- Research article
- Open Access
Addition of a sequence from α2-antiplasmin transforms human serum albumin into a blood clot component that speeds clot lysis
© Sheffield et al; licensee BioMed Central Ltd. 2009
Received: 19 August 2008
Accepted: 03 March 2009
Published: 03 March 2009
The plasma protein α2-antiplasmin (α2AP) is cross-linked to fibrin in blood clots by the transglutaminase factor XIIIa, and in that location retards clot lysis. Competition for this effect could be clinically useful in patients with thrombosis. We hypothesized that fusion of N-terminal portions of α2-antiplasmin to human serum albumin (HSA) and production of the chimeric proteins in Pichia pastoris yeast would produce a stable and effective competitor protein.
Fusion protein α2AP(13-42)-HSA was efficiently secreted from transformed yeast and purified preparations contained within a mixed population the full-length intact form, while fusions with longer α2AP moieties were inefficiently secreted and/or degraded. The α2AP(13-42)-HSA protein, but not recombinant HSA, was cross-linked to both chemical lysine donors and fibrin or fibrinogen by factor XIIIa, although with less rapid kinetics than native α2AP. Excess α2AP(13-42)-HSA competed with α2AP for cross-linking to chemical lysine donors more effectively than a synthetic α2AP(13-42) peptide, and reduced the α2AP-dependent resistance to fibrinolysis of plasma clots equally effectively as the peptide. Native α2AP was found in in vivo clots in rabbits to a greater extent than α2AP(13-42), however.
In this first report of transfer of transglutamination substrate status from one plasma protein to another, fusion protein α2AP(13-42)-HSA was shown to satisfy initial requirements for a long-lasting, well-tolerated competitive inhibitor of α2-antiplasmin predicted to act in a clot-localized manner.
Proteins contain domains, substructures with conserved sequence or structural similarity . The majority of eukaryotic proteins contain multiple domains, ones that have apparently arisen via "domain accretion" resulting from duplication and recombination of the domain-encoding genes . Biotechnological approaches mimic this principle, through the engineering of gene fusions to create expressed proteins, ones combining formerly separate components into a single polypeptide chain. Commonly, the rationale is to slow the clearance from the circulation of injectable protein-based therapeutic agents, or to otherwise stabilize such peptides or proteins. Either immunoglobulins or albumin have been widely used for this purpose .
Mammalian albumins are abundant proteins found in blood plasma at mean concentrations of 0.6 – 1.0 mM , of 584–585 amino acids in length [5, 6]. Unlike most plasma proteins, albumin is not glycosylated, but it nevertheless exhibits a long circulatory half-life [4, 7] of 19 days in humans . Recycling of albumin through the major histocompatibility complex-related Fc receptor for IgG (FcRn) has been shown to contribute to its circulatory longevity [8, 9]. Productive fusion to albumin has been demonstrated for such diverse proteins as interleukin-2 , atrial natriuretic peptide , interferon , butrylcholinesterase  and coagulation factor VII . In the context of improving therapies for thrombosis, our laboratory has explored albumin fusion to improve the pharmacokinetics of small proteins like the thrombin inhibitor hirudin [6, 15], the platelet aggregation antagonist barbourin [16, 17], and related peptides. Recently, we conceived a new strategy to use albumin fusion to counter thrombosis, by transferring a property of another plasma protein, α2-antiplasmin (α2AP), to albumin.
α2AP is a plasma glycoprotein of the serine proteinase (serpin) superfamily that regulates fibrinolysis, the natural process of blood clot lysis . It binds plasmin using a C-terminal lysine-rich domain and inhibits it by forming a stable, covalent serpin-enzyme complex . It is also cross-linked into fibrin clots, and in that location slows clot lysis by inhibition of plasmin . Chemical  or mutational  inactivation of the α2AP reactive centre loop (RCL) yields a protein that competes with native α2AP for cross-linking to fibrin, but which cannot inactivate plasmin. Consequently, clot lysis is accelerated. Activated factor XIII (fXIIIa) mediates the cross-linking of lysines on fibrin and glutamines on α2AP . The principal cross-linked residue on α2AP is Q14, although other residues can also be modified, and synthetic peptides containing this site are weaker substrates for transglutamination than intact or RCL-inactivated α2AP .
