Simulation, construction and characterization of a multi-functional thrombolytic agent with anti-thrombosis activity

Prourokinase (scu-PA), a thrombolytic agent, was inserted between Gly118 and Ile119 with foreign antithrombosis functional motif (Lys-Gly-Asp-Trp-motif) to construct a multi-functional chimeric molecule. The molecular model of a chimera was simulated and predicted. The recombinant chimeric protein was expressed by the baculovirus-insect cell expression system and purified by affinity chromatography. The physico-chemical characteristics of the chimeric molecule were assayed. The thrombolytic activity was determined to be 90000 IU/mg of fibrinolytic special activity by the fibrin-plate method. The anti-thrombosis activities were also assayed with IC₅₀ of 9.6 μM by an inhibition test of ADP-induced platelet aggregation.     

Construction and characterization of a recombinant chimeric plasminogen activator consisting of a fibrin peptide and a low molecular mass single-chain urokinase

A recombinant chimeric plasminogen activator (f beta/scuPA-32k), with a fibrin beta-chain peptide (comprising Gly15 through Arg 42) linked to the N-terminal of a low molecular mass (32 kDa) single-chain urokinase (scuPA-32k, comprising Leu144 through Leu 411) via a 50 amino acid linker sequence, was produced by expression the corresponding chimeric cDNA in Escherichia coli cells. After refolding in vitro, the chimeric protein was purified to homogeneity by zinc chelate-Sepharose chromatography, Sephacryl S200 chromatography and benzamidine-Sepharose chromatography in sequence. The apparent molecular mass was 36 kDa shown by SDS-PAGE analysis. The special activity was 87,000 IU/mg detected by fibrin plate determination. F beta/scuPA-32k could directly activate plasminogen following Michaelis-Menten kinetics with K(m) = 0.52 microM and k(2) = 0.0024 s(-1). Mediated by plasmin, the single-chain molecule could be converted to the active two-chain molecule. The chimeric protein had 3.3 times higher fibrin affinity than scuPA-32k in the fibrin concentration of 3.2 mg/mL, while the chimeric protein inhibited the fibrin clotting and platelet aggregation. F beta/scuPA-32k showed a higher thrombolytic potency in vitro plasma clot lysis than scuPA-32k and depleted less fibrinogen in plasma. These results showed that the chimeric protein had not only higher fibrinolytic activity but also anti-thrombus activity. Further evaluation of the thrombolytic potential in appropriate animal models is required.     

ut-PA抗PAI-1突变体的构建及在sf-9细胞中的表达

为使嵌合分子 ut- PA获得抗 PAI- 1抑制作用的性质 ,将删除了编码 u- PA中 R1 78- R1 79-H1 80 - R1 81的 1 2个核苷酸的 u- PA c DNA[u- PA( 1 ) ]的 Bam H - Eco R 部分酶切片段 ,克隆到含嵌合蛋白 ut- PA基因的转移载体 p VL 1 392 - ut- PA的相应位点中 ,构建了一个含有新的嵌合蛋白基因 ut- PA( 1 )的转移表达载体 p VL1 392 - ut- PA( 1 ) .在昆虫病毒表达系统 sf- 9细胞中表达该嵌合蛋白基因 ,表达上清具有纤溶性 ,用血纤维蛋白平板法和 S2 4 44 显色底物法分别测得活力为 2 4 8IU/ml和 380 IU/ml     

ut-PA抗PAI-1突变体的构建及在sf-9细胞中的表达

为使嵌合分子 ut- PA获得抗 PAI- 1抑制作用的性质 ,将删除了编码 u- PA中 R1 78- R1 79-H1 80 - R1 81的 1 2个核苷酸的 u- PA c DNA[u- PA( 1 ) ]的 Bam H - Eco R 部分酶切片段 ,克隆到含嵌合蛋白 ut- PA基因的转移载体 p VL 1 392 - ut- PA的相应位点中 ,构建了一个含有新的嵌合蛋白基因 ut- PA( 1 )的转移表达载体 p VL1 392 - ut- PA( 1 ) .在昆虫病毒表达系统 sf- 9细胞中表达该嵌合蛋白基因 ,表达上清具有纤溶性 ,用血纤维蛋白平板法和 S2 4 44 显色底物法分别测得活力为 2 4 8IU/ml和 380 IU/ml     

Inhibition of Platelet Aggregation of a Mutant Proinsulin Chimera Engineered by Introduction of a Native Lys-Gly-Asp-containing Sequence

An eight amino acid sequence, CAKGDWNC, from disintegrin barbourin, was introduced into an inactive human proinsulin molecule between the B28 and A2 sites to construct a chimeric, anti-thrombosis recombinant protein. The constructed Lys-Gly-Asp (KGD)-proinsulin gene was expressed in Escherichia coli and then purified. The KGD-proinsulin chimera protein inhibits human platelet aggregation, induced by ADP, with an IC₅₀ value (molar concentration causing 50% inhibition of platelet aggregation) of 830 nM and demonstrates also specific affinity to glycoprotein IIb/IIIa receptor. Its insulin receptor binding activity remaines as low as 0.04% with native insulin as a control.     

