©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Comparative Structural and Functional Features of the Human Fibrinogen C Domain and the Isolated C Fragment
CHARACTERIZATION USING MONOCLONAL ANTIBODIES TO DEFINED COOH-TERMINAL Aalpha CHAIN REGIONS (*)

(Received for publication, September 6, 1995; and in revised form, November 14, 1995)

Sergei Rudchenko Ilya Trakht Joan H. Sobel (§)

From the Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The alphaC domain of fibrinogen (Aalpha-(220-610)) plays a central role in maintaining hemostasis by serving as a substrate for factor XIII(a) and plasmin. Monoclonal antibodies that recognize eight distinct epitopes within the COOH-terminal two-thirds of the Aalpha chain were employed as structural probes to: 1) isolate the human alphaC domain, 2) compare the topography of the eight epitopes within the alphaC domain of intact fibrinogen and in purified alphaC fragments, and 3) explore the degree to which the alphaC domain's role as a factor XIII(a) substrate in intact fibrinogen is preserved within the structure of isolated alphaC fragments. Five antibodies were raised against small, synthetic peptide immunogens (Aalpha-(220-230), Aalpha-(425-442), Aalpha-(487-498), and Aalpha-(603-610)), and three were generated against larger cyanogen bromide (A)alpha chain derivatives with each epitope subsequently localized to discrete Aalpha chain sequences (Aalpha-(259-276), Aalpha-(529-539), and Aalpha-(563-578)). Human alphaC preparations were isolated from mild plasmin digests of fibrinogen by successive chromatography on concanavalin A-Sepharose, anti-Aalpha-(425-442)-Sepharose, and Superdex-75 fast protein liquid chromatography. Immunochemical characterization indicated that the NH(2)-terminal residue of alphaC fragments was either Aalpha-220 or Aalpha-231 and that, although the extreme COOH-terminal region, Aalpha-(603-610), was absent, all molecules were intact at least through Aalpha-(563-578). Solution phase competitive assays indicated that the release of the alphaC domain from intact fibrinogen was associated with several conformational changes, e.g. in the vicinity of Aalpha-(220-230), Aalpha-(259-276), Aalpha-(487-498), and Aalpha-(529-539), but that the relative accessibility of other localized structures remained unchanged, e.g. Aalpha-(425-442) and Aalpha-(563-578). Immunoblotting analysis of alphaC cross-linking in vitro revealed that isolated alphaC fragments could serve as a substrate for factor XIII(a). Immunoblotting studies of the Aalpha chain proteolysis that occurs during thrombolytic therapy indicated that alphaC fragments, similar in size and epitope content to those isolated from purified fibrinogen, were released in vivo early during fibrinolytic system activation. The collective findings provide new information about the fine structure of the fibrinogen alphaC domain and its functional implications and also draw attention to the as yet unexplored role of alphaC fragments in the pathophysiology of thrombosis and hemostasis.


INTRODUCTION

The past decade has witnessed a growing interest in the structure of the fibrinogen alphaC domain, i.e. the COOH-terminal two-thirds of the Aalpha chain that extends from the coiled-coil portion of each half of the dimeric fibrinogen molecule. Elucidation of this region's structural features is of considerable interest given its multifunctional role in maintaining hemostasis. Factor XIII(a) cross-linking sites included within the alphaC domain are responsible for the covalent bonds created between fibrin alpha chains and between alpha chains and alpha(2)PI (^1)that lead to the formation of a highly cross-linked alpha polymer network(1, 2, 3, 4, 5, 6) . The resulting ``stabilized'' fibrin is characterized by an enhanced resistance to lysis by plasmin, the hallmark of a physiologically effective thrombus(7, 8) . A number of plasmin-susceptible cleavage sites have been identified within the alphaC domain of fibrinogen(9) . Presumably, these same sites are responsible for the initial cleavages that eventually lead to disruption of the fibrin alpha polymer network, a process considered to be a prerequisite for efficient particulate clot lysis(10) . The fibrinogen Aalpha chain contains two RGD sites, and one of these, located within the alphaC domain, has recently been implicated in fibrinogen's binding to the endothelial cell vitronectin receptor (11) ; this suggests an additional role for the alphaC domain as a mediator of fibrinogen-vessel wall interaction.

Historically, the COOH-terminal two-thirds of the Aalpha chain (referred to here as alphaC) has been viewed as a ``free-floating'' appendage with little organized structure, based on primary structure considerations as well as observations from early biochemical and immunoelectron microscopy studies(12, 13, 14) . This concept has been challenged in recent years in light of newer electron microscopy and microcalorimetry findings which suggest that the alphaC portions of fibrinogen are, in fact, highly organized and interact to form a fourth globular domain that is centrally positioned over the E domain of the intact dimeric molecule(15, 16) . Additional evidence indicates that these intramolecular alphaC contacts are disrupted during fibrin formation, thus facilitating the intermolecular associations that must be in place before alpha chain cross-linking can occur(17) .

Apart from these structural studies which have provided important information about the general architecture of the alphaC domain as it exists in intact fibrin(ogen), a second area of related interest concerns the biochemistry of the isolated alphaC region, i.e. the large fragment representing nearly the entire COOH-terminal two-thirds of the Aalpha chain, that is released during the initial stages of plasmin cleavage. Structure-function studies of human alphaC fragments have been hampered to date because of the difficulties in obtaining homogeneous preparations due to the Aalpha chain's intrinsic COOH-terminal heterogeneity and its extreme lability during in vitro handling(18) . However, electron microscopy studies of isolated alphaC fragments from bovine fibrinogen (which are less susceptible to proteolytic degradation than their human counterpart) demonstrate that the structure of these derivatives can support the formation of linear arrays of alphaC polymers and can also promote the interaction of alphaC with intact fibrin molecules to inhibit their polymerization(17, 19) . These findings are consistent with earlier biochemical and immunologic observations suggesting the existence of a polymerization site within the COOH-terminal portion of the Aalpha chain (20, 21, 22) .

