Influence of hirudin on the consumption of antithrombin III in experimental DIC

The effect of hirudin and heparin on thrombin-induced consumption of antithrombin III, fibrinogen and platelets was studied in a rat model. Antithrombin III is consumed by tolerated thrombin doses by about 20 per cent. Hirudin and heparin ameliorate the consumption of fibrinogen and platelets at the low thrombin dose used. At high thrombin doses, tolerated only during simultaneous administration of exogenous inhibitors, heparin leads to markedly increased consumption of anti-thrombin III, whereas hirudin does not. With either kind of treatment, the thrombin effect on fibrinogen and platelets is inhibited, however, hirudin acts independently of a cofactor in contrast to heparin.     

Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases

Hirudin, a tyrosine-sulfated protein secreted by the leech Hirudo medicinalis, is one of the most potent anticoagulants known. The hirudin cDNA has previously been cloned and has been expressed in yeast, but the resulting recombinant protein was found to be produced in the unsulfated form, which is known to have an at least 10 times lower affinity for thrombin than the naturally occurring tyrosine-sulfated hirudin. Here we describe the in vitro tyrosine sulfation of recombinant hirudin by leech and bovine tyrosylprotein sulfotransferase (TPST). With both enzymes, in vitro sulfation of recombinant hirudin occurred at the physiological site (Tyr-63) and rendered the protein biochemically and biologically indistinguishable from natural hirudin. However, leech TPST had an over 20-fold lower apparent Km value for recombinant hirudin than bovine TPST. Further differences in the catalytic properties of leech and bovine TPSTs were observed when synthetic peptides were tested as substrates. Moreover, a synthetic peptide corresponding to the 9 carboxyl-terminal residues of hirudin (which include Tyr-63) was sulfated by leech TPST with a similar apparent Km value as full length hirudin, indicating that structural determinants residing in the immediate vicinity of Tyr-63 are sufficient for sulfation to occur.     

Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition.

Thrombin is a serine protease that plays a central role in blood coagulation. It is inhibited by hirudin, a polypeptide of 65 amino acids, through the formation of a tight, noncovalent complex. Tetragonal crystals of the complex formed between human alpha-thrombin and recombinant hirudin (variant 1) have been grown and the crystal structure of this complex has been determined to a resolution of 2.95 A. This structure shows that hirudin inhibits thrombin by a previously unobserved mechanism. In contrast to other inhibitors of serine proteases, the specificity of hirudin is not due to interaction with the primary specificity pocket of thrombin, but rather through binding at sites both close to and distant from the active site. The carboxyl tail of hirudin (residues 48-65) wraps around thrombin along the putative fibrinogen secondary binding site. This long groove extends from the active site cleft and is flanked by the thrombin loops 35-39 and 70-80. Hirudin makes a number of ionic and hydrophobic interactions with thrombin in this area. Furthermore hirudin binds with its N-terminal three residues Val, Val, Tyr to the thrombin active site cleft. Val1 occupies the position P2 and Tyr3 approximately the position P3 of the synthetic inhibitor D-Phe-Pro-ArgCH2Cl. Thus the hirudin polypeptide chain runs in a direction opposite to that expected for fibrinogen and that observed for the substrate-like inhibitor D-Phe-Pro-ArgCH2Cl.     

The comeback of hirudin--an old-established anticoagulant agent

Early studies dating back to 1884 revealed that extracts from medicinal leeches contain a substance which is able to prevent blood from clotting. Since our successful isolation of hirudin, the pure anticoagulant substance, in the late 1950s and its characterization as a selective thrombin inhibitor with polypeptide structure, hirudin preparations have been employed for diagnostic and scientific uses in haemostaseology. As early as 25 years ago we have shown in experimental pharmacotoxicological studies that hirudin is an anticoagulant of high quality. The antithrombotic effect of hirudin was demonstrated in several thrombosis models. But the clinical use of hirudin remained limited since it was not available in adequate amounts for therapeutic purposes. One hundred years after its discovery there is a renewed interest in this naturally occurring thrombin inhibitor. Advanced methods of peptide isolation and genetic engineering are about to provide sufficient quantities of hirudin in purified form. This prompted us to resume our investigations in hirudin and to represent new experimental and clinical pharmacological studies with natural hirudin prepared from medicinal leeches and genetically engineered recombinant hirudin, thus appreciating the comeback of hirudin into the focus of interest.     

Sulphation of hirudin in BHK cells

Hirudin, a thrombin inhibitor of the leech, was expressed in BHK cells; the alpha 1-antitrypsin signal peptide was used to direct secretion into the culture medium. The recombinant hirudin so produced inhibited thrombin and was shown by labelling experiments with [35S]sulphate to have been posttranslationally modified.     

Secretion of biologically active leech hirudin from baculovirus-infected insect cells.

