Zinc(II) Chelate Binds to Fibrinogen and Its αC Region

Fibrinogen is the immediate precursor of fibrin blood clots. When fibrinogen is cleaved by thrombin, it assembles into fibrin polymers through intermolecular interactions where calcium and zinc ions may both be involved. Fibrinogen’s αC domains play a major role in fibrin polymer assembly, and they contain a Zn 2+ binding sites based on peptide studies. However, Zn 2+ binding to natural αC and fibrin (ogen) remains to be demonstrated. The objective of this study was to directly demonstrate Zn 2+ interactions with purified fibrinogen and its αC region. Both human and bovine αC regions were investigated due to the significant differences in their protein sequences. To detect Zn 2+ binding, chelated Zn 2+ in the form of Zn 2+ nitrilotriacetic acid linked to alkaline phosphatase tracer (ZnTAP) was prepared and used in ELISA and Western blot style methods. Gel electrophoresis was used to monitor changes to fibrinogen after reaction with plasmin. Human and bovine αC regions were obtained by limited plasmin digests of fibrinogen and purified by Con-A and Zn-immobilized metal ion chromatography. The results showed ZnTAP binding to fibrinogen in solution and inhibition of binding to fibrinogen coated microplates by ELISA. Inhibition concentrations (IC50) of 0.58 μM for human fibrinogen and 0.26 μM for bovine fibrinogen were determined. Blot probes revealed ZnTAP binding to fibrinogen with disulfide bonds intact and to the Aα and Bβ chains with disulfide bonds reduced. Reduced Aα chain reaction was less than that of intact fibrinogen and the reduced Bβ chain was only weakly reactive. With plasmin treatment, ZnTAP binding decreased by ELISA in parallel with the degradation of αC region by gel electrophoresis analysis. Purified αC bound ZnTAP in ELISA and on Western blots. In this study we demonstrated that Zn 2+ binds to fibrinogen and its Aα and Bβ chains, and to the αC region of the Aα chain. The results support a role for protein conformation in Zn 2+ binding and demonstrate the utility of the ZnTAP complex with tracer for fibrinogen binding interactions.


Introduction
Fibrinogen functions in hemostasis as the precursor to fibrin blood clots. It is an elongated molecule with two sets of three protein chains (Aα, Bβ and γ) 2 [1]. The six chains are arranged head-to-head near their amino termini forming a central E region [2]. From E the two sets of chains extend in opposing directions, leading to two D regions [3,4]. The Aα chains split away from D regions as a connector sequence leading to the αC domains.
Fibrinogen is converted to fibrin monomers as thrombin cleaves peptides from the Aα and Bβ chains. End-to-end and staggered lateral association of fibrin monomers generate fibrin polymers through interactions among D and E domains [5,6]. The αC domains also are released from their intramolecular docking sites with thrombin cleavage of Aα and Bβ chains, and they form intermolecular associations with other αC domains during polymer assembly. The transglutaminase, factor XIII, catalyzes covalent cross linking between γ chains and among αC domains to strengthen and stabilize the fibrin clot [5].
Fibrin clots are broken down by plasmin. In-vitro studies on fibrinogen define a characteristic set of early plasmin cleavages whereby the αC region of the Aα chain is rapidly released and degraded before other observable cleavages (by SDS-PAGE) to Bβ and γ chains take place [2,7]. Fibrinogen des-αC, known as fragment X, degrades more slowly by plasmin cleavage between the D and E regions. Fragments X, D and E contain most of the mass of fibrinogen Bβ and γ chains and a small part of the Aα chain [8]. These fragments are cleaved further into smaller fragments by plasmin.
In vitro studies demonstrated that αC participates in intermolecular associations of fibrin promoted by calcium and zinc ions either separately or in combination, and at separate metal binding sites [9,10]. A source of the Zn 2+ is from platelets that aggregate on injured tissue where they release a localized bolus of Zn 2+ for fibrin assembly [11]. Indirect evidence for the role αC in fibrin assembly comes from fragment X which forms polymers with different physical properties than occur with intact fibrin [10,12]. Zn 2+ binding to αC from purified fibrinogen has yet to be characterized, however a peptide construct containing two histidine residues of human αC (H563-H564) binds to Zn 2+ and links to heparin [13]. It is not known whether or not this site also functions in polymerization of fibrin. Homologs of this site vary in amino acid sequence across species thus complicating the prospects of finding common sites of Zn 2+ involvement in fibrin function [13]. For example, bovine αC has a 21 amino acid gap at the human Zn 2+ binding site, as presented in Figure 1. However, bovine αC has another location His495-His496 which could serve a similar binding function ( Figure 1). Other differences between human and bovine αC are additional sequence gaps in bovine αC and its considerably smaller size [14].
Immobilized metal ion chromatography (IMAC) methods use chelated metal ions to bind His residues on proteins and is used for protein purification [15]. This method has been demonstrated for fibrinogen as well as fragment D [9,16]. αC ought to bind IMAC resins if there are exposed binding site(s) of sufficient affinity. ZnTAP is similar to Zn-IMAC resin with respect to the NTA-chelated Zn 2+ format, while differing in having alkaline phosphatase instead of the chromatography resin. On the assumption that ZnTAP would bind to Zn 2+ sites, the present study tested fibrinogen and αC by ELISA methods and by Western blot probes. αC was then purified by Zn-IMAC and used to directly demonstrate zinc ion binding to this region of fibrinogen.
The goals of this study were to 1) demonstrate Zn 2+ binding with ZnTAP to fibrinogen, and to the αC region prepared from natural fibrinogen, and 2) compare the binding of ZnTAP by human and bovine αC in view of their differences in αC.

