Comparison of bacterial attachment to platelet bags with and without preconditioning with plasma.

BACKGROUND AND OBJECTIVES: Canadian Blood Services produces apheresis and buffy coat pooled platelet concentrates (PCs) stored in bags produced by two different manufacturers (A and B, respectively), both made of polyvinyl chloride-butyryl trihexyl citrate. This study was aimed at comparing Staphylococcus epidermidis adhesion to the inner surface of both bag types in the presence or absence of plasma factors. MATERIALS AND METHODS: Sets (N = 2-6) of bags type A and B were left non-coated (control) or preconditioned with platelet-rich, platelet-poor or defibrinated plasma (PRP, PPP and DefibPPP, respectively). Each bag was inoculated with a 200-ml S. epidermidis culture adjusted to 0·5 colony-forming units/ml. Bags were incubated under platelet storage conditions for 7 days. After culture removal, bacteria attached to the plastic surface were either dislodged by sonication for bacterial quantification or examined in situ by scanning electron microscopy (SEM). RESULTS: Higher bacterial adhesion was observed to preconditioned PC bags than control containers for both bag types (P < 0·0001). Bacterial attachment to preconditioned bags was confirmed by SEM. Bacteria adhered equally to both types of containers in the presence of PRP, PPP and DefibPPP residues (P > 0·05). By contrast, a significant increase in bacterial adherence was observed to type A bags compared with type B bags in the absence of plasma (P < 0·05) [Correction added on 16 June 2017, after first online publication: this sentence has been corrected]. CONCLUSION: The ability of S. epidermidis to adhere to preconditioned platelet collection bags depends on the presence of plasma factors. Future efforts should be focused on reducing plasma proteins' attachment to platelet storage containers to decrease subsequent bacterial adhesion.

Early γ-irradiation and subsequent storage of red cells in SAG-M additive solution potentiate energy imbalance, microvesiculation and susceptibility to stress-induced apoptotic cell death.

γ-Irradiation of red blood cell (RBC) concentrates prevents transfusion-associated graft-versus-host disease but may diminish RBC quality. Herein, we show that early γ-irradiation (25 Gy) of RBC units and their subsequent storage in SAG-M additive solution altered membrane microvesiculation, supernatant haemoglobin and cytosolic ATP. γ-Irradiation did not influence phosphatidylserine externalization, a marker of erythrocyte apoptotic cell death (eryptosis), in RBC stored for 42 days. However, shorter periods (4-21 days) of storage accentuated eryptosis in γ-irradiated RBC versus untreated RBCs following energy depletion, suggesting that γ-irradiated RBC is primed for stress-induced eryptosis during storage.

Bacteria can proliferate in thawed cryoprecipitate stored at room temperature for longer than 4 h.

Although key coagulation factor activities are maintained in thawed cryoprecipitate stored for up to 24 h at ambient temperature, several jurisdictions limit such storage to 4-6 h. Here, we separately spiked thawed cryoprecipitate units with four bacterial strains: Staphylococcus epidermidis, Serratia liquefaciens, Pseudomonas putida and Pseudomonas aeruginosa. No strains grew in the first 4 h of storage, but by 24 h, three of four exhibited up to 1000-fold proliferation. Pathogen inactivation technologies could be explored to mitigate the safety risk posed by extending storage of thawed cryoprecipitate at room temperature.

Thrombolysis by chemically modified coagulation factor Xa.

