fibrinogen manchester

Biochem. J. (1985) 229, 723-730Printed in Great Britain

Fibrinogen Manchester

Detection of a heterozygous phenotype in the intraplatelet pool

Christopher SOUTHAN,* David A. LANE,* Irina KNIGHT,* Helen IRELAND*and Jill BOTTOMLEYt

*Department of Haematology, Charing Cross and Westminster Medical School, Hammersmith,London W6 8RP, U.K. and tDepartment of Haematology, Clinical Laboratories,

Manchester Royal Infirmary, Manchester M13 9WL, U.K.

(Received 27 November 1984/19 March 1985; accepted 18 April 1985)

Family members heterozygous for the congenitally abnormal fibrinogen designatedfibrinogen Manchester, Aal6Arg--His, have previously been shown by h.p.l.c. andamino acid analysis to release a variant fibrinopeptide, [Hisl6]fibrinopeptide A, fromplasma fibrinogen after the addition of thrombin. The present study was designed todetermine if the same abnormal phenotype was also present in the intraplateletfibrinogen pool. Fresh platelets were washed in buffers containing EDTA until itcould be shown that all washable plasma fibrinogen was removed. Normal plateletswere then lysed by freezing and thawing to release their intracellular proteins, whichwere then treated with thrombin. The fibrinopeptides, cleaved from the intraplateletfibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification ofthe intraplatelet fibrinogen gave a result (means+ S.D., n = 5) of 110+30 and90 + 30 pg/109 platelets, when determined by h.p.l.c. quantification of fibrinopeptideB content and fibrinogen fragment E radioimmunoassay respectively. Examinationof fibrinopeptides released from the platelet fibrinogen from the family withfibrinogen Manchester with the same techniques showed elution peaks in thesame positions as both [His'6]fibrinopeptide A and normal fibrinopeptide A. Theidentity of these peaks was further substantiated by analysis of the h.p.l.c. peaks byusing specific radioimmunoassay to fibrinopeptide A. Our results thereforedemonstrate that platelet fibrinogen expresses the heterozygous AacIl6His phenotype.This supports the view that the Aa chains of platelet and plasma fibrinogen are

produced from a single genetic locus.

The fibrinogen circulating in human plasma, ata concentration of 2-4mg/ml, is produced in thehepatocytes. A separate fibrinogen pool, contain-ing approx. 3% of total blood fibrinogen, is foundin the a-storage granules of the blood platelets(Niewiarowski, 1977). Previous investigators havecome to opposing conclusions as to whether thetwo types of fibrinogen are separate or common

gene products (for review see Niewiarowski, 1977).

Abbreviations used: FPA, all forms of fibrinopeptideA, including A (Aal-16), AY (Aa2-16) and AP (Aal-16), phosphorylated at serine in position 3; FPB, the twoforms of fibrinopeptide B, including B (BP 1-14) and des-Arg-B (B/I1-13); on Figures des-Arg-B is furtherabbreviated to B-R; [His'6]FPA, the abnormal FPAcleaved from plasma-derived fibrinogen Manchester,Aal6Arg-_His.

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One way in which it is possible to establishcommon genetic origins of the two pools is bystudying congenital fibrinogen variants. Some ofthese variants, which can readily be isolated fromplasma fibrinogen, have distinctive structuralcharacteristics. Their presence within the plateletfibrinogen pool would suggest that the existence ofseparate genes for the two pools is unlikely.We have examined the platelets from a hetero-

zygous case of a plasma fibrinogen Aa-chainvariant, fibrinogen Manchester (Lane et al., 1980),in which an abnormal FPA, [His'6]FPA, iscleaved slowly by thrombin and can be character-ized both as an abnormally eluted peak on h.p.l.c.(Southan et al., 1983) and by its altered immuno-reactivity towards anti-FPA sera (Lane et al.,1983). By developing sensitive h.p.l.c. techniques

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to quantify the release of fibrinopeptides directlyfrom platelet lysates, we have been able to showthat platelet fibrinogen from a member of thefibrinogen Manchester family exhibits the sameheterozygous phenotype as plasma fibrinogen.

