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.

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.

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.

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.

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 signal sequence domain essential for processing, but not import, of mitochondrial pre-ornithine carbamyl transferase.

Studies using deletion mutagenesis indicate that a processing recognition site lies proximal to the normal cleavage position between gln32 and ser33 of pre-ornithine carbamyl transferase (pOCT). pOCT cDNA was manipulated to delete codons specifying amino acids 22-30 of the signal sequence. The mutant precursor, designated pOCT delta 22-30, was imported to the matrix compartment by purified mitochondria, but remained largely unprocessed; the low level of processing that was observed did not involve the normal cleavage site. Several manipulations performed downstream of the normal pOCT processing site (deletion, substitution, and hybrid protein constructions) affected neither import nor correct processing. Our data suggest that domains specifying import and processing site recognition may be functionally segregated within the signal peptide; that processing is not requisite for import of pOCT; and that a proximal region, not involving the normal signal peptide cleavage site, is required for processing site recognition.

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.

Deletion mutagenesis of heparin cofactor II: defining the minimum size of a thrombin inhibiting serpin.

Heparin cofactor II (HCII) is a 66 kDa plasma glycoprotein that belongs to the serpin superfamily of protease inhibitors. Its natural target is thrombin. HCII inhibits thrombin in both a progressive reaction, and in an accelerated reaction catalyzed by a glycosaminoglycan, dermatan sulphate (DS). Both modes of inhibition result in the formation of a stable, denaturation-resistant complex. Using a cDNA clone encoding rabbit HCII recently isolated and characterized in our laboratory, we have employed deletion mutagenesis to identify amino-terminal regions of the molecular which are essential to the progressive reaction. PCR was employed to produce four deletion constructs: delta 58, delta 81, delta 106, and delta 169, all in an in vitro transcription vector plasmid background. Transcription of the full-length construct, and of the four deletion constructs, followed by in vitro translation in rabbit reticulocyte lysate, was used to produce the corresponding HCII-related polypeptides. The delta 106 and delta 169 mutants failed to react with thrombin, even in the presence of DS. In contrast, the delta 58 and delta 82 mutants retained the ability to form complexes with thrombin, although the rate of complex formation was decreased for the latter mutant compared to the full-length recombinant HCII; no acceleration of complex formation in the presence of 20 micrograms/ml DS was noted for either truncated recombinant HCII. Alignment of the rabbit HCII primary structure with secondary structural elements found in alpha 1 antitrypsin and other serpins showed that the non-functional delta 106 mutant lacks helix A, while the functional delta 82 mutant contains this element. Our results suggest that helix A is an essential part of a functional serpin, and define the limits of the amino-terminal region of HCII which is not essential for thrombin inhibition.

Site-directed mutagenesis of the P2 residue of human antithrombin.

Antithrombin (AT) is the principal inhibitor of thrombin in human plasma, and a member of the serine proteinase (serpin) family of proteins. Previously, we have described a point mutation in the human AT gene that converted amino acid 392 from glycine to aspartic acid which was associated with thrombotic disease in a Swedish family [(1992) Blood 79, 1428-1434]. This observation prompted us to investigate the consequences of other substitutions at this position, termed P2 with respect to the reactive centre. Site-directed mutagenesis was employed to generate seven mutants (Pro, Met, Gln, Val, Lys, Glu, and Asp), whose properties were compared with wild-type recombinant AT, following in vitro transcription and cell-free expression in a rabbit reticulocyte lysate system. With only one exception, the variant forms were less active than the wild-type in forming complexes with either alpha-thrombin, factor Xa, or trypsin. Hydrophobic (Val) or negatively charged (Asp or Glu) substitutions were particularly disruptive, in that these variants exhibited less than 10% wild-type antithrombin or antitrypsin activity. In contrast, the formation of complexes with the various proteases of the Pro variant was essentially unimpaired. We conclude that the P2 residue of AT plays a role in optimal presentation of the reactive centre to its cognate protease, and propose that the observed requirement of Gly or Pro at this position is suggestive of a bend in the polypeptide backbone that aids in this presentation.

Molecular cloning and expression of rabbit heparin cofactor II: a plasma thrombin inhibitor highly conserved between species.

Heparin cofactor II (HCII), a circulating plasma protein that inhibits thrombin, is a member of the serine proteinase (serpin) family of proteins. The extent to which HCII structure is conserved across species lines was investigated, by obtaining cDNA clones encoding rabbit HCII. Overlapping clones corresponding to rabbit HCII were obtained by the combined use of hybridization screening of a rabbit liver cDNA library, and by rapid amplification of cDNA ends (RACE). The consensus sequence obtained spans 2178 nucleotides, and is comprised of a 5' untranslated region of 77 nucleotides, an open reading frame of 1440 nucleotides, and a 3' untranslated region of 661 nucleotides that concludes with a poly A tract. The open reading frame is subdivided into a secretory signal sequence of 19 amino acids, and a mature protein of 461 amino acids. Within the region comprising the mature protein, 87% of the amino acid residues are identical to those seen in human HCII. Expression of an appropriately modified form of the rabbit HCII clone in an in vitro reticulocyte expression system yielded two major polypeptides, of 60 and 56 kD respectively, both of which were able to form SDS-stable complexes with human alpha-thrombin, in a reaction accelerated by dermatan sulphate. The remarkable degree of homology observed between rabbit HCII and its human counterpart, indicating a high degree of conservation of structure through evolution, suggests an important function of HCII in hemostasis.