As a glycoprotein and conformationally sensitive serpin , α2AP can be difficult to express efficiently in recombinant form. Recombinant human serum albumin (HSA), in contrast, is efficiently expressed at exceptionally high yield in Pichia pastoris yeast systems . We hypothesized that N-terminal portions of α2AP, when fused to HSA, would confer upon it the ability to be cross-linked to fibrin, and to interfere with α2AP-mediated delays in clot lysis. Furthermore, we thought that display on albumin could present Q14 in a more favourable conformation for cross-linking than when it is present in a short peptide. Reasoning that the cross-linking site(s) might require additional motifs from α2AP, we compared the production and properties of three overlapping, C-terminally truncated portions of α2AP fused to HSA.
Four plasmids were constructed to enable expression of HSA and three α2AP-HSA fusion proteins as C-terminally hexahistidine-tagged Pichia pastoris expression products. All oligonucleotides were synthesized and all constructs subjected to confirmatory DNA sequencing at MOBIX Lab, a McMaster University core facility. PCR employed Phusion™ high fidelity heat-stable DNA polymerase (NEB). Standard molecular biological protocols for purification of DNA from agarose gels, DNA restriction and ligation, transformation of E. coli Top10 (Invitrogen) to ampicillin resistance, and plasmid mini-DNA preparations were employed. First, the HSA DNA was manipulated for expression by PCR amplification, using oligonucleotides ML12007 (5'-CATGGAATTC TTAATGGTGA CGGTGATGGT GTAAGCCTAA GGCAGCTCGA CTTGCAGCAA C-3'), ML12008 (5'-GATCCTCGAG AAAAGAGACG CACACAAGAG TGAGGTTGC-3'), and plasmid pC3HFUS  as a template. The resulting 1788 bp amplification product was restricted with XhoI and EcoRI and the inserted between these sites in pPICZ9ssamp  to form pPICZ9ssHSAH6. PCR products A, B, and C were then generated using as a template the human α2AP-encoding plasmid pBAD-H6α2AP . Codons 13–42 were mobilized using oligonucleotides ML 17226 (5'-ACGTCTCGAGA AAAAGAAACC AGGAGCAGGT GTCCCC-3') and ML 17225 (5'-ACGTGGTAC CGACTCCTGG GGGACTCTTC AG-3') to form product A. Codons 13–73 were obtained using ML 17226 and ML 17227 (5'-AGCTGGTACCC CTGGTGAGCC ACCAGGGAGA AC-3') in product B. Codons 13–109 were amplified using ML 17226 and ML 17228 (5'-AGCTGGTACC CCCAGCACCT TTGCAGCCTC TG-3') to yield product C. The HSA cDNA was then PCR-amplified with oligonucleotides ML 12006 (5'-CATGCGGTAC CACAAGAGTG AGGTTGCTC-3') and ML 12007 (5'-GTTGCTGCAA GTCAGGCTGC CTTAGGCTTA CACCATCACC ATCACCATTA AGAATTCCAT G-3') to produce fragment D. A, B, and C were restricted with XhoI and KpnI and D with KpnI and EcoRI. D was then separately combined with A, B, and C, and inserted between the XhoI and EcoRI sites of pPICZ9ssamp , forming plasmids pPICZ9ss-α2AP(13-42)-HSAH6, pPICZ9ss-α2AP(13-73)-HSAH6, and pPICZ9ss-α2AP(13-109)-HSAH6. Each completed, sequence-validated plasmid listed above was used to transform Pichia pastoris strain X33 to Zeocin (Invitrogen) resistance as described .
