Genotype at the Secretor blood group locus is a determinant

of plasma von Willebrand factor level

James O'Donnell,1 Frank E. Boulton,2 Richard A. Manning1 and Michael A. Laffan1 1Department of

Haematology, Imperial College School of Medicine, Hammersmith Hospital, London, and 2National Blood Service,

Southampton, UK

Received 2 July 2001; accepted for publication 31 August 2001

Summary. Previous reports on the effect of Secretor andLewis blood groups on plasma factor VIII±von Willebrandfactor (FVIII±VWF) levels have produced con¯icting ®nd-ings. To determine whether either or both loci can in¯uenceplasma FVIII±VWF complex levels, we studied the rela-tionship between Secretor and Lewis genotypes, determinedde®nitively using polymerase chain reaction±restrictionfragment length polymorphism analysis, and plasma FVIIIcoagulant activity (FVIII:C) and VWF antigen (VWF:Ag)levels in 136 healthy volunteers. Overall, signi®cantlyhigher VWF:Ag levels were found in those individualshomozygous for the Se allele (genotype SeSe) than in thoseheterozygous for the Se allele (P < 0á001). To minimize anyconfounding in¯uence of ABO genotype/phenotype, weinvestigated the relationship between Secretor genotype and

plasma FVIII±VWF levels within individuals of the sameABO blood group genotype. In the subgroup analysis ofgroup O1O1 individuals alone, VWF:Ag levels were againsigni®cantly higher in those individuals with Secretorgenotype SeSe than in those either heterozygous or homo-zygous for the se null allele. Among A1O1 subjects, homo-zygous Secretors also had signi®cantly higher VWF:Aglevels. In contrast, we found no relationship between Lewisgenotype and either VWF:Ag or FVIII:C levels. This study isthe ®rst based on genotypic rather than serological analysis,and resolves the previously confounding effects of the Lewisand Secretor loci on plasma FVIII±VWF complex levels.

Keywords: Lewis and Secretor blood groups, von Willebrandfactor, factor VIII.

Factor VIII (FVIII) and von Willebrand factor (VWF) areplasma glycoproteins that perform vital roles in normalhaemostasis (Sadler, 1998). In plasma, FVIII and VWFcirculate as a non-covalent complex (Vlot et al, 1998). VWFacts as a carrier molecule for FVIII, protecting it frompremature dissociation or degradation (Vlot et al, 1996).The normal population distribution of plasma FVIII±VWFlevels shows a wide range, with a skewed distributiontowards higher levels (Gill et al, 1987). Plasma FVIII±VWFlevels are of clinical signi®cance. Low plasma levels of eitherFVIII (Haemophilia A) or VWF (von Willebrand disease)have long been recognized as causes of excess bleeding(Nichols & Ginsberg, 1997). Conversely, there is recentevidence that elevated FVIII±VWF levels may represent animportant and prevalent risk factor for both ischaemicheart disease (Rumley et al, 1999) and venous thrombo-embolism (Koster et al, 1995; Kyrle et al, 2000). It istherefore of interest to determine which genes in¯uence

plasma FVIII±VWF levels and to understand the mechanismby which these genes act.

It is well established that gene loci other than the FVIIIgene (Xq28) and the VWF gene (12p12) can exert majorquantitative effects on plasma FVIII±VWF complex levels.The most important of these loci is the ABO blood grouplocus on chromosome 9q34 (Shima et al, 1995). VWFantigen (VWF:Ag), ristocetin cofactor activity and botro-cetin cofactor activity (Gill et al, 1987; Shima et al, 1995),and FVIII coagulant activity (FVIII:C) and FVIII antigen(FVIII:Ag) (McCallum et al, 1983) are both approximately25% lower in group O individuals than in non-O individuals(groups A, B and AB). Effects of the Secretor blood grouplocus on plasma concentration of FVIII±VWF have also beenreported, albeit inconsistently (Orstavik et al, 1989; Greenet al, 1995). Furthermore, a relationship between Lewisblood group phenotype and FVIII±VWF levels has beenrecently described (Green et al, 1995).

