Recombinant Hirudin Research Articles

Expression, Purification and Characterization of the Recombinant Hirudin Variant iii in the Bacillus Subtilis

Hirudin is the most potent natural inhibitor of thrombin and a powerful anticoagulant. Large-scale production of recombinant hirudin is desirable for therapy. In this study, the gene encoding hirudin variant III was redesigned and synthesized by usingBacillus subtilispreferred codons, and the recombinant hirudin variant III (rHV3) was overexpressed inB. subtilisDB403 with strong anticoagulation activity for the first time. The hirudin activity from the supernatant of culture with optimized expression conditions could reach 210 ATU/ml. The protein in culture supernatant was precipitated by trichloroacetic acid, then desalted by ultrafiltration and purified by anion exchange chromatography. Strong anion Q F.F. performed better than weak anion DEAE F.F. The proper pH and conductivity was determined at pH 8 and 6 ms/cm, respectively. The maximum applied sample was 240 ATU/ml to medium of strong anion Q F.F. This optimized procedure was employed in strong anion exchange HiPrep 16/10Q with the 90% recovery rate and 70.2% purity. After gel filtration, the purity of rHV3 checked by HPLC could reach 95.1%, and the recovery rate was 93% for this step. The purified recombinant rHV3 showed a single band in SDS-PAGE. The rHV3 was stable at 100 °C and acidity condition, but was unstable under the condition of both heating and alkalinity. In conclusion, theses studies suggests thatB.subtilismight be useful for the production of biologically active medicine peptides in secretion facilitating purification procedures, and that this isolation method was suitable for scale-up purification process at a low cost.

Desirudin: a review of the pharmacology and clinical application for the prevention of deep vein thrombosis

Direct thrombin inhibitors (DTIs) are an emerging class of anticoagulants in routine clinical practice, although they have been under investigation for quite some time. Desirudin (Iprivask®, Canyon Pharmaceuticals™) is a recombinant hirudin derivative that directly inhibits free and fibrin-bound thrombin. Desirudin was the first DTI approved for deep vein thrombosis (DVT) prophylaxis in Europe (Revasc®) and is the newest injectable DTI commercially available in the USA. Desirudin is one of the few nonheparin subcutaneous drugs that has demonstrable efficacy for DVT prophylaxis. Subcutaneous desirudin has been shown to be more effective than both subcutaneous unfractionated heparin and enoxaparin, with comparable bleeding rates in the prevention of DVT in patients undergoing elective hip replacement. Desirudin also offers the advantage of a fixed dosing regimen while being less immunogenic than unfractionated heparin. However, owing to its pharmacokinetic properties, patients with renal dysfunction require dosing modifications. The favorable profile shown with desirudin thus far will allow its clinical use to grow and will promote further investigation into broadening its clinical applications.

Effects of natural and recombinant hirudin on superoxide dismutase, malondialdehyde and endothelin levels in a random pattern skin flap model

We have assessed the anti-inflammatory, anti-oxidative and anti-coagulant effects of locally applied natural and recombinant hirudin in a random skin flap rat model. Thirty Wistar rats with venous congested skin flaps were randomly divided into two treatment groups and a control group to receive subcutaneous injections of natural hirudin (6 U), recombinant hirudin (6 U) or physiological saline, respectively. Superoxide dismutase, malondialdehyde and endothelin levels as well as flap survival rates of the skin flaps were measured after surgery. Compared to the control group, the treatment groups had significant higher superoxide dismutase levels and lower malondialdehyde and endothelin levels in the skin flaps. The surviving areas of the flaps were larger in the treatment groups than the control group. Our results demonstrated that hirudin could improve skin flap survival through its anti-inflammatory, anti-oxidative and anti-coagulant activities.

Successful Use of Hirudin During Cardiac Surgery Using Minimized Extracorporeal Circulation in Patients With Heparin-Induced Thrombocytopenia

In this case series, we describe our successful use of a reduced hirudin dosage as an anticoagulant during cardiac surgery using minimized extracorporeal circulation in patients with heparin-induced thrombocytopenia.