UNLABELLED: Essentials Factor Xa (FXa) acquires cleavage-mediated tissue plasminogen activator (tPA) cofactor activity. Recombinant (r) tPA is the predominant thrombolytic drug, but it may cause systemic side effects. Chemically modified, non-enzymatic FXa was produced (Xai-K), which rapidly lysed thrombi in mice. Unlike rtPA, Xai-K had no systemic fibrinolysis activation markers, indicating improved safety. SUMMARY: Background Enzymatic thrombolysis carries the risk of hemorrhage and re-occlusion must be evaded by co-administration with an anticoagulant. Toward further improving these shortcomings, we report a novel dual-functioning molecule, Xai-K, which is both a non-enzymatic thrombolytic agent and an anticoagulant. Xai-K is based on clotting factor Xa, whose sequential plasmin-mediated fragments, FXaß and Xa33/13, accelerate the principal thrombolytic agent, tissue plasminogen activator (tPA), but only when localized to anionic phospholipid. Methods The effect of Xai-K on fibrinolysis was measured in vitro by turbidity, thromboelastography and chromogenic assays, and measured in a murine model of occlusive carotid thrombosis by Doppler ultrasound. The anticoagulant properties of Xai-K were evaluated by normal plasma clotting assays, and in murine liver laceration and tail amputation hemostatic models. Results Xa33/13, which participates in fibrinolysis of purified fibrin, was rapidly inhibited in plasma. Cleavage was blocked at FXaß by modifying residues at the active site. The resultant Xai-K (1 nm) enhanced plasma clot dissolution by ~7-fold in vitro and was dependent on tPA. Xai-K alone (2.0 µg g(-1) body weight) achieved therapeutic patency in mice. The minimum primary dose of the tPA variant, Tenecteplase (TNK; 17 µg g(-1) ), could be reduced by > 30-fold to restore blood flow with adjunctive Xai-K (0.5 µg g(-1) ). TNK-induced systemic markers of fibrinolysis were not detected with Xai-K (2.0 µg g(-1) ). Xai-K had anticoagulant activity that was somewhat attenuated compared with a previously reported analogue. Conclusion These results suggest that Xai-K may ameliorate the safety profile of therapeutic thrombolysis, either as a primary or tPA/TNK-adjunctive agent.

Stability of coagulation protein activities in single units or pools of cryoprecipitate during storage at 20-24°C for up to 24 h.

BACKGROUND AND OBJECTIVE: Cryoprecipitate is a concentrated source of fibrinogen and other plasma proteins. Cryoprecipitate must be transfused within 4-6 h of thawing and storage at 20-24°C. We compared plasma protein activities in single or pooled cryoprecipitate units stored at 20-24°C for 0, 4 or 24 h. MATERIALS AND METHODS: Individual cryoprecipitate units (n = 36) were thawed, diluted with sterile saline and sampled over time. Cryoprecipitate pools of eight individual units were assembled either by serial passage of diluent (Method A, n = 6 pools) or by separate dilution into a single collection bag (Method B, n = 6 pools). Fibrinogen, factor VIII, factor XIII and von Willebrand factor activities were measured. RESULTS: No significant losses in activities were found relative to at-thaw values after either 4 or 24 h of storage of individual cryoprecipitate units at 20-24°C; 35 of 36 units contained >150 mg of fibrinogen. No significant differences were found between activities in single vs. pooled units of cryoprecipitate assembled using either method, or between cryoprecipitate pools made by Method A (80-160 ml volume) or Method B (160-240 ml volume) at 0, 4 or 24 h post-thaw; freezing and thawing of pools did not lead to significant activity losses. CONCLUSION: The stability of fibrinogen and other factors in thawed cryoprecipitate stored at 20-24°C suggests that the shelf life may be safely extended to 24 h provided that sterility is maintained.

A plasmin-activatable thrombin inhibitor reduces experimental thrombosis and assists experimental thrombolysis in murine models.

