Platelet Glycoprotein Polymorphisms and Relationship to Function and Immunogenicity

Thomas J. Kunicki

Diane J. Nugent

In order to place the effect of glycoprotein gene polymorphisms on platelet function and immunogenicity into proper perspective, a brief summary of key aspects of platelet function is helpful.

Following vascular damage, platelets interact with specific components of the extracellular matrix, particularly collagen, and a complex series of receptor-ligand interactions ensue that ultimately lead to the formation of a stable platelet plug or thrombus. This process is a continuum of at least three phases that can be described as initiation, extension, and consolidation, each requiring the orchestrated cooperation of a group of receptors.

In the initiation phase, plasma von Willebrand factor (vWF) binds to collagen via its A3 domain and becomes structurally altered, enabling its A1 domain to bind to the platelet membrane receptor complex glycoprotein Ib-IX-V (GPIb complex). It is GPIbα, the larger subunit of GPIb, that makes direct contact with vWF. This association is a requisite step in platelet adhesion to exposed thrombogenic surfaces at sites of vessel wall injury or in regions of atherosclerotic plaque rupture. This leads to the formation of a more stable platelet monolayer on the collagen surface, mediated predominantly by the platelet-specific receptor glycoprotein VI (GPVI) and the platelet integrin α₂β₁.

The engagement of these receptors causes platelet activation leading to the extension phase, mediated largely by the conversion of prothrombin to thrombin on the activated platelet surface and the secretion of compounds from platelet granules (α-granules and δ-granules), which can further stimulate platelets. One of these compounds, adenosine diphosphate (ADP), augments platelet activation by binding to its cognate platelet receptors, the purinergic receptors P2Y₁ or P2Y₁₂. Activated platelets also produce and/or release additional agonists, including thromboxane A2 (TXA₂), which then binds to the platelet TXA₂ receptor. Most of the receptors involved in the events of the extension phase are members of the G-protein-coupled receptor family.

Other agonists that function during the extension phase include thrombin and epinephrine (EPI). Thrombin (also known as coagulation factor II) binds to a number of receptors, but of particular importance for this discussion is the protease-activated receptor 1 (PAR-1). EPI binds to the platelet α2A-adrenergic receptor (ADRA2A). EPI can contribute to the initiation phase and, in low doses, is thought to prime platelets for enhanced activation by other agonists. The ADRA2A is a G-protein-coupled receptor and activates heterotrimeric G proteins, including those containing the β3 subunit (GNB3).¹ Alternative splicing variants of GNB3 appear to enhance G-protein signal transduction²^,³ and could thus alter the effect of EPI on platelets. Thrombin contributes to the augmentation of platelet activation during the extension phase because the surface of activated platelets is a nidus for prothrombin conversion. Nonetheless, both thrombin and EPI, as well as ADP, are capable of binding to and activating the naïve platelet in an alternative initiation phase.

In the consolidation phase, platelet-platelet cohesion (aggregation), mediated by the binding of fibrinogen and/or vWF to the activated platelet integrin α_IIbβ₃ (also known as GPIIb-IIIa), together with the assembly of a fibrin network, results in the generation of platelet-rich aggregates or thrombi.

SINGLE NUCLEOTIDE POLYMORPHISMS AND GENE HAPLOTYPES

Receptor Glycoprotein Gene Haplotypes

This chapter is concerned with polymorphisms, defined as nucleotide sequence differences with a minor allele frequency (MAF) of ≥0.02 in any racial or ethnic group and that are detectable in unrelated normal subjects. Polymorphisms that are immunogenic and elicit a humoral response in normal subjects are defined as alloantigens and are designated by the prefix human platelet antigen (HPA-),⁴ as shown in Table 27.1. Immunogenic polymorphisms that are unique to a single group of related subjects are considered familial or private antigens and are not discussed in this chapter. Single nucleotide polymorphisms (SNPs) in platelet glycoprotein receptor genes can give rise to differences in expression levels, activity, and/or immunogenicity. Genes harboring SNPs that create alloimmunogenic epitopes include the integrin α₂ subunit gene ITGA2,⁵^,⁶ the integrin αIIb subunit gene ITGA2B,⁷^,⁸ the integrin β₃ subunit gene ITGB3,⁹^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸^,¹⁹^,²⁰ the GPIbα subunit gene GP1BA,²¹^,²² and the gene CD109.²³ In this chapter, protein and cDNA sequences are numbered from the ATG translation start codon, as recommended by the Human Genome Variation Society (http://www.hgvs.org/mutnomen/).