Fusion protein expression, purification, and characterization
HSA fusion proteins were purified from media conditioned by transformed Pichia pastoris cells and induced with 0.5% vol/vol methanol, as previously described for rabbit serum albumin (RSA) fusion proteins [18, 30]. Briefly, media was neutralized, separated from cells by centrifugation, treated with protease inhibitors (5 mM benzamidine and 0.1 mM phenylmethylsulfonyl fluoride), and purified using Ni-NTA agarose (Qiagen). Purified proteins were analyzed by SDS-PAGE and immunoblotting using polyclonal goat anti-human α2AP antibodies (Affinity Biologicals) and murine monoclonal anti-HSA antibodies (Genway Biotech). They were also characterized by automated Edman degradation at the Advanced Protein Technology Centre of the Hospital for Sick Children, Toronto, Canada. The same facility was the source for synthetic peptides corresponding to α2AP(13-42) and to a plasma protein unrelated to this study, heparin cofactor II residues 54–75. The former peptide was shown to contain a single main peak, corresponding to the predicted molecular mass of 3100 Da, by time-of-flight mass spectrometry performed by the manufacturing facility.
Transglutamination assays using chemical lysine donors
FXIIIa-catalyzed transglutamination of plasma-derived α2AP and recombinant α2AP-HSA fusion proteins was tested using both artificial (dansyl cadaverine and biotinylated pentylamine) and natural (fibrinogen) lysine donors, in protocols modified from the literature . Purified fXIII, thrombin, and α2AP were purchased from Enzyme Research Labs. Human proteins were used in all cases in reactions at 37°C. Using dansyl cadaverine (Sigma) at 0.5 mM, substrate proteins (7.14 μM) were combined with FXIII (1.52 μM) in Tris-buffered saline (20 mM Tris-Cl pH 7.5, 150 mM NaCl) containing 10 mM CaCl2 and 0.5 mM dithiothreitol (DTT). Reactions were initiated by the addition of thrombin to 5.0 IU/ml and terminated at specific intervals by addition of disodium EDTA to 10 mM. Transglutamination resulted in protein substrates becoming visible under ultraviolet illumination after SDS-PAGE. Biotinylated pentylamine (BPA; EZ-Link pentylamine-biotin, Pierce) was used under similar conditions with the following exceptions: BPA was substituted for dansyl cadaverine, at 10 mM, FXIII was 0.76 μM, CaCl2 was 5 mM and thrombin was 1.0 U/ml. Transglutamination in this case resulted in transfer of a biotin group to substrates. Reactions were visualized on 8% SDS-polyacrylamide gels following transfer to nitrocellulose and blocking with 4.0 mg/ml BSA in TBS/0.2% Tween 20 (TBST), using streptavidin conjugated to horseradish peroxidase (Cedarlane Laboratories) at a 1:5000 dilution. Chemiluminescent substrate cleavage was captured on Biomax XAR scientific imaging film (Kodak). In some experiments band intensity was quantified using UN-Scan-it software (Silk Scientific) and a scanning unit (Canon).
Transglutamination of fibrin(ogen)
FXIIIa-catalyzed transglutamination of the natural substrate fibrinogen was assessed using SDS-PAGE and immunoblotting of cross-linking reactions. FXIII (100 nM) was quantitatively activated by incubation with 5.0 IU/ml thrombin for 5 minutes at 37°C prior to thrombin inactivation by addition of Phe-Pro-Arg chloromethylketone (FPRck; Calbiochem) to 10 μM. This step eliminated thrombin cleavage of chromogenic substrate S2238 (Diapharma). FXIIIa (at 50 nM final concentration) was then introduced into crosslinking reactions containing 6.0 μM fibrinogen (Sigma), and 1.0 μM substrate proteins in TBS containing 10 mM CaCl2. Reactions were stopped by addition of SDS to 1% w/vol, DTT to 50 mM, and urea to 4 M, followed by addition of 0.25 volumes of 4 × SDS loading buffer (125 mM Tris-Cl pH 6.8, 5% SDS, 25% (vol/vol) glycerol, 400 mM DTT, 0.1 mg/ml bromophenol blue). Reactions were electrophoresed on 8% SDS polyacrylamide gels under reducing conditions, and either stained with Coomassie Brilliant Blue or probed with antibodies on immunoblots as described above.