Construction of novel bifunctional chimeric proteins possessing antitumor and thrombolytic activities

Based on their respective antitumor and thrombolytic activities, the superantigen staphylococcal enterotoxin C2 (SEC2) and staphylokinase (Sak) were chosen for the construction of the novel chimeric proteins Sak-linker- SEC2 and SEC2-linker-Sak using a linker composed of nine Ala residues. Both chimeric proteins possessed nearly the same PBMC proliferation stimulating activity and antitumor activity as SEC2 and thrombolytic activity as Sak. Neither the SEC2 or Sak component of each chimeric protein affected the activity of the other component. The results presented in this study provide a possible strategy to prevent and cure tumor thrombus.     

A bi-functional anti-thrombosis protein containing both direct-acting fibrin(ogen)olytic and plasminogen-activating activities

Direct-acting fibrin(ogen)olytic agents such as plasmin have been proved to contain effective and safety thrombolytic potential. Unfortunately, plasmin is ineffective when administered by the intravenous route because it was neutralized by plasma antiplasmin. Direct-acting fibrin(ogen)olytic agents with resistance against antiplasmin will brighten the prospect of anti-thrombosis. As reported in ‘Compendium of Materia Medica’, the insect of Eupolyphaga sinensis Walker has been used as traditional anti-thrombosis medicine without bleeding risk for several hundreds years. Currently, we have identified a fibrin(ogen)olytic protein (Eupolytin1) containing both fibrin(ogen)olytic and plasminogen-activating (PA) activities from the beetle, E. sinensis. Objectives: To investigate the role of native and recombinant eupolytin1 in fibrin(ogen)olytic and plasminogen-activating processes. Methods and Results: Using thrombus animal model, eupolytin1 was proved to contain strong and rapid thrombolytic ability and safety in vivo, which are better than that of urokinase. Most importantly, no bleeding complications were appeared even the intravenous dose up to 0.12 µmol/kg body weight (3 times of tested dose which could completely lyse experimental thrombi) in rabbits. It is the first report of thrombolytic agents containing both direct-acting fibrin(ogen)olytic and plasminogen-activating activities. Conclusions: The study identified novel thrombolytic agent with prospecting clinical potential because of its bi-functional merits containing both plasmin- and PA-like activities and unique pharmacological kinetics in vivo.     

Construction of novel chimeric proteins through the truncation of SEC2 and Sak from Staphylococcus aureus

It is an usual clinical phenomenon that cancer patients are prone to thrombosis. Until now, there have been no efficient methods or appropriate drugs to prevent and cure tumor thrombus. Therefore, the construction of a bifunctional chimeric protein for the treatment of cancer, complicated with thrombosis, is of great significance. Utilizing the superantigenic activity of staphylococcal enterotoxin C2 (SEC2) and the thrombolytic activity of staphylokinase (Sak), Sak-linker-SEC2 and SEC2-linker-Sak were constructed which had good anti-tumor and thrombolytic activities at the same time. Due to the intrinsic emetic activity of SEC2 and high molecular weight (MW) of chimeric proteins (44?kDa), their clinical applications will be restricted. In this study, novel chimeric proteins including ΔSEC2–ΔSak and ΔSak–ΔSEC2 were constructed through the truncation of SEC2 and Sak without 9-Ala linker and His-tag. Compared with the former, both the truncated proteins preserved nearly the same anti-tumor and thrombolytic activities. In addition, their MWs were only 29?kDa and their immunoreactivities were slightly lower than that of Sak-linker-SEC2 and SEC2-linker-Sak, respectively. Therefore, the novel chimeric proteins possessed merits and characteristics, such as low MS, low immunogenicity, and difunctionality which the former had not. It will be of great interest if the above-mentioned proteins can be used to cure Trousseau syndrome in clinic.     

