Von Willebrand Factor But Not α-Thrombin Binding to Platelet Glycoprotein Ibα Is Influenced by the HPA-2 Polymorphism
Objective— Glycoprotein (GP) Ibα is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex. The N-terminal domain of the GPIbα chain contains binding sites for α-thrombin and von Willebrand factor (VWF). The human platelet alloantigen (HPA)-2 polymorphism of the GPIbα gene is associated with a C/T transition at nucleotide 1018, resulting in a Thr/Met dimorphism at residue 145 of GPIbα. To study the structural and functional effects of this dimorphism, N-terminal fragments (AA1-289) of the HPA-2a and HPA-2b alloform of GPIbα expressed in CHO cells were used.
Methods and Results— Of 74 moAbs directed against human GPIbα, 2 antibodies with epitope between AA1-59 could differentiate between both alloforms. In addition, VWF bound with a higher affinity to the recombinant HPA-2a fragment or to homozygous HPA-2a platelets. In contrast, no difference was found in the binding of α-thrombin to the recombinant alloform fragments or of antibodies directed against the α-thrombin binding anionic sulfated tyrosine sequence (AA269-282).
Conclusions— Whereas the Thr145Met dimorphism does not affect α-thrombin binding, it does influence the conformation of the N-terminal flanking region and first leucine-rich repeat of GPIbα and by this has an effect on VWF binding.
The platelet glycoprotein (GP) Ib-IX-V complex has a vital role in primary hemostasis by mediating initial platelet adhesion to von Willebrand factor (VWF) bound to the subendothelium under conditions of high shear stress. This interaction leads to the transient capture of flowing platelets1 and triggers activation of platelet GPIIb/IIIa,2 leading to irreversible platelet adhesion and subsequent thrombus growth.3 In the absence of high shear stress, VWF is not able to interact with GPIb-IX-V. However, binding can be initiated in vitro by the nonphysiological modulators botrocetin4 and ristocetin.5 The physiological shear-dependent interactions between VWF and GPIb-IX-V more closely correlate with ristocetin-dependent rather than botrocetin-dependent binding under static conditions.6 Within GPIb-IX-V, the GPIbα polypeptide is the functionally dominant subunit of the receptor complex with binding sites for VWF and α-thrombin in its N-terminal domain (AA1-282).7,8⇓ At least 3 regions in the N-terminal domain of GPIbα are important for VWF binding9,10⇓: the region encompassed by the sulfated tyrosine sequence (Asp269-Glu282), the disulphide loop between Cys209-Cys248, and the N-terminal flanking/leucine-rich repeat region. The major thrombin recognition sequence is also located in the sulfated tyrosine sequence.11
Three polymorphisms are associated with GPIbα; the first is a single nucleotide substitution (T/C) in the Kozak sequence,12 and a second is associated with variations in the number of 13 amino acid tandem repeats (1 through 4) (VNTR) in the macroglycopeptide region.13 Finally, the third polymorphism, the human platelet antigen-2 (HPA-2) system, is characterized by a C/T transition at nucleotide 1018, resulting in an amino acid dimorphism (Thr/Met) at residue 145.14 The HPA-2 system is strongly linked to the VNTR polymorphism. Alleles with 1 or 2 repeats are linked to the Thr-isoform (HPA-2a allele), whereas alleles with 3 or 4 repeats are linked to the Met-isoform (HPA-2b allele).15 The HPA-2a and HPA-2b alleles have respective allelic frequencies in the white population of approximately 0.89 and 0.11.16
The HPA-2 system is of clinical interest, because platelet-specific alloantibodies attributable to exposure to nonself alloforms have been implicated in the pathogenesis of neonatal alloimmune thrombocytopenic purpura, posttransfusion purpura, and refractoriness to HLA-matched platelet transfusion.17 Recently, several clinical studies were performed to determine whether the HPA-2 system is associated with an increased risk for thrombosis. Although earlier studies failed to demonstrate the HPA-2a/2b or HPA-2b/2b genotype as a risk factor,18,19⇓ more recent ones found an association of the HPA-2a/2b or HPA-2b/2b genotype with ischemic stroke20 or cardiovascular diseases.21–23⇓⇓
Because the HPA-2 polymorphism is located close to the VWF- and the thrombin-binding sites, this amino acid dimorphism could cause a conformational variation in the structure of GPIbα that might affect the ligand binding. To be able to study the putative structural or functional effects of the HPA-2 system without the influence of the VNTR polymorphism, soluble HPA-2a and HPA-2b GPIbα fragments were expressed in CHO cells and screened with a panel of anti-GPIbα antibodies, and the binding to VWF and α-thrombin was studied and compared with HPA-2 typed platelets.
