We present here the results of meta-analyses of GWAS results, performed in the CHARGE consortium, that identified significant and replicated associations of sICAM-1 and sP-selectin levels with variants in the genes encoding these proteins, ICAM-1
, respectively. In addition, both markers were associated with variants in the well-known ABO
blood group locus, which encodes glycosyltransferase enzymes that transfer sugar residues to the H antigen and determine an individual's blood group (28
The observed association of sICAM-1 and sP-selectin with ABO variants could be explained by the A1 blood group allele, that determines A blood group. The other A blood group allele, A2, was also associated with decreased levels of sP-selectin, however, with much lower level of significance. Both A1 and A2 alleles encode for the same form of glycosyltransferase that adds the N
-acetylgalactosamine to the H antigen, however, with lower activity in case of A2 allele (29
). These findings suggest a possible role of glycosylation in ABO's association with sP-selectin and sICAM levels.
There was no observed association between the ABO
locus with platelet-bound P-selectin although the flow cytometry analysis was limited to P-selectin on platelets, and does not rule out the possibility that endothelial P-selectin levels may be influenced by the ABO locus. Soluble forms of P-selectin arise in part from shedding or active proteolytic cleavage of membrane molecules (8
). It is known that both soluble and cellular forms of ICAM-1 (31
) and probably of P-selectin are glycosylated. The glycosylation is also critical for the binding activity of the P-selectin receptor, P-selectin glycoprotein ligand-1 (32
). The observed association of ABO blood group markers with sP-selectin and sICAM-1 and not platelet-bound P-selectin suggests that the ABO
gene product related glycosylation influences shedding/cleavage of these markers from the endothelium, probably by glycosylation of P-selectin and ICAM-1. Glycosylation could also affect the clearance rate of sP-selectin and sICAM-1 from blood as has been suggested for von Willebrand factor (vWF), a large circulating glycoprotein involved in hemostasis (33
), whose levels are also influenced by the ABO
). It is interesting that both P-selectin and vWF are stored in the same granules, Weibel-Palade bodies, prior to being released on endothelial cells (35
). Moreover, the vWF receptor, glycoprotein (GP)Ibα, has been shown to act as a counter-receptor for P-selectin (37
). vWF carries N-linked ABO antigens, and it has been suggested that vWF levels vary according to vWF glycosylation that influence its secretion and/or clearance rate (34
). The similarity in P-selectin and vWF physiology underscores the inter-related nature of hemostatic and inflammation responses, both of related to CVD risk and now reported to be influenced by the ABO
blood group locus. In a recent GWA study, variation in ABO locus was also associated with serum soluble E-selectin levels (38
)—another member of selectin family of adhesion molecules.
Additional insights may come from ABO's association with malaria infection susceptibility and severity (39
). Recent studies of Plasmodium falciparum
infection show that the infected erythrocytes mimic the leukocytes’ attachment to endothelium by binding to ICAM-1 and P-selectin on endothelium (41
). Erythrocytes also bind to the A and B antigens (but not O) on endothelial cells as an additional contributor to cytoadherence (42
). This functional homology suggests the hypothesis that leukocytes might also interact with ABO antigens on endothelium. The A antigen might promote stronger (longer) binding of leukocytes to P-selectin and ICAM-1 on the vascular wall, thereby protecting the protein from enzymatic cleavage which in turn would lead to decreased levels in circulation. Decreased cleavage of adhesion molecules from endothelial cells associated with A allele would mean more adhesion molecules on the endothelial cells, increased adhesion and inflammation (21
). This corresponds well with the findings of studies relating ABO
blood group alleles with CVD that have been of interest for many years (43
) and most of which suggested A allele association with increased risk of CVD (46
). However, multiple epidemiologic studies have reported that increased levels of sP-selectin and sICAM-1 are associated with increased risk of CVD (12
). Consequently, the A allele's association with decreased levels of sICAM-1 and sP-selectin but increased risk of CVD seems as a paradox and underscores the complex nature of CVD, its risk factors and their interrelationships (21).