Effects of recombinant hirudin variant III on expression of apoptosis-related genes during galactose-mediated human lens epithelial cells damage

Background Recombinant hirudin variant Ⅲ(rHV3) can effectively prevent galactose-induced human lens epithelial cells LECs injury,but little is known about the molecular mechanism of its action.Objective The present study was to investigate the effects of rHV3 on the expression of apoptosis-related genes in damaged LECs induced by galactose.Methods The rHV3 was extracted by our research group,and the biological activity of rHV3 was identified by titration of thrombase according to Markwardt's method.Human LECs (SRA01/04) were cultured using 125×10-3 mol/L D-galactose+10% FBS+D/F12 medium to establish the damaged human LECs model.rHV3 was added into the medium of the damaged human LECs model.Human LECs were cultured in D/F12 medium containing 10% FBS as normal control.The expression of apoptosis-related genes,such as aldose reductase (AR),bax,bcl2 and p53,in LECs at the mRNA level was detected using RT-PCR.The abundance ratio of target genes was presented with the absorbance (A) of gene mRNA/GAPDH mRNA.Results Compared to the normal control group,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were significantly elevated in model group (t=3.90E-06,t=8.44E-04,t=5.15E-08,P＜0.01).However,in the rHV3-treated group,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were lower than those of model group (t=5.90E-06,t=1.51E-04,t=3.42E-06,P＜0.01).The bcl2 mRNA/GAPDH mRNA was markedly downregulated in the model group when compared with the normal control group (t=1.86E-05,P＜0.01);while after rHV3 addition,bcl2 mRNA/GAPDH mRNA increased in comparison with the model group (t=8.56E-05,P＜0.01).Conclusion 125×10-3mol/L D-galactose induces the damage and apoptosis of human LECs.rHV3 likely plays a protective function on D-galactose-induced damage of human LECs by inhibiting the polyol pathway and mitochondria-mediated pathway. Key words: Human lens epithelial cells; Recombinant hirudin variant Ⅲ; Galactose; Apoptosis; Gene

Protein Chip Array Profiling of Molecular Variants of Hirudins Using Surface Enhanced Laser Desorption Ionization (SELDI) Technique

Various molecular variants of natural hirudin are developed for pharmaceutical purposes. Most of these are molecular variants, their conjugates with PEG Hirudin and fragment conjugates. Desirudin and Refludin represent recombinant hirudin variants with minor structural differences. PEG Hirudin represents a polyethylene glycol conjugate. Angiomax represents a terminal decapeptide of hirudin which is complex with a tripeptide and linker polypeptide. Although these products are homogeneous, their immunogenic responses reportedly vary. The purpose of this investigation was to investigate the molecular profile of each of these agents utilizing the SELDI technique employing a gold chip. Desirudin showed a single peak at 6.97kda, where is Refludin showed a single peak at 6.98kda. PEG Hirudin showed a heterogeneous peak at 17.8kda Angiomax showed a major peak at 2.18kda and a minor peak at 2.39kda. All of these products were free of other minor components below 1kda. These results show that ProteinChip array can be used to determine the molecular weight profile of each of these proteins and to check the presence of other impurities such as peptide fragment and other contaminants.

Effect of Recombinant Hirudin Variant III Emulsive Mi-croparticles on Thrombosis and the Study on Its Oral Ab-sorption Mechanism

Effect of Recombinant Hirudin Variant III Emulsive Mi-croparticles on Thrombosis and the Study on Its Oral Ab-sorption Mechanism

Simple and efficient methods for isolation and activity measurement of the recombinant hirudin variant 3 from Bacillus subtilis

A simple purification approach of the recombinant hirudin variant 3 from the Bacillus subtilis was established, by which the hirudin could be purified to the purity of 95% through one-step chromatography with the total recovery rate of 83.9%. A modified Markwardt thrombin titration method for measuring hirudin activity was also set up. Briefly, a series of concentrations of thrombin was prepared and titrated to hirudin sample, respectively and the anti-thrombin activity-range of hirudin was narrowed down by several thrombin solutions at high or low concentration and the optimum group of thrombin concentrations was determined for titration of the hirudin sample. In this modified method, the hirudin activity was determined more accurately, concisely and promptly than the classic Markwardt method.