The leech protein hirudin is a potent natural thrombin inhibitor. Its potential as an antithrombotic agent is limited by its promotion of bleeding. We attempted to modify this profile by positioning albumin and a plasmin cleavage site on its N-terminus, in recombinant protein HSACHV3 [comprising hirudin variant 3 (HV3) fused to the C-terminus of human serum albumin (HSA) via a plasmin cleavage site (C)], Previously we showed that HSACHV3 inhibited thrombin in a plasmin-dependent manner, and that, unlike HV3, it did not increase bleeding in vivo when administered to mice. Here we tested HSACHV3 for the ability to reduce thrombosis and assist enzymatic thrombolysis in animal models. Intravenous administration of HSACHV3, but not a control protein lacking the plasmin cleavage site (HSAHV3), reduced thrombus weight by 2.1-fold in the ferric chloride-injured mouse vena cava. Similarly, thrombi formed in a rabbit jugular vein stasis model were 1.7-fold lighter in animals treated with HSACHV3 compared to those receiving HSAHV3. Administration of 60 mg/kg body weight HSACHV3 prolonged the time to occlusion in the ferric chloride-injured mouse carotid artery by threefold compared to vehicle controls, while equimolar HSAHV3 had no effect. HSACHV3 had no ability to restore flow to the murine carotid arteries occluded by ferric chloride treatment, but combining HSACHV3 (60 mg/kg) with recombinant mutant tissue plasminogen activator (TNKase) significantly reduced the time to restore patency to the artery compared to TNKase alone. Unlike unfused HV3, HSACHV3 did not increase bleeding in a mouse liver laceration model. Our results show that HSACHV3 acts as an antithrombotic agent that does not promote bleeding and which speeds the time to flow restoration when used as an adjunct to pharmacological thrombolysis in animal models.

γT -S195A thrombin reduces the anticoagulant effects of dabigatran in vitro and in vivo.

BACKGROUND: Dabigatran etexilate (DE) is an oral direct thrombin inhibitor used to prevent strokes in patients with atrial fibrillation. No licensed DE antidote is currently available. We hypothesized that active site-mutated S195A thrombin (S195A-IIa) and/or its trypsinized derivative (γT -S195A-IIa) would sequester dabigatran, the active form of DE, and reduce its anticoagulant effects. OBJECTIVE: To assess active site-mutated S195A or γT -S195A-IIa as dabigatran reversal agents in vitro and in vivo. METHODS: Diluted thrombin time (dTT) assays were performed using human or murine plasma containing dabigatran, combined with S195A-IIa, γT -S195A-IIa or FPR-chloromethyl ketone-treated thrombin (FPR-IIa). Bleeding times were determined in anesthetized DE-treated mice also receiving γT -S195A-IIa or vehicle 15 min prior to tail transection. The time to occlusion of carotid arteries of DE-treated mice also receiving S195A-IIa, γT -S195A-IIa, prothrombin complex concentrate (PCC) or vehicle, 15 min prior to topical FeCl3 , was determined using Doppler ultrasound. RESULTS: γT-S195A-IIa reduced dTT values of dabigatran-containing human and murine plasma more effectively than S195-IIa; FPR-IIa had no effect. A dose of 13 mg kg(-1) DE abrogated occlusive thrombus formation in the carotid arteries of FeCl3 -treated mice; γT -S195A-IIa (6 mg kg(-1) ) or PCC (14.3 IU kg(-1) ), but not saline vehicle or S195A-IIa (6 mg kg(-1) ), was equally effective in restoring thrombus formation. Bleeding times of mice treated with 60 mg kg(-1) DE and γT -S195A-IIa (6 mg kg(-1) ) or saline vehicle did not differ. CONCLUSIONS: Our data suggest that γT -S195A-IIa decreases the anticoagulant effects of dabigatran in vitro and is partially effective at restoring hemostasis-related thrombus formation in DE-treated mice in vivo.

Prothrombin complex concentrates reduce blood loss in murine coagulopathy induced by warfarin, but not in that induced by dabigatran etexilate.

