Recombinant Hirudin Research Articles

A new signal peptide useful for secretion of heterologous proteins from yeast and its application for synthetis of hirudin

The BGL2 gene from Saccharomyces cerevisiae encodes a β-glucanase which is localized to the yeast cell wall. The ability of a 23-amino acid (aa) signal peptide derived from the BGL2 gene to direct a heterologous protein to the secretory pathway of yeast has been compared to that of the MFα1-encoded signal peptide in a series of gene fusions. As a model protein, the leech anticoagulant, recombinant hirudin variant 2-Lys 47 (HIR) has been studied. From a multicopy plasmid chimaeric proteins were produced which carry the BGL2 signal peptide (or the artificial BGL2 pre-Val 7 variant) ( i) in front of the MFα1 pro sequence (or modified versions of MFα1 pro), i.e., a prepro signal, or ( ii) joined directly to the heterologous protein. Accumulation of active HIR in yeast culture supernatants was observed when the BGL2 (or the BGL2 pre-Val 7) signal peptide were used in combination with either of three versions of the MFα1 pro peptide: the authentic MFα1 pro, a partially deleted MFα1 pro-Δ22–61, or a pro bearing an aa change (MFα1 pro-Gly 22. In each case the BGL2 signal peptide (or its variant) has proven equally productive to the corresponding MFα1 peptide. Four times more active HIR was detected in the culture supernatant when either signal peptide was fused directly to the recombinant protein, as compared to a prepro protein version. Correct signal peptide cleavage was obtained when HIR was produced as a BGL2 pre-Val 7:: fusion protein.

Biological changes following infusion of recombinant hirudin (HBW 023) in 10 patients with severe thromboembolism

Biological changes following infusion of recombinant hirudin (HBW 023) in 10 patients with severe thromboembolism

Intravenous recombinant soluble human thrombomodulin prevents venous thrombosis in a rat model

Thrombomodulin is an endothelial surface thrombin receptor. Thrombin bound to thrombomodulin loses all procoagulant activity and instead activates the protein C anticoagulant pathway. We developed a recombinant thrombomodulin analog and compared the effects of recombinant thrombomodulin (100 μg/ea), saline (controls), recombinant hirudin (1.0 mg/kg), and heparin (100 units/kg) on thrombus formation, activated partial thromboplastin time, and tail transection bleeding time in a rat model of stasis-induced venous thrombosis. Results showed that thrombus was detected in the vena cava in six of the six rats treated with saline solution, in zero of the six rats treated with recombinant thrombomodulin ( p < 0.05), in one of six rats treated with recombinant hirudin ( p < 0.05), and in zero of six rats treated with heparin ( p < 0.05). The activated partial thromboplastin time in rats receiving recombinant thrombomodulin was slightly longer than controls (22 ± 8 vs 37 ± 6, p < 0.05). The bleeding times in rats receiving recombinant thrombomodulin were approximately twice as long as controls (215 ± 68 vs 545 ± 173, p = 0.037). In all rats treated with recombinant hirudin or heparin, activated partial thromboplastin times were >120 seconds and bleeding times were >1200 seconds. We conclude that recombinant thrombomodulin inhibits venous thrombosis in a rat model with less prolongation of activated partial thromboplastin time and bleeding time than heparin or hirudin.

Characterization, stability and refolding of recombinant hirudin

A recombinant variant of hirudin, the blood-clotting inhibitor of the leech Hirudo medicinalis, has been characterized employing spectroscopic and hydrodynamic techniques. Conditions have been defined for efficient reconstitution of the native, disulfide-bonded inhibitor from completely unfolded, reduced polypeptide chains. The spectral properties of the native inhibitor are consistent with previous results on the solution structure of hirudin. Extremely low circular dichroism in the far ultraviolet ([theta]Mr,220 nm = -8 +/- 1 x 10(2) deg.cm2.dmol-1) indicates a very low content of regular secondary structure. Although both tyrosine residues of the recombinant inhibitor titrate around pH 10.6, typical for solvent-exposed tyrosines, fluorescence emission and near-ultraviolet circular dichroism suggest that at least one of the tyrosines is partially shielded from solvent quench, and immobilized in an asymmetric environment. Reversible thermal unfolding of hirudin around 65 degrees C is indicated by the disappearance of its dichroic absorption in the near ultraviolet and by a fourfold increase in ellipticity at 225 nm. The transition can be approximated by a two-state model with a transition enthalpy of delta Hvan't Hoff = 159 kJ/mol and a transition entropy of 464 J.mol-1.K-1. Reduced hirudin at room temperature is largely unfolded and inactive as an inhibitor of thrombin assayed with a low-molecular-mass substrate. Refolding and reoxidation are observed at alkaline pH in the presence of a mixture of glutathione and glutathione disulfide. Spectroscopy, thrombin inhibition, and reversed-phase HPLC indicate reconstitution yields close to 100% and that the reconstituted inhibitor is identical to the native starting material.

