C4BPB/C4BPA is a new susceptibility locus for venous thrombosis with unknown protein S–independent mechanism: results from genome-wide association and gene expression analyses followed by case-control studies

Abstract

Through its binding with protein S (PS), a key element of the coagulation/fibrinolysis cascade, the C4b-binding protein (C4BP) has been hypothesized to be involved in the susceptibility to venous thrombosis (VT). To identify genetic factors that may influence the plasma levels of the 3 C4BP existing isoforms, ␣ 7 ␤ 1 , ␣ 6 ␤ 1 , and ␣ 7 ␤ 0 , we conducted a genomewide association study by analyzing 283 437 single nucleotide polymorphisms (SNPs) in the Genetic Analysis of Idiopathic Thrombophilia (GAIT) study composed of 352 persons. Three SNPs at the C4BPB/C4BPA locus were found genomewide significantly associated with ␣ 7 ␤ 0 levels. One of these SNPs was further found to explain approximately 11% of the variability of mRNA C4BPA expression in the Gutenberg Heart Study composed of 1490 persons, with no effect on C4BPB mRNA expression. The allele associated with increased ␣ 7 ␤ 0 plasma levels and increased C4BPA expression was further found associated with increased risk of VT (odds ratio [ IntroductionProtein S (PS), a vitamin K-dependent plasma protein, is a key regulator of the coagulation/fibrinolysis cascade. PS has no enzymatic activity but acts mainly as a cofactor for activated protein C (APC). 1 It participates in the inactivation of factors Va (FVa) and FVIIIa, leading to inhibition of thrombin generation. [2][3][4] Even in absence of APC, PS could still inhibit thrombin generation by decreasing the conversion of prothrombin to thrombin and that of FX to FXa. 5 PS is also a cofactor for tissue factor pathway inhibitor (TFPI) for inhibiting FXa. 6 In plasma, PS circulates either in a free form (fPS; ϳ 40%) or as a complex with the C4b-binding protein (C4BP; ϳ 60%). [7][8][9][10] It was initially thought that the C4BP-bound PS was not active and that only the circulating fPS was. [7][8][9] However, more recent works shed new light on a more complex mechanism, as C4BP-PS complex was shown to directly participate in the FVa and FVIIIa inactivation, 11 and C4BP could inhibit APC-catalyzed FVa inactivation even in the absence of PS. 12 Such complex and elusive mechanisms could explain why, despite the key role of PS in the regulation of thrombin generation, its involvement in the susceptibility of venous thrombosis (VT) remains obscure. Although PS deficiency is considered a risk factor for VT, 13-15 total PS and fPS show very little correlation with VT. 16,17 C4BP is a large plasma protein existing in 3 different isoforms, ␣ 7 ␤ 1 , ␣ 7 ␤ 0 , and ␣ 6 ␤ 1 , according to the number of identical ␣-chains (6 or 7) and the presence/absence of a single ␤-chain. 18 The main isoform is ␣ 7 ␤ 1 , whereas the ␣ 7 ␤ 0 isoform lacking the ␤-chain represents approximately 17% of the C4BP molecules. 7,18 Whereas the ␣-chain binds for many ligands, 19 the ␤-chain is specific for the binding of PS. As a consequence, the amount of ␤-chaincontaining isoforms can be viewed as a surrogate marker for fPS levels. Plasma levels of all 3 isoforms have documented high heritability estimates (ϳ 40% each), 20 but little is know ab...

Inherited Thrombophilia: Past, Present, and Future Research

“…Carriers of combinations of defects also presented with thrombosis earlier in life and more frequently. The same was observed in protein S deficient families, where combined defects of the protein S gene and either the Factor V Leiden mutation or the prothrombin 20210G>A were found in 40% (Koeleman et al, 1995;Zoller et al, 1995) and 30% (Castaman et al, 2000) of families, respectively. As a result, thrombophilia was then suggested to be an oligogenetic disease in which inherited predisposition results from 2 or more mutations in genes involved in blood clotting (Miletich et al, 1993).…”

“…Later, a scan employing 307,984 SNPs was performed (Buil et al, 2010;Malarstig et al, 2009). The investigators focussed on associations between genetic markers and intermediate phenotypes of VTE, like lipoprotein(a) levels (Lopez et al, 2008), factor XII levels (Soria et al, 2002), total plasma homocysteine (Malarstig et al, 2009), and C4BP plasma levels (Buil et al, 2010). In these studies quantitative trait loci were discovered, but these loci often included the structural gene for the investigated intermediate phenotype.…”

“…For the first scan 363 microsatellite markers were genotyped (Soria et al, 2002) while 485 microsatellite markers were used in the second scan (Lopez et al, 2008). Later, a scan employing 307,984 SNPs was performed (Buil et al, 2010;Malarstig et al, 2009). The investigators focussed on associations between genetic markers and intermediate phenotypes of VTE, like lipoprotein(a) levels (Lopez et al, 2008), factor XII levels (Soria et al, 2002), total plasma homocysteine (Malarstig et al, 2009), and C4BP plasma levels (Buil et al, 2010).…”

Inherited Thrombophilia and the Risk of Vascular Events

“…The authors concluded that this variation is evolutionary neutral and that T allele appeared approximately 100,000 years ago, reaching high frequencies by genetic drift. Buil et al (2010) reported that a new locus, C4BPB/C4BPA (coding for C4-binding protein), is involved in susceptibility to VT through a still unknown, but protein S-independent mechanism. Bezemer et al (2008) found SNPs significantly associated with VT in CYP4V2/KLKB1/F11 gene cluster, as well as in the GP6 and SERPINC1 genes.…”

VT study disassociates C4BP from protein S

“…1 Although this feat falls short of the conclusive identification of causative single nucleotide polymorphisms (SNPs), 2 a strength of the study lies in the efforts to explore the contribution of candidate SNPs to venous thrombosis. C4b-complementary binding protein (C4BP) circulates in the blood in the different isoforms comprising ␣ and ␤ subunits.…”