Reagent Sources
Millipore corporation was the source of human fibrinogen and some of the bovine fibrinogen. Bovine plasma was from PelFreez Biologicals. Plasmin was from Haematologic Technologies, Inc. Ni-His-60 resin was from Clontech Laboratories. Precast stain-free gels and other electrophoresis reagents were from Bio-Rad. Con-A Sepharose was from Sigmaaldrich. Nickel-NTA-alkaline phosphatase and Western blot substrate were obtained from KPL. Bovine fibrinogen was prepared by salt precipitation and IMAC as described [16]. Antibody sc-398806 (C-7) to human αC was from Santa Cruz Biotechnology.

Preparation of ZnTAP
ZnTAP was derived from Ni-NTA-alkaline phosphatase. One-tenth ml of Ni-NTA-alkaline phosphatase was diluted to 2 ml with TBS, 0.1 mg/ml bovine serum albumin, pH 7.4. Then 0.1 ml of 0.1 M EDTA was added and the mixture was dialyzed into 3 changes of TBS. The sample was next dialyzed into 2 changes of the same buffer containing 10 mM zinc sulfate, 5 mM magnesium chloride at pH 6.0 in order to load NTA with zinc and alkaline phosphatase with magnesium. The product was finally dialyzed into several changes of TBS, pH 7.4. Control samples without zinc were prepared in parallel.
Complexometric titration of ZnTAP was done with EDTA to demonstrate specificity of the reagent and to measure zinc content. ZnTAP at a concentration giving greater than 75% of maximum response to fibrinogen by ELISA was incubated with varying concentrations of EDTA. The mixtures were applied to fibrinogen coated microplates and then processed and tested for alkaline phosphatase hydrolysis of ρNPP. The concentration of EDTA that completely inhibited ZnTAP was recorded.
Zn-IMAC resin was prepared from Ni-IMAC resin by metal ion exchange according to the Clontech User Manual.

Plasmin Digestion of Fibrinogen
Fibrinogen was dialyzed into TBS, pH 7.4 and plasmin was added to give a mass ratio of plasmin to fibrinogen of 1:1000 for electrophoresis and ELISA inhibition studies. A 1:15,000 dilution of 0.42 mg/ml of plasmin was used for experiments where fibrinogen was first coated onto microplates and the reacted with plasmin. Plasmin reactions were terminated by addition of PMSF to 1 mM, followed by addition of 6-aminohexanoic acid (εACA) in TBS to 0.1 M. For SDS-PAGE, these samples were mixed with 2 volumes of SDS-sample buffer and heated to 60°C for 10 minutes. Some reaction samples were heated to 75° for 10 minutes to precipitate residual fibrinogen and fragment X [17]. The precipitate and soluble αC fractions were analyzed by SDS-PAGE. The soluble fraction was also used for the purification of αC.

