D-dimer specific monoclonal antibodies react with fibrinogen aggregates

Thrombosis Research, Vol. 82, No. 2. pp. 169-176,1996 Couwieht Q 1996 Elsetier Science Ltd P&&d-h the USA. All rights reserved ~9-3~8~6 $12.00 + .OO

Pergamon PII S~9-~(%)~63-~

D-DIMER

SPECIFIC MONOCLONAL ANTIBODIES FIBRINOGEN AGGREGATES

REACT WITH

Anne Bennick, Unni Haddeiand and Frank Brosstad. Research Institute for Internal Medicine, Rikshospi~le~ Oslo, Norway.

(Received 2 1 September 1995 by Editor 8. Osterud; revised/accepted

6 March 1996)

Abstract Human fibrinogen exposed to 46S”C was subjected to gel permeation chromatography. The protein eluted in two distinct peaks. The first peak appeared in the void volume containing soluble fibrinogen aggregates, while the other peak represented monomeric fibrinogen. In contrast to the monomeric peak material, the aggregate fraction reacted with a panel of mon~lonal antibodies specific for fragment D-dimer using an ELISA system. Edman degradation showed that both the aggregate and the monomeric fractions were devoid of soluble fibrin, and immunoblo~ of SDS-PAG electrophoretic profiles disclosed no sign of stabilized high molecular weight derivatives. We have previously shown that the aggregate fraction of similarly treated fibrinogen, in contrast to the monomeric fraction, stimulates the t-PA catalyzed conversion of plasminogen to plasmin and concomitantly exposes the sequences Aa-( 14% 160) and y-(3 12-324) involved in t-PA stimulation. Our present and previous findings suggest that soluble fibrinogen aggregates possess a fibrin-like structure, and that fibrin or fibrinogen polymer formation is a prerequisite for the enhancing effect on t-PA-mediated plasminogen to plasmin conversion which is seen even with the polymers in the soluble state.

Recently, we showed that fibrinogen exposed to heat stimulates the t-PA catalyzed conversion of plasminogen to plasmin (1). This stimulating effect was closely associated with the aggregate form as it was lost upon removal of the aggregates by gel filtration (2). Moreover, we observed that the soluble aggregates, in contrast to the monomeric fibrinogen fraction, concomitantly exposed the Aa-( 14X-160) and y-(312-324) epitopes known to be involved in t-PA stimulation (2). Since the availability of these epitopes are known to be fibrin specific (3,4), we found it of interest to investigate the possibility that soluble fibrinogen aggregates would react with monoclonal antibodies specific for fragment D-dimer, which may be considered the hallmark of fibrin protofibril assembly. Key words: Fibrinogen, aggregates, DD-anti~dies, t-PA. Corresponding author: Anne Bennick, Research Institute for Internal Medicine, 0027 Oslo, Norway.

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AND METHODS

Fibrinagen was purified from fresh normal #D-plasma by P-ala&e (Sigma Chemical Co., St.Louis, USA) pre~ipi~tion as previously described (S-7) and stored at -86°C at 5.6 rn~~ for maximum 3 weeks. The protein concentration was estimated by determination of absorbancy at 280 nm (X,9).

A subsample of fibrinogen as above was exposed to 46.5”C in a glass tube immersed in a water bath for 25 minutes. Minute amounts of visible precipitates that formed were removed by f&ration through cotton, and the solution sub~uendy kept at room temperature until used. Gel l)esnigurif)~~ ~bsf)nlurugsu~by of heat-exposedfibrinoge~. A subsample of fibrinogen exposed to heat as above was subjected to gel permeation chromatography on a 2.5 x 26 cm bed of Ultrogel AcA 22 (Pharmacia LKB Biotechnology, Uppsala, Sweden). The column was developed at 24°C with buffer (7.9 mmovl Na,HPO,, 1.6 mol/l KH,PO,, 0.15 mol/l NaCI, 0,058 (v/v) Tween 80, pH 7.41, at a flow rate of 0.7 mUmin, under continuous registration of absorbancy at 280 nm. Fractions of 3.6 ml were collected and the protein ~on~en~~tion determined as above. The void volume was determined by ~hro~tog~phy of Biue Dextran 2m.

Edman deg~ddation of subsamples of the monomeric and aggregate fractions of heat-exposed fibrinogen was pe~ormed on the first ten N-terminal amino acids. A 477 A Sequencer ~Ap~Ii~ Biosystems, USA) equipped with an on-line 120A PTH analyzer was used.

SDS-PAGE was performed by the method of Laemmli (10) on 5% gels applying non-reduced samples in buffer (0.5 mol/l Tris-HCl, lO%(v/v) glycerol, 2%(w/v) SDS, 5 mol/l urea, O.O5%(w/v~ bromophe~olbIue, pH 6.8). The following samples were run: 1: titrated normal plasma (5 pl, dilution 1: loo), 2: human fibrinogen grade L (0.1 pg, Ph~macia, St~kholm, Sweden), 3: fibrinogen prepared as above, not exposed to heat (0.1 pg), 4: monomeric fraction of gel filtrated, heat-exposed ~brinogen (0.1 pg), 5: aggregate fraction of gel filtrated heatexposed ~brinogen (0.1 pg), 6: pre-stained high molecular weight markers (0.i pg, BioRad, USA~.Immunoblotting was performed by methods described by Gron fl 1).