The plasmid pcDNA3,24 containing the full-length GPIbα gene (HPA-2a allotype), was a kind gift of Dr K.J. Clemetson (Theodor Kocher Institut, Bern, Switzerland). The CHO/dhfr− cell line and the pSV2-dhfr plasmid were from ATCC. HPA-2a2a and HPA-2b2b platelets were purchased from the National Institute for Biological Standards and Controls, Potters Bar, UK. Of a panel of 74 anti-GPIbα monoclonal antibodies (moAbs), 21 moAbs recognize the N-terminal (1 to 289) part of GPIbα, of which moAbs 2D4, 12G1, 27A10, 24G10, 6B4, and 12E4 were described previously.25,26⇓ Anti-GPIIb/IIIa moAb 16N7C2 was prepared by immunizing mice with human platelets as described.27 The anti-GPIbα moAbs LJ-Ib1 and LJ-Ib10 were a kind gift of Dr Z.M. Ruggeri (Scripps Research Institute, La Jolla, Calif).28 The anti-GPIbα moAb TM60 was a kind gift of Dr N. Yamamoto (Department of Hematology and Oncology, Hiroshima University, Japan).8 Botrocetin was purified from crude Bothrops jararaca venom (Sigma) as described.29
Construction of Expression Vectors
The DNA fragment, coding for ΑΑ1-289 of GPIbα containing the HPA-2a alloform (rHPA-2a), was amplified by polymerase chain reaction from the plasmid pcDNA3. The DNA fragment, coding for the rHPA-2b, was constructed through overlap polymerase chain reaction. Both DNA fragments were ligated in pCMV-Script (Stratagene). DNA sequence analysis was performed to confirm the correct orientation and sequence of the inserts.
Expression of rHPA-2a and rHPA-2b
CHO/dhfr− cells were cotransfected with the plasmids and the pSV2-dhfr plasmid using lipofectin (Invitrogen).30 Transfected cells were subcloned, and in the positive clones, identified in a sandwich immunoassay, DNA fragments were amplified by stepwise increasing concentrations of methotrexate (10 nmol/L to 10 μmol/L; Sigma).
Culture medium was concentrated using a CH2PR Concentrator S1Y3 (Amicon), and rHPAs were purified using an affinity column coupled with the moAb 12E4.26
Purified rHPAs were separated on a 7.5% SDS-polyacrylamide gel under reducing conditions, transferred to a nitrocellulose membrane (Schleicher & Schuell), and detected with biotinylated anti-GPIbα (b-anti-GPIbα) moAb 27C11, 24G10, or 14B11, followed by horseradish peroxidase (HRP)-labeled streptavidine (streptavidin-HRP) (Roche Molecular Biochemicals). Visualization was performed with ECL reagent (Amersham Life Science).
Indirect moAb-Specific Immobilization of Platelet Antigens Assay
Human serum containing anti-HPA-2b alloantibody was incubated with 100 to 300 ng of rHPA-2a or rHPA-2b. Subsequently, HPA-2a2b and HPA-2b2b platelets were incubated with the preabsorbed serum in the presence of anti-GPIX moAb FMC25 (CLB, Amsterdam) for 30 minutes at 37°C. Platelets were washed and then lysed in 0.5% Triton X-100 (Sigma). After centrifugation, the supernatants were diluted 1:5 and transferred to microtiter plates coated with goat anti-mouse IgG (Dianova, Hamburg, Germany). Bound human antibodies were detected with goat anti-human IgG-HRP (Dianova).31,32⇓
Binding of Anti-GPIbα moAbs to rHPAs
Microtiter plates (Greiner) were coated with the panel of 21 anti-GPIbα (1 to 289) moAbs and incubated with a dilution series of rHPA-2a or rHPA-2b. Bound rHPAs were detected with b-6B4 and b-12G1, followed by streptavidin-HRP.