ELISA
For direct ELISA, microplates were coated with 5mg/ml fibrinogen or αC in PBS, blocked with 0.1 mg/ml of bovine serum albumin, and then incubation with ZnTAP. Wells were then washed with TBST, incubated with 1 mg/ml ρNPP in 0.1 M Tris-HCl, 5 mM MgCl 2 , pH 9.3, and monitored at 415 nm. A concentration of ZnTAP of greater than 75% of maximum response in the direct assay was used for inhibition assays. Direct ELISAs using plasmin reacted fibrinogen at E:S of 1:000 was performed and terminated with PMSF and εACA over a time course. In other experiments, microplates were first coated with fibrinogen, then blocked and incubated with plasmin over a time sequence. At the desired incubation times, reactions were stopped with PMSF and εACA and then processed as described above.
For inhibition ELISA using fibrinogen, a constant pre-determined concentration of ZnTAP was incubated for 60 minutes with varying concentrations of fibrinogen and the mixtures were transferred to microplates that had been coated with 5 µg/ml of fibrinogen, blocked with serum albumin, and processed as above to estimate IC50 [18]. After 60 minutes incubation time the wells were emptied, washed and ρNPP was added. Absorbance of the NPP product was monitored at 415 nm. Plasmin-digested fibrinogen was similarly tested for inhibition of ZnTAP on fibrinogen coated microplates.

αC Preparation
αC from human and bovine fibrinogen was purified by Con-A chromatography [19] and Zn-IMAC. Typically, 20 mg of fibrinogen in 6 ml TBS, pH 7.4 was digested for 10 minutes with 2 ug of plasmin. Digests were terminated with PMSF and εACA and heated to 75° for 10 minutes [17]. The heat-treated products were spun for 5 minutes at 12,000 x g, and the supernatant containing αC was re-spun. The soluble fraction was applied to 2 ml columns of Con-A in TBS and unbound protein containing αC was further purified on Zn-IMAC columns. NaCl (1 M) in TBS, pH 7.4 was added to bring the concentration of NaCl to 0.3 M and the samples were applied to 2 ml columns of Zn-IMAC. Bound αC was released in the same buffer with added 0.005 M and then 0.1 M imidazole.

Characterization of the ZnTAP Reaction with Fibrinogen
ZnTAP was prepared for the purpose of directly demonstrating Zn 2+ binding to fibrinogen and to the αC region of fibrinogen. To prepare ZnTAP, EDTA was added to Ni-NTA-alkaline phosphatase to remove Ni 2+ . Then Zn 2+ was added back to NTA through dialysis changes. ZnTAP bound to fibrinogen by ELISA while control reagent lacking Zn 2+ did not bind. Alkaline phosphatase in the control hydrolyzed ρNPP, indicating that Zn 2+ had been exchanged for zinc ions in the functioning reagent. Complexometric titration of ZnTAP with EDTA demonstrated complete displacement of Zn 2+ . Characterization of fibrinogen and αC by SDS-PAGE and Western blot probes is shown in Figure 3. The high molecular weight bands of bovine and human fibrinogen are seen in Figure 3A, lanes 1 and 2. Upon reduction of disulfide bonds, bovine Aα, Bβ and γ chain molecular weights were 60 kD, 52 kD and 47 kD, lane 3. Human Aα, Bβ and γ chain sizes were 66 kD, 53 kD and 48 kD, lane 4. Figure 3, Panels B-D represents ZnTAP probes of Western blots of human fibrinogen with disulfide bonds intact (Panel B) and with disulfide bonds reduced (Panel C). Panel D represents αC from the heat-soluble fraction of plasmin digested human fibrinogen. The darker background in C resulted from longer exposure to the substrate compared to B and D. Blots of intact fibrinogen develop quickly, while reduced chains are less reactive based on slower blot development times. For intact fibrinogen the reaction took about 1 minute, and about 5 minutes was typically required in order to visualize reduced Aα chain. A signal from the Bβ chain was consistently detected at 10-minute incubation time. Comparable results were observed with fibrinogen of human and bovine origin. Fibrinogen γ chains were generally not observed to produce a signal except occasionally trace reactions were detected with prolonged incubation. Trace reactions at the γ position of the blot were attributed to partial degradation of the Bβ chain that migrated close to the γ chain. The γ chain was therefore considered to be negative.