BACKGROUND: Both established oral anticoagulants such as warfarin and newer agents such as dabigatran etexilate (DE) effectively prevent thromboembolic disease, but may provoke bleeding. Limited clinical data exist linking oral anticoagulant reversal and bleeding tendency, as opposed to surrogate laboratory markers. OBJECTIVE: To quantify bleeding in warfarin-anticoagulated and DE-anticoagulated mice by tail transection with or without pretreatment with potential reversal agents: prothrombin complex concentrate (PCC); activated PCC (APCC); recombinant factor VIIa (rFVIIa); or murine fresh-frozen plasma (FFP). METHODS: CD1 mice were given warfarin or DE by gavage, and the effects on in vitro coagulation assays, volume of blood loss and the bleeding time following tail transection injury were evaluated with different reversal agents. RESULTS: PCC (14.3 IU kg(-1) ), but not rFVIIa (3 mg kg(-1) ) or FFP (12 mL kg(-1) ), normalized blood loss and bleeding time in mice with warfarin-induced elevations of mean prothrombin time at two intensities (prothrombin time ratios of either 4.3 or 24). Neither separate nor combined PCC and/or rFVIIa treatment nor APCC (100 U kg(-1) ) treatment significantly reduced blood loss in mice anticoagulated with 60 mg kg(-1) DE 75 min prior to tail transection. Both combined PCC plus rFVIIa treatment and APCC treatment significantly reduced bleeding time in the DE-treated mice. CONCLUSIONS: Our data suggest that PCC treatment prevents excess bleeding much more effectively in warfarin-induced coagulopathy than in DE-induced coagulopathy.

Retention of thrombin inhibitory activity by recombinant serpins expressed as integral membrane proteins tethered to the surface of mammalian cells.

BACKGROUND: Serpins form a widely distributed protein superfamily, but no integral membrane serpins have been described. OBJECTIVES: To anchor three serpins -α(1) -proteinase inhibitor (α(1) PI) (M358R), antithrombin (AT), and heparin cofactor II (HCII) - in the plasma membranes of transfected mammalian cells and assess their ability to inhibit thrombin. METHODS: Serpin cDNAs were altered to include N-terminal, non-cleavable plasma membrane-targeting sequences from the human transferrin receptor (TR) (TR-serpin) or the human asialoglycoprotein receptor (AR) (AR-serpin), and used to transfect COS-1 or HEK 293 cells. Cells were analyzed for serpin expression by immunoblotting of subcellular fractions, by immunofluorescence microscopy, or by flow cytometry, with or without exposure to exogenous thrombin; AR-serpins and TR-serpins were also compared with their soluble recombinant counterparts. RESULTS: Both TR-α(1) PI (M358R) and AR-α(1) PI (M358R) were enriched in the integral membrane fraction of transfected COS-1 or HEK 293 cells, and formed inhibitory complexes with thrombin, although less rapidly than soluble α(1) PI (M358R). Thrombin inhibition was abrogated by an additional T345R mutation in AR-α(1) PI (M358R). Surface-displayed AR-AT also formed serpin-enzyme complexes with thrombin, but to a lesser extent than AR-α(1) PI (M358R); AR-HCII inhibitory function was not detected. Immunofluorescence detection and flow cytometric quantification of bound thrombin also supported the status of AR-α(1) PI (M358R) and AR-AT as thrombin inhibitors. CONCLUSIONS: Two of three thrombin-inhibitory serpins retained functionality when expressed as integral membrane proteins. Our findings could be applied to create and screen hypervariable serpin libraries expressed in mammalian cells, or to confer protease resistance on engineered cells in vivo.

Lipoprotein enrichment in orange insoluble particulate matter reproducibly appearing in cryoprecipitate.

BACKGROUND AND OBJECTIVE: Cryoprecipitate prepared from two whole blood donations from the same donor contained insoluble orange particulate material (OPM). We sought to identify the OPM. MATERIALS AND METHODS: OPM was recovered from the blood product by centrifugation, dissolved in sodium dodecyl sulphate (SDS) and analysed by SDS-polyacrylamide gel electrophoresis and immunoblotting. RESULTS: Solubilized OPM was enriched in apolipoproteins B and E, but not apolipoprotein A1, immunoglobulin G or albumin, suggesting lipoprotein enrichment in OPM. Subsequent clinical laboratory blood tests confirmed low-density lipoprotein hyperlipidaemia with normal triglyceride levels. Further, cryoprecipitate production from this donor was prevented by implementation of national predominantly male plasma policies. CONCLUSION: Cryoprecipitate produced from hyperlipidaemic donors may contain insoluble particles that render it inappropriate for transfusion.