RGD多肽与尿激酶原嵌合分子的构建及性质研究

利用定点突变及DNA重组技术 ,将编码RGD多肽 (GPRGDWRMLG)的双链DNA片段 ,定向插入到相对于编码尿激酶原的Gly118 Leu119的cDNA分子中 ,构建了尿激酶原的嵌合体基因 ,并在甲醇酵母表达系统中进行了分泌表达。经过Zn²⁺ 螯合柱及SP阳离子柱两步层析后 ,目的蛋白质被纯化。经纤维蛋白平板测活 ,嵌合分子的比活为 6 5 0 0 0IU mg。嵌合分子与尿激酶相比 ,对显色底物S2 44 4作用的催化效率偏低 ,但具有较强的抑制血小板聚集的功能 ,抑制常数IC5 0为 2 1μmol L。以上结果表明嵌合分子不但具有较强的溶栓功能 ,而且具有抗栓功能 ,很可能是一种具有发展前景的双功能溶栓 抗栓分子     

嵌合体纤溶酶的研究进展

嵌合体纤溶酶是采用基因重组技术或化学偶联的方法将纤溶酶与其它的功能多肽(舍有酶原结构域,具有抗凝血酶、抗血小板聚集活性,能特异识别纤维蛋白的多肽)结合起来所形成的嵌合体蛋白质．它尽可能地保留了分子中每一组分的生物活性,增强了纤溶酶的溶栓功效,较大程度地克服了临床溶栓药物在某些方面的不足,是目前溶栓药物研究领域中的热点。     

Development of thrombolytic agents

Despite their widespread use in patients with acute myocardial infarction, all currently available thrombolytic agents suffer from a number of significant limitations, including resistance to reperfusion, the occurrence of acute coronary reocclusion and bleeding complications. Furthermore, the therapeutic use of plasminogen activators as thrombolytic agents requires intravenous infusion of relatively large amounts of material. Therefore, the quest for thrombolytic agents with a higher thrombolytic potency, specific thrombolytic activity and/or a better fibrin-selectivity continues. Several lines of research towards improvement of thrombolytic agents are being explored, including the construction of mutants and variants of plasminogen activators, chimeric plasminogen activators, conjugates of plasminogen activators with monoclonal antibodies, or plasminogen activators from animal or bacterial origin.     

Receptor functions and ligand-dependent transforming potential of a chimeric kit proto-oncogene.

The c-kit proto-oncogene, the cellular homolog of the transforming gene of a feline retrovirus, encodes a transmembrane tyrosine kinase homologous to receptors for growth factors. To study the cellular function of c-kit, we constructed a chimeric molecule composed of the extracellular portion of the receptor for epidermal growth factor (EGF) and the transmembrane and cytoplasmic domains of p145kit. The hybrid molecule was properly expressed in murine fibroblasts and displayed specific binding of EGF (Kd, 3 x 10(-8) M). Activation of the chimeric receptor by EGF stimulated the tyrosine kinase activity of kit and led to the generation of a potent mitogenic signal. Moreover, cells expressing the chimeric receptor acquired a transformed phenotype once they were stimulated with the heterologous ligand.     

Soluble Epoxide Hydrolase as an Anti-inflammatory Target of the Thrombolytic Stroke Drug SMTP-7

Although ischemic stroke is a major cause of death and disability worldwide, only a small fraction of patients benefit from the current thrombolytic therapy due to a risk of cerebral hemorrhage caused by inflammation. Thus, the development of a new strategy to combat inflammation during thrombolysis is an urgent demand. The small molecule thrombolytic SMTP-7 effectively treats ischemic stroke in several animal models with reducing cerebral hemorrhage. Here we revealed that SMTP-7 targeted soluble epoxide hydrolase (sEH) to suppress inflammation. SMTP-7 inhibited both of the two sEH enzyme activities: epoxide hydrolase (which inactivates anti-inflammatory epoxy-fatty acids) and lipid phosphate phosphatase. SMTP-7 suppressed epoxy-fatty acid hydrolysis in HepG2 cells in culture, implicating the sEH inhibition in the anti-inflammatory mechanism. The sEH inhibition by SMTP-7 was independent of its thrombolytic activity. The simultaneous targeting of thrombolysis and sEH by a single molecule is a promising strategy to revolutionize the current stroke therapy.     

Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by in vitro gene fusion

Two different lengths of the gene encoding Escherichia coli heat-stable toxin (STa) were fused to the carboxy end of the gene coding for the E. coli heat-labile toxin A-subunit (LTA). The hybrid genes directed expression of chimeric LTA-STa proteins. Association of these chimeras with native heat-labile toxin B-subunit (LTB) resulted in protein complexes that bound to GM1 ganglioside and thereby could be assayed in a GM1 ELISA. The complexes reacted with monoclonal antibodies against either LTA, LTB or STa indicating that the STa and LT epitopes remained immunologically intact after fusion. Genetically constructed chimeric proteins exhibiting LT and STa antigens on the same molecule may represent a promising approach to development of broadly protective immunoprophylactic agents and/or useful immunodiagnostic reagents for diarrhoeal diseases caused by enterotoxinogenic E. coli.     