Some Objective Considerations for the Use of Heparins and Recombinant Hirudin in Percutaneous Transluminal Coronary Angioplasty

Some Objective Considerations for the Use of Heparins and Recombinant Hirudin in Percutaneous Transluminal Coronary Angioplasty

Specificity determinants for tyrosine protein kinases

Hirudin, the powerful anticoagulant agent of leech (Hirudo medicinalis) saliva, was readily phosphorylated by two spleen tyrosine protein kinases (TPK-IIB and TPK-III) at Tyr63 with Km values of 238 microM and 74 microM, respectively. The synthetic tridecapeptide DGDFEEIPEEYLQ, corresponding to the hirudin 53-65 C-terminal fragment, was phosphorylated even more efficiently than hirudin itself. Four hirudin mutants, in which one or more of the glutamic acids at positions 57, 58, 61 and 62 have been replaced by glutamines, were poorer substrates than hirudin. The mutant in which all four glutamates were substituted, [Gln57,58,61,62]hirudin, was virtually not phosphorylated by either TPK-IIB and TPK-III. Substitution of Glu57 and Glu58 was less deleterious than substitution of the two glutamic acids adjacent to Tyr63: [Gln61,62]hirudin exhibited a 20-fold lower phosphorylation efficiency with TPK-IIB. With TPK-III, however, the Km value of [Gln61,62]hirudin was slightly lower, while the Vmax decreased sixfold. The substitution of Glu62 alone was also more detrimental with TPK-IIB than with TPK-III. The behaviour of a third spleen TPK, named lyn TPK-I and belonging to the src family, was markedly different in that it did not phosphorylate hirudin but exhibited significant activity towards [Gln57,58,61,62]hirudin. Taken together, these data confirm and extend with a protein substrate the results obtained with short model peptides which indicated the stringent substrate requirements of TPK-IIB (and of TPK-III to a lesser extent) for N-terminal acidic residues. In contrast, such residues are deleterious with lyn TPK-I. These observations also support the concept that tyrosine protein kinases recognize specificity determinants situated in the vicinity of the target residue rather than requiring higher-order structural features.

Radioimmunoassay for for the detection of active-site specific thrombin inhibitors in biological fluids. I. Assay characteristics and quantitation of recombinant hirudin

A sensitive radioimmunoassay (RIA) for the quantitation of recombinant (r) hirudin in biological fluids is described. Taking advantage of the highly specific hirudinthrombin interaction, a monoclonal antibody to human α-thrombin was used to capture hirudin-thrombin complexes in a competitive binding assay. Quantitation of r. hirudin in buffer, plasma or urine at concentrations ranging from 0.17 to 20 ng/ml (1.7×10 −3 to 2×10 −2 antithrombin units/ml) was achieved. In the absence of competing unlabelled r.hirudin the assay also measured α-thrombin (from 2×10 −4 to 1× l0 −2 NIH units/ml) in citrated or defibrinated human plasma. A series of peptides corresponding to the carboxyl-terminal region of hirudin and with varying anticoagulant activities did not displace 125I- r.hirudin in the RIA described, confirming published data that these hirudin fragments bind to a site distant to the catalytic site of thrombin. The assay was used to test hirudin clearance after bolus i.v. injections of 0.1mg r.hirudin [Val 1-Val 2] into human volunteers. The plasma concentrations and elimination kinetics of r. hirudin were in good agreement with published data and a close correlation between hirudin plasma concentration and prolonged clotting time was observed.