A barbourin-albumin fusion protein that is slowly cleared in vivo retains the ability to inhibit platelet aggregation in vitro.

Barbourin is a 73 amino acid venom protein that inhibits platelet aggregation. Recombinant barbourin (BARH6), rabbit serum albumin (RSAH6), and a barbourin-RSA fusion protein (barbourin-linker-albumin; BLAH6) were secreted from Pichia pastoris yeast, and purified by nickel-chelate affinity chromatography via their C-terminal hexahistidine (H6) tags. BARH6 and BLAH6 did not differ in their IC50s for inhibition of platelet aggregation using either human platelets stimulated with thrombin or ADP, or rabbit platelets stimulated with ADP. BARH6 and BLAH6 were also effective in inhibiting platelet aggregation in whole blood, and formed complexes with platelet integrin alphaIIbbeta3. The terminal catabolic half-life of BLAH6 approached that of RSAH6 [3.4 +/- 0.2 versus 4.0 +/- 0.1 days (n = 4 +/- SD)], but was substantially increased relative to that of BARH6 [0.15 +/- 0.03 days (n = 3 +/- SD)]. Our results suggest that fusion to albumin slows the clearance of barbourin in vivo, while preserving its ability to inhibit platelet aggregation.

Prolonged in vivo anticoagulant activity of a hirudin-albumin fusion protein secreted from Pichia pastoris.

Hirudin is a small, proteinaceous thrombin inhibitor that clears rapidly from the circulation. A hexahistidine-tagged hirudin-rabbit serum albumin (RSA) fusion protein, HLAH6, was characterized following secretion from Pichia pastoris. HLAH6 bound to immobilized nickel, anti-RSA, and anti-hexahistidine antibodies, and contained the expected (ITYTD) N-terminus. Its spectrometric mass was 74,490 (versus the theoretical mass of 74,410 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of 84 kDa). The terminal catabolic half-life in rabbits of HLAH6, recombinant Pichia-derived His-tagged RSA, or plasma-derived RSA did not differ. Injection of 2 mg/kg HLAH6 into rabbits raised the activated partial thromboplastin time (aPTT) above initial values for 4-24 h, while the equimolar dose of unfused hirudin was without significant effect. A higher dose of HLAH6 (3 mg/kg functional HLAH6, equivalent to 37.6 thrombin-inhibitory units/g) raised the aPTT by 2.0- to 2.5-fold; the elevation persisted for > 48 h. Importantly, both HLAH6 and unfused hirudin inhibited clot-bound thrombin. Our results suggest that HLAH6 exhibits not only delayed clearance, but also prolonged biological activity in vivo compared with unfused hirudin.

Modification of clearance of therapeutic and potentially therapeutic proteins.

Advances in biochemistry, protein chemistry and molecular biology over the last twenty-five years have spurred the increased use and development of proteins as injectable therapeutic agents. Introduction of proteins into the circulation exposes them to numerous different cells, enzymes and routes of extravasation that contribute to their clearance and their catabolism. Overly rapid clearance, particularly of small proteins, can limit therapeutic efficacy. Many strategies have been devised to retard the clearance of therapeutic or potentially therapeutic proteins, but relatively few proteins with clearance-retarding modifications are in clinical use. Proteins have been chemically modified towards this end by covalent attachment of polyethylene glycol or dextran chains or by protein-protein cross-linking. Genetic modification has also been employed to fuse proteins of interest to long-lived plasma proteins like albumin or immunoglobulins, or portions of these proteins. While all modifications may reduce the biological activity of the protein of interest or elicit antibody formation in recipient animals or patients, there now exists sufficient experience in this area that an optimal clearance-extending strategy can often be designed and successfully executed. With the explosive growth of genomic and proteomic information, an exponentially increasing number of engineered proteins are likely to be developed, with a probable need for clearance-related modification.