The fact that isolated alphaC fragments retain their parent protein's capacity for polymerization raises the possibility that other functionalities unique to the fibrin(ogen) alphaC domain may be preserved as well. To date, little information is available regarding the extent to which conformational features of isolated alphaC fragments support the various functions predicted for these derivatives based on their primary structure alone, e.g. factor XIII(a) cross-linking, plasmin susceptibility, and endothelial cell interaction. This is of particular significance when one considers that alphaC fragments are expected to be released as natural by-products of fibrinolytic system activation and, therefore, their relative prothrombotic or anticoagulant activity in this milieu may have important clinical ramifications.

The studies in this report focus on the isolation and structural characterization of the human alphaC domain and consider the extent to which the functional capacity for alpha chain cross-linking is preserved within the native conformation of the isolated fragment. The studies feature an immunochemical approach, employing a unique panel of monoclonal antibodies to defined COOH-terminal Aalpha chain regions as structural probes for localized sequences within the alphaC domain of intact fibrinogen and within isolated alphaC. The results obtained provide preliminary information about the comparative topography of selected regions within the native structure of the two alphaC forms and demonstrate that conformational features unique to the isolated fragment are compatible with Factor XIII(a) cross-linking. The collective findings, which include observations to indicate that alphaC fragments are released in vivo during fibrinolytic system activation, provide new insights into the structure-function relationships of the fibrinogen alphaC domain and suggest an as yet unexplored role for the isolated fragment in the pathophysiology of hemostasis.


MATERIALS AND METHODS

Monoclonal Antibodies to Defined Aalpha Chain Regions

The development and characterization of mAbs F-102 (anti-Aalpha-(563-578)) and F-103 (anti-Aalpha-(259-276)), raised against a purified CNBr derivative of the alpha polymer component of cross-linked fibrin, have been previously described(23, 24) . mAb 5A2 (anti-Aalpha-529-539)) was produced following immunization with a preparation of partially purified CNBr fibrin derivatives and selected based on its preferential binding to fibrin, as distinct from fibrinogen, in a direct-binding screening ELISA on antigen-coated wells. The development and characterization of mAb 5A2 will be detailed in a separate report. (^2)Five monoclonal antibodies were raised against small synthetic peptides modeled after sequences within the COOH-terminal two-thirds of the Aalpha chain. Among these, mAb F-48 (anti-Aalpha-(603-610)) was kindly provided by Dr. Gary Matsueda (Bristol-Myers Squibb, Princeton, NJ) for use in these studies. General procedures employed for the isolation and characterization of mAbs F-106 and F-106f (anti-Aalpha-(220-230)), F-105 (anti-Aalpha-(425-442)), and F-104 (anti-Aalpha-(487-498)) are included here in abbreviated form since detailed descriptions of these antibodies and their application in studies of fibrinogenolysis will be described elsewhere. (^3)

Peptide Synthesis

The linear peptides, SQLQKVPPEWKC (Aalpha-(220-230 + C)) and TGKEKVTSGSTTTTRRSC (Aalpha-(425-442)) were synthesized using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) and t-Boc chemistries, respectively, according to programs provided by the instruments' manufacturer (Applied Biosystems, Foster City, CA). The crude peptide preparations were purified by reversed phase high performance liquid chromatography on a preparative C-8 column (2.1 times 25 cm; Rainin; Woburn, MA) using an increasing gradient of acetonitrile (4-60%) in 0.1% trifluoracetic acid/water for elution. Amino acid analysis confirmed the molar composition expected for the purified peptide preparation in each case. The peptide, LDGFRHRHPDEA (Aalpha-(487-498)) was synthesized as a multiple antigenic derivative comprised of eight copies of the desired sequence attached to a seven-membered polylysine core (TAM peptide)(25) . Synthesis was conducted using standard t-Boc chemistry and the resin, [alpha-N-t-Boc-lys(-N-t-Boc)](4)-lys(2)-lys-ala-OCH(2)-PAM (Applied Biosystems, Foster City, CA). The crude peptide preparation was subsequently employed without further purification.

Immunization

The peptides corresponding to Aalpha-(220-230 + C) and Aalpha-(425-442) were coupled to keyhole limpet hemocyanin (KLH) (Pierce, Rockford, IL) via their COOH-terminal cysteine residue using the heterobifunctional cross-linker, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Pierce) as previously detailed(26) . Peptide:carrier ratios of 200-300:1 were typically obtained, based on subtractive compositional data derived from amino acid analysis of the conjugate, carrier, and free peptide. The Aalpha-(487-498) TAM peptide was employed as an immunogen without prior conjugation to KLH. Balb/c mice were immunized with the two linear peptide conjugates using multiple low dose intraperitoneal injections (30 µg/dose) over an approximate 1-month period, as described previously(26) . For the TAM peptide immunization, Balb/c mice were injected weekly (50 µg/dose). Serum titers were determined in direct binding ELISAs on peptide-coated wells (Aalpha-(220-230 + C), 0.13 µg/well; Aalpha-(425-442), 0.015 µg/well) or, in the case of the TAM peptide immunization, on wells coated with a partially purified preparation of Aalpha FDPs (20 pmol/well), prepared as described previously(24) . Comparative binding to fibrinogen on the solid phase (1 µg/well) was determined in parallel. Bound immunoglobulins were detected based on their relative 414 nm absorbance following successive incubations with GAM-IgG-HRP (Kierkegaard & Perry; Gaithersburg, MD), diluted 1:2000, and the HRP substrate, o-phenylenediamine.

