Protein S Deficiency 

In 1979, researchers in Seattle, Wash, first discovered protein S and arbitrarily named it after the city of its discovery. Protein S is a vitamin K–dependent anticoagulant protein. Its major function is as a cofactor to facilitate the action of activated protein C (APC) on its substrates, activated factor V (FVa) and activated factor VIII (FVIIIa). Protein S deficiencies are associated with thrombosis.

Protein S deficiency may be hereditary or acquired, the latter is usually due to hepatic diseases or a vitamin K deficiency. Protein S deficiency usually manifests clinically as venous thromboembolism (VTE). The association of protein S deficiency with arterial thrombosis appears coincidental or weak at best. Arterial thrombosis is not evident with other hereditary anticoagulant abnormalities (eg, protein C or antithrombin III deficiency, factor V Leiden gene mutation). Protein S deficiency manifests as an autosomal dominant trait; manifestations of thrombosis are observed in both heterozygous and homozygous genetic deficiencies of protein S.

To understand how thrombosis occurs in protein S deficiency, its physiological function should be briefly reviewed. Protein S is part of a system of anticoagulant proteins that regulate normal coagulation mechanisms in the body.^[1] Under most normal circumstances, the anticoagulant proteins prevail and blood remains in a liquid nonthrombotic state. Whenever procoagulant forces are locally activated to form a physiologic or pathologic clot, protein S participates as part of one mechanism of controlling clot formation.^{[2, 3, 4]}

Protein S functions predominantly as a nonenzymatic cofactor for the action of another anticoagulant protein, activated protein C (APC). This activity occurs via a coordinated system of proteins, termed the protein C system. The image below shows a simplified outline of the function of protein S in the protein C system.

During the process of clotting, multimolecular complexes are formed on membrane surfaces. These membranes are usually negatively charged phospholipids and/or activated platelets. These multimolecular complexes are referred to as the tenase and prothrombinase complexes for their key activities of activation of factor X and prothrombin, respectively. Anchoring these two complexes are the activated form of factor VIII (FVIIa) used for the tenase complex and the activated form of factor V (FVa) for the prothrombinase complex. These two large proteins are homologous in structure and are cofactors, not enzymes, in the clotting process.

In one of many examples of nature's efficiency, the same enzyme that clots blood, thrombin, is converted from clotting to an anticoagulant mechanism on the surface of the endothelium and it then activates protein C to its active enzymatic form, APC. APC requires protein S as a cofactor in its enzymatic action on its 2 substrates, FVa and FVIIIa. Thus, this process is designed to dampen and shut off clotting by switching off the key cofactor proteins FVa and FVIIIa. Protein S and APC are sufficient to inactivate FVa. However, for the inactivation of FVIIIa, APC and protein S require the help of the nonactivated clotting protein, factor V. This is another example of dual use of a protein in this same process.

Factor Va as noted above is cleaved by APC to an inactive form. However, in assisting protein S to inactivate FVIIIa, it is the inactive FV that is cleaved by APC. An important consequence of this dual procoagulant and anticoagulant property of factor V, is that the mutant factor V Leiden, which resists APC cleavage, cannot be switched off but also cannot function here at this step as an anticoagulant protein (see factor V Leiden gene mutation in Race). In addition to its cofactor role in the protein C system, protein S functions independently of protein C to directly inhibit the factor X clotting factor–activating complex and the prothrombin-activating complex.

Furthermore, protein S interacts with the complement system and may play a role in phagocytosis of apoptotic cells. It has been recently observed that protein S binds to phosphatidyl serine on apoptotic cells and stimulates macrophage phagocytosis of early apoptotic cells. The physiological impact of protein S deficiencies on these nonanticoagulant roles of protein S is not yet known.

Protein S is a single-chain glycoprotein, and it is dependent on vitamin K action for posttranslational modification of the protein to a normal functional state. Vitamin K–dependent proteins are synthesized with a unique recognition propeptide piece. The propeptide sequence serves as a recognition site for the vitamin K–dependent gamma-carboxylase enzyme that modifies the nearby glutamic acid residues to gamma-carboxyglutamic acid (Gla) residues. Gla residues are responsible for calcium-dependent binding to membrane surfaces. Structural studies indicate that protein S contains 10-12 Gla residues, a loop region sensitive to thrombin (ie, thrombin-sensitive region [TSR]), 4 epidermal growth factor (EGF)–like modules, and a carboxy-terminal portion that is homologous to a sex hormone-binding globulin (SHBG)–like region.

In blood plasma, protein S exists in both a bound and a free state. A portion of protein S is noncovalently bound with high affinity to the complement regulatory protein C4b-binding protein (C4BP). The C4BP molecule consists of 7 alpha chains that bind to the complement protein, C4b, and one beta chain. The beta chain of the C4bBP molecules contains the binding sites for protein S. The role of protein S in complement regulation by C4BP is not completely understood.

In healthy individuals, approximately 30-40% of total protein S is in the free state. Only free protein S is capable of acting as a cofactor in the protein C system. This distinction between free and total protein S levels is important and gives rise to the current terminology regarding the deficiency states. Type I protein S deficiency is a reduction in the level of free and total protein S. Type III deficiency is a reduction in the level of free protein S only. Type II deficiency is a reduction in the cofactor activity of protein S, with normal antigenic levels.