Modulation of clearance of recombinant serum albumin by either glycosylation or truncation.

Albumin is an abundant non-glycosylated plasma protein with a slow clearance profile. It has been employed as a fusion partner in efforts to slow the clearance of small antithrombotic proteins like hirudin. In the present study, the in vivo clearance of recombinant rabbit serum albumin (rRSA), of mutant rRSAs containing consensus sequences for N-linked glycosylation (D494N and V14T variants), and of mutant mini-proteins truncated at albumin domain boundaries (rRSAs 1-185, 1-377, or 378-584) was examined. Mean terminal catabolic half-lives (t(0.5)cat) in rabbits for plasma-derived RSA, rRSA, and the V14T variant did not differ significantly (range 4. 32-4.76 days). In contrast, mean t(0.5)cat was reduced to 2.87 days for the D494N variant and to less than 0.071 days for all mini-proteins. The mini-proteins were found in the urine in tissue distribution experiments, suggesting a renal route of clearance. Our results suggest that all three internally repeated albumin domains are required to maintain the slow in vivo clearance profile of albumin, and that albumin glycosylation can be associated with an acceleration of clearance. This information could be used to design fusion proteins, including those with antithrombotic properties, with predictably altered in vivo half-lives less than that of serum albumin.

Mutation of any site of N-linked glycosylation accelerates the in vivo clearance of recombinant rabbit antithrombin.

Antithrombin (AT) is a plasma protein with four sites of N-linked glycosylation. Asn 135 is incompletely glycosylated, and the resulting 3-glycan AT is cleared more rapidly in vivo than the 4-glycan form. The Asn codons in each of the four sites of glycosylation were altered in turn, to create four mutant rabbit AT cDNAs. Permanently transfected CHO cell lines were generated following transfection of the resulting constructs, encoding either the wild-type rabbit AT (AT-WT) or one of the four underglycosylated variants (AT-N96Q, AT-N135Q, AT-N155Q, and AT-N155Q). Comparison of the five resulting recombinant AT proteins revealed that the major AT species of each variant co-migrated on SDS gels, and migrated more rapidly than the major form of AT-WT. The shift in mobility, from 60 to 57 kDa, was consistent with the loss of one fully sialylated complex N-linked glycan. Neither the amount of AT secreted (range: 1.25 to 4.2 microg/10(6) cells/day) nor the kinetics of secretion differed significantly between cell lines expressing AT-WT or any of the AT variants. All forms of recombinant rabbit AT were capable of forming denaturation-resistant complexes with thrombin. Purification and radioiodination of each of the five recombinant AT proteins permitted pharmacokinetic analysis of their individual clearance in rabbits. While neither the equilibration half-life (t(0.5)alpha) nor the terminal catabolic half-life (t(0. 5)beta) differed significantly between plasma-derived rabbit AT and AT-WT, the t(0.5)beta of all the underglycosylated variants was decreased relative to that of AT-WT (maximum reduction in mean: from 70.1+/-3.2 h to 52.4+/-2.5 h). These results suggest that the overall extent of glycosylation, rather than the location within AT of the glycan chains, is a primary determinant of AT clearance.

Cytokeratin 18 is expressed on the hepatocyte plasma membrane surface and interacts with thrombin-antithrombin complexes.

During experiments to identify putative hepatic receptors for thrombin-antithrombin (TAT) complexes, a 45-kDa protein was identified by ligand blotting. Following gel purification, amino acid sequencing revealed the 45-kDa TAT-binding polypeptide to be cytokeratin 18 (CK18). The presence of CK18 on the surface of intact rat hepatoma cells was demonstrated by binding of 125I-anti-CK18 antibodies. Anti-CK18 antibodies reduced the binding and internalization of 125I-TAT by rat hepatoma cells. Immunocytochemical analysis, to determine the location of CK18 in vivo, revealed a periportal gradient of CK18 staining; with hepatocytes around the portal triads demonstrating striking pericellular staining. In addition, anti-CK18 IgG associated with perfused livers to a significantly greater extent than preimmune IgG. Taken together, these data provide evidence that CK18 is found on the extracellular surface of hepatocytes and could play a role in TAT removal. Finally, these data, in conjunction with recent reports of CK8 (Hembrough, T. A., Li, L., and Gonias, S. L. (1996) J. Biol. Chem. 271, 25684-25691) and CK1 cell membrane surface expression (Schmaier, A. H. (1997) Thromb. Hemostasis 78, 101-107), indicate a novel role for these proteins as putative cellular receptors or cofactors to cellular receptors.