Hybridoma Development

Mice whose serum exhibited preferential binding either to Aalpha chain peptides or to intact fibrinogen and mice whose serum appeared to recognize both types of antigen equally were taken for fusion. Hybridoma lines were selected according to standard procedure (27) except that an extract prepared from nonimmune mouse spleens was employed as a growth supplement in place of the fetal calf (or horse) serum traditionally added to hybridoma cultures. Hybridoma lines producing anti-COOH-terminal Aalpha chain immunoglobulins of interest were selected in a direct binding ELISA on antigen coated wells, as described above, and in a secondary screening assay that evaluated antigen recognition in solution phase (see below). Two cell lines that exhibited preferential binding to Aalpha chain peptides, referred to as F-106 (anti-Aalpha-(220-230)) and F-105 (anti-Aalpha-(425-442)), one cell line specific for intact fibrinogen, F-104 (anti-Aalpha-(487-498)), and one cell line that recognized both fibrinogen and peptide, F-106f (anti-Aalpha-(220-230)), were cloned by limiting dilution(28) . The cloned cell lines were expanded in ascites (29) and the respective IgGs purified by affinity chromatography on protein A-Sepharose (Bio-Rad) as recommended by the manufacturer. (mAb F-106f was not purified for these studies and was employed in the form of expanded cloned culture supernatant.)

Solution Phase Competitive ELISAs

Detailed methods for this ELISA format as conducted in our laboratory have been previously described(24, 26) . Briefly, serial dilutions of antigen were mixed with monoclonal antibody, then preincubated for 1.5 h at 37 °C, delivered into blocked, antigen-coated wells, and incubated for an additional 18 h at 4 °C. Bound immunoglobulin was detected at 414-nm absorbance following successive incubations with RAM-Ig-HRP (Dako, Carpenteria, CA), diluted 1:500, and o-phenylenediamine. The ELISAs for mAbs F-106f, F-103, F-104, 5A2, F-102, and F-48 were conducted on fibrinogen-coated wells (1 µg/well), using highly purified material prepared from Kabi fibrinogen (Pharmacia-Hepar, Franklin, OH) as previously detailed(30) . IgG concentrations in the solution phase ranged from 0.09-0.14 nM, depending on the antibody. Purified fibrinogen was employed as the assay standard for these six ELISAs. The ELISAs for F-106 and F-105 were conducted on peptide-coated wells (Aalpha-(220-230 + C), 0.13 µg/well; Aalpha-(425-442), 0.015 µg/well) at IgG concentrations of 0.17 nM and 0.27 nM, respectively. The appropriate peptide was employed as the assay standard for these two ELISAs. The percent of antibody binding to the solid phase in the presence and absence of solution phase competitor (percent bound) was calculated for each data point with the ELISA software, Automate (Flow-ICN; Huntsville, AL) and plotted as the logit-transformed percent bound versus dose using the scientific graphing software, SigmaPlot (Jandel Scientific, Sausalito, CA). The dose of each purified competitor was determined by amino acid analysis and expressed as molar concentration of Aalpha chain equivalents. Additional details appear in the legends to Fig. 4and Table 2.


Figure 4: Relative epitope expression in the intact fibrinogen alphaC domain and in isolated alphaC determined by ELISA. Purified alphaC (solid symbols) and fibrinogen (open symbols) were employed as competitors in solution phase competitive ELISAs developed for mAbs F-102 (anti-Aalpha-(563-578); Panel A), F-105 (anti-Aalpha-(425-442); Panel B), F-103 (anti-Aalpha-(259-276); Panel C), and F-106 (anti-Aalpha-(220-230); Panel D), as detailed under ``Materials and Methods.'' The assay standards for the F-105 and F-106 ELISAs were the appropriate synthetic peptide; 50% bound = 0.90 ± 0.21 nM and 0.24 ± 0.06 nM, respectively (mean, ±S.D.; n = 3). Fibrinogen was the assay standard for the F-102 and F-103 ELISAs (see Table 2for additional details). Immunoreactivity is plotted as the logit-transformed percent bound versus dose, where dose is expressed as the molar concentration of competitor in the solution phase determined from amino acid analysis of the purified standard preparation. The molar concentration of fibrinogen, expressed as Aalpha chain equivalents, was calculated assuming a molecular mass of 340 kDa and 2 mol of Aalpha chain/mol of fibrinogen.





Isolation and Purification of alphaC

Plasmin Digestion

In a typical procedure, approximately 40 mg of purified fibrinogen (in 10 ml of TBS containing 1 mM CaCl(2); pH 7.6) were digested with plasmin (0.0015 unit/mg fibrinogen) (Pharmacia-Hepar) for 6 h at room temperature and proteolysis interrupted by addition of phenylmethylsulfonyl fluoride to 0.5 mM, final.

ConA Affinity Chromatography

To remove residual fibrinogen and its glycolysated derivatives, the digest was incubated with ConA Sepharose (Pharmacia Biotech Inc.) in TBS (3 mg of fibrinogen/ml of ConA) for 45 min at room temperature. Nonbound components, including alphaC, were collected in the flow-through for further purification.

F-105 Immunoaffinity Chromatography

Preliminary studies identified a major group of Aalpha FDPs emerging in close proximity to alphaC during size-exclusion chromatography. In order to minimize contamination by these fragments in the final alphaC preparations, an immunoaffinity step was introduced that took advantage of the observation that these Aalpha FDPs, but not alphaC, exhibited anti-Aalpha-(425-442) immunoreactivity in the F-105 ELISA. The ConA flow-through was incubated with F-105-Sepharose in TBS (1.2 mg of IgG/ml of gel), prepared according to previously described methods (31) , for 18 h at 4 °C. Nonbound components, including alphaC, smaller Aalpha FDPs, and other carbohydrate-free fibrinogenolytic derivatives, were collected in the flow-through for final purification after concentration to 0.125 volume by either vacuum centrifugation or Amicon filtration using a 30-kDa cutoff membrane (Amicon, Beverly, MA).