Figure 3. A) SDS-PAGE of intact bovine and human fibrinogen, 1-2, and reduced 3,4. B-C) Intact and reduced human fibrinogen blots probed with
ZnTAP, and Panel D, probe of αC. Panels are from separate gels.

Reaction of ZnTAP with Fibrinogen Fragments
Plasmin reactions with fibrinogen generated the characteristic early fragments, αC and fibrinogen-des αC (fragment X). Plasmin digests of fibrinogen at 2 mg/ml were obtained by incubation with 2 µg/ml of plasmin and samples taken at various reaction times were terminated with plasmin inhibitors. Reactions were heated at 75° for 10 minutes to precipitate residual fibrinogen and fragment X, leaving αC in solution. The heat-insoluble samples taken over 32 minutes of plasmin treatment were analyzed by SDS-PAGE as shown in Figure 4A insert. SDS-PAGE of fibrinogen with disulfide bonds reduced is the best way demonstrate changes in the separate chains.
Scans of the Coomassie Blue stained gel at time 0 (Panel A), and at 16 minutes in Panel B illustrate rapid degradation of the Aα chain while changes to Bβ occur more slowly and γ chain appears slightly elevated by 16 minutes. The results by SDS-PAGE are consistent with the known pattern for fibrinogen reaction with low levels of plasmin where the Aα chain is degraded before Bβ and γ chains degradation is detected [7,8].
The αC bands of the heat-soluble plasmin digests are closely spaced bands from 37-39 kDa for human, and 31-34 kDa for bovine αC.  Plasmin digests of bovine fibrinogen were first analyzed by direct ELISA. Over a 64-minute reaction time, the reactivity with ZnTAP decreased rapidly ( Figure 6). The decrease in the ELISA values is similar to the decrease over time of αC by SDS-PAGE suggesting that αC continues to bind ZnTAP after it is cleaved from Aα chain. To confirm the results by other approaches, fibrinogen was first bound to microplates and then digested with plasmin over a time course and analyzed by ELISA. The results were qualitatively the same when fibrinogen was digested with plasmin before being coated to the microplate as when fibrinogen was first applied to the microplate and then digested with plasmin (not shown).
An inhibition experiment was next performed on a plasmin digest whereby samples were inactivated over 60 minutes and then mixed with ZnTAP and applied to fibrinogen coated microplates (Figure 7). Control sample, C, represents ZnTAP in BSA without fibrinogen. The other samples contained ZnTAP mixed with 250 ug/ml of bovine fibrinogen digests. At one minute, activity was inhibited by 82%, having 18% signal compared to the control, and after 60 minutes. reaction, just 12% inhibition capacity remained (the signal is 88% of control). Numbers above the bars in Figure 7 indicate percent of control value for each sample. The A415 of the control was 1.4. ELISA results collectively suggest that the decrease in ZnTAP binding to plasmin-reacted fibrinogen resulted from plasmin degradation of the ZnTAP binding entity.

Purification and Confirmation of αC
αC was isolated in order to confirm ZnTAP binding to the purified protein. Fibrinogen was digested with plasmin for 10 minutes and the reaction was terminated with PMSF and εACA. The sample was heated to precipitate non-αC fragments and the soluble fraction was passed through a Con-A column to remove residual carbohydrate-containing material. αC was present in the Con-A unbound fraction, and this fraction was applied to Zn-IMAC columns as presented in Figure 8. Most low molecular weight peptides failed to bind to the column, while αC bound and then was released when imidazole was added to the column buffer. SDS-PAGE of the peak samples shown in Figure 8 inset reveal that αC is present in column fractions 2-4. Both human and bovine αC were successfully prepared by Zn-IMAC using identical conditions. ZnTAP reacted with both purified human and bovine αC by ELISA and blotting methods (not shown).
Monoclonal antibody, C-7, to human αC was used to confirm the identity of Zn-IMAC purified αC.
ELISA values for purified human αC were 13 x to 19 x background and were 80% of the values obtained with a human fibrinogen control. ELISA values for bovine αC were 2.5-times background with C-7 antibody. Western blots probed with C-7 showed reactivity with human αC and trace reaction with bovine αC.