To human fibrinogen as above (56 Mimi) was added CaCI, mmoh’l and bovine thrombin to a final concentration of 0.8 incubated at 37°C overnight. The clot was removed and buffer NaGi, pH 7.4) added to l/3 of the starting volume. Streptokinase the mixture incubated at 37°C until complete visual clot lysis fragment D-dimer was then performed as earlier described (11).

to a final condensation of 10 NIH U/ml. The mixture was (50 mmol/l TRIS, 150 mmol/l (100 KIUIrnl) was added and was obtained. P~i~~ation of

The monoclonat antibody S4H9 was produced in our laboratory as p~vio~sly described fI 1) and is of the IgG1 subtype. It has been shown by ELISA-based assays to be specific for D-dimer configurated molecules like DD and X-oligomers in human plasma (I I).

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The monoclonal antibody DgHl was produced by NycoMed, Osio, Norway, and is of the IgG immunoglobuline class. It is specific to human DD. The rnon~lo~~1 anti~y DD3B4~2 detects a plasmin sensitive site on the N-terming half of the y-chain in cross-linked DD or fibrin-derivatives. This antibody was a kind gift from Dr.D.Rylatt, Agen Biomedical Limited, Australia (12-17). The monoclonal antibody MAD8D3 was purchased from Biopool (Umel, Sweden) and is of the IgGl subtype (1X). ELISA. Immunoplates (Maxi Sorp, Nunc A/S, Roskilde, Denmark) were coated witb 10 uglml mAB to D-dimer as above in phosphate-buffers saline (PBS) overnight followed by blocking further adsorbancy with PBS-Tween 20 containing 1% bovine serum albumin (BSA) for 30 min. The immunoplates were now incubated with increasing amounts of D-dimer to assess the D-dimer concentration at saturation (100% binding), which corresponded to 5 us/ml. A comparison between the D-dimer-related immunoreactivity of the fibrinogen-specimens to that of D-dimer was accordingly performed at concentrations twice this amount (i.e.10 pg/ml>. The fibrinogen samples, plasma samples and D-dimer samples in PBS-Tween containing l/10 volume of 0.11 mol/l of sodium citrate were added and the mixture incubated for 1 hour. As secondary antibodies were used rabbit anti-human fibrinogen (A080 from Dako, Copenhagen, Denmark) in PBS-Tween with 0.1% BSA for 1 hour. Subsequently, a tertiary antibody, swine anti-rabbit Ig conjugated with alkaline phosphatase (D306 from DAKO, Copenhagen, Denmark) in PBSTween with 1% BSA was added, followed by incubation for 2 hours. To visualize the binding of the fibrinogen to insolubilized mABs, p-nitrophenyl disodium phosphate (Sigma Chemical Co., St. Louis, MO, USA) in buffer (1 moh’l diethanolamine, 0.5 mmol/l MgCl,*6H,O, 3 mmol/l NaN,) was used. The colour reaction was stopped with 50 pl of 1 mol/i NaOH per well and read at 405 nm in a Titertek Multis~n ELISA-reader (Flow Laboratories, Irvine, Scotiand, UK). RESULTS

AND DISCUSSION

Gel permeation chromatography (Fig. 1) of heat-exposed fibrinogen disclosed two elution peaks. The first peak eluted in the void volume (i.e. identical to Blue Dextran 2000), indicated the presence of high molecular weight fibrinogen aggregates; the other peak represented monomeric fibrinogen. lmmunoblots after unredu~ed SDS-PAGE (Fig.2 panel A) of the material from the two peaks demonstrated no difference in electrophoretic mobility, indicating that the fibrinogen aggregates were non-crosslinked. Reduced SDS-PAGE gels that were blotted and subsequently immunovisualized with a polyclonal antibody against human fibrinogen (Fig.2 panel B) showed no y-y dimer formation during heating. Both the monomeric and the aggregate fractions were found to be virtually free of fibrin as revealed by Edman degradation. Recently, we showed that fibrinogen that had been exposed to heat under identical conditions stimulated t-PA conversion of plasminogen to plasmin (1). When the heat-expose fibrinogen was subjected to gel perm~tion ~hro~to~raphy as above, it became evident that the t-PA stimulat~g effect was associated only with the aggregate fraction. Moreover, the fibrinogen aggregates, in contrast to the monomeric fraction, showed a concomitant exposure of the sites Aol-(148-160) and y-(312324) involved in t-PA stimulation (2). Based on these findings, we hypothesized that the t-PA stimulatory effect of aggregated fibrinogen might reside in a fibrin-like structure of the fibrinogen aggregates, and that exposure of polymerization sites and t-PA stimulating sites could be interlinked. The present findings seem to further support this hypothesis, as soluble fibrinogen aggregates react with mono~iona1 antibodies specific for ~brinderiv~ D-dimerized mofecules (Table I). To be able to compare the immuno~activity of the ~b~nogen-specimens to that of D-