FPLC

Approximately 30% of the total F-105 flow-through material was applied per run in a 0.5-ml volume to a Superdex-75 column (1.6 times 62 cm; Pharmacia) equilibrated in TBS. One-ml fractions were collected at a flow rate of 1 ml/min and absorbance monitored at 280 nm. A mixture of standard molecular weight proteins was chromatographed for reference (Bio-Rad). Fractions of interest, based on their elution position and SDS-PAGE profiles (see below), were pooled and rechromatographed; 0.5-ml fractions were collected at a flow rate of 0.5 ml/min. Those containing alphaC fragment(s) free of contaminating Aalpha FDPs based on SDS-PAGE analysis were pooled for use in subsequent characterization studies.

alphaC Cross-linking

Purified fibrinogen or alphaC (2.5 nmol/ml in TBS) was incubated for 3 h at 37 °C together with preactivated plasma factor XIII(a), purified as described elsewhere(32) , human thrombin (Sigma), and 10 mM CaCl(2); 9 µg of factor XIII(a) and 0.042 unit of thrombin/ml were present in the fibrinogen incubations, and four times these concentrations were employed for alphaC. Cross-linking was also conducted in the presence of the recently reported factor XIII(a) donor lysine peptide probe, NQEQVSPLTLLK, included here at a 40 times molar excess over fibrinogen Aalpha chains(26) . Reduced SDS-PAGE sample buffer (see below) containing 9 M urea was added at the end of all incubations to inhibit further cross-linking activity. Cross-linking was visualized by immunoblotting, employing the anti-alpha(2)PI-(1-12) peptide antibody, mAb AP-102(26) , and the anti-Aalpha chain antibody, mAb F-103 (anti-Aalpha-(259-276)) as primary immunoglobulins. Additional details appear below and in the legend to Fig. 6.


Figure 6: Immunovisualization of alphaC release in vivo during thrombolytic therapy. Plasmas (5 µl) from two patients undergoing thrombolytic therapy with rt-PA and streptokinase were applied to the lanes of 12.5% gels, and electrophoresis was conducted under nonreducing conditions. Duplicate nitrocellulose transfers were incubated with anti-Aalpha-(563-578) (mAb F-102) and anti-Aalpha-(259-276) (mAb F-103), employing expanded cloned culture supernatants diluted 1:20 and 1:100, respectively. Immunoreactive components were visualized as described in the legend to Fig. 3. The arrows identify the migration of intact and partially degraded fibrinogen. The migration of Aalpha FDPs (right) and standard molecular mass markers (left) are indicated for reference (see Fig. 2legend for identification of marker proteins).




Figure 3: Immunologic characterization of purified alphaC by immunoblotting with anti-Aalpha chain mAbs. Ten pmol of purified alphaC were applied to each of seven lanes of a 12.5% gel, and electrophoresis was conducted under nonreducing conditions. Nitrocellulose transfers of each lane were incubated with purified IgGs from the antibody panel and immunoreactive components subsequently visualized using RAM-Ig-HRP as described under ``Materials and Methods.'' The migration of standard molecular markers is indicated at the left for reference (ovalbumin, 43 kDa; carbonic anhydrase, 29 kDa; beta-lactoglobulin, 18.8 kDa; lysozyme, 16.5 kDa; bovine trypsin inhibitor, 6.4 kDa; insulin alpha and beta chains, 3.0 kDa).




Figure 2: FPLC purification of isolated alphaC fragment(s). 1.6 mg of partially purified alphaC obtained following successive adsorption of plasmin-treated fibrinogen on ConA-Sepharose and anti-Aalpha-(425-442)-Sepharose were chromatographed on Superdex-75 as described (see ``Materials and Methods''). The 280-nm absorbance profile of the column effluent is shown; AUFS, absorbance full scale. The elution positions of standard molecular mass markers are indicated at the top for reference (-globulin, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa; and vitamin B-12, 1.35 kDa). SDS-PAGE/Western blotting: approximately 1% of the material in fractions 17-22 was subjected to SDS-PAGE on 12.5% gels under nonreducing conditions. Nitrocellulose transfers were stained for total protein with Amido Black. The migration of standard molecular mass markers is indicated at the right for reference (phosphorylase b, 92.5 kDa; bovine serum albumin, 69 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 21.5 kDa; and lysozyme, 14.3 kDa). Note that the high molecular mass components eluting in fraction 17 (as well as 15 and 16) were barely detectable by Amido Black staining indicating that they comprised a negligible proportion of the total material recovered. Moreover, there was no COOH-terminal Aalpha chain immunoreactivity associated with this material based on ELISA and immunoblotting.



SDS-PAGE/Immunoblotting

Discontinuous SDS-PAGE was conducted on Laemmli and Favre (33) gels under nonreducing and/or reducing conditions. Prestained molecular weight markers (Amersham Corp.; Life Technologies, Inc.) were included on each run. Electrophoresed components were transferred onto nitrocellulose membranes (Schleicher & Schuell), and immunoblotting was conducted according to standard methodology(34, 35) . Transfers were stained for total protein with Amido Black. Purified anti-Aalpha chain mAbs were employed at concentrations of 0.5-2 µg/ml, depending on the antibody, and in some cases, expanded cloned culture supernatants, diluted 1:20-1:300, were used. Immunoreactive components were detected following successive incubations with either RAM-Ig-HRP (1:500) or GAM-IgG-HRP (1:2000) and the HRP substrate, 4-chloro-1-naphthol (Bio-Rad). Additional details appear in the legends to Fig. 2, 3, 5, and 6.

NH(2)-terminal Sequencing

Automated Edman degradation was conducted on an Applied Biosystems model 470A sequencer equipped with an on-line model 120A phenylthiohydantoin analyzer.

Amino Acid Analysis

Samples were hydrolyzed in 6 N HCl for 24 h under vacuum at 110 °C in a Pico Tag workstation (Waters, Milford, MA), and amino acid analysis was conducted on a Beckman model 6300 amino acid analyzer (Beckman Instruments). The molar compositions of purified alphaC preparations were derived from these data (and molecular weight estimates by SDS-PAGE) employing the peptide data manipulation software, PROCOMP (Dr. P. C. Andrews, University of Michigan Medical School, Ann Arbor, MI).