Materials and methods

MaterialsUnless otherwise stated all chemicals were

AnalaR grade from BDH Chemicals and allprocedures were carried out at room temperature(approx. 22°C). Human thrombin (3000 NIHunits/mg) was a gift from Dr. J. W. Fenton (NewYork State Department of Health, New Albany,NY, U.S.A.).

Isolation of blood plateletsAfter collection, citrated blood was centrifuged

at lOOg for 20min. From the resulting platelet-richplasma, washed platelets were prepared by themethod of Broekman et al. (1974). The final pelletwas washed three times before resuspension in0.15M-NaCl/5OmM-Tris/HCl buffer, pH7.4, con-taining 1 mM-NaN3 and 5mM-EDTA for storage inliquid N2 . At various stages samples were removedfor counting of platelets (Coulter model S-plus) anddetermination of fibrinogen content (see below).

Release offibrinopeptidesPlatelet concentrates were subjected to three

cycles of freezing in liquid N2 and thawing at37°C. Thrombin (200units/ml final concentration)was then added to the platelet lysates, andthe suspension was incubated for 10min. Sampleswere then boiled for 3 min, centrifuged for 5 min inan Eppendorf 5414 bench centrifuge and filteredon a 0.22,um-pore-size membrane filter (GelmanSciences, Northampton, U.K.) before applicationto h.p.l.c. Control lysates not treated by thrombinwere treated in parallel. Control fibrinopeptidesolutions were prepared as described by Southan etal. (1983) from purified normal plasma fibrinogenand from plasma fibrinogen isolated from thefamily with fibrinogen Manchester.

Determination of concentration of extracellular andintracellular fibrinogenThe fibrinogen concentration released from

washed and disrupted platelet lysates was deter-mined from the fibrinopeptide content by compar-ing the FPB peak heights with those obtained bythrombin treatment of a known fibrinogen stan-dard analysed under the same conditions. A secondmethod of fibrinogen quantification, using aradioimmunoassay for fibrinogen fragment E, wasperformed as described previously (Lane et al.,1984), and standardized with purified fibrinogen

solutions (KabiVitrum, Uxbridge, Middx., U.K.).Additionally, 1 251-labelled fibrinogen was pre-pared by the iodogen method (Fraker & Speck,1978) to a specific radioactivity of lOOmCi/mg, aportion (200000c.p.m.) was added to platelet-richplasma and the radioactivities of supernatants ofplatelet washings and the platelet pellets werecounted. Finally, washed platelet pellets fromnormal volunteers were resuspended in plasmaobtained from the family member with thefibrinogen Manchester variant and subjected to afurther cycle of washings, followed by h.p.l.c.analysis as described above.

H.p.l.c. techniquesThe h.p.l.c. equipment from Spectra-Physics

(St. Albans, Herts., U.K.) employed an SP8700solvent delivery system and an SP3750 organizerwith a Rheodyne 7125 injector fitted with a 1.0 mlsample loop. A Pye-Unicam LC871 UV-Visdetector with an SP4270 computing integrator wasused at 210nm and 0.08 absorbance units full-scale. The column was a Shandon (Runcorn,Cheshire, U.K.) 3 ju Hypersil ODS column(120mm x 4.6mm) with a Shandon 5 j HypersilODS pre-column (50mm x 4.6mm). The solventsand conditions for fibrinopeptide analysis were asdescribed by Kehl et al. (1981) except for the use ofsteeper elution gradient of 0% to 40% solvent Bover 25min.

Radioimmunoassay with anti-FPA seraImmunological identification of peptides in

h.p.l.c. fractions was achieved by minor modifica-tion (Lane et al., 1982) of the assay described byNossel et al. (1974), with reagents purchased fromIMCO Corp. (Stockholm, Sweden) (anti-FPA serabatch 133-8-1).