Molecular cloning and cell-free expression of mouse antithrombin III.

The homology between antithrombin III (AT-III) of mouse, of man, and that of other species was investigated. Preliminary experiments showed that mouse AT-III inhibited human alpha-thrombin efficiently (second order rate constant [K2nd] 5.8 x 10(3) M-1 s-1) as compared to human AT-III (K2nd 6.7 x 10(3) M-1), but was not recognized on immunoblots by antibodies that recognized both human and rabbit AT-III. In order to compare AT-III from different species at the molecular level, a cDNA clone for murine AT-III was isolated from a lambda ZAP mouse liver cDNA library on the basis of hybridization to a rabbit AT-III cDNA probe. The 1509 bp murine AT-III cDNA consists of a 1398 bp open reading frame, preceded by a 15 bp 5' untranslated region, followed by a 75 bp 3' untranslated region. The deduced primary protein structure consists of a 32 amino acid signal sequence, with a mature portion of 433 residues. Mature murine AT-III is 89% identical to its human counterpart, 86% identical to bovine AT-III, and 82% identical to that of the rabbit. Constructs lacking the nucleotides encoding the signal sequence were engineered and expressed in a cell-free system. The resulting 47 kDa non-glycosylated translation product was capable of being cleaved by human alpha-thrombin, of forming SDS-stable complexes with the protease, and of binding to immobilized heparin. Isolation of the murine AT-III cDNA will make feasible molecularly defined experiments with murine AT-III in the mouse system.

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.

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.

Amino acid substitutions of the P2 residue of human antithrombin that either enhance or impair function.

Recombinant forms of human antithrombin (AT) were expressed in COS-1 cells, and their interaction with human thrombin characterized by comparing the reactivity of two engineered mutant forms of AT with the wild-type recombinant. Both mutant forms contained single amino acid substitutions of Asp (G392D) or Pro (G392P) for the wild-type Gly, at residue 392, termed the P2 position with reference to the adjacent reactive centre bond. All three forms of AT co-migrated on Western blots, with an apparent molecular weight of 58 kD, with endoglycosidase F treatment reducing their mobility to 47 kD. The two mutant forms of AT reacted with thrombin differently from the wild-type molecule, in that the G392D substitution abrogated the thrombin inhibitory capacity of the protein, while the G392P substitution enhanced the reactivity of the recombinant mutant AT with thrombin. Under pseudo-first order conditions, the second order rate constants for the reaction of the recombinant wild-type and G392P mutant AT were determined to be 1.4 x 10(4) L-mol-1 sec-1 and 3.0 x 10(4) L-mol-1 sec-1, respectively, a difference of 210%. In contrast, in the presence of heparin, the reaction rates of the G392P and wild-type AT forms with thrombin, differed by less than 25%. We conclude that the P2 position of AT is an important residue for AT to express its inhibitory activity, alterations to which can either enhance or impair the inhibition of thrombin by AT.

Inhibition of thrombin by hirudin genetically fused to wild-type or mutant antithrombin.

Recombinant fusion proteins consisting of hirudin variant 3 (HV3) fused at its C-terminus to either of two forms of mature rabbit antithrombin (AT) were generated in COS-I cells. HV3 fused to wild-type AT was designated HAT, while a similar chimeric protein in which the P12 residue of AT was mutated from Ala to Thr, was designated HAT(H) for the Hamilton (A382T) mutation. Addition of the HV3 domain resulted in a decreased mobility of both HAT and HAT(H) relative to COS-derived AT (68 kDa versus 60 kDa). Both proteins had a greatly increased ability to inhibit thrombin in amidolytic activity assays, relative to recombinant AT. Addition of heparin to these reactions was without effect. Incubation of conditioned media containing recombinant AT with 125I-labelled thrombin resulted in the formation of SDS-stable AT-IIa complexes; no such complexes were detected in identical reactions containing either HV3-AT fusion protein. The two proteins did not differ significantly in their ability to compete for the binding of 125I-labelled thrombin to immobilized HV1 (CGP 39393). Both proteins were found to bind to heparin-Sepharose, but less tightly than unfused AT. This property was demonstrated by the peak elution of the fusion proteins at 0.65 M NaCl, as compared to that of COS-derived AT at 1.05 M NaCl. We conclude that the fusion proteins inhibit thrombin with similar affinity to unfused hirudin via their hirudin and not their antithrombin domains. The heparin-binding capability of these proteins may indicate the acquisition of vessel wall binding capacity by these novel forms of recombinant hirudin.

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.

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.

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.

Complete nucleotide sequence of the cDNA encoding rabbit coagulation factor VII.

Rabbit coagulation factor VII cDNA has been cloned and sequenced. Two rabbit liver cDNA libraries in lambda gt11 were screened using both random-primed, 32p-labelled, factor VII cDNAs and polymerase chain reaction technology. Three overlapping cDNA clones were isolated comprising a sequence of 1572 base pairs of DNA encoding an open-reading-frame of 443 amino acids. Mature circulating rabbit factor VII is predicted to contain 404 amino acids, two less than its human counterpart and three less than bovine factor VII.