Apart from ABO, sP-selectin and sICAM-1 levels are largely determined by variants within the genes encoding the two biomarker proteins, SELP
. The strong association of rs6136 with sP-selectin has been previously reported by ourselves (22
) and others (13
). Although heterogeneity test for any of the top SNPs in our sP-selectin and sICAM-1 meta-analyses was not statistically significant after accounting for multiple comparison, the I2
statistic (percentage of total variation across studies that is due to heterogeneity) for rs6136 was high (82%). This high level of heterogeneity in our meta-analysis for this SNP should be investigated further. rs6136 (Thr715Pro) has a functional effect of changing the conformation of P-selectin near the cleavage site (22
). Besides Thr715Pro, the P-selectin gene is characterized by 2 LD blocks that are additionally associated with sP-selectin levels. Another SELP variant, Val599Leu (rs
6133), reported to be associated with sP-selectin in previous studies (13
), was not in high LD with any of the 3 LD blocks and did not reach genome-wide significance in our study. In total, the three SELP
regions accounted for 9.3% of the variance of sP-selectin levels in the FHS sample, the largest in our study. However, only Thr715Pro remained significantly associated with platelets P-selectin, a result that has already been reported (48
). The lack of association of the other two SNPs could be explained by smaller sample size and insufficient power to detect the association for platelet P-selectin. Alternatively, it is also possible that different variants contribute to cellular and soluble P-selectin levels.
We report here suggestive evidence for a novel locus influencing soluble P-selectin levels on chromosome 3. The region contains SCAP
(SREBP cleavage-activating protein), PTPN23
(protein-tyrosine phosphatase), KIF9
(kinesin family member 9) and TMEM103
(transmembrane protein). SCAP cleaves the active domain of SREBP (sterol regulatory element binding protein) which acts as a transcription factor and participates in cellular cholesterol homeostasis (49
). Given SCAP
variants association with sP-selectin, it could be hypothesized that SCAP is also capable of cleaving P-selectin from cell membrane. However, SCAP is only known for its intracellular activities and its extracellular presence is doubtful.
The top SNP associated with ICAM-1 levels, rs3093030, is in the 3′ end of the ICAM- 1
gene and is in high LD with rs5498 (Lys469Glu) that causes an amino-acid change from glutamic acid to lysine and is located in the fifth Ig-like domain of ICAM-1 (51
). Lys469Glu has previously been reported to influence sICAM-1 levels (21
). This variant influences the dimerization of the protein which promotes enhanced binding to leukocytes (53
). Another variant in ICAM-1 gene (rs1799969 or Gly241Arg) is independently associated with ICAM-1 levels. This variant changes the amino-acid in the third immunoglobulin domain of ICAM-1 protein and has been shown to be of importance in binding to the leukocyte integrin Mac-1 (52
Although this study was well-suited to identify loci with common variation associated with sP-selectin and sICAM-1 levels detectable by GWAS in large samples, it was limited to European ancestry population and its generalizability to other ethnic groups remains to be investigated. As with other GWAS, we were able to narrow down the association of sP-selectin and sICAM-1 to a particular genomic region, but further analyses, beginning with DNA resequencing in large well-phenotyped cohorts, will be necessary to detect functional variants within those genes. Future projects with larger data sets will be required to examine any gene–gene and gene–environment interactions and the identification of additional variants that may have been undetected in this study. Finally, the possible heterogeneity of the effect of rs6136 on sP-selectin levels is a limitation and deserves further attention.
In summary, this study suggests the importance of the ABO blood group in influencing circulating sP-selectin and sICAM-1 levels. The probable mechanism involves glycosylation that interacts with P-selectin/ICAM-1 shedding from the cell membrane. These results contribute to the knowledge of the adhesion molecules physiology that is necessary to understand the link between the inflammation and atherosclerotic process and the development of new anti-inflammatory therapies for cardiovascular complications.