ITGA2. Polymorphisms in the ITGA2 promoter and regulatory regions generate a fourfold range of α₂β₁ expression on platelets and other cells. The six most common haplotypes are defined by three SNPs, C-52T (rs28095), C759T (rs1126643), and G1600A (rs1801106). The rs1801106 alleles result in the nonconservative amino acid substitution E534K, creating the clinically relevant HPA-5 alloantigen system, but these alleles do not affect α₂β₁ binding to collagen.⁵ G1600A is in linkage disequilibrium (LD) with C-52T that has a substantial effect on the rate of ITGA2 transcription. However, unlike C-52T, it is not associated
with regulation of a2 expression.²⁴ However, selected gene association studies indicate that the minor allele of rs1801106 (1600A) is associated with recurrent stroke²⁵ and an increased risk for breast cancer in women.²⁶ Even though this SNP does not alter α₂β₁ binding to collagen, it is possible that it modulates the binding of other physiologically relevant α₂β₁-specific ligands, such as C1q, decorin, endorepellin, or perlecan.²⁷^,²⁸^,²⁹^,³⁰

Table 27.1 Human platelet alloantigens

Antigen	Synonym	Gene	dbSNP	cDNA	Amino Acid	Reference
HPA-1	Pl^A, Zw	ITGB3	rs5918	T176C	L59P	9,10
HPA-2	Ko, Sib	GP1BA	rs6065	C482T	T161M	21
HPA-3	Bak, Lek	ITGA2B	rs5911	T2621G	I874S	7
HPA-4	Yuk, Pen	ITGB3	rs5917	G506A	R169Q	12
HPA-5	Br, Zav, Hc	ITGA2	rs1801106	G1600A	E534K	5
HPA-6bw	Ca, Tu	ITGB3	rs13306487	G1544A	Q515R	13
HPA-7bw	Mo	ITGB3	rs121918448	C1297G	A433P	14
HPA-8bw	Sr	ITGB3	nd	C1984T	C662R	15
HPA-9bw	Max	ITGA2B	rs74988902	G2602A	M868V	8
HPA-10bw	La	ITGB3	nd	G263A	Q88R	16
HPA-11bw	Gro	ITGB3	nd	G1976A	H659R	17
HPA-12bw	Iy	GP1BA	nd	G119A	Q31G	22
HPA-13bw	Sit	ITGA2	rs79932422	C2483T	T828M	6
HPA-14bw	Oe	ITGB3	nd	1909-1911 delAAG	L637del	18
HPA-15	Gov	CD109	rs10455097	C2108A	Y703S	23
HPA-16bw	Duv	ITGB3	rs74708909	C497T	T166I	19
HPA-17bw	Va	ITGB3	nd	C662T	T195M	11,20
nd, not designated.

The ITGA2 mutation T828M (rs79932422) produces the HPA-13bw alloantigen,⁶ but has a MAF of 0.0025 and is not of clinical significance. Although T828M has no effect on platelet adhesion to collagen I, II, or IV, four individuals have been reported who are at least heterozygous 828M and whose platelets exhibit a diminished aggregation response to low-dose collagen (2.5 mg/mL) but a normal response to high-dose collagen (10 mg/mL).⁶ Nonetheless, these reports must be interpreted with caution because platelet levels of α₂β₁ in the affected individuals were not measured.

ITGA2B. Two major ITGA2B haplotypes are characterized by the SNP T2621G (rs5911) giving rise to the substitution I874S that defines the HPA-3 alloantigen system.⁷ I874S is adjacent to the binding site of the murine monoclonal antibody PMI-1 that inhibits platelet adhesion and spreading on collagen,³¹ suggesting that the region of α_IIb encompassing the HPA-3 and PMI-1 epitopes participate in aIIbb3-mediated platelet adhesion. The precise impact of the T2621G substitution on platelet function has not been determined, but homozygosity for the 2621G haplotype is associated with a fivefold increase in the risk of ischemic stroke among women with hypertension or diabetes.³²

ITGB3. The β₃ SNP T176C (rs5918) results in an L59P substitution that defines the HPA-1 alloantigen system.¹⁰ In whites, sensitization to HPA-1a is the leading cause of alloimmunization against platelets (discussed below). There has been considerable debate about the influence of ITGB3 HPA-1b on platelet function and the risk of acute coronary disease or cerebrovascular disease. Several reports from one group of investigators indicated that HPA-1b confers either enhanced α_IIbβ₃ function or resistance to inhibitors of platelet function,³³^,³⁴^,³⁵^,³⁶^,³⁷ but not all studies have observed an increased biologic activity associated with the HPA-1b haplotype.³⁸^,³⁹ After considerable conflicting evidence from several gene association studies, most of which were seriously underpowered, subsequent meta-analyses failed to clarify the potential association of HPA-1b with risk for coronary artery disease: One meta-analysis⁴⁰ concluded that there is a weak but significant association of HPA-1b with overall cardiovascular disease in the general population and a stronger association in subgroups, such as younger cohorts or a restenosis subset with stents; the second meta-analysis⁴¹ simply found that HPA-1b is not associated with an increased risk of developing myocardial infarction. As regards cerebrovascular disease, a recent meta-analysis found that HPA-1b does not represent a useful marker of increased risk⁴².