Results

Determination of concentration of extracellular andintracellular fibrinogenThe results in this section are given as the

means+S.D. of measurements from five differentwashed platelet preparations. The yield of plate-lets, expressed as the percentage recovery fromplatelet-rich plasma, was 59.5+8.9%. The finalpellet was resuspended to a platelet concentrationof approx. 1.3 x 101 2/1. Addition of 1 25I-labelledfibrinogen to platelet-rich plasma and counting ofradioactivity in platelet suspensions after washingshowed that the fibrinogen concentration externalto and associated with the platelet had beendecreased to 0.14 + 0.04% of that present in theplasma. The radioactivity in the supernatant of thefinal wash was not appreciably above backgroundvalues. From an estimated intraplatelet fibrinogen

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Fibrinogen Manchester in the intraplatelet pool

concentration of 125 ug/ 109 platelets and a plasmaconcentration of 2mg/ml this indicates a possiblecontamination by plasma fibrinogen of less than2% of the intraplatelet pool. The radioimmuno-assay for fibrinogen fragment E also revealed aprogressive decrease in supernatant fibrinogenduring platelet washing to a final concentration of1.05 ± 0.12pg/ml. The lysed platelet suspensionhad a fibrinogen content equivalent to90 + 30jpg/109 platelets. From the known volumesof supernatant and final suspension, the totalextracellular fibrinogen in the final supernatantwas calculated to be 6.2 + 2.2% of the amount inthe lysed platelet suspension. This is probably anoverestimate of plasma fibrinogen contaminationbecause of the likelihood of some platelet lysis

10 15 20Elution time (min)

Fig. 1. H.p.l.c. anali'sis of normal platelet extractsThese were analysed (a) before and (b) afterthrombin treatment. The injected sample of lysatewas derived from approx. 6 x 108 platelets. Controlfibrinopeptides (c) were 10pl of supernatant from a7.6mg/ml plasma fibrinogen solution clotted withthrombin. The identity of each fibrinopeptidespecies is indicated according to Kehl et al. (1981).

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during the washing procedure. The results of athird method of contamination assessment aredescribed below.

H.p.l.c. analysis of normal platelet IysatesThe control lysate was analysed for peptide

content by h.p.l.c. (Fig. la). After thrombintreatment of the washed platelet lysate (Fig. lb),several additional peaks were observed that wereeluted in positions identical with those of thecontrol fibrinopeptides (Fig. lc). The peak elutedat 18min may represent a peptide hydrolysed bythrombin that is unrelated to the fibrinopeptides.Sectional enlargements of the traces in Figs. l(a)and 1(b) are shown in Figs. 2(a) and 2(b). Thisclearly illustrates peaks in the thrombin-treatedlysate that are eluted in positions identical withthose of fibrinopeptides AP, A, AY and B. Themixing of 3 p1 of control fibrinopeptides with500 pl of thrombin-treated lysate caused a relativeincrease in peak height only for the fibrinopeptideslabelled in Fig. 2(b) (results not shown). The lysatewithout thrombin treatment (Fig. 2a) also con-tained small amounts of peptides eluted in the

Elution time (min)Fig. 2. Sectiotnal enlargement olh.p.l.c. traces of thrombin-

treated and non-treated platelet extractsThese are identical with traces in Figs. I (a) and 1(b)but are enlargements of the elution profiles between12min and 22min elution time.

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position of the fibrinopeptides and these may havebeen caused by small amounts of thrombin-likeproteolytic activity in the lysates.A calculation of the fibrinogen content of

washed platelet lysates was made by comparingthe FPB peak areas with known amounts of thecontrol fibrinopeptide. This gave results of109+29.4pg/109 platelets. The proportions

Table 1. Relative proportions ofjfibrinopeptides cleaved from normal plasma fibrinogen and platelet fibrinogenThese were determined with platelet lysates, purified fibrinogen from normal individuals and a commercialfibrinogen preparation from pooled blood (KabiVitrum). Peptides were quantified according to their integratedpeak areas and results are expressed as means + S.D. The fibrinopeptide AP, A and AY contents are expressed as apercentage of the total FPA.