Rapid and Quantitative Disulfide Bond Formation for a Polypeptide Chain Using a Cyclic Selenoxide Reagent in an Aqueous Medium

To elucidate the reaction mechanism of the disulfide (SS) bond formation reaction of a polypeptide molecule with a water-soluble selenoxide reagent, trans-3,4-dihydroxyselenolane oxide (DHS(ox)), short-term oxidation experiments were carried out for the reduced state (R) of a recombinant hirudin CX-397 variant at pH 7.0 and 25 °C. In the reaction, R was oxidized sequentially to one-SS, two-SS, and three-SS intermediate ensembles within 1 min. The kinetic analysis revealed that the three second-order rate constants for the SS formation are proportional to the number of thiol groups existing in the reactant SS intermediates, indicating the stochastic nature of the SS formation. Ab initio calculation at the HF/6-31++G(d,p) level in water by using the polarizable continuum model suggested that the SS formation reaction is highly exothermic and proceeds via a reactive thioselenurane intermediate with a distorted linear O-Se-S linkage. The results clearly demonstrated that the rate-determining step of the SS formation reaction is the first bimolecular process between a thiol substrate and DHS(ox) rather than the subsequent process to release a SS product.

New parenteral anticoagulants in development

The therapeutic armamentarium of parenteral anticoagulants available to clinicians is mainly composed by unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), fondaparinux, recombinant hirudins (i.e. bivalirudin, desirudin, lepirudin) and argatroban. These drugs are effective and safe for prevention and/or treatment of thromboembolic diseases but they have some drawbacks. Among other inconveniences, UFH requires regular anticoagulant monitoring as a result of variability in the anticoagulant response and there is a risk of serious heparin-induced thrombocytopaenia (HIT). LMWH, fondaparinux and recombinant hirudins are mainly cleared through the kidneys and their use in patients with severe renal insufficiency may be problematic. LMWH is only partially neutralized by protamine while fondaparinux and recombinant hirudins have no specific antidote. Novel anticoagulants in development for parenteral administration include new indirect activated factor Xa (FXa) inhibitors (idrabiotaparinux, ultra-low-molecular-weight heparins [semuloparin, RO-14], new LMWH [M118]), direct FXa inhibitors (otamixaban), direct FIIa inhibitors (flovagatran sodium, pegmusirudin, NU172, HD1-22), direct FXIa inhibitors (BMS-262084, antisense oligonucleotides targeting FXIa, clavatadine), direct FIXa inhibitors (RB-006), FVIIIa inhibitors (TB-402), FVIIa/tissue factor inhibitors (tifacogin, NAPc2, PCI-27483, BMS-593214), FVa inhibitors (drotrecogin alpha activated, ART-123) and dual thrombin/FXa inhibitors (EP217609, tanogitran). These new compounds have the potential to complement established parenteral anticoagulants. In the present review, we discuss the pharmacology of new parenteral anticoagulants, the results of clinical studies, the newly planned or ongoing clinical trials with these compounds, and their potential advantages and drawbacks over existing therapies.