Radioimmunoassay for the detection of active-site specific thrombin inhibitors in biological fluids. II. Heparin affects the binding of hirudin to α-thrombin

A radioimmunoassay for recombinant (r.) hirudin was used to study the effect of heparin and other glycosaminoglycans (GAG's) on interactions between hirudin and thrombin. Binding of 125I-r.hirudin to thrombin in the presence or absence of heparin (and other GAG's) was monitored by immunoprecipitation of thrombin- 125I-r.hirudin complexes with a monoclonal antibody to human a-thrombin. Heparin and dextran sulfate substantially reduced hirudin binding to thrombin in human plasma. Inhibition by heparin was restored by addition of increasing amounts of antithrombin (AT) to samples containing constant amounts of heparin, thrombin and 125I-r.hirudin and was not neutralized by protamine sulfate. At very low heparin concentrations competitive displacement of 1251-r.hirudin was observed, while in the presence of heparin-AT complexes some 125I-r.hirudin remained bound to thrombin, suggesting that two or more binding sites for hirudin on the thrombin molecule may be occupied simultaneously.

Refined structure of the Hirudin-thrombin complex

The structure of a recombinant hirudin (variant 2, Lys47) human α-thrombin complex has been refined using restrained least-squares methods to a crystallographic R-factor of 0·173. The hirudin structure consists of an N-terminal domain folded into a globular unit and a long 17-peptide C-terminal in an extended chain conformation. The N-terminal domain binds at the active-site of thrombin where Ilel' to Tyr3' penetrates to the catalytic triad. The α-amino group of Ilel' of hirudin makes a hydrogen bond with OG of Ser195 of thrombin, the side-chains of Ilel' and Tyr3' occupy the apolar site. Thr2' is at the entrance to, but does not enter, the S1 specificity site and Ilel' to Tyr3' form a parallel β-strand with Ser214 to Gly219. The latter interaction is antiparallel in all other serine proteinase-protein inhibitor complexes. The extended C-terminal segment of hirudin, which is abundant in acidic residues, makes many electrostatic interactions with the fibrinogen binding exosite while the last five residues are in a 3 10 helical turn residing in a hydrophobic patch on the thrombin surface. The precision of the complementarity displayed by these two molecules produces numerous interactions, which although independently generally weak, together are responsible for the high degree of affinity and specificity. Although hirudin-thrombin and d-Phe-Pro-Arg-chloromethyl ketone-thrombin differ in conformation in the autolysis loop (Lys145 to Gly150), this is most likely due to different crystal packing interactions and changes in circular dichroism between the two are probably due to the inherent flexibility of the loop. An RGD sequence, which is generally known to be involved in cell surface receptor interactions, occurs in thrombin and is associated with a long solvent channel filled with water molecules leading to the surface from the end of the S1 site. However, the RGD triplet does not appear to be able to interact in concert in a surface binding mode.

Human α- and ζ-thrombin inhibition by recombinant HV1 and HV2k47 hirudins

Human α- and ζ-thrombin inhibition by recombinant HV1 and HV2k47 hirudins

Thrombin regulation of platelet interaction with damaged vessel wall and isolated collagen type I at arterial flow conditions in a porcine model: effects of hirudins, heparin, and calcium chelation

The role of thrombin inhibition in platelet vessel wall interaction and thrombus growth was studied under controlled flow conditions. Natural hirudin and recombinant hirudin (r-hirudin), which are specific thrombin inhibitors, were compared with heparinized blood (1.8 +/- 0.2 U/mL) and Ca(2+)-chelated blood in their potential to inhibit platelet interaction and thrombus growth on two biologic vascular surfaces and one immobilized vessel wall component. The substrates were perfused by flowing blood at shear rates typical of patent and stenosed arteries (212 to 1,690/s) for 5 minutes. Platelet deposition was measured by In-111-labeled platelets. We found that both natural and r-hirudin have similar effects on platelet-substrate interaction. As compared with heparin, platelet deposition to mildly damaged vessel wall and digested collagen type I was not reduced by hirudin or citrate. However, hirudin and citrate significantly reduced platelet deposition to severely damaged vessel wall (platelets x 10(6)/cm2: 93 +/- 10 in heparinized blood v 50 +/- 7 in blood treated with 100 U/mL r-hirudin). Therefore, thrombus growth on areas of severe wall damage is in part dependent on local thrombin production at the site of vascular damage. We also found that hirudin added to heparinized blood reduced platelet deposition to severely injured wall but not to subendothelium or collagen-coated slides. Hirudin added to citrated blood did not affect platelet deposition. Our study indicates that local thrombin generation at the site of severe injury will induce platelet activation and deposition even in the presence of average therapeutic heparin levels that inhibit blood coagulation.