Ristocetin- and Botrocetin-Induced Binding of VWF to the rHPAs
Microtiter plates coated with moAb 2D4 were incubated with a dilution series of rHPA-2a or rHPA-2b followed by either 1/32 diluted plasma containing (1) 760 μg/mL ristocetin (abp) or (2) 1500 μg/mL ristocetin with 0.5 μg/mL purified VWF (Red Cross) containing 400 μg/mL ristocetin or with 1 μg/mL VWF containing 0.1 μg/mL botrocetin. Bound VWF was detected with anti-VWF-Ig-HRP (Dako).26
Apparent dissociation constants (Kd,app) were determined by fitting the data to the specific binding model characterized by the following equation: y=Bmax.x/(Kd,app+x), where y represents the absorbance at 490 nm and x the rHPA-2a or rHPA-2b concentration.33
Binding of α-Thrombin to the rHPAs
The binding of α-thrombin (Kordia) to the rHPAs was determined according to a method adapted from De Cristofaro et al.34 Microtiter plates coated with 12G1 were incubated with rHPA-2a or rHPA-2b and next with a dilution series of α-thrombin. Bound α-thrombin was visualized with the chromogenic substrate S-2238 (Chromogenix). Kd,app was determined as described above.
Botrocetin-Induced Binding of VWF to HPA-2a2a and HPA-2b2b Platelets
Microtiter plates were coated with 16N7C2 and incubated with washed HPA-2a2a and HPA-2b2b platelets. Next, wells were incubated with a dilution series of VWF containing 0.1 μg/mL botrocetin or with a dilution series of b-27C11. Bound VWF was detected with anti-VWF-Ig-HRP and bound b-27C11 was detected with streptavidin-HRP as described above. Kd,app was determined and normalized as above.
Cross-Blocking Analysis for moAb Binding to Human Platelets
Microtiter plates were coated with poly-l-lysine (Sigma); platelets were added, fixed with 0.5% formaldehyde (Merck), and incubated with a dilution series of the anti-GPIbα moAbs. b-anti-GPIbα moAb 27C11 or 14B11 was added to determine their residual binding with streptavidin-HRP.
Construction and Expression of rHPA-2a and rHPA-2b
Expression plasmids coding for the first 289 amino acids of either the rHPA-2a or rHPA-2b allotype were constructed and used to transfect CHO cells. Stable cell lines were established and rHPAs were purified by affinity chromatography. Purity of both rHPAs was approximately 90%, as determined by SDS-PAGE and silver stain (not shown). Both fragments had the expected molecular mass of ≈42 kDa (Figure 1). rHPA-2b competed dose dependently with HPA-2a2b and HPA-2b2b platelets for the binding of anti-HPA-2b antiserum, whereas rHPA-2a had no effect, demonstrating that both recombinant GPIbα alloforms are allele specific (Figure 2). Furthermore, binding of VWF (Figure 3A) and α-thrombin (Figure 3B) to rHPA-2a and rHPA-2b showed that both recombinant fragments were functionally active.
Influence of the Thr/Met Allotype on the Conformation of GPIbα
The conformation of both HPAs was additionally studied using 21 anti-GPIbα moAbs. As a reference, 27C11, recognizing a linear epitope on both alloforms, as shown in immunoblot (Figure 1), was used. Nineteen moAbs (n=2), including 27C11 (n=3, P>0.5), showed no difference in binding between rHPA-2a and rHPA-2b (Figure 4). However, 24G10 and 14B11 bound significantly better to rHPA-2a (P<0.03 and P<0.01, respectively, n=3) and to HPA-2a2a than to HPA-2b2b platelets (not shown).
MoAb 24G10 recognizes a conformational epitope within the 1 to 89 amino-terminal domain of GPIbα and inhibits the GPIbα-VWF interaction.25 MoAb 14B11 also recognizes a conformational epitope but did not inhibit VWF binding (not shown). MoAbs 12G1, 12E4, 27A10, 24G10, 6B4,25 LJ-Ib1,35 LJ-Ib10,28 and TM608 were used to map the epitope of 14B11 and 27C11. Only 12G1, 12E4, and 27A10, which recognize an epitope in the 1 to 59 region of GPIbα,25 completely inhibited the binding of 14B11 to human platelets (Figure 5). The platelet binding of 27C11 in contrast was blocked by LJ-Ib10, which binds to the 268 to 282 anionic sulfated tyrosine sequence of GPIbα and inhibits the α-thrombin interaction with GPIbα.28 Because 27C11 also completely and dose-dependently inhibits the α-thrombin–induced platelet aggregation (not shown), it is likely that 27C11 recognizes the same 268 to 282 sequence.