Relative proportions of fibrinopeptides (%)

AP A AY FPA/FPB ratio

Platelets (n = 5) 28.2 + 2.5 56.9 +4.4 14.6 + 2.4 1.10 + 0.13Normal individuals (n = 18) 18.9 +4.2 67.2+ 2.4 13.2+ 3.7 1.03 +0.09KabiVitrum fibrinogen (n = 5) 20.6+ 1.2 65.9+0.9 13.7 + 1.6 1.06+0.05

15Elution time (min)

20

Fig. 3. H.p.l.c. anall'sis of' a thrombin-treated plateletIusate from a family member with fibrinogen ManchesterThis is shown in trace (a), and the fibrinopeptidesreleased from a sample of plasma fibrinogenpurified from a family member are shown in trace(b). These traces are sectional enlargements between12 min and 22 min elution time. The sample for trace(a) was derived from approx. 4 x 108 platelets, andthat for trace (b) was 1Ol of supernatant fromthrombin-treated plasma fibrinogen at a concentra-tion of 3mg/ml.

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to

4-

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:3Es

6 10 15 20Elution time (min)

Fig. 4. H.p.l.c. analysis of' the fibrinopeptides fromfibrinogen Manchester plasma fibrinogen and parallel

radioimmunoassayFor the h.p.l.c. analysis (b) approx. 3nmol offibrinopeptides was injected. Radioimmunoassaywas carried out on the h.p.l.c. fractions of 20sintervals marked with vertical lines, and theapparent FPA concentration was plotted in (a).

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ctl

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.E

Elution time (min)Fig. 5. H.p.l.c. anall.sis ol thrombin-treated platelet lysates and parallel radioimmunoassay

The h.p.l.c. trace in (a) shows part of the FPA peak from normal platelets. The vertical lines mark the fractionscollected at 20s intervals. In (b) is illustrated a plot of FPA immunoreactivity in the h.p.l.c. fractions collected fromthe experiment (a). The corresponding analysis from the platelets of the family member of fibrinogen Manchester isshown in trace (c) and the plot of FPA immunoreactivity from the h.p.l.c. fractions in (d).

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between fibrinopeptides AP, A, AY and B foundin the platelets of individuals (Table 1) weresimilar to those determined for plasma fibrinogenfrom individuals and pooled commercial fibrino-gen, except that the relative content of plateletfibrinopeptide AP was somewhat higher than thatof plasma fibrinopeptide AP.

H.p.l.c. analysis of platelet fibrinopeptides from afamily member with fibrinogen Manchester

The h.p.l.c. trace from this lysate, after throm-bin treatment, is illustrated as a sectional enlarge-ment in Fig. 3(a). The elution profiles of controlfibrinopeptides obtained from the plasma-derivedfibrinogen Manchester are shown in Fig. 3(b).These demonstrate the characteristic FPA peak-splitting caused by the presence of the [Hisl6]FPApeptide (Southan et al., 1983). The elution profileobtained from the washed, lysed and thrombin-treated platelets (Fig. 3a) contains peaks that areeluted in the same positions as fibrinopeptides[His16]A, A and B.

Radioimmunoassay of h.p.l.c. fractionsConfirmation of the identity of the platelet-