Impact of mutations at the P4 and P5 positions on the reaction of antithrombin with thrombin and elastase.

Antithrombin (AT) is a serpin capable of trapping thrombin (IIa) in a stable and covalent complex. Complex formation is prevented by leukocyte elastase (LE) cleavage near the AT reactive centre. We mutated the known LE cleavage sites of AT to explore the possibility of producing an LE-resistant AT molecule. Initially, six rabbit AT variants differing only at residue 390 (P4) were generated in a cell-free system, and gel-based assays were used to assess IIa-mediated complex formation and LE-mediated cleavage of the variants. Substitution of charged residues (Glu or Arg) reduced complex formation by 50-60%, while the Ser variant was incapable of inhibiting thrombin; LE reactivity was less affected. The least (Trp) and most (Ser) affected variants were expressed in COS-1 cells. Again, the Ser variant was incapable of detectably reducing the rate of thrombin-mediated amidolysis while the Trp variant inhibited thrombin at a slightly reduced rate (-28%). LE inactivated the Trp variant and the wild-type AT to a similar extent. Recreation of the Trp mutation in COS-derived human AT showed similar results. Since retention of LE-sensitivity could have arisen due to cleavage at Val389 (P5), we produced and characterized a human AT substitution mutant with Trp at both P4 and P5. This variant showed a slight reduction in thrombin inhibitory activity (-22%), but remained susceptible to LE inactivation. These results suggest either that LE cleaves at secondary sites if its primary cleavage sites are blocked, or that the substrate specificity of LE differs in polypeptides as compared to peptide substrates.

Potent antithrombin activity and delayed clearance from the circulation characterize recombinant hirudin genetically fused to albumin.

In this study we sought to extend the plasma half-life while maintaining the potent antithrombin activity of hirudin. We hypothesized that gene fusion of hirudin to albumin would result in the expression of a slowly cleared hirudin molecule. A hirudin variant 3 (HV3) cDNA was obtained by gene synthesis, while a 1,996-bp full-length rabbit serum albumin (RSA) cDNA was selected from a rabbit liver cDNA library. Expression of the former in COS-1 cells conferred antithrombin activity on media conditioned by the cells, while expression of the latter resulted in the secretion of a 67-kD protein that reacted with mono-specific anti-RSA antibodies. Having shown independent expression of the two proteins, we next expressed two fusion proteins: HV3 linked via its C-terminus to albumin (HLA), and HV3 linked via its N-terminus to albumin (ALH). The former, but not the latter, inhibited both the amidolytic and fibrinogenolytic activities of thrombin. HLA also retained the dye-binding characteristics of RSA, as judged by Affi-Gel Blue chromatography. Highly similar concentrations of either commercial HV1 (40 nmol/L) or HLA (30 nmol/L) were required to halve the initial rate of thrombin reaction with chromogenic substrate S2238, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein. An His-tagged form of HLA was purified by Ni2+-chelate affinity and heparin-Sepharose chromatography. The purified, radioiodinated protein was injected into rabbits, and demonstrated a catabolic half-life of 4.60 +/- 0.16 days. This represents an extension of hirudin half-life in vivo of greater than two orders of magnitude; gel analysis of HLA(H)6 recovered from rabbits showed that it circulated in intact form. Our results provide a rationale for future testing of the biological effects of HLA, and support our initial hypothesis.