Discussion
It was of interest in this study to confirm Zn 2+ binding to αC prepared from isolated fibrinogen, and to enhance the understanding of the roles of Zn 2+ in coagulation [11]. For this purpose, a zinc chelate tag (ZnTAP) was prepared and used to demonstrate Zn 2+ binding to fibrinogen and its αC region.
By probing Western blots and by ELISA methods, ZnTAP was shown to bind fibrinogen and αC. In addition, Zn-IMAC resin bound αC and was used as the final step in its purification. αC was released from Zn-IMAC resin with imidazole, indicating that histidine is likely to be involved in interactions between αC and Zn 2+ . ZnTAP binding by ELISA decreased in parallel with the release and further degradation of the αC region from fibrinogen by plasmin. The collective observations indicate the αC region binds Zn 2+ . The location of the binding site(s) within the protein sequence is not identified in this study but the separate His-His sequences in the human and bovine proteins seem likely to be involved.
Other studies by indirect methods previously concluded that αC interacts with Zn 2+ [12]. Additionally, synthetic peptides corresponding to a sequence from human αC demonstrated that His545-His546 is a site where Zn 2+ links heparin to αC [13]. If ZnTAP binds to the human His545-His546 site, it seems reasonable that it also binds to the His493-His494 site in bovine αC. Besides linking heparin, Zn 2+ promotes fibrin polymer formation [5,10]. However further studies are needed to reveal the interactions that take place between Zn 2+ and fibrin as blood clots.
From investigations using recombinant αC protein, its interactions with the E region at the N-terminal of the Bβ chain are defined [5] This interaction is disrupted upon cleavage of fibrinopeptide B, and then αC associates intermolecularly with αC regions of adjacent fibrin molecules. The switch allows for aggregation of fibrin protofibrils and fibrin assembly. While these interactions can occur in the absence of metal ions it is of interest to examine the roles of zinc and calcium ions in the process since fibrin polymer formation is accelerated by the ions [20]. In addition, possible roles for metal ions in αC functions such as cell adhesion, wound healing and fibrinolysis [21] are of interest. Moreover, mutations in αC that result in clotting disorders and amyloid deposition in some cases, could be investigated further [22].
Elucidation of the role of metal ions in fibrin formation will permit a better understanding of the mechanisms involved in abnormal states.
Although bovine and human αC differ substantially in their amino acid sequences and size, their reactions with ZnTAP were essentially identical in this study. However, the picture becomes less intuitive when considering sequence homology among species. For example, while most primate species are homologous to human Aα chain at the site represented by His545-His546, chimpanzee and orangutan each lack one of these His residues [13]. Adjacent His residues aren't a requirement for Zn 2+ binding, as closely spaced His residues can function in this capacity [14]. Therefore, closely spaced His in the αC regions of the latter two species could involve Zn 2+ binding.
In contrast to primates, just 3 of 15 mammals contain His-His sites within their αC regions [13]. Highly conserved His residues that exist in αC and are shared by both primates and other mammals are potential Zn 2+ interaction sites. Further laboratory and predictive modeling studies are necessary to discover the modes and sites of Zn 2+ binding to αC and their functions [23]. The Bβ chain reacted with ZnTAP and it lacks a His-His sequence while γ chain has a His-His site but is unreactive by Western blot analysis. These observations suggest that the conformation of binding sites is significant in metal ion binding.
Although ZnTAP is bulky compared to Zn 2+ , the reagent worked well judging by its affinity for fibrinogen, and this is consistent with the fact that αC could be purified by Zn-IMAC. The IC50 values for ZnTAP were similar for human (0.58 µM) and bovine (0.26 µM) fibrinogen and should be close to the Kd for this interaction. Values of Kd = 0.67 µM and 1.0 µM were reported using FluoZin-1 with human fibrinogen [10]. In studies where ZnSO 4 or ZnCl 2 , were used, different values of Kd = 18 µM [5] and Kd = 9.4 µM were reported [9]. Zinc salts produce zinc oxide and hydroxides that may generate complicating side reactions which could contribute to the differences in Kd measured by the different methods [24,25].

Conclusions
1. Zn 2+ binding to intact fibrinogen, reduced Aα and Bβ chains of fibrinogen, and αC region of the Aα chain was directly demonstrated with the chelate, ZnTAP.
2. The αC region of fibrinogen accounts for most of the ZnTAP reaction with fibrinogen.
3. ZnTAP binding to human and bovine fibrinogen and αC was comparable in spite of their amino acid sequence differences.