Effect of Recombinant Hirudin Variant III Emulsive Microparticles on Thrombosis and the Study on Its Oral Absorption Mechanism

AimTo evaluate the antithrombosis of recombinant hirudin variant III emulsive microparticles (rHVIII EP) and disclose its mechanism via oral administration. MethodsNormal and DIC (disseminated intravascular coagulation) rats as well as IVC (inferior caval vein) thrombosis rats were used to determinate the pharmacodynamics of rHVIII. rHVIII was labeled with FITC (fluorescein isothiocyanate) to test the absorption of emulsive microparticles using Caco-2 monolayer model and everted gut sacs model. ResultsrHVIII EP was able to prolong thrombin time, clotting time and inhibit thrombus formation in vivo. rHVIII EP could be absorbed in the whole intestinal sections. Transport experiment indicated that the Papp value was (2.68 ± 0.14) ×10-6 cm·s−1 from AP side to BL side, and (3.02 ± 0.66) ×10-6 cm·s−1 from BL side to AP side, respectively, indicating that rHVIII is absorbed in the intestine through passive transport. Compared with intravenous route, the absolute bioavailability of rHVIII EP via oral route was 11.42%. ConclusionrHVIII EP has anti-thrombosis and anticoagulation effects with proper bioavailability. Our study here elucidated for the first time that rHVIII EP delivered via oral route has potentiality in development.

Overproduction of 15N-Labeled r-RGD-Hirudin in Pichia pastoris for NMR Studies

The novel recombinant hirudin, r-RGD-hirudin, inhibits thrombin and platelet aggregation. Here, we reported over-expression of (15)N-labeled r-RGD-hirudin by Pichia pastoris in minimal medium. After extensive optimization, the yield of active r-RGD-hirudin reached ≈600 mg/L when the yeast cells were culture in a fermenter. The purified (15)N-labeled r-RGD-hirudin retained full biological activity and was uniformly labeled. Heteronuclear NMR of the (15)N-labeled r-RGD-hirudin was performed for the first time, and all signals in the heteronuclear single quantum coherence (HSQC) spectrum were successfully assigned.

Nanoscaled Polyion Complex Micelles for Targeted Delivery of Recombinant Hirudin to Platelets Based on Cationic Copolymer

Polyion complex (PIC) micelles based on methoxy poly(ethylene glycol)-grafted-chitosan (mPEG-g-chitosan) and Arg-Gly-Asp conjugated poly(ethylene glycol)-grafted-chitosan (RGD-PEG-g-chitosan) were designed as carriers for platelet-targeted delivery of recombinant hirudin variant-2 (rHV2). The rHV2-loaded plain PIC micelles (mPIC micelles) and RGD conjugated PIC micelles (RGD-PIC micelles) were successfully prepared with mean size of 30.9 +/- 0.5 nm and 41.9 +/- 1.8 nm, and their encapsulation efficiencies were 76.90 +/- 0.84% and 81.08 +/- 0.85%, respectively. The pharmacokinetics experiments showed that the mean retention time (MRT) of rHV2 encapsulated in both kinds of micelles was significantly prolonged, especially for mPIC micelles. The confocal laser scanning microscopy intuitively proved the specific binding of RGD-PIC micelles to platelets. The efficacies of rHV2-loaded RGD-PIC micelles were greatly better than those of rHV2-loaded mPIC micelles and rHV2 solution in aspect of anticoagulation and inhibition of platelet aggregation. These results suggested platelet-targeting specificity of RGD-PIC micelles and that RGD-PIC micelles could potentially be used as a carrier for platelet-targeted delivery and long circulation of rHV2.

Recombinant Factor VIIa to Manage Major Bleeding from Newer Parenteral Anticoagulants