Influence of HPA-2 Polymorphism on VWF Binding
The influence of the HPA-2 polymorphism on the VWF-GPIbα interaction was studied by determining the ristocetin- and botrocetin-induced binding of VWF to the rHPAs.26 Whereas no binding of VWF to either rHPA-2a or rHPA-2b could be observed in the absence of ristocetin (not shown), both dose dependently interacted with VWF in the presence of 1500 μg/mL ristocetin (Figure 3A) with a Kd,app of, respectively, 0.69±0.06 and 0.67±0.03 nmol/L (mean±SE) (P=0.80), showing no statistical difference in VWF interaction at this ristocetin concentration. At 760 μg/mL ristocetin, however, plasma VWF bound with a Kd,app of 3.35±0.41 nmol/L to rHPA-2a and a Kd,app of 5.67±0.75 nmol/L to rHPA-2b (Figure 3C). This statistically significant (P<0.02) difference shows a stronger affinity of plasma VWF for rHPA-2a in the presence of 760 μg/mL ristocetin. A similar difference was also observed when purified VWF was used in the presence of 400 μg/mL ristocetin, with a Kd,app of 1.66±0.68 and 2.49±1.47 nmol/L for rHPA-2a and rHPA-2b, respectively.
A difference in affinity of VWF for rHPA-2a and rHPA-2b was also seen in the presence of 0.1 μg/mL botrocetin, where VWF bound with a Kd,app of 2.49 and 7.47 nmol/L to rHPA-2a and rHPA-2b, respectively (mean of 2 experiments), which could be confirmed with HPA-2a2a and HPA-2b2b platelets (P<0.05) when anti-GPIbα moAb 27C11 was used as a reference to normalize for GPIbα numbers (Figure 6). Ristocetin could not be used in this ELISA assay as modulator, because this resulted in a too high background signal.
Influence of HPA-2 Polymorphism on α-Thrombin Binding
The Kd,app for the α-thrombin binding to rHPA-2a and rHPA-2b was 0.493±0.045 and 0.428±0.099 nmol/L (mean±SE), respectively (P=0.38), pointing out that the HPA-2 polymorphism does not influence the α-thrombin interaction with GPIbα (Figure 3B).
The HPA-2 system within GPIbα of platelet GPIb-IX-V is associated with a Thr/Met dimorphism at position 145. In this study we investigated the structural and functional effects of the HPA-2 system without the VNTR polymorphism. The N-terminal domain (AA1-289) of the HPA-2a (Thr-form) and HPA-2b alloform (Met-form) were expressed in CHO cells. Purified proteins with the expected size were recognized by a panel of 21 anti-GPIbα moAbs. Moreover, the rHPA-2b competed dose-dependently with anti-HPA-2b antiserum for the binding to HPA-2b–positive platelets, whereas the rHPA-2a had no effect, demonstrating the allele specificity of these recombinant GPIbα isoforms. Both rHPA-2a and rHPA-2b bound to both VWF and α-thrombin. These results together demonstrated the correct folding of both alloforms.
Most of the anti-GPIbα moAbs had a similar affinity for rHPA-2a and rHPA-2b. However, 24G10 and 14B11, which bind to the N-terminal flanking region and first leucine-rich repeat of GPIbα (AA1-59) and recognize a conformational epitope, bound with a higher affinity to rHPA-2a, suggesting that the conformation of the amino-terminal region AA1-59 is important for the discrimination between HPA-2a and HPA-2b. Recently, the crystal structure of the VWF A1-domain bound to the N-terminus of GPIbα was elucidated.10 Intriguingly, no direct interaction of the Thr/Met-145 residue with the N-terminal region of GPIbα is observed, suggesting that the structural effect of this dimorphism is caused by a long-range conformational change. In contrast, 27C11, which likely binds to the anionic sulfated tyrosine sequence (AA268-282), bound equally well to both alloforms, suggesting that no different conformation was induced in this region. Together with the region comprising the polymorphic amino acid, this different conformation of the N-terminal region could be the molecular basis for the alloantibody combining sites. Moreover, both 14B11 and 24G10 might be useful reagents to substitute the very rare HPA-2 antisera for phenotyping of the HPA-2 alloantigen system. However, because both moAbs bind quite well to both alloforms, normalization for receptor density might be needed, and additional studies are needed to validate the potential applicability.