In vitro clot lysis
Human plasma immunodepleted to < 1% normal α2AP levels (ERL) was diluted 1:1 in TBS to final concentrations of 10 mM CaCl2, 5 nM thrombin, and 0.125 nM tissue-type plasminogen activator (tPA; single-chain, recombinant tPA, 60 kDa, carrier-free, purchased from Genentech by Dr. Ed Pryzdial, Canadian Blood Services, and University of British Columbia Centre for Blood Research, and kindly donated for our use). Reactions (50 μl) were carried out in microtiter plate wells, and clot formation and lysis was monitored by following changes in turbidity, using an ELx808 plate reader (BioTek Instruments) set to take absorbance readings at 340 nm every 30 seconds for 4 hours. The area under the curve of the resulting profile was measured using data from such experiments transferred to GraphPad Prism 4.0 (GraphPad Software).
Wessler-type in vivo clot incorporation
The propensity of injected proteins to remain in an in vivo clot exposed to flowing blood was determined in anesthetized New Zealand White rabbits using a Wessler-type model, in a protocol modified from the literature [32–34]. Purified human fibrinogen (Sigma) and recombinant proteins were iodinated using the Iodogen method as described by the manufacturer (Pierce), using either sodium 125I or 131I. Unincorporated radioactivity was removed by exhaustive dialysis versus phosphate-buffered saline. Specific activities of labelling exceeded 1 × 109 cpm/mg. Anaesthetized rabbits were cannulated via the carotid artery and subjected to a medial incision in the neck to expose the jugular veins. Three ml of whole blood was drawn via the cannula, and anticoagulated with 1/9th volume of 3.8% w/vol sodium citrate. Two centimetre long sections of the right and left jugular veins were isolated, emptied of blood, and blocked off by bulldog clamping. Clotting of the anticoagulated, autologous whole blood was initiated by combining it with warm (37°) human thromboplastin reagent Thromborel S (Dade Behring) in a 1:4 (vol:vol) ratio, supplementing it to 33 × 106cpm/ml 125I-fibrinogen and 131I-recombinant protein, and the clotting blood (0.15 ml) was re-introduced into the isolated segments. The process was then repeated for the other jugular vein. After 30 minutes of stasis, the clamps were removed, and blood flow restored. One hour later, the jugular veins were opened by longitudinal incision, and the clots were removed, weighed, and γ-counted in a Auto Gamma 5530 Minaxi γ counter (Perkin Elmer), with windows set to discriminate between 125I and 131I. Values were summed for left and right jugular veins for each animal.
Statistical tests were performed using GraphPad Instat version 3.06 (GraphPad Software). Multiple comparisons used one-way parametric analysis of variation (ANOVA) with Tukey-Kramer post-tests for assignment of group-specific differences, while single comparisons employed Student's t-tests
Expression and characterization of fusion proteins
Amino acid sequencing of the three preparations shown in Fig. 1C was attempted. No sequence of α2AP(13-109)-HSA was obtained. For α2AP(13-73)-HSA, a single sequence of VAQTGY, corresponding to residues 70-73 of α2AP and a GY dipeptide introduced by DNA manipulations was obtained. This result indicated near-complete proteolysis of the intended α2AP(13-73) extension. In contrast, α2AP(13-42)-HSA was found to contain a mixture of 4 termini depicted in Fig. 1D. These included the intended full-length sequence, as well as 3 smaller products with N-termini corresponding to L25, Q28, and S38.