The thrombin inhibitor, hirudin, from the leech Hirudo Medicinalis, is the most powerful natural anticoagulant known. It has been characterized as a polypeptide of 65 amino acids which exhibits its anticoagulant properties by binding tightly and specifically to alpha-thrombin. The potency and specificity of hirudin have generated interest on its possible use in the treatment or prophylaxis of various thrombotic diseases. We have used the baculovirus expression system to efficiently produce active hirudins in insect cells. The Autographa californica nuclear polyhedrosis virus has proved useful as a helper-independent viral expression vector for high-level production of recombinant proteins in cultured insect cells. Hirudin variants (HV1 and HV2) were produced in infected insect cells as secreted proteins by joining their coding sequences to the leader peptide sequence of the vescicular stomatitis virus G protein. The recombinant products were biologically active and, interestingly, N-terminal sequencing of HV1 revealed that the heterologous leader peptide is correctly removed.     

Effect of heparin on the interaction between thrombin and hirudin

The effect of heparin on the interaction between thrombin and hirudin has been examined by kinetic methods. Three forms of heparin fractionated on the basis of their affinity for antithrombin III and unfractionated heparin were found to act as noncompetitive inhibitors of the formation of the thrombin-hirudin complex. A three--four fold increase in the dissociation constant of the complex was observed at saturating heparin concentrations. This increase in the dissociation constant was due to a twofold decrease in the rate of association of thrombin and hirudin together with a similar increase in the rate of dissociation of the complex. Implications for the location of the heparin binding site on thrombin and the possible therapeutic use of the hirudin are discussed.     

水蛭素抑制人脐静脉内皮细胞基质金属蛋白酶-2的生成及活化

研究凝血酶对人脐静脉内皮细胞表达基质金属蛋白酶的影响及重组水蛭素的作用。将原代培养人脐静脉内皮细胞(HUVEC)的第2～5代分组后与凝血酶(4．0ku／L)共同温育,同时分别加入水蛭素(6．0ku/L)和肝素(6．0ku／L),在不同时间,用逆转录聚合酶链反应和免疫组织化学分析的方法评价基质金属蛋白酶-2的表达情况。结果显示凝血酶促进血管内皮细胞产生和活化基质金属蛋白酶-2。重组水蛭素可以有效地阻断凝血酶的上述作用。     

重组水蛭素的突变及突变体部分性质研究

以基因突变结合动力学分析的方法研究了水蛭素空间结构及其与凝血酶的相互作用．采用基因定点突变和随机突变的方法得到两个重组水蛭素突变体,并从抗酰胺水解活性,抗凝血酶活力和稳定性三个方面,比较研究了重组水蛭素ｒＨＶ２中４７位和１１位两个氨基酸残基对其稳定性和抑制能力的影响．将ｒＨＶ２中Ｇｌｎ１１和Ａｓｎ４７分别突变为Ｈｉｓ１１和Ｌｙｓ４７后,ｒＨＶ２－Ｈ１１生物活力降低３０％,ｒＨＶ２－Ｋ４７生物活力提高６１％．测定抑制常数Ｋｉ表明,ｒＨＶ２－Ｈ１１突变体Ｋｉ值升高１４倍,ｒＨＶ２－Ｋ４７突变体Ｋｉ值降低１４倍,两个突变体的热稳定性均有所增强,ｒＨＶ２－Ｈ１１在酸性和碱性条件的稳定性降低．分析实验结果,可以认为：①４７位的Ｌｙｓ可能是通过氢键和静电两种作用力同时影响着水蛭素的三维结构和其与凝血酶的结合．②１１位氨基酸可能是水蛭素分子中另一个重要位点．     

Incorporation of noncoded amino acids into the N-terminal domain 1-47 of hirudin yields a highly potent and selective thrombin inhibitor.

Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potency (Kd = 0.2-1.0 pM) and selectivity. Hirudin is composed of a compact N-terminal region (residues 1-47, cross-linked by three disulfide bridges) that binds to the active site of thrombin, and a flexible C-terminal tail (residues 48-64) that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeutic profile, several N-terminal fragments have been prepared as potential anticoagulants. However, the practical use of these fragments has been impaired by their relatively poor affinity for the enzyme, as given by the increased value of the dissociation constant (Kd) of the corresponding thrombin complexes (Kd = 30-400 nM). The aim of the present study is to obtain a derivative of the N-terminal domain 1-47 of hirudin displaying enhanced inhibitory potency for thrombin compared to the natural product. In this view, we have synthesized an analogue of fragment 1-47 of hirudin HM2 in which Val1 has been replaced by tert-butylglycine, Ser2 by Arg, and Tyr3 by beta-naphthylalanine, to give the BugArgNal analogue. The results of chemical and conformational characterization indicate that the synthetic peptide is able to fold efficiently with the correct disulfide topology (Cys6-Cys14, Cys16-Cys28, Cys22-Cys37), while retaining the conformational properties of the natural fragment. Thrombin inhibition data indicate that the effects of amino acid replacements are perfectly additive if compared to the singly substituted analogues (De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A, 1998, Biochemistry 37:13507-13515), yielding a molecule that inhibits the fast or slow form of thrombin by 2,670- and 6,818-fold more effectively than the natural fragment, and that binds exclusively at the active site of the enzyme with an affinity (Kd,fast = 15.4 pM, Kd,slow = 220 pM) comparable to that of full-length hirudin (Kd,fast = 0.2 pM, Kd,slow = 5.5 pM). Moreover, BugArgNal displays absolute selectivity for thrombin over the other physiologically important serine proteases trypsin, plasmin, factor Xa, and tissue plasminogen activator, up to the highest concentration of inhibitor tested (10 microM).     