derived FPA was obtained by using a specificradioimmunoassay for this peptide. Initial calibra-tion experiments were performed with fibrino-peptides obtained from plasma fibrinogen fromthe fibrinogen Manchester family. The four majorFPA-related peaks, fibrinopeptides [His1 6]AP,AP, [His'6]A and A, as indicated on the h.p.l.c.trace in Fig. 4(b), could be clearly resolved asimmunoreactive peaks in Fig. 4(a). A lowerimmunoreactivity of [Hisl6]FPA compared withFPA has been previously reported (Lane et al.,1983). From the relative h.p.l.c. peak areas in Fig.4(b), immunoreactivity ratios for fibrinopeptidesA/[His'6]A of 2.7 :1 and for fibrinopeptidesAP/[His16]AP of 7.0:1 were calculated from Fig.4(a). The same type of analysis was then performedwith thrombin-treated platelet lysates. Normalplatelets (Fig. 5a) released two immunoreactivepeaks (Fig. 5b), whereas the equivalent h.p.l.c.traces from the fibrinogen Manchester familymember (Fig. 5c) contained four immunoreactivepeaks (Fig. 5d). The fibrinopeptide [His16]A and[His' 6]AY peaks eluted at 15.2 and 15.8minrespectively (Fig. 5c) could be positively identifiedby their lower immunoreactivity compared withthe fibrinopeptide A and AY peaks eluted at 16.2and 16.8 min respectively from the same lysate(Figs. Sc and Sd). The elution time shift of 1 minbetween these peaks is also the same as that ofplasma fibrinogen fibrinopeptides shown in Fig.4(b). The fibrinopeptide A/[His'6]A immunoreac-tivity ratio of 2.6 :1 was essentially the same as thatfound with fibrinopeptides derived from plasma

fibrinogen (Fig. 4a). These experiments thusconfirmed the identity of platelet-derived FPA and[Hisl 6]FPA.

H.p.l.c. determination ofJfibrinogen associated withplatelets

H.p.l.c. methods were applied to eliminatefurther the possible contamination by plasmafibrinogen of the intraplatelet pool. Normalwashed platelets were resuspended in plasma fromthe fibrinogen Manchester patient and then sub-jected to a further cycle of washes before h.p.l.c.analysis as described above. Any small amounts ofabnormal plasma fibrinogen adsorbed on thesurface of the normal platelets would be quantifi-able from the amounts of [Hisl6]FPA present. Ascan be seen in sectional enlargements of the h.p.l.c.

Elution time (min)

Fig. 6. H.p.l.c. traces from normalplatelets resuspended inplasma.from the affected family member with fibrinogen

ManchesterAfter a washing, the platelets were lysed and treatedwith thrombin as described in the Materials andmethods section. An enlargement of the h.p.l.c.trace is shown in (a). An elution profile of controlfibrinopeptides released by thrombin from plasmafibrinogen obtained from a member of the fibrino-gen Manchester family is shown in trace (b).

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traces (Fig. 6a), only trace amounts of peptide wereeluted in the [His'6]FPA position of the controlfibrinopeptides (Fig. 6b).

Discussion

Any study of platelet fibrinogen must include anassessment of contamination from the plasmapool. We employed three separate approaches tothis problem. The use of '251-labelled fibrinogen,added to plasma, suggested a contamination of lessthan 2% of the intracellular platelet pool. Theradioimmunoassay for fibrinogen fragment E gavea value of 6.2%, but, for the reason given in theResults section, we believe this to overestimate thecontamination. The suspension of platelets inplasma containing the congenitally abnormalfibrinogen with [His'6]FPA also suggested thatonly minimal amounts of plasma fibrinogen werecontaminating the intraplatelet pool.

Structural investigations of platelet fibrinogenhave been hindered by the problem of preparingthe protein pure and intact. In particular, un-degradedAa chains have not yet been demonstrated(Kunicki et al., 1984; Teige et al., 1985). To avoidthese difficulties, we have developed a procedurefor the direct identification of fibrinopeptides fromthrombin-treated platelet lysates by using h.p.l.c.All forms of fibrinopeptide can be detectedbecause their elution positions are identicalwith those of the fibrinopeptides derived fromplasma fibrinogen. The identity of platelet-derivedFPA was confirmed by using radioimmunoassay.These results are in agreement with the work ofDoolittle et al. (1974), who found identical aminoacid compositions for FPA and FPB isolated fromplasma fibrinogen and platelet fibrinogen. Wehave noted an increase in the proportion offibrinopeptide AP in the platelet, but the signifi-cance of this observation is uncertain at the presenttime. Measurement of the amount of the intra-platelet fibrinogen content from the amount ofFPB released was in close agreement with thatdetermined by radioimmunoassay for fibrinogenfragment E, and both values are within the range50-200 jug/109 platelets, previously reported fromdeterminations with different techniques(Niewiarowski, 1977).The results presented in this work have demon-