Characterization of recombinant fusion proteins as fXIIIa substrates
In the experiment shown in Fig. 2C, BPA-detected transglutamination of α2AP was carried out in either the presence of a homogeneous synthetic peptide corresponding to α2AP (13-42), or the preparation of α2AP(13-42)-HSA shown to contain mixed N-termini by sequencing. Ten- and 100-fold molar excesses of the former reduced but did not eliminate fXIIIa-dependent transglutamination of α2AP. An unrelated peptide corresponding to heparin cofactor II residues 54–75 was without effect at 100-fold molar excess. In contrast, a 24-fold molar excess of α2AP(13-42)-HSA abrogated transglutamination of plasma-derived α2AP, in spite of its heterogeneity at its N-termini.
Fusion protein α2AP(13-42)-HSA competes with α2AP in a plasma clot
Association of α2AP and recombinant proteins with an in vivo clot
While it has been known for some time that the principal site of cross-linking of α2AP resides in the N-terminal section of the molecule [31, 38], short peptides recreating this motif are less efficiently incorporated into fibrin than native α2AP. This difference is shown by the finding that 100- to 1000-fold molar excesses of a synthetic peptide corresponding to residues 13–24 of α2AP were required to diminish α2AP cross-linking to fibrin by 50% . Hypothesizing that anchoring the N-terminal sequence to a protein would conformationally improve its fitness as a fXIIIa substrate, we designed three fusion proteins containing overlapping N-terminal portions of α2AP fused to HSA. These segments were rationally selected on the basis of the conserved and aligned secondary structural elements in serpins . Thus, the first selected segment, α2AP(13-42), terminates at the beginning of helix A, the second, α2AP(13-73), at the beginning of helix B, and the third, α2AP (13-109), at the end of combined helices B and C. In addition, choice of the 13–42 segment deliberately avoided residue C43 and the possibility of interfering with the correct pairing of the 17 disulphide bonds in HSA. Finally, Q33 has been reported to be transglutaminated to fibrin or fibrin surrogates, albeit at a 10-fold reduced rate compared to Q14 , and it was postulated that its inclusion might increase the reactivity of the chimeric proteins. We had also previously expressed not only small proteins such as hirudin variant 3 (66 amino acids) and barbourin (73 amino acids), as well as smaller barbourin- or RGD-containing derivative peptides as N-terminal fusions to rabbit albumin in the Pichia pastoris system employed in this study, without encountering either inefficiency or instability of expression [15, 16, 18].
In spite of this rational design and previous examples of the soundness of the strategy, the two larger α2AP-HSA chimerae were inefficiently expressed in Pichia pastoris. While the extreme nature of this expression problem prevented characterization of α2AP(13-109)-HSA, α2AP(13-73)-HSA was purified and shown to have been proteolyzed at the L69-V70 bond. While we cannot rule out the possibility that this cleavage occurred during the purification process, in spite of the presence of serine protease inhibitors in all solutions, the diminished yield and apparent lack of full-length products in conditioned media samples suggests intracellular proteolysis. In contrast, at least some of α2AP(13-42)-HSA protein was produced in full-length form. In two of the three N-terminally truncated chimeric proteins also detected in the purified preparation by N-terminal amino acid sequencing, Q33 was also present and located closer to the N-terminus than in its natural setting, increasing its potential to contribute as a substrate. For these reasons we continued to investigate the α2AP(13-42)-HSA, while realising that its functional concentration was probably being underestimated in our assays.
We found that the α2AP(13-42)-HSA fusion protein was readily transglutaminated by fXIIIa to both artificial lysine donors like BPA and dansyl cadaverine, and to the natural lysine donor fibrinogen. While the rates of these reactions were noticeably less rapid than that of the fusion protein's natural counterpart, α2AP, it was clearly a more effective substrate than its synthetic peptide counterpart, α2AP(13-42). This finding was shown through the lower concentrations of fusion protein required to compete α2AP cross-linking under at least some conditions.