Patient Plasmas

Plasma aliquots from patients undergoing thrombolytic therapy for myocardial infarction were archival and originated from the TIMI (Phase I) Trial held at the Columbia Presbyterian Medical Center. Details regarding blood collection and processing of these samples have been reported elsewhere(36) .


RESULTS

Monoclonal Antibodies to Defined Regions within the COOH-terminal Two-thirds of the Aalpha Chain

Fig. 1illustrates the panel of eight characterized antibodies applied in these studies and indicates the position of each epitope relative to known functional domains along the length of the Aalpha chain. The panel includes three antibodies generated against large (A)alpha chain derivatives (Fig. 1A) and five antibodies raised against synthetic peptide-KLH conjugates (Fig. 1B). Among the anti-peptide antibodies, two recognize unique determinants within the same 11-residue sequence, Aalpha-(220-230); mAb F-106 requires a free NH(2) terminus at Aalpha-220 for immunoreactivity while mAb F-106f does not share this structural specificity.


Figure 1: mAbs to defined regions within the human fibrinogen alphaC domain. The Aalpha chain is illustrated schematically from residues 1-610. The alphaC domain is shown extending from the NH(2)-terminal third of the molecule which is included within the E and D domains of fibrinogen. The various Aalpha chain functional domains, including RGD sites, plasmin cleavage sites (P), acceptor Gln residues (Q), the alpha(2)PI lysine cross-linking site and the lysine-rich region associated with alpha chain donor cross-linking activity (K), are indicated for reference. mAbs raised against large CNBr (A)alpha chain derivatives, with their respective epitopes subsequently localized within small defined sequences, are shown in A. mAbs raised against predefined peptide conjugates are shown in B. The inclusive Aalpha chain residues that contain the respective epitopes recognized by each of the eight antibodies in the panel are: 1) Aalpha-(220-230) (mAbs F-106, 106f); 2) Aalpha-(259-276) (mAb F-103); 3) Aalpha-(425-442) (mAb F-105); 4) Aalpha-(487-498) (mAb F-104); 5) Aalpha-(529-539) (mAb 5A2); 6) Aalpha-(563-578) (mAb F-102); and 7) Aalpha-(603-610) (mAb F-48) (see also Table 2).



Isolation and Characterization of Purified Human alphaC Fragment(s)

Table 1summarizes the results obtained when selected antibodies from the panel shown in Fig. 1were employed to monitor the recovery of COOH-terminal Aalpha chain derivatives, Aalpha FDPs, during the initial steps of the alphaC isolation and purification procedure. Plasmin digestion had different effects on the four epitopes examined. While the localized regions, Aalpha-(220-230) (as recognized by mAb F-106) and Aalpha-(425-442), were occult in fibrinogen they became exposed as a result of proteolysis, with 14.6 and 9.1 times the initial immunoreactivity observed after 6 h of plasmin treatment, respectively. The epitope within Aalpha-(563-578) was not affected, with 97% of the original fibrinogen-associated immunoreactivity preserved among the various digest products formed. In contrast, a significant decrease in anti-Aalpha-(603-610) immunoreactivity, to 20.4% of its initial level was observed, indicating plasmin-mediated destruction or altered accessibility of this localized region within the COOH-terminal end of the Aalpha chain. Affinity chromatography on ConA-Sepharose provided a rapid and efficient method for isolating Aalpha FDPs (including alphaC) free of residual intact and partially degraded fibrinogen, as well as fragment X, that were present in the plasmin digest under the experimental conditions employed. Aalpha FDPs, which do not contain carbohydrate, were recovered in excellent yield (72.8-98.2%) in the ConA flow-through fraction. The high molecular weight derivatives, retained by the lectin by virtue of their glycosylated D and E domains, were eluted in the bound fraction and based on their COOH-terminal Aalpha chain-associated immunoreactivities accounted for less than 4% of the original, intact fibrinogen population. Subsequent immunoaffinity chromatography of the Aalpha FDPs on anti-Aalpha-(425-442)-Sepharose led to the removal of a significant proportion of fragments whose structures included the Aalpha-(425-442) (alone or in combination with the Aalpha-(563-578) and/or Aalpha-(603-610) epitopes) but were missing the more NH(2)-terminal region, Aalpha-(220-230). Aalpha FDPs, including alphaC, that contained this NH(2)-terminal portion of the alphaC domain within their structures were efficiently recovered in the immunosorbent flow-through fraction where they comprised an enriched population relative to the fragments present in the previous step.



Fig. 2(left) illustrates the 280-nm absorbance profile obtained when Aalpha FDPs in the anti-Aalpha-(425-442) flow-through fraction were separated by size exclusion chromatography on Superdex-75 FPLC. Analysis of the column effluent with the anti-Aalpha chain antibody panel indicated the emergence of (at least) five discrete peaks of COOH-terminal Aalpha chain immunoreactivities, reflecting a heterogeneous population of Aalpha FDPs ranging in size from approximately <1.3 to approx40 kDa (data not shown). SDS-PAGE (Fig. 2, right) identified two closely migrating components in fractions 18 and 19 with the approximate mass of 40 kDa expected for alphaC.