The anionic sulfated tyrosine sequence of GPIbα (AA268-282) is the most important α-thrombin binding domain.11 Because 27C11 recognizes this sequence, inhibits α-thrombin binding, and does not discriminate between both HPA-2 alloforms, it therefore was not completely surprising that α-thrombin also binds with the same affinity to rHPA-2a and rHPA-2b.
The AA1-59 region of GPIbα not only contains the epitope for 24G10 but also is important for VWF binding. In agreement with earlier results,36 no difference in binding of VWF to rHPA-2a and rHPA-2b was observed in the presence of 1500 μg/mL ristocetin. Kd,app is in good agreement with the Kd,app of 0.89 nmol/L described by Li et al.33 In the presence of 760 μg/mL ristocetin, however, or in the presence of 0.1 μg/mL botrocetin, VWF bound stronger to rHPA-2a. This was confirmed with HPA-2a2a and HPA-2b2b platelets in the presence of 0.1 μg/mL botrocetin. At ristocetin concentrations equal to or above 1 mg/mL, maximal VWF binding to glycocalicin, the extracellular part of GPIbα, is observed.37 Therefore, it is likely that in the presence of such high ristocetin concentrations, differences in VWF/GPIbα interaction might be overlooked. Earlier results also showed no difference in VWF binding to HPA-2a2a and HPA-2b2b platelets16 induced by 2.5 μg/mL botrocetin, a concentration that also might have been too high to see a difference.
Our results show a stronger VWF interaction with the HPA-2a alloform in the presence of ristocetin or botrocetin. Clinical association studies, however, suggest that the HPA-2b alloform might be a genetic risk factor for thrombotic events, which seems to be in contradiction with our results, unless the effect of the HPA-2 system is similar to platelet-type von Willebrand disease (PT-vWD). PT-vWD is an autosomal dominant bleeding disorder characterized by abnormally enhanced binding of VWF by patient platelets. PT-vWD is attributable to a G233V38 or a M239V39 mutation in GPIbα. These mutations cause a conformational change, resulting in spontaneous binding of high multimers of VWF such that the latter are cleared from the plasma, finally resulting in bleeding problems. One could hypothesize that, compared with HPA-2b, the HPA-2a phenotype might behave as a mild PT-vWD phenotype. This hypothesis is corroborated by the fact that 24G10, which binds stronger to the HPA-2a alloform, also binds stronger to PT-vWD GPIba.25 However, care has to be taken in drawing this conclusion, because in this study VWF binding to GPIbα is only investigated in the presence of ristocetin and botrocetin and nothing is yet known on the effect of the HPA-2 polymorphism on shear-induced VWF binding. Another hypothesis could be that the VNTR polymorphism, which is in linkage disequilibrium with the HPA-2 polymorphism, is the true genetic risk factor. A better presentation of the weaker HPA-2b VWF-binding site by 3 to 4 VNTRs might overrule the VWF binding differences because of the HPA-2 polymorphism. However, the association of the VNTR polymorphism with thrombotic events also is unclear.19,20,23,40⇓⇓⇓ Finally, because GPIbα is also a receptor for P-selectin, Mac-1, FXI, FXII, and high-molecular-weight Kininogen,41 the HPA-2 polymorphism could also affect these interactions, which could be the basis for the thrombotic risk.
In conclusion, our results show that the HPA-2 system has no influence on the conformation of the anionic sulfated tyrosine sequence of GPIbα and by this has no influence on the α-thrombin interaction. However, the HPA-2 system does modify the conformation in the AA1-59 region of GPIbα, resulting in a stronger interaction of VWF with HPA-2a. The present studies on the functional and structural relevance of platelet GPIb polymorphisms may provide an explanation for the clinical data linking this polymorphism to thrombotic and immune-mediated disorders.
Note Added in Proof
Recently, Boncler et al42 indicated that aurintricarboxylic acid inhibits VWF-mediated platelet aggregation more efficiently in VNTR B/Met145(+) carriers than in VNTR B/Met145(−) carriers. This also suggests a stronger VWF interaction of HPA-2a2a platelets.
This study is supported by F.W.O.-Flanders grant G.0168.02. H.U. is a F.W.O.-Flanders Aspirant. The authors thank Drs Z.M. Ruggeri and N. Yamamoto for generously providing anti-GPIb antibodies and Dr K.J. Clemetson for the pcDNA3-plasmid.
- Received March 26, 2003.
- Accepted May 13, 2003.
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