GP1BA. The GPIb complex is a heptamer composed of four distinct gene products and consists of two molecules of GPIbα, two of GPIbβ, two of GP IX, and one of GP V. Each GPIbα molecule is disulfide-linked to a GPIbβ, whereas the interactions of GPIbα with GPIX and GPV are noncovalent. vWF binds directly to the N-terminal portion of GPIbα. Clinically relevant polymorphisms have been associated with the gene GP1BA encoding the GPIbα subunit, and two major haplotypes are distinguished by the SNP C482T (rs6065), giving rise to a T161M substitution within the GPIbα ligand-binding region that defines the HPA-2 alloantigen system.²¹^,⁴³

T161M is in LD with a variable number of tandem repeats (VNTRs) in the mucin-like macroglycopeptide region of GPIbα. The VNTR region results from duplication of a 13-amino acid sequence once (VNTR A), twice (VNTR B), thrice (VNTR C), or four times (VNTR D), leading to polypeptide lengths of 610, 623, 636, or 649 amino acids, respectively.⁴⁴^,⁴⁵ Because the repeats are rich in serine and threonine, they can be O-glycosylated, and each repeat adds 32 Å to the length of the GPIbα extracellular domain.⁴⁴ This progressively increases the distance of the vWF- and thrombin-binding sites on GPIbα from the plane of the platelet plasma membrane, increasing the accessibility of these binding sites and perhaps accounting for the reported increased risk for acute coronary artery disease associated with the GPIbα longer variants, VNTR C and VNTR D, which are in LD with M161.⁴⁶^,⁴⁷

In addition, the SNP C-5T (rs2243093) at a position 5 nucleotides upstream from the GPIbα initiator codon ATG appears to enhance its translational efficiency.⁴⁸^,⁴⁹ Thus, the presence of the -5C allele increases the mean level of GPIbα on the platelet plasma membrane (roughly, a 50% increase in homozygous individuals and a 33% increase in heterozygous individuals).⁴⁸ The C-5T alleles and the M161T alleles are in complete LD.⁵⁰

CD109. The CD109 SNP C2108A (rs10455097) gives rise to the HPA-15 alloantigen system.²³^,⁵¹ The resulting Y703S amino acid substitution is carried by the glycosylphosphatidylinositollinked protein CD109, a negative regulator of the transforming growth factor (TGF)-β system in keratinocytes.⁵²^,⁵³ Currently there are no published data concerning the effect of anti-HPA-15 antibodies on platelet function.

Table 27.2 Minor allele frequencies

	Brazil		Europe	Africa		North America
	Amerindian	Black	White	Berber	Tunisian	Black	White	Indian
HPA-1	0	0.11	0.16	0.25	0.25	0.08	0.17
HPA-2	0.04	0.17	0.09	0.18		0.18	0.09	0.12
HPA-3	0.36	0.34	0.39	0.32	0.31	0.37	0.41
HPA-5	0.04	0.16	0.10	0.14	0.22	0.18	0.09	0.02
HPA-15	0.28	0.43	0.40				0.53
References	99,100,101,102	99,100,102,103	50,56,104,105,106,107,108,109,110,111,112,113	114	115	116,117	23, 50,116	117
	Australia		Asia					Bahrain
	Aboriginal	White	Korean	Japanese	Chinese	Taiwanese	Thais	Arabs
HPA-1	0.005	0.14	0.008	0.002	0.004	0.001	0.002	0.24
HPA-2	0	0.07	0.08	0.14	0.03	0.05	0.06	0.23
HPA-3	0.07	0.38	0.44	0.46	0.43	0.39	0.48	0.43
HPA-4				0.008				0.07
HPA-5	0.20	0.09	0.02	0.06	0.02	0.18	0.04	0.13
HPA-6w				0.02
HPA-15						0.46	0.52
References	118,119	119	116,120	61,103,121,122	123,124	125,126	125,127,128	129