To evaluate the use of recombinant factor VIIa (rFVIIa) to reverse major bleeding from newer parenteral anticoagulant therapy. MEDLINE/PubMed was searched from January 2000 through December 2009 using the terms recombinant factor VIIa, rFVIIa, NovoSeven, enoxaparin, argatroban, fondaparinux, lepirudin, bivalirudin, idraparinux, nadroparin, hirudin, and desirudin. References of identified articles were reviewed. Data evaluating the role of rFVIIa to reverse major bleeding from newer parenteral anticoagulant therapy is limited to case reports and small laboratory investigations. Laboratory investigations suggest that rFVIIa may be effective in reversing the hemostatic effects of newer parenteral anticoagulants. In most case reports analyzed, standard interventions for bleeding (eg, fresh frozen plasma, packed red blood cells) were attempted prior to using rFVIIa. Sixteen published cases describe the use of rFVIIa to reverse major bleeding from low-molecular-weight heparins, synthetic pentasaccharides, and direct thrombin inhibitors. Initial doses ranged from 20 to 120 mug/kg. rFVIIa was considered effective or partially effective based upon clinical response in 13 cases. Use was not effective in 3 cases because of a thrombotic event, no change in hemostasis, and death from bleeding complications. As thrombosis is the major safety concern, an individualized risk-benefit assessment is required prior to the use of rFVIIa therapy to restore hemostasis. rFVIIa may be considered to manage major refractory bleeding from newer parenteral anticoagulant agents when the benefit is thought to outweigh the thrombotic risk.

Abstract C187: Antitumoral efficacy of the novel thrombin inhibitor Revasc©/Desirudin in an orthotopic metastatic AsPC-1 pancreas tumor model

Abstract Background: Thrombo-embolism is one of the most common secondary diseases in cancer patients and is the second leading cause of death of these patients. In pancreatic cancer patients, thrombosis occurs more frequently than in other cancer patients. Thus anticoagulants are widely used in cancer therapies mainly to prevent the development of thrombo-embolisms. However there is increasing evidence that anticoagulants have also antitumoral and anti-angiogenic effects. The aim of this study was to evaluate the anti-tumoral, anti-metastatic and anti-angiogenic efficacy of the novel anticoagulant Revasc© a recombinant hirudin and direct thrombin inhibitor, in an orthotopic mouse model of pancreatic cancer. Material and Methods: To evaluate the anti-tumoral, anti-metastatic and anti-angogenic efficacy of Revasc©, luciferase labeled pancreatic AsPC-1-tumor cells were orthotopically implanted into the pancreas of NMRI nude mice. After onset of tumor growth, mice were randomized into six groups. Group 1 received vehicle control (0.9% NaCl) twice daily, Group 2 received Gemcitabine (240 mg/kg) once weekly; Group 3 received Revasc at a concentration of 1 mg/kg twice daily, Group 4 received Revasc© at a concentration of 3 mg/kg twice daily, Group 5 received Revasc© at a concentration of 10 mg/kg daily and Group 6 received a combination therapy of twice daily 10 mg/kg Revasc© plus a weekly dose of 240 mg/kg Gemcitabine. At the end of the study, tumor weight and volume were determined and luciferase activity was monitored in the primary tumor and selected organs (spleen, liver, stomach, intestine, lung and inguinal lymph nodes). In addition, microvessel densities and vessel maturation was determined by immunohistochemisty. Results: In general, Revasc© was very well tolerated. The primary tumor weight was significantly reduced in the groups receiving Revasc© at a concentration of 3 mg/kg and 10 mg/kg as well as in the control group treated with Gemcitabine. The combination of Gemcitabine and Revasc©, however, did not show any additive effect. No significant reduction of metastasis could be observed in the Revasc© Groups, whereas Gemcitabine efficiently inhibited metastasis into the lung. However, Revasc© showed a significant anti-angiogenic potential at the lowest concentration of 1mg/kg, corroborating findings of other groups showing that thrombin inhibition acts anti-angiogenic. Conclusion: Taken together the study showed that Revasc© has an anti-tumoral and a mild anti-angiogenic effect. The data suggest that Revasc© has a potential to be used for the treatment of patients with pancreatic cancers; primarily to prevent thromboembolism but secondarly also to inhibit tumor growth. It remains to be shown if Revasc will lead to a solid anti-tumoral effect in patients and if the relatively short mean elimination half life of Revasc© (2–3 h) will allow to identify a convenient dosing scheme. Alternatively a pegylated hirudin derivative (PEG-hirudin) which has a longer half life and which has proven to have a very efficient antitumoral activity could be of high therapeutic interest. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C187.