Similar effects from the ABO, Lewis and Secretor bloodgroup systems are plausible because they are closely related(Watkins, 1996). All three systems are characterized by thepresence or absence of speci®c terminal carbohydrate deter-minants on the oligosaccharide structures of glycoproteins

Correspondence: Dr James O'Donnell, Department of Haematology,ICSM, Hammersmith Hospital, Du Cane Road, East Acton, London

W12 ONN, UK. E-mail: james.o'donnell@virgin.net

British Journal of Haematology, 2002, 116, 350±356

350 Ó 2002 Blackwell Science Ltd

or glycolipids. In the case of Secretor, blood group O (H) andLewis, the terminal sugar is a fucose. The Secretor bloodgroup locus maps to chromosome 19q13 and has recentlybeen cloned (Kelly et al, 1995). The gene encodes an a(1,2)fucosyltransferase (FUT2) similar to that encoded by theclosely related H gene locus (FUT1) necessary for expressionof ABO blood groups. Individuals with an Se allele (genotypesSeSe or Sese respectively) are Secretors, and can secrete ABHand Lewis structures in their plasma and secretions.Approximately 20% of Caucasians have the genotype sese,and are non-Secretors (Kukowska-Latallo et al, 1990). Themolecular basis underlying polymorphism at the Secretorlocus has recently been established. In Caucasians, these null allele results from an enzyme inactivating pointmutation (W143X; Trp 143 ® ter) (Kelly et al, 1995;Vestergaard et al, 1999).

The Lewis blood group maps to chromosome 19p, andencodes an a(1,3) fucosyltransferase (FUT3) which canmodify secreted sugar chains (Orntoft et al, 1997). Indi-viduals carrying an Le allele express this glycosyltransfer-ase, while individuals homozygous for le (genotype lele) donot. In Caucasians, two common mutations (T59G and theC314T-T202C haplotype respectively) have been shown toproduce an le null allele. C314T and T202C are incomplete linkage disequilibrium but virtually never occuron the same allele as the T59G (Elmgren et al, 1996;Orntoft et al, 1997). The molecular genetic mechanismbehind non-functional Lewis alleles is not fully understood,but these mutations appear to cause changes in pFUT3membrane insertion and folding respectively (Elmgrenet al, 1996).

Lewis blood group antigens (Lea and Leb respectively) areproduced by interaction of FUT2 and FUT3 (Watkins,1980). Individuals expressing FUT 3 (LeLe or Lele at theLewis locus) can synthesize Lea structures. Those individualsalso expressing FUT 2 (SeSe or Sese at the Secretor locus) canconvert Lea into Leb. Consequently, individuals with red cellphenotype Le(a+b±) are non-Secretors, while individualswith phenotype Le(a±b+) are Secretors. Lewis-negativeindividuals Le(a±b±) may or may not be Secretors.

Previous reports on the effect of Secretor and Lewis bloodgroups on plasma FVIII±VWF levels have producedcon¯icting ®ndings, strongly dependent on ABO bloodgroup (Orstavik et al, 1989; Green et al, 1995). However,these studies used serologically determined red blood cellLewis phenotypes to infer probable Lewis and Secretorgenotypes respectively. Recent evidence has shown thisapproach to be unreliable (Henry et al, 1995; Svenssonet al, 2000). Moreover it does not permit discernment ofdosage effects. To determine whether either or both loci canin¯uence plasma FVIII±VWF complex levels, we studied therelationship between Secretor and Lewis genotypes, deter-mined de®nitively using polymerase chain reaction±restric-tion fragment length polymorphism (PCR±RFLP) analysis,and plasma FVIII:C and VWF:Ag levels in 136 healthyvolunteers. The validity of this genotyping methodology inEuropean Caucasians has previously been established(Vestergaard et al, 1999).

