effective in hybridoma culture, where a 4-fold increase in viable cell index yielded a 4-fold increase in antibody concentration. The half-life of t-PA decreased from 43 to 15 days with decreasing cell viability (from 92% to 71%), but this was not sufficient to explain the apparent t-PA threshold. Instead, the CHO results were explained by a reduction in t-PA production at higher extracellular t-PA concentrations that limited the fed-batch maximum at 35 mg/L for the cell line investigated. Analysis of both the total and t-PA mRNA levels revealed no response to increasing extracellular t-PA concentrations upon exogenous additions. Instead, intracellular t-PA levels were increased, revealing a possible secretory pathway limitation. A new reactor configuration was developed using an acoustic filter to retain the cells in the reactor while an ultrafiltration module stripped t-PA from the clarified medium before the permeate was returned to the reactor. By adding this harvesting step, the t-PA fed-batch production was increased over 2-fold, up to a yield of 80 mg/L.     

Tissue plasminogen activator catalyzed Lys-plasminogen activation on heparin-inserted phospholipid liposomes

We prepared heparin-inserted phospholipid liposomes as a functional model of heparan sulfate present on the vascular surface and examined tissue plasminogen activator (t-PA) catalyzed plasminogen activation on the liposome surface. Kinetic analyses showed a marked increase in the affinity of t-PA for Lys-plasminogen in the presence of heparin-inserted phosphatidylcholine (PC) liposomes. The catalytic efficiency (kcat/Km) of t-PA for the plasminogen activation on the surface of heparin-inserted PC liposomes was 5.4 times that on the surface of heparin-free PC liposomes. This stimulatory action of immobilized heparin was apparently affected by changing the phospholipid component of liposomes. Phosphatidylethanolamine or stearylamine, having a positively charged group, reduced the catalytic efficiency of t-PA by raising its Km value (10-fold), whereas negatively charged phospholipids, phosphatidylserine and phosphatidylinositol, did not affect the efficiency. t-PA and generated plasmin bound to the liposome surface heparin were protected from inhibition by plasminogen activator inhibitor type 1 and alpha 2-plasmin inhibitor, respectively. t-PA-induced clot lysis of euglobulin or whole plasma, which contained native (Glu-) plasminogen and the above inhibitors, was also accelerated by addition of heparin-inserted PC liposomes. These results suggest that the vascular surface heparin-like molecules may play an important role in modulating fibrinolytic events. The principles of conjugation of t-PA with a biologically active liposome will be applied to the construction of better thrombolytic agents.     

Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2

Highly purified plasminogen-activator inhibitors of type 1 (PAI-1) and type 2 (PAI-2), low-Mr form, were compared with respect to their kinetics of inhibition of tissue-type (t-PA) and urokinase-type plasminogen activator (u-PA). The time course of inhibition of plasminogen activator was studied under second-order or pseudo-first-order conditions. Residual enzyme activity was measured by the initial rate of hydrolysis of a chromogenic t-PA or u-PA substrate or by an immunosorbent assay for t-PA activity. PAI-1 rapidly reacted with single-chain t-PA as well as with two-chain forms of t-PA and u-PA. The second-order rate constant k for inhibition of single-chain t-PA (5.5 x 10(6) M-1 s-1) was about three times lower than k for inhibition of the two-chain activators. PAI-2 reacted slowly with single-chain t-PA, k = 4.6 x 10(3) M-1 s-1. The association rate was 26 times higher with two-chain t-PA and 435 times higher with two-chain u-PA. The k values for inhibition of single-chain t-PA, two-chain t-PA and two-chain u-PA were respectively, 1200, 150 and 8.5 times higher with PAI-1 than with PAI-2. The removal of the epidermal growth factor domain and the kringle domain from two-chain u-PA did not affect the kinetics of inhibition of the enzyme, suggesting that the C-terminal proteinase part of u-PA (B chain) is responsible for both the primary and the secondary interactions with PAI-1 and PAI-2. The k values for inhibition of single-chain t-PA and endogenous t-PA in plasma by PAI-1 or PAI-2 were identical indicating that t-PA in blood consists mainly in its single-chain form.