Abstract C183: Antitumor efficacy of PEG-Hirudin (LU 87981), administered three times weekly, in an orthotopic AsPC-1 pancreas carcinoma model

Abstract Background: Since anticoagulants could be shown as promising novel therapeutics in oncology, direct thrombin inhibitors like recombinant Hirudin (Revasc©/desirudin) were assessed in a preclinical orthotopic AsPC-1 mouse model in vivo. The challenges of desirudin for the treatment of cancer are its complex pharmacodynamics and a short half life (mean elimination half-life of 2–3h), requiring at least twice daily subcutaneous administration of the drug. PEG-Hirudin, the conjugate of recombinant Hirudin with two molecules of polyethylene glycol (PEG)-5000 represents a potential alternative to Revasc© with a longer half-life and more stable plasma levels. In order to test this compound, the pancreatic cancer cell line, AsPC-1 was chosen, for which preliminary results with Revasc© were available. AsPC-1 cells implanted orthotopically into the mouse pancreas reflect hallmarks of the human disease such as early invasion of the surrounding pancreatic tissue and the formation of metastases in the spleen and liver as well as the peritoneum. Material and Methods: Luciferase expressing AsPC-1 cells were orthotopically implanted into mouse pancrei. The growth of the tumors was followed via bioluminescence measurements (whole body imaging). 13 days after surgery, 12 animals each were randomized into 5 treatment groups according to their bioluminescence signal. Group 1 served as vehicle control receiving 100µl saline solution three times weekly s.c. from day 15 to day 38. The application scheme was comparable to treatment groups receiving 1mg/kg (group 3), 3 mg/kg (group 4) and 0.3mg/kg (group 5) PEG-Hirudin three times weekly s.c. starting at day 15. The animals of group 2 received Gemcitabin at 240mg/kg administered i.v. once weekly starting at day 15. At the end of the study tumor weight and volume were determined and luciferase activity was monitored in the primary tumor and selected organs (spleen, liver, stomach, intestine, lung and inguinal lymph nodes). Results: PEG-Hirudin was well tolerated, no bleeding at the injection site or other side effects could be observed. Only one animal of group 4 was found dead during the study, all other animals reached the end of the study. Gemcitabin, did not affect tumor growth according to in vivo bioluminescence imaging, whereas PEG-Hirudin at 1 and 3 mg reduced the increase of the bioluminescence signal during the treatment period significantly. Once the animals were sacrificed and tumor sizes measured, treatment with PEG-Hirudin at 1 and 3 mg/kg showed significant reduction of the average primary tumor size (Group 3: *p= 0.0129, Group 4:**p=0.0015). The positive control, Gemcitabin showed as well a significant antitumor efficacy (***p=0.0002). The differing results of in vivo imaging and primary tumor sizes are reflecting the different efficacy of the test items on primary tumor and peritoneal metastases. Conclusion: The results presented here suggest a promising anti-tumoral efficacy of the PEG-Hirudin compound. Due to its extended half-life and the resulting relaxation of the application scheme, PEG-Hirudin is a promising alternative to Revasc© (desirudin) in oncology. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C183.

Expression, purification and characterization of recombinant targeting bifunctional hirudin in Pichia pastoris

A recombinant targeting bifunctional hirudin was expressed in the yeast Pichia pastoris. In order to decrease the side effects of hirudin and increase its activity to prevent arterial thrombus, we fused a factor Xa (FXa) recognition sequence into N’ of hirudin, while maintaining the activity of natural hirudin. In addition, an Arg-Gly-Asp (RGD) sequence was fused into appropriate genetic locus of hirudin. Furthermore, we added a 9 × His - Tag to make its separation and purification conveniently. The recombination hirudin gene was successfully cloned and ligated into the P. pastoris vector pPIC9K to form an expression vector, which was transferred into P. pastoris GS115. A transformant strain was selected and expressed efficiently in suitable conditions. Then, the fusion protein was purified by affinity chromatography. Through anti-thrombin activity analysis and anti-platelet aggregation activity analysis, we found that the anti-thrombin activity of the fusion protein did not change comparing with the natural hirudin, whereas its anti-platelet capability was enhanced.