MATERIALS AND METHODS

Sample collectionOne hundred and thirty-six healthy volunteers wererecruited from routine blood donors at the Wessex RegionalTransfusion Centre, Southampton, UK. All donors wereeither blood group A or O and aged between 18 and70 years. Each donor provided written informed consent.Blood was collected from the antecubital vein into Becton-Dickinson (Oxford, UK) VacutainerÒ tubes containing0á105 mol/l trisodium citrate. Platelet-poor plasma wasobtained by centrifugation at 2000 g for 20 min, within90 min of collection. The plasma was then aliquoted andstored at )70°C.

ABO, Secretor and Lewis genotypingABO genotyping. For each individual the red blood cell

ABO phenotype was con®rmed by routine serologicaltesting, using monoclonal anti-A and -B respectively (Bio-test AG, Solihull, UK). The ABO genotype was alsoestablished. Genomic DNA was extracted from 1 ml ofcitrated whole blood using a commercial kit (Scotlab, Luton,UK). ABO genotyping was performed by PCR ampli®cationof exons 6 and 7 of the ABO gene, followed by diagnosticrestriction enzyme digestion, as previously described(O'Donnell et al, 2000).

Secretor genotyping. A single fragment of genomic DNAspanning the W143X Secretor polymorphism was ampli®edby PCR using the following primer pair (Sigma-Genosys,Pampisford, UK): SEC2P2 (5¢-ATGGACCCCTACAAAGGTG-CCCGGCCGGCT-3¢); and SEC2P3 (5¢-GAGGAATACCGCCA-CATCCCGGGGGAGTAC-3¢).

The PCR protocol was the same as previously described,with an annealing temperature of 64°C. Restriction enzymedigestion was performed on a 25-ll aliquot of each PCRproduct, using 20 units of the enzyme Ava II.

Lewis genotyping. Two fragments of genomic DNA wereampli®ed by PCR, each spanning one of the two Lewispolymorphisms. Primer sequences (Sigma-Genosys) were asfollows:

Lewis fragment 1 (including T59G polymorphism): VE1MHS (5¢-CCATGGCGCCGCTGTCTGGCCGCCC-3¢); and EL3(5¢-GGGAGTGGTGTCCTGTCGGGAGGACGGACT-3¢).

Lewis fragment 2 (including C314T polymorphism):VE1 MHS (5¢-CCATGGCGCCGCTGTCTGGCCGCCC-3¢); andVE4AS (5¢-GTTGGACATGATATCCCAGTGGTGCACGAT-3¢).

The PCR protocol was as previously described, with 10%dimethyl sulphoxide (DMSO, Sigma). Restriction enzymedigestion was performed on a 25-ll aliquot of each PCRproduct, using 20 units of either Msp I (fragment 1) or NlaIII (fragment 2).

VWF:Ag and FVIII:CPlasma VWF:Ag levels were determined using a standardsandwich enzyme-linked immunosorbent assay (ELISA)technique as previously described (O'Donnell et al, 1997).All samples were tested in duplicate at three differentdilutions. Dilutions of 100% reference plasma (VWF:Ag

Secretor Blood Group/Von Willebrand Factor 351

Ó 2002 Blackwell Science Ltd, British Journal of Haematology 116: 350±356

1á05 iu/ml; Immuno, Vienna, Austria) were used to con-struct standard curves for calibration. Plasma FVIII:C levelswere measured by the one-stage clotting method using aFVIII-de®cient substrate (Immuno), as previously described(O'Donnell et al, 1997).

A and H blood group antigenic determinants on VWFA (N-acetylgalactosaminyl a-1 ® 3[fucosyl a-1 ® 2]galactose) antigenic determinants on plasma VWF weremeasured using a modi®ed sandwich ELISA. ELISA plateswere coated with rabbit anti-human VWF antibody (Dako),diluted 1:500 in 0á05 mol/l (pH 9á6) carbonate buffer,overnight at room temperature. After washing with Tris-buffered saline (TBS) containing 0á05% Tween, the plateswere blocked using TBS containing 1% bovine serumalbumin (Sigma). After three further washes, the plasmasamples were added to the wells and incubated for 2 h atroom temperature. Each plasma was tested in duplicate atthree dilutions. The plates were washed with TBS/Tweenand then incubated with murine anti-A monoclonal anti-body (Ortho Diagnostics, Raritan, New Jersey, USA), diluted1:10 in TBS, for 1 h. After a further three washes, the plateswere incubated with goat anti-mouse IgM peroxidaseconjugate (Sigma), diluted 1:1000 in TBS, for 1 h. Afteranother TBS/Tween wash, peroxidase substrate solutionwas added and incubated in the dark for 20 min. Thereaction was stopped with 1 mol/l H2SO4 after 3 min, andthe optical density measured at wavelength 492 nm usingan ELISA reader. Pooled group A plasma was assayed toproduce a standard curve for each ELISA. The poolednormal A plasma was assigned a value of 1 u/ml for theamount of A antigen expressed per unit of VWF.