Fig. 3shows the immunoblotting profile typically obtained for alphaC preparations isolated and purified as described above. Characterization using the antibody panel confirmed the material's purity, based on the predominant 40-kDa component observed in each lane. These data, obtained from a denaturing system, also provided qualitative structural evidence to indicate that although a significant proportion of alphaC fragments included the NH(2) terminus of the alphaC region, i.e. Aalpha-(220-230) (Fig. 3, lane 1), none contained the extreme COOH-terminal epitope within Aalpha-(603-610) (Fig. 3, lane 7). (Small Aalpha FDPs, representing approx10- and approx3-kDa cleavage products released from the Aalpha chain COOH terminus, were identified in the FPLC column effluent as discrete peaks of anti-Aalpha-(603-610) immunoreactivity; data not shown). Amino-terminal sequencing revealed that two major components were included in our alphaC preparations and that these were present at approximately equimolar level. Comparison of the phenylthiohydantoin-derivatives recovered at each cycle with the known primary structure of the Aalpha chain indicated that the NH(2) terminus of these fragments originated from plasmin cleavage at residues Aalpha-(219-220) and Aalpha-(230-231), respectively (data not shown). Based on the structural information obtained from the immunoblotting and NH(2)-terminal sequencing findings, the molar composition derived from amino acid analysis of the alphaC preparation (data not shown) was most consistent with both fragments extending at least through residue Aalpha-583.

Immunologic Characterization of Conformational Features within the Fibrinogen alphaC Domain and Isolated alphaC

In the next series of studies we applied the antibody panel to obtain information about the native structure of purified alphaC and to compare the fragment's topographical features with those of its counterpart in intact fibrinogen. Purified alphaC and fibrinogen were employed as solution phase competitors in ELISAs developed for the various antibodies in the panel and the dose of each antigen required to achieve 50% binding (compared to the assay standard) was taken as an index of relative epitope accessibility, i.e. a ``buried'' or ``exposed'' conformation. Fig. 4illustrates the paired dose-response curves obtained for several of the assay systems examined, with the four panels shown representative of the four types of epitopes revealed by this analysis. These include epitopes that are equally exposed in alphaC and fibrinogen (Panel A); epitopes that are buried in both antigens (Panel B); epitopes whose accessibility is decreased in alphaC (Panel C); and neo-epitopes, occult in fibrinogen but revealed in alphaC (Panel D). Table 2summarizes the molar cross-reactivity findings obtained for each of the eight Aalpha chain regions characterized. Molar ratios (alphaC:fibrinogen) of approx1.0, approx0.1, and geq10.0 are assumed to reflect conformations that are unchanged, newly exposed, and newly masked, respectively, when the structures of alphaC and fibrinogen are compared. The findings obtained indicate the following: 1) the conformations of one epitope within the sequence Aalpha-(220-230) (as recognized by mAb F-106f) and another within Aalpha-(563-578) are similarly exposed in both alphaC and fibrinogen; 2) the localized region Aalpha-(425-442), is relatively inaccessible in the two antigens; 3) the release of alphaC is accompanied by conformational changes that serve to mask previously accessible epitopes within Aalpha-(259-276), Aalpha-(487-498), and Aalpha-(529-539); and 4) a second, distinct epitope within the localized region Aalpha-(220-230) (as recognized by mAb F-106) becomes newly exposed in alphaC as a result of plasmin cleavage at residues Aalpha-(219-220).

Factor XIII(a) Cross-linking in Isolated alphaC Fragments

Based on its primary structure alone, isolated alphaC includes the two acceptor glutamine residues at Aalpha-328 and Aalpha-366 as well as the lysine-rich donor region within Aalpha-(518-584) believed responsible for factor XIII(a)-mediated alpha chain cross-linking (see Fig. 1). In view of the findings summarized in Fig. 4and Table 2, which demonstrated that several conformational differences exist in the structure of isolated alphaC when compared with the alphaC domain of intact fibrinogen, we were interested to evaluate the functional ramifications of these differences on the fragment's capacity for factor XIII(a) cross-linking. Fig. 5illustrates the results obtained when a newly developed factor XIII(a) lysine-labeling system was applied to determine whether isolated alphaC could serve as a plasma transglutaminase substrate. This labeling system features a small, synthetic glutamine-containing peptide, modeled after the NH(2)-terminal acceptor cross-linking domain of alpha(2)PI, which becomes cross-linked by factor XIII(a) to susceptible lysine residues in transglutaminase-sensitive proteins. Peptide incorporation into control fibrin(ogen) (Fig. 5, left) and alphaC (Fig. 5, right) was visualized by immunoblotting with two antibodies, one specific for the localized region Aalpha-(259-276), within the alphaC domain (anti-(A)alpha-(259-276); mAb F-103), and the other specific for the peptide probe (anti-alpha(2)PI-(1-12); mAb AP-102). In this analysis, cross-linking was inferred from an apparent increase in the molecular weight of alpha chain monomers or alphaC fragments, coincident with the appearance of anti-peptide immunoreactivity. As shown in the last two panels of Fig. 5, purified alphaC (applied alone in lanes 1), in the presence of thrombin, factor XIII(a), and the peptide probe, formed a heterogeneous array of approx40-67-kDa peptide-decorated products (lanes 3). Furthermore, when the peptide was omitted from the incubation mixture natural alphaC-alphaC cross-linking was observed based on the appearance of a higher molecular weight band of anti-Aalpha chain immunoreactivity consistent with alphaC dimer formation (lane 2 in the F-103 alphaC panel). The findings obtained in Fig. 5indicate that isolated alphaC retains the conformational features required for factor XIII binding and for the subsequent alignment of cross-linking partners in both synthetic (peptide) and natural (alphaC) substrates.


Figure 5: Factor XIII(a)-mediated alphaC cross-linking visualized by immunoblotting. Purified fibrinogen (left; lanes 1) and alphaC fragment(s) (right; lanes 1) were subjected to in vitro cross-linking in the presence (lanes 3) or absence (lanes 2) of a synthetic peptide probe for factor-XIII(a) donor lysine residues, as described under ``Materials and Methods.'' Approximately 2.5 µg of fibrin(ogen) and 5.2 µg of alphaC from the respective incubation mixtures were applied to 9% gels, and electrophoresis was conducted under reducing conditions. Duplicate nitrocellulose transfers were treated with anti-Aalpha-(259-276) (mAb F-103; first three lanes in each panel) and anti-alpha(2)PI-(1-12) peptide (mAb AP-102; last three lanes in each panel), employing expanded cloned culture supernatants diluted 1:100 and 1:300, respectively. Immunoreactive components were visualized using GAM-IgG-HRP, as described under ``Materials and Methods.'' The faint bands of immunoreactivity observed in the first two lanes of each AP-102 panel are due to nonspecific binding by the HRP-IgG conjugate, as described previously(26) . The migration of standard molecular mass markers is indicated at the extreme left for reference (myosin, 215 kDa; phosphorylase b, 105 kDa; bovine serum albumin, 70 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 28 kDa).