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dimer, the ELBA-tests listed in Table I were performed using antigen concentrations equal to twice the concentration of D-dimer that ensured 100 percent saturation of the binding capacity of the insolubili~ monoclonal ~tib~y to D-dimer. A s~tistic~ly signi~cant difference (pc 0.05) was found between the immunorea~vity of the aggregate fraction and that of the other fibrinogen-related antigen sources. As seen from Table I, the immunoreactivity of the antigens versus the four different monoclonal antibodies varies slightly. This variability is probably related to differences in epitopes and avidity. Our previous (2) and present findings are thus in accordance with other observations(l9-23) that fibrinogen may form structures similar to polymeric fibrin by binding end-to-end in D-dimer configuration, forming flexible polymer chains, readily ~rosslin~ble by FXIIfa (23). Mosesson et al. (23) used scanning electron microscopy to show that nei~h~~ng D domains in each fibrinogen fibril strand were aligned end-to-end, forming “DD” regions that were in register with an E domain in the opposite strand. Our present observation that heat-exposed fibrinogen prior to gel permeation chromatography demonstrated a distinctly lower immunoreactivity in the ELBA assay, is interesting. We speculate that one of the reasons for this might be due to the large excess of monomeric fibrinogen over fibrinogen aggregates. This may suppress aggregate formation in the total preparation, while the aggregates readily form during gel separation, a phenomenon that parallels the behaviour of soluble fibrin under similar conditions (24-26). Thus, when the rnono~~c fibrinogen fraction were mixed with the aggregate fraction, the i~unoreactivity was reduced to the same level as in the total preparation (data not shown). Recently, we showed that in order to stimulate t-PA, even fibrin had to exist in the form of polymers, so it seems that a polymer form of both fibrinogen and fibrin is a prerequisite for such stimulation (2).

FIG 1. Gel permeation chromatography of human fibrinogen exposed to 46S”C for 25 min. A 2.5~26 cm gel bed of Ultrogel AcA 22 was developed at 24°C. Flow rate was 0.7 ml/min and the protein load was 12.5 mg. The ” ” mark indicates void volume.

-e

63 ELUTION

VOLUME

(ml)

t-PA STIMU~TION

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116.500 80.000~~ 49.500~,, 12

FIG 2. Panel A.

34

5

6

7

Immunoblot after SDS-PAGE on a 5% gel of unreducedsamplesof plasmadiluted 1:LOO(lane 2), human fibrinogen, grade L, (Chromogenix, Sweden) (lane 3), D-alaninepurified fibrinogen (lane 4), gel-fractionated, heat-exposedBalanine purified fibrinogen, monomeric fraction {lane 5) and aggregatefraction (lane 6). High MW standardsapplied in lane 1 and 7. Blot visualized with polyclonal anti-human fib~nogen.

MW

101.000--.-..83.00050.60035.500-

29.10020.900-

FIG 2. Panel B.

I

234

567

Immunoblot of the same samplesas in Panel A after reduced SDS-PAGE on a 10% gel. Low MW standardswere applied in lane 1 and 7. The blot was visualized with polyclonal anti-human fibrinogen.

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TABLE I EL~SA-bard Immuuor~~tivity of Native and ~~at”Expos~ Human Fibrinogen using a Panel of Monoclonal Antibodies specific for Fragment D-Dimer. S4H9 and D8Hl are Domestic maBs, “Agen” and “Biopool” are Co~ercia~ mABs. Plasma and Fragment DDimer are used as Negative and Positive Controls, respectively, Median Values of Absorbaney at 405 nm are given with Range of Values in Brackets. Figures for Immunoreactivity of the Aggregate Fraction are marked with *, indicating statistically signifi~nt Difference (p~.O5) from the ~o~esponding Figures of Unexposed Fibrinogen as well as Heat-Exposed Fibrinogen (Total Preparation and Monomeric Fraction)

Antigen:

Monoclanal Antibody _-**_-*-_--*-____*-_--------------------------*-----S4H9(n=4) DSHl(n=4) Agen(n==4) Biopool(n==4)

Heat-exposed fibrinogen total prepa~tion

0.10 (0.07-O. 16)

0.11 (0.08-O. 16)

0.15 (0.07-~.25)

0.15 (0.07-0.3 1)

Monomeric fraction of heat-exposed fibrinogen

0.12 (0.1 l-0.13)

0.08 ~0.06-0.09~

~~-0.05~

0.02 (0.01-0.03~

Aggregate fraction of heat-expose ~brino~e~

0.39*

0.56* (0.53-0.57~

0.39” (0.35-0.39)

0.44*

0.03 {0.02-0.05~

0.05 {0.04-0.~)

0.04 (0.03-0.05~

(0~05-0.06)

0.04 (0.03-0,~~

0.05 (0.02-0.05)

0.03 (0.01-0.04~

0.67 ~O.~-0.75}

0.96 (0.83-1.2)

0.61 (0.55-0.70)

0.80 ~0,75-0.92)

~0.36-0.41~ 0.09

Unexposed (native) fibrinogen

(0.06-o. 1)

Citrated normal plasma

0.06

Fragment D-dimer

(0*~-0*45~

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