H (fucosyl a-1 ® 2 galactose) antigenic determinants onplasma VWF were measured using a similar modi®ed ELISAmethodology. After incubation with the plasma samples, theplates were washed and incubated with biotin-conjugatedUlex europaeus (Vector Laboratories, Peterborough, UK)diluted 1:500 for 1 h. After further washing, the plateswere then incubated with streptavidin conjugated to peroxi-dase (Vector Laboratories) diluted 1:1000 for 45 min. Pooledgroup O plasma was assayed to produce a standard curve.The pooled normal O plasma was assigned a value of 1 u/mlfor the amount of H antigen expressed per unit of VWF.

RESULTS

ABO, Secretor and Lewis blood group genotype distributionsWe investigated a total of 136 normal Caucasian blooddonors. Previous serological testing had shown that all wereeither blood group A or O. The ABO genotype distribution ofthese individuals was as follows (9 A1A1; 53 A1O1; 13 A2O1

and 61 O1O1). The Secretor and Lewis blood group genotypedistributions are shown in Tables 1A and B respectively. Atthe Secretor locus, the allele frequencies were 54á8% for thewild-type and 45á2% for the W143X allele. At the Lewislocus, the allele frequencies were 73á2% for the wild-typeallele, 11á0% for the T59G allele and 15á8% for the C314Tallele. These frequencies are consistent with those previ-ously reported in Caucasian populations (Vestergaard et al,

1999; Svensson et al, 2000) and are close to Hardy±Weinberg equilibrium.

ABO genotype and plasma VWF:Ag/FVIII:Ag levelsIn keeping with previous reports, VWF:Ag and FVIII:C levelswere higher in A1A1 individuals (means 109á8 and 175 iu/dlrespectively) than in A1O1 (means 97á3 and 168 iu/dlrespectively). Moreover VWF:Ag and FVIII:C were bothsigni®cantly higher in A1O1 than either A2O1 or O1O1

individuals (VWF:Ag: P � 0á03 and P < 0á01; FVIII:C:P � 0á02 and P � 0á01: Mann±Whitney). There was nosigni®cant difference in VWF:Ag or FVIII:C level betweenA2O1 and O1O1 individuals.

Secretor genotype and plasma VWF:Ag/FVIII:C levelsThe effect of Secretor genotype on plasma FVIII-VWFconcentrations was initially studied across all A and Ogenotypes. Over all groups considered together, signi®cantlyhigher VWF:Ag levels were found in those individualshomozygous for the Se allele (genotype SeSe) than in thoseheterozygous for the Se allele; P < 0á001 (Fig 1).

ABO blood group phenotype and genotype are known toexert a major in¯uence on plasma FVIII±VWF levels.Furthermore, the A and B transferases can modify the Hsubstance produced by the Secretor gene product. Tominimize any confounding in¯uence of ABO genotype/phenotype, we also investigated the relationship betweenSecretor genotype and plasma FVIII±VWF levels withinindividuals of the same ABO blood group genotype. In thesubgroup analysis of group O1O1 individuals alone, VWF:Ag

Table I. A. ABO and Secretor genotype distributions deter-

mined using polymerase chain reaction±restriction frag-

ment length polymorphism (PCR±RFLP) analysis.