DNA改组技术在水蛭素实验进化中的应用

蛋白质的改造是生物工程的重大研究课题.由于结构和功能预测的不精确性,而使按照三维结构信息进行定位诱变往往达不到预期的目的.近年来,另一条改造蛋白质的途径有较大的发展,即在实验室条件下模拟生物分子的自然进化,通过变异和靶功能的选择来获得改进性能的蛋白质[1],此过程称为生物分子实验定向进化.DNA改组(DNAshuffling)是一种改造基因和蛋白质的有效实验进化技术[2].它是在体外进行基因随机片段的重组,从而增加基因的多样性,促使有利变异与不利变异分离,通过选择使有利变异得到优化组合[3].DNA改组包含3个步骤:基因的随机片段化,自身引发PCR和重组合PCR.经过DNA改组的突变体库有可能选择到性能更优的突变体.为进行亲和淘选,需将突变体展示在噬菌体的表面[4].     

Hirudin variants production by genetic engineered microbial factory

Hirudin was discovered as an active anticoagulant in leech extracts almost 60 years ago. Since their initial discovery, hirudin and its variants have been produced with various anti-thrombotic, cancer cell inhibition, diabetic cataract treatment and anti-fatigue activities. Some hirudin variants have been approved for clinical use and released into the marketplace. Recent progress has seen made in relation to hirudin variants expressed in several well-established microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and others, with high levels of activity and yield. This review summarizes the current progress on hirudin production using microbial producers, and considers the outlook for future development.     

Production and Characterization of Hirudin Variant-1 by SUMO Fusion Technology in E. coli

Hirudin is the most potent non-covalent inhibitor of thrombin. Several expression systems have been used to produce recombinant hirudin for pharmaceutical purposes. However, high expression of active hirudin in Escherichia coli cytoplasm has not been successful owing to the fact that heterogenetic small peptide is easily degraded in the cell. To solve this problem, we constructed a recombinant form of the hirudin variant-1 (HV1) as a fusion protein with the small ubiquitin-related modifier gene (SUMO) by use of over-lap PCR. The fusion gene His₆-SUMO-HV1 was highly expressed in E. coli BL21 (DE3) in which the SUMO-HV1 accounts for over 30% of the soluble fraction. The fusion protein was purified by Ni?CNTA affinity chromatography and cleaved by a SUMO-specific protease Ulp1 to release the HV1 with natural N-terminal. The recombinant HV1 (rHV1) was further purified by Ni?CNTA affinity chromatography and then by Q anion-exchange chromatography. N-terminal sequencing result demonstrated the purified rHV1 had the same N-terminal sequence as the native hirudin. MALDI-TOF/MS analysis indicated that the molecular weight of the purified rHV1 protein was 6939.161 Da, which was similar to the theoretical molecular weight of rHV1 6,944 Da. The Chromozym TH assay result showed that the anti-thrombin activity of purified rHV1 was 8,800 ATU/mg and comparable to the specific activity of native hirudin.     

Antithrombin activity of the hirudin N-terminal core domain residues 1–43

Hirudin N-terminal core domain residues 1–43 (r-Hir^1–43) were prepared from limited proteolysis of recombinant hirudin by V8 Staphylococcal protease followed by purification with reversed-phase HPLC. r-Hir^1-43 lacks the putative reactive site of hirudin (Lys⁴⁷), but binds to thrombin (with K_i of 300 nM) and blocks the catalytic activity of the protease. The structural element which accounts for the thrombin inhibitory activity of r-Hir^1–43 is analyzed in this report.     