The specific mechanism by which presentation of α2AP(13-42) on the N-terminus of a large protein such as albumin, rather than in untethered free peptide form, would increase its reactivity is not clear. Sugimura et al. also reported productive transfer of peptide sequences as fXIIIa substrates to the N-terminus of another protein, in this instance glutathione sulfotransferase . The requirements for efficient cross-linking of sequences by fXIIIa to lysine donors are not well understood, although the suggestion that the reactive glutamine should ideally be positioned in a highly flexible portion of the substrate protein is consistent with our findings [39, 41]. It is also likely that the peptide substrates fluctuate between many different conformations in solution, only some of which are optimal for interaction with the transglutaminase, whereas the tethered sequences in the fusion protein context could be constrained in a more optimal conformation.
Although previous reports have highlighted the possibility of using chemically or mutationally modified α2AP as antithrombotic agents, by virtue of their ability to reduce α2AP-mediated fibrinolytic resistance [22, 23], albumin fusion proteins such as α2AP(13-42)-HSA, with similar properties, could have significant advantages. α2AP has been reported to be inefficiently secreted from Pichia pastoris yeast , would likely have to be prepared in more expensive mammalian cell culture systems, and has not been investigated clinically or preclinically as a recombinant product in vivo. In contrast, HSA is particularly efficiently expressed in Pichia pastoris yeast, a scalable system ideal for economical large-scale pharmaceutical production, and Pichia-derived HSA has been shown to have equivalent effects to plasma-derived HSA in patients . Recombinant HSA and HSA fusion proteins exhibit the same or similar plasma longevity to plasma-derived HSA [12, 27]. Moreover, there may be immunogenicity advantages to albumin fusion proteins, particularly ones with minimized fusion domains. In a comparison of a 17 amino acid peptide-RSA fusion protein containing the same KGD active antiplatelet sequence found in the 73 amino acid barbourin-RSA fusion protein, the former, but not the latter, elicited antibody formation .
In spite of its biotechnological advantages, α2AP(13-42)-HSA appears less active in reducing α2AP-mediated fibrinolytic resistance in clots than modified α2AP proteins, in that an approximately 10-fold molar excess of fusion protein, as opposed to equimolar amounts of modified α2AP, are required to halve fibrinolytic resistance in plasma clots in vitro [22, 23]. Moreover, α2AP(13-42)-HSA demonstrated less ability to become clot-associated in an animal model of thrombosis employed in this study, than native α2AP. In this regard, α2AP(13-42)-HSA can be viewed as a prototype fusion protein, one that could likely readily be improved in several ways: by mutating residues prone to proteolysis, such as K24 and K37; or by substituting the α2AP-derived N-terminal extension with more readily transglutaminated peptides such as those identified by phage display . Such an optimized modified HSA could prove therapeutically useful as an antithrombotic agent, or as an adjunctive drug used in association with thrombolytic therapy.
An α2AP(13-42)-HSA fusion protein was secreted more efficiently by transformed Pichia pastoris yeast than chimerae containing longer stretches of α2AP. Fusion of the α2AP motif changed HSA into a substrate for transglutamination by fXIIIa. The order of reactivity with chemical and physiological lysine donors was α2AP > α2AP(13-42)-HSA > α2AP(13-42) synthetic peptide. The fusion protein reduced α2AP-mediated fibrinolytic resistance when present at 14-fold molar excess in plasma clots, but was unable to localize to clots as effectively as α2AP in rabbits in vivo. The fusion protein could be the prototype of a well tolerated, long lasting protein drug that counters thrombosis in patients
The authors gratefully acknowledge Dr. Ed Pryzdial, Canadian Blood Services and University of British Columbia Centre for Blood Research, for his generous sharing of both recombinant tPA (originally purchased from Genentech) and his expertise in fibrinolysis. This study was made possible by a Grant-In-Aid from the Heart and Stroke Foundation of Ontario (award number T5827) to WPS.
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