Immunologic Identification of alphaC Fragments in Vivo

The observation that purified alphaC preparations retain at least a partial capacity for factor XIII(a) cross-linking prompted us to consider whether naturally occurring alphaC fragments could be demonstrated in vivo. While fibrinogenolytic products do not circulate at significant level under physiologic conditions, their release is expected in clinical states associated with fibrinolytic system activation. Fig. 6illustrates the immunoblotting results obtained when two of the antibodies from the panel, anti-Aalpha-(259-276) and anti-Aalpha-(563-578), were employed as structural probes to monitor the Aalpha chain proteolysis expected during thrombolytic therapy with rt-PA and streptokinase as a result of the systemic lytic state created during treatment. A heterogeneous series of Aalpha FDPs (approx18-41 kDa) was rapidly released, within 15-30 min after administration of each lytic agent. Prominent among the population of fragments observed was one group whose size (38-41 kDa) and coincident epitope distribution suggested the presence of alphaC. Based on the relative band intensities observed over the course of therapy, alphaC fragment(s) appeared to comprise a significant proportion of the initial Aalpha FDP population released and persisted at high level during the first half (at least) of the infusion periods employed for rt-PA (180 min) and streptokinase (60 min) treatment, respectively.


DISCUSSION

The studies described in this report illustrate the application of a unique panel of characterized monoclonal antibodies to address questions related to the biochemistry of the fibrinogen alphaC domain. The antibodies in the panel recognize eight distinct epitopes that span the COOH-terminal 391 residues of the Aalpha chain (Fig. 1). These include three epitopes within the ``loosely'' organized portion of the alphaC domain (Aalpha-(220-390)) and five within the portion characterized as ``compact'' (Aalpha-(390-610)), based on biophysical observations and inferences drawn from the region's primary structure (12, 16) . To date, characterization of the topography of the alphaC domain and its relationship to other domains within the trinodular fibrinogen molecule, have provided a general view of this region's structural features(15, 17) . As demonstrated here, the availability of monoclonal antibodies to defined Aalpha chain regions provides the opportunity to refine this view by characterizing localized conformations within the alphaC domain.

Several groups have previously reported the use of anti-Aalpha chain antibodies as conformational probes of native fibrinogen and its structural modulation during plasmin digestion(22, 37) . In both studies, antisera were raised against native fibrinogen or isolated Aalpha chains, with antibody selection methods contributing to the recovery, primarily, of immunoglobulin populations that recognized two classes of immunodominant epitopes. One of these was localized to the mid-section of the Aalpha chain, within Aalpha-(239-429), and the other to the COOH-terminal end of the Aalpha chain, within Aalpha-(518-584). The three antibodies in our panel, anti-Aalpha-(259-276) (mAb F-103), anti-Aalpha-(529-539) (mAb 5A2), and anti-Aalpha-(563-578) (mAb F-102), which were each raised against large Aalpha chain derivatives, more than likely reflect this same phenomenon. Their isolation highlights the fact that repertoires of anti-COOH-terminal Aalpha chain antibodies will be restricted to immunodominant epitopes unless rational approaches to immunogen design and antibody selection are considered. In view of this, we have focused on the use of small peptide immunogens, modeled after defined COOH-terminal Aalpha chain sequences, to produce structural probes other than those represented in the traditional schemes employed for the development of anti-fibrinogen monoclonal antibodies. Peptides were chosen based on hydropathy criteria (for example, Aalpha-(487-498) is predicted to be one of the most hydrophilic Aalpha chain regions) or functional considerations (the reported early plasmin cleavage sites at Aalpha-(219-220) and Aalpha-(424-425) (9) would be expected to produce at least some antibodies to neo-epitopes). This approach, together with a dual primary screening assay employing both peptide and intact fibrinogen as solid phase antigens, led to the isolation of two anti-fibrinogen antibodies (mAbs F-106f and F-104) and two anti-peptide antibodies (mAbs F-106 and F-105), each characterized by unique specificities (Fig. 1). (A similar strategy resulted in recovery of mAb F-48, anti-Aalpha-(603-610)). (^4)

In developing in vitro methods for the isolation of alphaC fragment(s) from purified fibrinogen, we were aware of the heterogeneity of plasmin cleavage sites within the COOH-terminal two-thirds of the Aalpha chain (38) and anticipated that alphaC would represent one of a series of overlapping Aalpha chain derivatives, each isolated in reduced yield. In fact, while characterization with our antibody panel indicated the efficient recovery of COOH-terminal Aalpha chain immunoreactivity, i.e. Aalpha FDPs, during the initial purification steps (Table 1), alphaC typically accounted for only 5% of the total Aalpha FDP population based on amino acid analysis of the purified material isolated following the FPLC step (Fig. 2). This finding highlights the difficulties associated with the preparation of human alphaC fragments in vitro and explains, in part, the choice of bovine fibrinogen as a starting substrate for most of the previously reported structural studies of alphaC. However, the substitution of bovine for human fibrinogen does not consider the potential for conformational differences imposed by nonconserved sequences within the Aalpha chains of various animal species. As two examples, 1) the region corresponding to human Aalpha-(529-539) is missing in bovine fibrinogen as part of a 26-residue deletion and 2) hydrophobic residues appear in the bovine Aalpha chain at the position corresponding to the early plasmin cleavage site, Aalpha-(424-425). Structural differences such as these may partially explain why bovine alphaC fragments form long linear polymers at neutral pH (17) while human alphaC fragments do not appear to aggregate under similar conditions (Fig. 2).