A Role for Gab1/SHP2 in Thrombin Activation of PAK1

To understand the role of epidermal growth factor receptor (EGFR) transactivation in G protein-coupled receptor (GPCR) agonist-induced signaling events, we have studied the capacity of thrombin in the activation of Gab1-SHP2 in vascular smooth muscle cells (VSMCs). Thrombin activated both Gab1 and SHP2 in EGFR-dependent manner. Similarly, thrombin induced Rac1 and Cdc42 activation, and these responses were suppressed when either Gab1 or SHP2 stimulation is blocked. Thrombin also induced PAK1 activation in a time- and EGFR-Gab1-SHP2-Rac1/Cdc42-dependent manner. Inhibition of activation of EGFR, Gab1, SHP2, Rac1, Cdc42, or PAK1 by pharmacological or genetic approaches attenuated thrombin-induced VSMC stress fiber formation and motility. Thrombin activated RhoA in a time-dependent manner in VSMCs. LARG, a RhoA-specific GEF (guanine nucleotide exchange factor), was found to be associated with Gab1 and siRNA-mediated depletion of its levels suppressed RhoA, Rac1 and PAK1 activation. Dominant negative mutant-mediated interference of RhoA activation inhibited thrombin-induced Rac1 and PAK1 stimulation in VSMCs and their stress fiber formation and migration. Balloon injury induced PAK1 activity and interference with its activation led to attenuation of SMC migration from media to intima, resulting in reduced neointima formation and increased lumen size. Inhibition of thrombin signaling by recombinant hirudin also blocked balloon injury-induced EGFR tyrosine phosphorylation and PAK1 activity. These results show that thrombin-mediated PAK1 activation plays a crucial role in vascular wall remodeling and it could be a potential target for drug development against these vascular lesions.

Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII

The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were approximately 50 and approximately 10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an approximately 340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed approximately 4- and 11-fold reductions in A2 subunit generation and approximately 3- and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.

A long-lasting, plasmin-activatable thrombin inhibitor aids clot lysis in vitro and does not promote bleeding in vivo

The leech protein hirudin is a potent inhibitor of thrombin, but clinical use of recombinant hirudin is restricted by haemorrhagic risks, and complicated by hirudin's rapid clearance from the circulation. We previously employed albumin fusion to slow hirudin variant 3 (HV3) clearance. In this study, we hypothesized that reconfiguration of the chimera, appending human serum albumin (HSA) to the N-terminus of HV3, with an intervening plasmin cleavage site, would create a slowly cleared, plasmin-activatable HV3. Potential plasmin cleavage sites were screened by expression in Escherichia coli, interposed between glutathione sulfotransferase and HV3 domains. The most reactive sequence (GSGIYR-ITY) was recreated in C-terminally His-tagged albumin fusion protein HSACHV3, expressed in Pichia pastoris yeast and purified by nickel-chelate affinity chromatography. HSACHV3 showed no thrombin inhibitory activity in the absence of plasmin, but liberated active HV3 in a time- and concentration-dependent manner in its presence. In a discontinuous clot assay involving clot-bound thrombin, HSACHV3 assisted clot lysis by limiting clot extension in a tPA- and concentration-dependent manner. Similar results were obtained in plasma at higher concentrations of HSACHV3. The chimeric protein exhibited much slower clearance in mice than unfused HV3, and indistinguishable pharmacokinetics from unfused recombinant HSA. In a mouse tail transection bleeding model, doses of HSACHV3 identical to those of HV3 that elicited a four-fold increase in the volume of shed blood were without effect. Our results suggest that HSACHV3 is a fully latent, plasmin activatable, long-lasting hirudin, of potential benefit in thrombotic disorders resistant to natural or pharmacological clot lysis.