Thrombin Regulation of Platelet Interaction With Damaged Vessel Wall and Isolated Collagen Type I at Arterial Flow Conditions in a Porcine Model: Effects of Hirudins, Heparin, and Calcium Chelation

Abstract The role of thrombin inhibition in platelet vessel wall interaction and thrombus growth was studied under controlled flow conditions. Natural hirudin and recombinant hirudin (r-hirudin), which are specific thrombin inhibitors, were compared with heparinized blood (1.8 +/- 0.2 U/mL) and Ca(2+)-chelated blood in their potential to inhibit platelet interaction and thrombus growth on two biologic vascular surfaces and one immobilized vessel wall component. The substrates were perfused by flowing blood at shear rates typical of patent and stenosed arteries (212 to 1,690/s) for 5 minutes. Platelet deposition was measured by In- 111-labeled platelets. We found that both natural and r-hirudin have similar effects on platelet-substrate interaction. As compared with heparin, platelet deposition to mildly damaged vessel wall and digested collagen type I was not reduced by hirudin or citrate. However, hirudin and citrate significantly reduced platelet deposition to severely damaged vessel wall (platelets x 10(6)/cm2: 93 +/- 10 in heparinized blood v 50 +/- 7 in blood treated with 100 U/mL r-hirudin). Therefore, thrombus growth on areas of severe wall damage is in part dependent on local thrombin production at the site of vascular damage. We also found that hirudin added to heparinized blood reduced platelet deposition to severely injured wall but not to subendothelium or collagen-coated slides. Hirudin added to citrated blood did not affect platelet deposition. Our study indicates that local thrombin generation at the site of severe injury will induce platelet activation and deposition even in the presence of average therapeutic heparin levels that inhibit blood coagulation.

Interaction of hirudin with the dysthrombins Quick I and II

The interaction of hirudin with the dysfunctional enzymes thrombin Quick I and II has been investigated. Natural and recombinant hirudin caused nonlinear competitive inhibition of thrombin Quick I. The results were consistent with thrombin Quick I existing in two forms that have different affinities for hirudin. The affinities of these forms for natural hirudin were respectively 10(4)- and 10(6)-fold lower than that of alpha-thrombin. In contrast, truncated hirudin molecules lacking the C-terminal tail of the molecule caused linear inhibition of thrombin Quick I. These results indicate that different modes of interaction of the two forms of thrombin Quick I with the C-terminal tail of hirudin were the cause of the nonlinear inhibition. Comparison of the dissociation constants of thrombin Quick I with the truncated and full-length forms of hirudin suggested that the interactions that normally occur between the C-terminal tail of hirudin and thrombin were completely disrupted with the low-affinity form of thrombin Quick I. Thrombin Quick II displayed an affinity for natural hirudin that was 10(3)-fold lower than that observed with alpha-thrombin. In contrast, it bound a mutant hirudin with altered N-terminal amino acids only 16-fold less tightly. These results are discussed in terms of structural alterations in the active-site cleft in thrombin Quick II.

Thromboxane and cysteinyl-leukotriene formation are differentially activated in spontaneously clotting whole human blood in vitro