ABO genotype

Secretor genotype

SeSe Sese sese Total

A1A1 3 4 2 9A1O1 17 25 11 53

A2O1 4 9 0 13

O1O1 16 31 14 61Total 40 69 27 136

B. ABO and Lewis genotype distributions determined byPCR±RFLP analysis.

ABO genotype

Lewis genotype

LeLe Lele lele Total

A1A1 6 2 1 9

A1O1 25 24 4 53

A2O1 5 6 2 13O1O1 40 15 6 61

Total 76 47 13 136

352 J. O'Donnell et al

Ó 2002 Blackwell Science Ltd, British Journal of Haematology 116: 350±356

levels were again signi®cantly higher in those individualswith Secretor genotype SeSe than in those either heterozy-gous or homozygous for the se null allele (Fig 2, Table 2A).Similarly, among subjects with genotype A1O1, homozygousSecretors had signi®cantly higher plasma VWF:Ag levelsthan heterozygous Secretors (Table 2B). The A1A1 and A2O1

subgroups were too small (n � 9 and n � 13 respectively)to permit meaningful subgroup analysis.

Despite the relationship between VWF:Ag levels andhomozygosity for the Se allele at the Secretor locus, nodifference in VWF:Ag levels was found between thoseheterozygous for the Se allele and those individuals homo-zygous for the se allele. This ®nding was consistent in thegroup as a whole, and in the separate ABO genotypesubgroup analyses. Although FVIII:C levels were alsoconsistently highest in SeSe individuals, this trend failed toreach statistical signi®cance in any ABO group.

Lewis genotype and plasma VWF:Ag/FVIII:C levelsThe effect of Lewis genotype on plasma FVIII±VWF levelswas also investigated. Overall, there was no relationshipbetween Lewis genotype and either VWF:Ag or FVIII:Clevels. Moreover, within the O1O1 and A1O1 subgroups,

Lewis genotype did not signi®cantly in¯uence VWF:Ag orFVIII:C plasma concentrations (Tables 3A and 3B respec-tively).

Secretor genotype and the amount of N-linked ABHdeterminant expressed on circulating VWFABH antigenic determinants have been identi®ed on theN-linked glycans of circulating VWF and FVIII. We havepreviously shown that the amount of H structure expressedon VWF is a major determinant of plasma VWF levels. Toinvestigate whether Secretor genotype in¯uences plasmaVWF:Ag levels by altering the amount of ABH expressed oncirculating VWF, we have quanti®ed the amount ofN-linked ABH structure present on VWF using a modi®edsandwich ELISA. In group O1O1 individuals, the amount ofH antigen expressed per unit of VWF (HVWF) did not varybetween the different Secretor genotypes. Furthermore,among A1O1 individuals, the amount of A antigenicdeterminant expressed per unit of VWF was not in¯uencedby Secretor genotype (data not shown).

DISCUSSION

Orstavik et al (1989) were the ®rst to describe an effect ofSecretor and Lewis blood groups on plasma concentrations of

Fig 1. Plasma VWF:Ag levels against Secretor genotype. Plasma

VWF:Ag levels were measured using enzyme-linked immunosor-bent assay (ELISA) in 136 healthy volunteers and the Secretor

genotype of each subject was determined using polymerase chain

reaction (PCR) ampli®cation of genomic DNA. The mean VWF:Ag

level for each genotype is shown. VWF:Ag levels were signi®cantlyhigher in SeSe than in Sese individuals.

Fig 2. Plasma VWF:Ag levels against Secretor genotype in groupO1O1 individuals. Plasma VWF:Ag levels were measured using

enzyme-linked immunosorbent assay (ELISA) in 136 healthy

volunteers and the Secretor and ABO genotypes of each subject were

determined using polymerase chain reaction (PCR) ampli®cation ofgenomic DNA. The mean VWF:Ag level for each genotype is shown.

VWF:Ag levels were signi®cantly higher in SeSe than Sese or sese

individuals.