strated that an individual heterozygous for theAal6Arg-+His substitution in plasma fibrinogendisplays the same phenotype within the plateletfibrinogen pool. It has already been pointed out, onthe basis of precise determinations of the normaland abnormal fibrinopeptides of fibrinogensManchester (Southan et al., 1983) and Sydney I(Southan et al., 1985), that the symmetricalheterozygous phenotype (i.e. a normal/abnormal

peptide ratio of 1: 1) observed in both these cases ofan AaArg--His substitution is consistent with asingle genetic locus for the plasma Aa chain. Thedemonstration of the same symmetrical phenotypein platelet fibrinogen is further evidence that theplasma and platelet Aa chains are products of thesame gene. Additional evidence that permitsextension of this conclusion to all three genes isprovided in a study of fibrinogen variants withabnormalities of the other polypeptide chains. Thestructural defects of fibrinogen Oslo I (alteredcharge on B,B chain) and Oslo III (extended ychain) are also present in both the plasma and theplatelet fibrinogen pools (Teige et al., 1985). More-direct evidence is provided by two studies thathave mapped single copies of all three genes to aspecific locus on human chromosome 4 (Henry etal., 1984; Kant et al., 1984). Furthermore the Aachains of foetal fibrinogen, which were originallythought to be structurally distinct from the adultchains, have now been shown to have the sameprimary structure as those of adult fibrinogen(Kaiser et al., 1984).

Earlier studies in apparent contradiction withthe single-gene hypothesis include those of fibrino-gens Metz (Soria et al., 1976) and Paris I (Jandrot-Perrus et al., 1979), in which both variants werereportedly absent from the platelet pool. In thehomozygous case of fibrinogen Metz, anAal6Arg-*Cys substitution (Southan et al., 1982),the conclusions drawn from the platelet study werebased upon small differences in electrophoreticmobility, which may have been caused by theproblems of isolating intact pure fibrinogen fromplatelets. Alternative explanations may apply tofibrinogen Paris I, which has an extended y chain(Budzynski et al., 1974), and these are also relevantto three more-recent studies of the polypeptidechain composition of normal platelet fibrinogen. Ithas been concluded that the B,B and y chains ofnormal platelet fibrinogen are structurally identi-cal with those of plasma fibrinogen. However, anormal extended-y-chain variant could not bedetected in the platelet (Francis et al., 1984;Mosesson et al., 1984; Teige et al., 1985). Thenormal variant chain, termed y', comprises 5-10%of human plasma fibrinogen and has a 22-amino-acid-residue C-terminal extension produced from acommon y-chain gene by alternative mRNAsplicing (Chung & Davie, 1984; Fornace et al.,1984), which results in expression of part of theninth intron of the gene. Since Fornace et al.(1984) have postulated that the fibrinogen Paris Ichain extension may be caused by an mRNA-processing defect, tissue-specific differencesbetween the megakaryocyte and the hepatocytemay account for the absence of both fibrinogenParis I and normal y' chains from the platelet.

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Alternatively, both types of C-terminally extendedchain may be produced but not be subsequentlypackaged into the platelet ax granules because oftheir increased negative charge (Francis et al.,1984; Mosseson et al., 1984).

This work was supported by a grant from theWellcome Trust to D. A. L. We express our thanks to themember of the fibrinogen Manchester family, S. J., fordonating her blood for this work.

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Soria, J., Soria, C., Samama, M., Poirot, E. & Kling, C.(1976) Pathol. Biol. 24, 15-17

Southan, C., Henschen, A. & Lottspeich, F. (1982) inFibrinogen: Recent Biochemical and Medical Aspects(Henschen, A., Graeff, H. & Lottspeich, F., eds.), pp.153-166, Walter de Gruyter, Berlin and New York

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