Identification of domains and amino acids essential to the collagen galactosyltransferase activity of GLT25D1

Collagen is modified by hydroxylation and glycosylation of hydroxylysine residues. This glycosylation is initiated by the β1,O galactosyltransferases GLT25D1 and GLT25D2. The structurally similar protein cerebral endothelial cell adhesion molecule CEECAM1 was previously reported to be inactive when assayed for collagen glycosyltransferase activity. To address the cause of the absent galactosyltransferase activity, we have generated several chimeric constructs between the active human GLT25D1 and inactive human CEECAM1 proteins. The assay of these chimeric constructs pointed to a short central region and a large C-terminal region of CEECAM1 leading to the loss of collagen galactosyltransferase activity. Examination of the three DXD motifs of the active GLT25D1 by site-directed mutagenesis confirmed the importance of the first (amino acids 166-168) and second motif (amino acids 461-463) for enzymatic activity, whereas the third one was dispensable. Since the second DXD motif is incomplete in CEECAM1, we have restored the motif by introducing the substitution S461D. This change did not restore the activity of the C-terminal region, thereby showing that additional amino acids were required in this C-terminal region to confer enzymatic activity. Finally, we have introduced the substitution Q471R-V472M-N473Q-P474V in the CEECAM1-C-terminal construct, which is found in most animal GLT25D1 and GLT25D2 isoforms but not in CEECAM1. This substitution was shown to partially restore collagen galactosyltransferase activity, underlining its importance for catalytic activity in the C-terminal domain. Because multiple mutations in different regions of CEECAM1 contribute to the lack of galactosyltransferase activity, we deduced that CEECAM1 is functionally different from the related GLT25D1 protein.     

The C-terminal 165 amino acids of the plasma membrane Ca(2+)-ATPase confer Ca2+/calmodulin sensitivity on the Na+,K(+)-ATPase alpha-subunit.

The C-terminal 165 amino acids of the rat brain plasma membrane (PM) Ca(2+)-ATPase II containing the calmodulin binding auto-inhibitory domain was connected to the C-terminus of the ouabain sensitive chicken Na+,K(+)-ATPase alpha 1 subunit. Expression of this chimeric molecule in ouabain resistant mouse L cells was assured by the high-affinity binding of [3H]ouabain. In the presence of Ca2+/calmodulin, this chimeric molecule exhibited ouabain inhibitable Na+,K(+)-ATPase activity; the putative chimeric ATPase activity was absent in the absence of Ca2+/calmodulin and activated by Ca2+/calmodulin in a dose-dependent manner. Furthermore, this chimeric molecule could bind monoclonal IgG 5 specific to the chicken Na+,K(+)-ATPase alpha 1 subunit only in the presence of Ca2+/calmodulin, suggesting that the epitope for IgG 5 in this chimera is masked in the absence of Ca2+/calmodulin and uncovered in their presence. These results propose a direct interaction between the calmodulin binding auto-inhibitory domain of the PM Ca(2+)-ATPase and the specific regions of the Na+,K(+)-ATPase alpha 1 subunit that are structurally homologous to the PM Ca(2+)-ATPase. A comparison of the deduced amino acid sequences revealed several possible regions within the Na+,K(+)-ATPase that might interact with the auto-inhibitory domain of the PM Ca(2+)-ATPase.     

Anti-thrombosis effect of diosgenin extract from Dioscorea zingiberensis C.H. Wright in vitro and in vivo

Thrombus formation in blood vessel plays an important role in the pathogenesis and progression of atherosclerosis and cardiovascular diseases. Extract of Dioscorea zingiberensis C.H. Wright (D. zingiberensis) is demonstrated to posses activities for curing cardiovascular diseases such as coronary heart disease and angina pectoris. However, there were few studies on anti-thrombosis activity of it. We investigated the anti-thrombosis effect of diosgenin from D. zingiberensis (Dio) in vitro and in vivo on inferior vena cava ligation thrombosis rat model and pulmonary thrombosis mice model. We evaluated the protective effect of Dio by measuring the platelet aggregation, activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and the venous thrombosis in rats and the bleeding time, clotting time and protection rate in mice. Results showed that Dio inhibited platelet aggregation, thrombosis and prolonged APTT, PT and TT in rats in a dose-dependent manner. They also prolonged the bleeding time, clotting time and increased protection rate in mice in a dose-dependent manner. Taken together, these findings suggested that Dio which contained 95% diosgenin had anti-thrombosis activity. Dio executives the anti-thrombosis activity through improving the anticoagulation function, inhibiting platelet aggregation and thrombosis.