Secretor Blood Group/Von Willebrand Factor 353

Ó 2002 Blackwell Science Ltd, British Journal of Haematology 116: 350±356

VWF±FVIII. In a study of 323 twins and 58 blood donors,they found signi®cantly higher VWF:Ag and FVIII:Ag levelsin group O individuals with the red cell phenotype Le(a+b±)than in group O individuals with phenotypes Le(a±b+) orLe(a±b±) respectively. However, this relationship was notobserved among the non-O (A, B and AB respectively)individuals studied. Moreover, because this was a serolog-ically based study, 80% of those with Le (a±b±) phenotypewould have been Secretors, with H substance present inplasma.

Green et al (1995) also reported an association betweenred cell Lewis phenotype and plasma FVIII±VWF levels. Inhealthy white men with blood group A, B or AB, both FVIIIand VWF:Ag levels were signi®cantly higher in those withLewis phenotype Le(a±b±) than in Le(a+b±) and Le(a±b+)individuals. This effect of Lewis status on FVIII±VWF levelswas not reproduced in group O individuals, women or blackmen. Moreover, the widely cited conclusion of Orstavik et al

(1989) that higher levels of FVIII±VWF are present in groupO individuals with Lewis phenotype Le(a+b±) was notveri®ed by this much larger study.

The discrepant ®ndings of these two studies may in partbe attributable to differences in the populations studied.However, both groups also determined Lewis and Secretorstatus by routine serological testing, making it dif®cult todisentangle the separate effects of the Lewis and Secretorgene products which are obscured in such tests. Moreover,recent reports have demonstrated that such serologicaldeterminations can themselves be unreliable, particularly inblood group A1 individuals and in those of non-Europeanorigin (Henry et al, 1995; Svensson et al, 2000). Even inEuropean populations, the use of serological Lewis pheno-typing to deduce Secretor status has been shown to produceerroneous results in up to 10% cases (Svensson et al, 2000).With the recent description of the molecular bases under-lying polymorphisms at both the Lewis and Secretor blood

Table II. A. Relationship between Secretor genotype and plasma VWF:Ag and FVIII:C levels in group O1O1 individuals.

Secretor genotype nPlasma VWF:Ag

(mean � 1SD iu/dl) (mean � 1SD iu/dl) Plasma FVIII:C

SeSe 16 89á1 � 21á6 P � 0á02 145 � 41á0 P � 0á18

Sese 31 71á5 � 27á1 P � 0á04 128 � 33á1 P � 0á10sese 14 72á8 � 20á9 P � 0á86 124 � 21á0 P � 0á68

B. Relationship between Secretor genotype and plasma VWF:Ag and FVIII:C levels in group A1O1 individuals.

Secretor genotype nPlasma VWF:Ag

(mean � 1SD iu/dl) (mean � 1SD iu/dl) Plasma FVIII:C

SeSe 17 108á7 � 22á9 P � 0á01 174 � 35á0 P � 0á28

Sese 24 87á3 � 22á6 P � 0á58 159 � 51á0 P � 0á83sese 12 102á6 � 27á5 P � 0á16 177 � 41á0 P � 0á27

Table III. A. Relationship between Lewis genotype and plasma VWF:Ag and FVIII:C levels in group O1O1 individuals.

Secretor genotype nPlasma VWF:Ag

(mean � 1SD iu/dl)

Plasma FVIII:C

(mean � 1SD iu/dl)

LeLe 40 73á0 � 25á3 P � 0á31 134 � 38á0 P � 0á63

Lele 15 81á6 � 27á1 P � 0á16 126 � 24á9 P � 0á93lele 6 86á5 � 16á9 P � 0á78 130 � 23á4 P � 0á76

B. Relationship between Lewis genotype and plasma VWF:Ag and FVIII:C levels in group A1O1 individuals.

Secretor genotype nPlasma VWF:Ag

(mean � 1SD iu/dl)

Plasma FVIII:C

(mean � 1SD iu/dl)

LeLe 25 102á1 � 26 P � 0á58 173 � 43á9 P � 0á66

Lele 24 91á3 � 24á6 P � 0á64 160 � 44á4 P � 0á68lele 4 104á3 � 20á5 P � 0á95 179 � 49á4 P � 0á91

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354 J. O'Donnell et al

Ó 2002 Blackwell Science Ltd, British Journal of Haematology 116: 350±356

group loci, accurate genotyping is now possible and iswidely regarded as de®nitive (Vestergaard et al, 1999). Notonly is this approach free from the above pitfalls of serology,it allows the assessment of dosage effects at the loci, lendingpower to the analyses. We have therefore used a genotypingstrategy to de®nitively determine whether the Secretor orLewis loci can exert quantitative effects on plasma FVIII±VWF levels.