We have previously demonstrated that clotting of whole human blood in vitro not only triggers the production of thromboxane (TX) B 2 but is also accompanied by formation of 5-lipoxygenase-derived cysteinyl-leukotrienes (LT). In order to further characterize the mechanisms leading to activation of the cysteinyl-LT production, we have now investigated the effects of thrombin on cysteinyl-LT as well as TXB 2 formation in whole human blood. Addition of exogenous human α-thrombin (0.1 – 3.0 U/ml) to whole human blood incubated in vitro led to a concentration- and time-dependently increased release of TXB 2 into the serum samples. The serum contents of cysteinyl-LT were, however, not significantly affected. Inactivation of endogenously generated thrombin by inhibitors such as recombinant hirudin (HBW 023, 0.43–1.43 μM) or the peptidyl chloromethyl ketone, D-Phe-Pro-Arg-CH2Cl (1.0 – 100 μM) concentration- and time-dependently inhibited the release of TXB2 into the serum or plasma samples. In contrast, however, serum contents of cysteinyl-LT remained unchanged. The identity of immunoreactive material was confirmed by thin-layer chromatography of immunoreactive TXB 2 and by reversed phase HPLC of immunoreactive cysteinyl-LT. As expected, washed human platelets stimulated with α-thrombin were identified as the major source of TXB 2 generation but purified monocytes were also found to release some TXB 2 upon α-thrombin stimulation. Release of TXB 2 by isolated human polymorphonuclear leukocytes (PMN) was negligible in the presence of this stimulus. None of the cells which are known to possess 5-lipoxygenase activity such as PMN or monocytes did release neither cysteinyl-LT nor LTB4 upon stimulation with human α-thrombin up to 10 U/ml. These data demonstrate that TXB 2 production and cysteinyl-LT formation are differentially activated in spontaneously clotting whole human blood in vitro, the former being dependent on endogenously generated thrombin the latter being dependent on a stimulus yet to be identified.

A new class of potent thrombin inhibitors that incorporates a scissile pseudopeptide bond

A synthetic hirudin peptide analog corresponding to N′-acetyl [D-Phe 45, ArgΨ(COCH 2) 47, Gly 48]desulfo hirudin 55–65 (P79) was synthesized. Comparative kinetic studies showed that while recombinant hirudin (HV2) is a slow-tight binding inhibitor, P79 behaves as a classical competitive inhibitor of human α-thrombin ( K i=3.7±0.3 × 10 −10 M) and bovine α-thrombin (1.8±0.7 × 10 −9 M). P79 showed saturable inhibition of plasma APTT. The P 1 subsite of P79 is isosteric with the glycine residue of the natural thrombin substrate fibrinogen, but is proteolytically stable due to the incorporation of a ketomethylene pseudopeptide bond. The model active site-directed tripeptide [D-Phe-Pro-ArgΨ(COCH 2)CH 3COOCH 3, P79L] corresponding to the amino terminal of P79 also binds competitively to the active site of α-thrombin and inhibited the proteolysis of a tripeptidyl substrate with a K 1=17.9±2.1 μM (human) and 10.3±3.6 μM (bovine) α-thrombin. NMR experiments indicated that P79L and the corresponding amino terminal residues of P79 occcupy a mutually exclusive binding site on bovine α-thrombin while the carboxyl terminal tail of the latter adopts a similar bound conformation as the fragment hirudin??? which is known to interact with the ‘anion’ exosite. Taken together these results provide conclusive evidence that the high antithrombin activity of N???-acetyl[D-Phe 45, ArgΨ(COCH 2) 47, Gly 48]desulfo hirudin 45–65 stems from the concurrent interaction with the catalytic site and the putative ‘anion’ exosite through its respective NH 2- and COOH-terminal recognition sites.

Neutralization of Recombinant Hirudin: Some Practical Considerations

The developments in the area of glycosaminoglycan derived drugs can be summarized in the following: 1. Currently available glycosaminoglycan-derived drugs represent a first generation of clinically useful agents. At present these agents are not optimized and their basic and clinical pharmacology is poorly understood. 2. Most of these agents represent mixtures of different types of glycosaminoglycans. They can be purified to isolate their active components and perhaps many new drugs may be developed from the existing products. 3. Chemical derivatization and fractionation methods can be used to obtain more defined products. These products will be developed for specific clinical indications. 4. Each glycosaminoglycan derived product should be considered as an individual drug and should be developed separately. 5. Many semisynthetic and synthetic analogs of glycosaminoglycans will be introduced for various clinical indications.

Report on a Discussion Forum: “Medical and Surgical Application of Recombinant Hirudin”

Report on a Discussion Forum: “Medical and Surgical Application of Recombinant Hirudin”

Recombinant hirudin reduces the incidence of thrombotic occlusion in a canine model of coronary vascular injury

Recombinant hirudin reduces the incidence of thrombotic occlusion in a canine model of coronary vascular injury

Relative efficacy of antithrombin compared with antiplatelet agents in accelerating coronary thrombolysis and preventing early reocclusion.

