Successful Hunt for Quantitative Trait Locus in Thrombophilia
In recent years, our knowledge of the genetic mechanisms of thrombophilia has advanced considerably. The elucidation of the protein C system with the identification of protein C and protein S deficiencies and activated protein C (APC) resistance due to a factor V gene mutation (FV Leiden) as major risk factors of thrombosis has been instrumental for the rapid progress.1–3⇓⇓ Despite these achievements, our understanding of the genetic contribution to thrombophilia is incomplete, and new technological approaches are tried to elucidate unknown genetic factors. The GAIT (Genetic Analysis of Idiopathic Thrombophilia) project is an effort to use genome-wide linkage screening to identify unknown genetic risk factors. This project in addition allows the identification of genes influencing the normal variation of the concentrations of certain plasma proteins.
See page 508
In this issue, Almasy and colleagues4 describe a genome-wide screen aiming at identification of genes affecting the level of free protein S (fPS) in plasma. The positive results of this exercise are of interest not only from the point of view of protein S biology but also because it proves the experimental approach useful. The authors investigated a large number (398) of individuals from 21 Spanish families using many (363) informative DNA markers. Highly significant linkage (logarithm of odds [LOD] score around 4) was observed between the fPS level and a distinct region on chromosome 1q. The likelihood that this represents a biologically significant association is high because the identified chromosomal region houses the C4BPA and C4BPB genes. These genes code for the C4b-binding protein (C4BP), a complement regulatory protein that forms a high-affinity complex with protein S in plasma.5 C4BP has a fascinating molecular architecture; the molecule is composed of seven identical 70-kDa α-chains and a unique 45-kDa β-chain (Figure). These chains are disulfide-linked in their C-termini, and the molecule has a characteristic octopus-like shape with multiple tentacles, seven long (α-chains) and one short (β-chain), extending from a central core. Each of the α-chains carries a binding site for the complement protein C4b, whereas protein S binds with high affinity to the shorter β-chain.6,7⇓
The free form of protein S is an important component of the protein C anticoagulant system, functioning as a cofactor to the activated protein C (APC) in the degradation of coagulation factors Va and VIIIa.1 Like other vitamin K–dependent proteins, protein S binds phosphatidyl serine–containing phospholipids. Phosphatidyl serine is normally not presented on the cell surface but is exposed on activated (eg, platelets) or apoptotic cells. Recently, both free and C4BP-complexed forms of protein S have been found to bind to apoptotic cells thus providing potential for local control of complement and coagulation systems.8 In addition, protein S binding has been shown to stimulate phagocytosis of the apoptotic cells.9
Even though both forms of protein S have important biological functions, for the diagnosis of protein S deficiency, fPS assays are preferred over assays of total protein S.10 The background for the higher predictive value of fPS assays can be found in the basic biology of protein S and the two C4BP chains and of their gene regulation. C4BP circulates in blood in different isoforms, the major form being composed of seven α-chains and a single β-chain. However, there are also C4BP isoforms in blood which lack the β-chain and therefore are unable to bind protein S.11 The concentration of fPS is determined by the concentrations of protein S and the β-chain containing C4BP isoforms (C4BPβ+). Under normal conditions, the molar concentration of protein S is ≈40% higher than that of C4BPβ+ and the molar surplus of protein S constitutes fPS.12 As a consequence, there is little or no free C4BPβ+ in the circulation. From these basic data, it follows that the concentration of C4BPβ+ greatly influences the plasma level of fPS. In this context, is it interesting that the C4BPA and C4BPB genes are differentially regulated by the inflammatory cytokines. C4BPA behaves as an acute phase gene, whereas the C4BPB gene does not. Therefore, even though the plasma levels of C4BP increase up to 4-fold during inflammatory reactions, most of these molecules lack the β-chain, ie, they are composed of only α-chains and cannot bind protein S.13–15⇓⇓ As a consequence of the coordinated regulation of expression of protein S and C4BPB genes, the concentration of fPS remains stable during acute phase reactions.
The individuals that take part in the GAIT project are healthy and are expected to have normal regulation of the C4BPA and C4BPB genes. The results of the GAIT investigation suggest that there is a genetic variability in the chromosome 1q region, which influences the concentration of fPS. It is likely that this genetic component specifically affects the basal level of expression of the β-chain gene (C4BPB). Increased expression of the C4BPB gene is expected to yield higher levels of the C4BPβ+ isoform in plasma and, as a result, slightly lower fPS levels. Whether these normal variations in regulation of C4BPB gene expression affect the fPS level to an extent that it will constitute a risk factor of thrombosis remains to be elucidated. It is not only this perspective that makes the report by Almasy et al4 interesting. In addition, the report provides a proof of concept that the genome-wide screening approach is useful for the identification of genetic components affecting circulating levels of important plasma proteins.
- ↵Almasy L, Soria JM, Souto JC, Coll I, Bacq D, Faure A, Mateo J, Borrell M, Muñoz X, Sala N, Stone WH, Lathrop M, Fontcuberta J, Blangero J. A quantitative trait locus influencing free plasma protein S levels on human chromosome 1q: results from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) project. Arterioscler Thromb Vasc Biol. 2003; 23: 508–511.
- ↵Dahlback B, Stenflo J. High molecular weight complex in human plasma between vitamin K- dependent protein S and complement component C4b-binding protein. Proc Natl Acad Sci U S A. 1981; 78: 2512–2516.
- ↵Dahlback B, Smith CA, Muller-Eberhard HJ. Visualization of human C4b-binding protein and its complexes with vitamin K-dependent protein S and complement protein C4b. Proc Natl Acad Sci U S A. 1983; 80: 3461–3465.
- ↵Hillarp A, Dahlback B. Novel subunit in C4b-binding protein required for protein S binding. J Biol Chem. 1988; 263: 12759–12764.
- ↵Webb JH, Blom AM, Dahlback B. Vitamin K-dependent protein S localizing complement regulator C4b-binding protein to the surface of apoptotic cells. J Immunol. 2002; 169: 2580–2586.
- ↵Zoller B, Garcia de Frutos P, Dahlback B. Evaluation of the relationship between protein S and C4b-binding protein isoforms in hereditary protein S deficiency demonstrating type I and type III deficiencies to be phenotypic variants of the same genetic disease. Blood. 1995; 85: 3524–3531.
- ↵Griffin JH, Gruber A, Fernandez JA. Reevaluation of total, free, and bound protein S and C4b-binding protein levels in plasma anticoagulated with citrate or hirudin. Blood. 1992; 79: 3203–3211.
- ↵Sanchez Corral P, Criado Garcia O, Rodriguez de Cordoba S. Isoforms of human C4b-binding protein. I. Molecular basis for the C4BP isoform pattern and its variations in human plasma. J Immunol. 1995; 155: 4030–4036.
- ↵Garcia de Frutos P, Alim RI, Hardig Y, Zoller B, Dahlback B. Differential regulation of alpha and beta chains of C4b-binding protein during acute-phase response resulting in stable plasma levels of free anticoagulant protein S. Blood. 1994; 84: 815–822.
- ↵Criado Garcia O, Sanchez Corral P, Rodriguez de Cordoba S. Isoforms of human C4b-binding protein. II. Differential modulation of the C4BPA and C4BPB genes by acute phase cytokines. J Immunol. 1995; 155: 4037–4043.