Our results clearly demonstrate that genotype at theSecretor blood group locus is a determinant of plasmaVWF:Ag concentration. Among group O1O1 subjects, thoseindividuals homozygous for the Se allele (homozygousSecretors) had VWF:Ag levels signi®cantly higher thanthose individuals with genotypes Sese or sese (P � 0á02and P � 0á04 respectively). Moreover, in contrast to previ-ous reports linking Secretor status and FVIII±VWF levels(Orstavik et al, 1989), we found this relationship to be alsopresent in A1O1 individuals, and to remain when both Aand O were considered as one group. Although FVIII:Clevels also demonstrated a trend towards highest levels inSeSe individuals, this association failed to reach statisticalsigni®cance, suggesting that any effect of Secretor genotypeon FVIII levels is probably secondary to a direct effect onVWF:Ag levels.

It is important to note that this effect would not havebeen detected by a serological analysis because Se hetero-zygotes and homozygotes would have been grouped togeth-er as Secretors. The lack of a dominant Se effect is somewhatsurprising and leads us to conclude that it represents adosage effect such that the products of two functional Segenes are required to in¯uence VWF levels.

The mechanism by which Secretor genotype in¯uencesplasma VWF:Ag levels remains unclear. Theoretically,Secretor status may alter the rate of VWF synthesis orsecretion within endothelial cells. Alternatively, Secretorstatus may affect VWF plasma clearance rates. We havepreviously found a close association between the amount ofH substance expressed on circulating VWF and plasmaVWF:Ag concentration, suggesting that terminal fucoseresidues may be important in mediating VWF clearance(unpublished observations). In homozygous Secretors,plasma H substance could theoretically compete with theH determinants expressed on the N-linked glycans of VWFfor this clearance mechanism.

It is also possible that that the relationship betweenSecretor locus and plasma VWF levels is not owing to adirect functional effect of the Secretor gene. Rather theSecretor locus may exist in linkage disequilibrium withanother unidenti®ed VWF regulatory locus. The proximityof the Secretor (FUT2) gene to the H (FUT1) gene at 19qraises the possibility that the Se gene is in linkagedisequilibrium with a quantitative variant at the latterlocus. If this were the case then we would expect theamount of H antigen on VWF in group O individuals to varybetween Secretors and non-Secretors. We therefore proceed-ed to assess this variable but found no difference betweenthe groups.

In contrast to the in¯uence of Secretor genotype onplasma VWF:Ag levels, we found no relationship between

Lewis genotype and plasma FVIII±VWF levels. The previ-ously identi®ed association between Le(a±b±) and increasedFVIII±VWF was not reproduced. Previous studies may havebeen confounded by the inability to detect the Secretor statusof these individuals. However, the number of individualswith genotype lele in our Caucasian population waspredictably low (13/136, 9á6%).

In conclusion, we have shown that individualshomozygous for the functional Se gene have higher plasmalevels of VWF and that there is no effect from Lewisgenotype. The Secretor effect is seen in both blood group Aand O individuals. This study is the ®rst based on genotypicrather than serological analysis and resolves the confound-ing effects of the Lewis and Secretor loci interaction that maybe responsible for the previously con¯icting reports of thisphenomenon. We postulate that the effect is mediated bycompetition for clearance mechanisms between H substanceon VWF and that in plasma.

ACKNOWLEDGMENTS

The authors would like to thank Professor W. M. Watkinsand Dr J. L. Clarke (Imperial College School of Medicine,London) and the staff of the Wessex Regional TransfusionCentre, Southampton for their help.

James O'Donnell was sponsored by a Medical ResearchCouncil Training Fellowship award.

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