Optimal coronary thrombolysis should be prompt and persistent. Although activation of platelets and increased thrombin activity have been associated with clinical thrombolysis, the role of each in delaying thrombolysis or inducing early coronary reocclusion has been difficult to define. In conscious dogs with coronary thrombosis induced by electrical current, we assessed the impact on the rapidity of thrombolysis and the incidence of reocclusion of two types of adjunctive treatment given concomitantly with intravenous tissue-type plasminogen activator (t-PA): 1) inhibition of platelet function with a peptide mimetic antagonist of platelet glycoprotein IIb/IIIa receptors or with lysine acetylsalicylic acid (ASA) and 2) inhibition of thrombin activity with recombinant hirudin or with heparin. ASA but not the receptor antagonist shortened the time to thrombolysis with t-PA (20 +/- 13 [mean +/- SD] minutes with ASA, 36 +/- 15 minutes with receptor antagonist, and 43 +/- 16 minutes with the saline control). Reocclusion occurred promptly after completion of the infusion of t-PA in all seven dogs given saline. Reocclusion was delayed and prevented in some dogs within 90 minutes after the end of the infusion of t-PA by both antiplatelet agents but still occurred in 42% despite continued inhibition of platelet function (i.e., three of six dogs given ASA and two of six given receptor antagonist). In contrast, inhibition of thrombin activity with recombinant hirudin in a dose that prolonged the partial thromboplastin time modestly (1.5-2-fold) resulted in accelerated lysis (19 +/- 10 minutes) and prevention of reocclusion in each of six dogs. Heparin given in doses that elicited similar prolongation of the partial thromboplastin time did not accelerate lysis nor prevent reocclusion, which occurred in five of six dogs. Inhibition of thrombin by recombinant hirudin facilitates thrombolysis and maintains patency of coronary arteries recanalized with t-PA particularly effectively. The benefit conferred may reflect direct anticoagulant effects plus diminished activation of platelets secondary to decreased thrombin activity.

Hirudin interruption of heparin-resistant arterial thrombus formation in baboons

To determine the role of thrombin in high blood flow, platelet- dependent thrombotic and hemostatic processes we measured the relative antithrombotic and antihemostatic effects in baboons of hirudin, a highly potent and specific antithrombin, and compared the effects of heparin, an antithrombin III-dependent inhibitor of thrombin. Thrombus formation was determined in vivo using three relevant models (homologous endarterectomized aorta, collagen-coated tubing, and Dacron vascular graft) by measuring: (1) platelet deposition, using gamma camera imaging of 111In-platelets; (2) fibrin deposition, as assessed by the incorporation of circulating 125I-fibrinogen; and (3) occlusion. The continuous intravenous infusion of 1, 5, and 20 nmol/kg per minute of recombinant hirudin (desulfatohirudin) maintained constant plasma levels of 0.16 +/- 0.03, 0.79 +/- 0.44, and 3.3 +/- 0.77 mumol/mL, respectively. Hirudin interrupted platelet and fibrin deposition in a dose-dependent manner that was profound at the highest dose for all three thrombogenic surfaces and significant at the lowest dose for thrombus formation on endarterectomized aorta. Thrombotic occlusion was prevented by all doses studied. In contrast, heparin did not inhibit either platelet or fibrin deposition when administered at a dose that maximally prolonged clotting times (100 U/kg) (P greater than .1), and only intermediate effects were produced at 10-fold that dose (1,000 U/kg). Moreover, heparin did not prevent occlusion of the test segments. Hirudin inhibited platelet hemostatic function in concert with its antithrombotic effects (bleeding times were prolonged by the intermediate and higher doses). By comparison, intravenous heparin failed to affect the bleeding time at the 100 U/kg dose (P greater than .5), and only minimally prolonged the bleeding time at the 1,000 U/kg dose (P less than .05). We conclude that platelet-dependent thrombotic and hemostatic processes are thrombin-mediated and that the biologic antithrombin hirudin produces a potent, dose-dependent inhibition of arterial thrombus formation that greatly exceeds the minimal antithrombotic effects produced by heparin.