Use of fragments of hirudin to investigate thrombin-hirudin interaction

Site-directed mutagenesis was used to create hirudin in which Asn52 was replaced by methionine. Cyanogen bromide cleavage at this unique methionine resulted in two fragments. These fragments have been used to study the kinetic mechanism of the inhibition of thrombin by hirudin and to identify areas of the two molecules which interact with each other. The binding of the C-terminal fragment (residues 53-65) to thrombin resulted in a decrease in the Michaelis constant for the substrate D-phenylalanylpipecolylarginyl-p-nitroanilide (DPhe-Pip-Arg-NH-Ph). The N-terminal fragment (residues 1-52) was a competitive inhibitor of thrombin. There was a small amount of cooperativity in the binding of the two fragments. Whereas hirudin and its C-terminal fragment protected alpha-thrombin against cleavage by trypsin, the N-terminal fragment did not. Hirudin and the N-terminal fragment completely prevented the cleavage of alpha-thrombin by pancreatic elastase while the C-terminal fragment afforded a lesser degree of protection. The results of these experiments with trypsin and elastase are discussed in terms of interaction areas on thrombin and hirudin.     

Interaction of hirudin with thrombin: identification of a minimal binding domain of hirudin that inhibits clotting activity

Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, we have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace 125I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. We show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, we found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin.     

Rapid purification and revised amino-terminal sequence of hirudin: a specific thrombin inhibitor of the bloodsucking leech

Hirudin is a specific polypeptide thrombin inhibitor consisting of 65 amino acids that is produced by the leech, Hirudo medicinalis. We describe a rapid method for the purification of hirudin from a leech extract. Crude hirudin, purchased from a commercial source, was first fractionated on a DEAE-HPLC column using a salt gradient. Hirudin activity was monitored by inhibition of the thrombin-mediated hydrolysis of a synthetic substrate H-D-Phenylalanyl-Pipecolyl-Arginine-p-Nitroanilide. The fractions containing antithrombin activity were pooled and further purified by reverse-phase HPLC. The homogeneity of purified hirudin was confirmed by a single amino-terminal sequence for 43 residues with Val-Val as the first two amino acids. Residue 33 was Asn rather than Asp as reported previously.     

Chimeric antithrombin peptide. Characterization of an Arg-Gly-Asp (RGD)- and hirudin carboxyl terminus-containing synthetic peptides

We investigated the properties of an artificial chimeric peptide that contains an Arg-Gly-Asp (RGD)-tripeptide, the versatile cell recognition signal of extracellular matrix protein components, coupled to a carboxyl-terminal fragment of the highly specific alpha-thrombin inhibitor, hirudin (residues 53-64): WGRGDSANGDFEEIPEEYL (RGD-hirudin53-64). Hirudin53-64 and RGD-hirudin53-64 inhibited the fibrinogen clotting activity of alpha-thrombin and prolonged the activated partial thromboplastin time of human plasma. In addition, both peptides afforded total protection to thrombin from trypsionolysis. Neither hirudin53-64 nor RGD-hirudin53-64 dramatically interfered with the thrombin-antithrombin inhibition reaction either in the absence or presence of added heparin. alpha-Thrombin-induced platelet aggregation was effectively inhibited by hirudin53-64 and RGD-hirudin53-64. Unlike hirudin53-64, RGD-hirudin53-64 in solution inhibited integrin-mediated endothelial cell and fibroblast cell attachment to polystyrene wells in the presence of fetal bovine serum. Collectively, our results demonstrate that RGD-hirudin53-64 has anticoagulant/antiplatelet aggregation activity attributable to its hirudin sequence and integrin-directed cell attachment activity due to its RGD site. Our results suggest that this chimeric motif may serve as a prototype for a new class of anticoagulants where an integrin-specific sequence "targets" the peptide to a cell (ultimately through the platelet integrin alpha IIb beta 3) trapped amid a thrombus with ensuing proteinase inhibition.     

The stabilization and release of hirudin from liposomes or lipid-assemblies coated with hydrophobically modified dextran

Hirudin is a 65-amino acid peptide and the most potent and specific known inhibitor of thrombin (K_i=0.2 pM). The short elimination half-life of hirudin from the body (1 hour) necessitates the use of a sustained and controlled delivery system. A proliposome method was used to entrap hirudin in liposomes coated with palmitoyl dextran-coated liposomes and lipid-assemblies. In vitro release studies of hirudin were performed using the lipid systems enclosed in dialysis membranes or deposited in the pores of a vascular graft. The activity of hirudin and released hirudin was measured using a thrombin chromogenic substrate assay.     

嵌合水蛭肽的构建与活性分析

血管成形术或动脉粥样斑块破裂等因素所致血管壁损伤而引起的血栓形成过程中 ,血小板的激活和凝血酶的形成起着关键作用 .因此 ,抗血小板和抗凝是治疗血栓的两个重要方面 .血小板膜糖蛋白GPⅡb Ⅲa受体拮抗剂 ,如含Arg Gly Asp(RGD)序列的多肽 ,在临床上已显示了良好的抗血小板