Application of the antibody panel to characterize localized conformations within the fibrinogen alphaC domain and the isolated alphaC fragment was conducted in an aqueous environment that presumably preserved the native form of each molecule. (In this regard, the potential for structural distortion introduced during electron microscopy sample preparation should not be overlooked.) Immunologic analysis was predicated on the fact that epitopes that are oriented in an exposed conformation on the surface of antigen molecules will be readily accessible for antibody binding and, therefore, will require less antigen to effect a given response than epitopes contained within hydrophobic regions or regions that are sterically masked. Since the goal of this immunologic characterization was to detect whether or not conformational differences existed within respective regions of fibrinogen and alphaC, all the information required to address this question was provided by the comparative dose response curves obtained for the two antigens in each assay (Fig. 4, Table 2). (Issues related to antibody affinity or the degree to which either antigen achieved maximal epitope expression, were, therefore, not pertinent here.) Results of this immunologic analysis indicated that the release of alphaC from intact fibrinogen was accompanied by conformational changes in the isolated fragment, as reflected in the altered accessibility of several of the epitopes examined. Although speculative, one structural implication drawn from the findings is that the newly exposed and loosely organized NH(2)-terminal connector portion of alphaC may fold back and wrap around the more globular portion of the molecule, thereby obscuring a number of localized COOH-terminal regions that were surface-oriented on the alphaC domain of intact fibrinogen.

The conformational profiles obtained for several different alphaC preparations using these immunologic methods were similar, except for a significant difference in expression of the localized region, Aalpha-(425-442). This difference appeared to be associated with the F-105 immunoaffinity step since alphaC preparations obtained by direct FPLC following ConA chromatography exhibited enhanced expression of this localized region compared to fragments isolated without prior immunoadsorption. Immunoblotting analysis confirmed that a group of contaminating 17-25 kDa F-105-immunoreactive Aalpha FDPs originating from cleavage at the Aalpha-(424-425) bond were responsible (data not shown). Curiously, although the anti-Aalpha-(425-442) immunoaffinity step was subsequently found to remove a major proportion of these fragments from the total Aalpha FDP population (thereby permitting a more effective separation of alphaC in the final gel-filtration step), 47% failed to bind to the immunosorbent despite the relatively high molar excess (5:1) of antibody employed (Table 1). The reason for this apparent heterogeneity in F-105 epitope expression on the solid phase matrix and in solution remains unclear although a contributing factor may be the fact that the region Aalpha-(425-442) is in the vicinity of the Aalpha chain's single intrachain disulfide (involving the cys residues at Aalpha-442 and Aalpha-472; see Fig. 1). The reported lability of this disulfide (39) suggests that perturbations in its structure may affect adjacent local conformations.

The structural modulations revealed for alphaC coincident with its release from intact fibrinogen did not interfere with the fragment's capacity to serve as a factor XIII(a) substrate based on the findings obtained employing a synthetic peptide probe for plasma transglutaminase donor lysine residues(26) . Up to 8 mol of peptide became incorporated into purified alphaC fragments based on the molecular weight differences observed for monomeric alphaC and its peptide-decorated products (Fig. 5). This represents cross-linking at 35% of the potentially available donor lysines within the region, Aalpha-(220-610). Moreover, alphaC fragments could undergo cross-linking by factor XIII(a) in the absence of the peptide probe to produce a significant, albeit minor, proportion of dimeric alphaC forms. The degree to which natural alphaC cross-linking can be extended in vitro to involve a greater proportion of alphaC monomers and/or result in the formation of polymeric species remains to be explored. In addition, the role of intact fibrin(ogen) as a partner for alphaC cross-linking requires evaluation since the structural features intrinsic to the intact molecule could serve to potentiate what may be only a partial capacity for cross-linking on the part of isolated alphaC fragments alone.

The cross-linking of alphaC fragments, particularly in a fibrin(ogen)-containing milieu may have important potential implications with respect to the thrombotic and hemorrhagic complications often associated with current thrombolytic therapies. Immunologic characterization of the Aalpha chain proteolysis that reflects the lytic state created during treatment clearly demonstrates, for the first time, that alphaC fragments are naturally occurring products of fibrinolytic system activation (Fig. 6). Since these Aalpha chain derivatives circulate together with varying concentrations of intact fibrinogen over the course of therapy, their capacity to serve as a cross-linking partner may provide a functional reserve in a plasma milieu where the conventional substrate is facing continual depletion.

The findings described here, together with the unique panel of anti-Aalpha chain monoclonal antibodies developed, offer new approaches for investigating other structure-function relationships associated with the fibrinogen alphaC domain in basic research as well as clinical applications.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL 45936 (to J. H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medicine/Irving Center for Clinical Research, 630 W. 168th St., College of Physicians and Surgeons of Columbia University, New York, NY 10032. Tel.: 212-305-9298; Fax: 212-305-3213.

(^1)
The abbreviations used are: alpha(2)PI, alpha(2)-antiplasmin; alpha(2)PI-(1-12) peptide, the 12-residue peptide corresponding to the NH(2)-terminal portion of alpha(2)PI; FDP, fibrinogen degradation product; Aalpha FDPs, COOH-terminal fibrinogen Aalpha chain degradation products released by plasmin; ConA, concanavalin A; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; GAM, goat anti-mouse; FPLC, fast protein liquid chromatography; KLH, keyhole limpet hemocyanin; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; RAM, rabbit anti-mouse; rt-PA, recombinant tissue plasminogen activator; t-Boc, t-butoxycarbonyl.

(^2)
O. V. Mitkevitch, J. H. Sobel, and J. R. Shainoff, manuscript in preparation.

(^4)
G. Matsueda, personal communication.

(^3)
J. H. Sobel, I. Trakht, and S. Rudchenko, manuscript in preparation.


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