APC and protein S require negatively charged phospholipids (PL) and Ca²⁺ for normal anticoagulant activity. Studies of the structure and function relationships of protein S demonstrate that the APC interaction sites are located in the Gla, TSR, and first EGF-like modules of protein S. The binding site for C4BP is located in the SHBG-like region, which is also important for full anticoagulant activity.

A recent study by Heeb et al reported protein S has APC-independent anticoagulant activity, termed PS-direct, that directly inhibits thrombin generated by the factor Xa/factor Va prothrombinase complex,^[5] a process made possible by the presence of zinc (Zn²⁺) content in protein S. The investigators found Zn²⁺ content positively correlated with PS-direct in prothrombinase and clotting assays, but the APC-cofactor activity of protein S was independent of Zn²⁺ content. In addition, protein S that contained Zn²⁺ bound factor Xa more efficiently than protein S without Zn²⁺, and, independent of Zn²⁺ content, protein S also efficiently bound tissue factor pathway inhibitor.^[5]

Heeb et al suggested that conformation differences at or near the interface of 2 laminin G-like domains near the protein S C terminus may indicate that Zn²⁺ is necessary for PS-direct and efficient factor Xa binding and could have a role in stabilizing protein S conformation.^[5]

Researchers have identified 2 genes for human protein S and both are linked closely on chromosome 3p11.1-3q11.2. One gene is the active gene, PROS -b (ie, PROS1), and the other, PROS- a, is an evolutionarily duplicated nonfunctional gene, which is classified as a pseudogene because it contains multiple coding errors (eg, frameshifts, stop codons). The expressed (alpha) PROS1 gene is more than 80 kb long and contains 15 exons and 14 introns. The protein S pseudogene (beta) has 97% homology to the PROS -a gene.

Molecular studies into the genetic causes of protein S deficiency are complicated by the presence of the pseudogene, PROS- b, and phenotypic variation. Deletions of large portions of the PROS- a gene are associated with protein S deficiency and thrombophilia.^[6] Researchers located the first such deletion in the central portion of the PROS- a gene. The second deletion described (5.3 kb) was a deletion of coding exon XIII, which resulted in a truncated protein product.

Family members with either deletion exhibit protein S deficiency and thrombophilia; however, subsequent studies indicate that the most common genetic defects in the protein S gene are point mutations rather than gene deletions. Phenotypic variation has been observed in protein S deficiency. The coexistence of type I deficiency and type III deficiency in families with the same protein S mutation has been shown at least in one family to be due to an age-related increase in total protein S level. In this family, the apparent type III variant with only low free S blood levels, was explained by the age increase in total protein S. Younger persons in the family when tested for blood levels still had low total and free protein S.

Frequency

United States

Congenital protein S deficiency is an autosomal dominant disease, and the heterozygous state occurs in approximately 2% of unselected patients with VTE.

Protein S deficiency is rare in the healthy population without abnormalities. Frequency is approximately 1 out of 700 based on extrapolations from a study of over 9000 blood donors who were tested for protein C deficiency. When looking at a selected group of patients with recurrent thrombosis or family history of thrombosis, the frequency of protein S deficiency increases to 3-6%.^[7]

Very rarely, protein S deficiency occurs as a homozygous state, and these individuals have a characteristic thrombotic disorder, purpura fulminans. Purpura fulminans is characterized by small vessel thrombosis with cutaneous and subcutaneous necrosis, and it appears early in life, usually during the neonatal period or within the first year of life.^[8]

International

Data for European studies indicated the same frequencies for protein S deficiency as in the United States. Recent studies have indicated that the prevalence of protein S deficiency is particularly high in the Japanese population. In several reported series of patients with VTE in the United States, protein S deficiency was seen in 1-7% of patients. The deficiency is rare in population surveys of Caucasians, at approximately 0.03%. However, Japanese patients with VTE have reported a frequency of approximately 12.7% protein S deficiency and similarly elevated population frequencies of approximately 0.63%.

Mortality/Morbidity

• VTE develops in 60-80% of patients who are heterozygous for protein S deficiency. The remaining patients are asymptomatic, and some heterozygous individuals never develop VTE. Though it is controversial, no clear association exists between protein S deficiency and arterial thrombosis. Many case reports and small case series describe protein S deficiency as one factor found in patients with arterial thrombosis most commonly stroke. However, prospective and cohort studies have not shown convincing increased risk for arterial thrombosis.
• Protein S deficiency is also associated with fetal loss in women, in the absence of VTE. Some authors suggest that as many as 40% of women with obstetric complications other than VTE may carry some form of thrombophilia. Protein S deficiency is one of these factors along with several other more common genetic thrombophilic states.
• Mortality is from pulmonary embolism. In several studies, the 3-month mortality rate of pulmonary embolism ranged from 10.0-17.5%. In a study of Medicare recipients with pulmonary embolism, men had a 13.7% mortality rate compared to 12.8% in women; the mortality rate was 16.1% in blacks, compared to 12.9% in whites.

Race

Race-related variations exist in thrombophilic disorders, as one may expect from genetic-based population traits. In general, a significant difference exists in the frequency of thrombophilic disorders in whites compared with thrombophilic disorders in Japanese (Asian) and black African persons. Current research indicates that protein S deficiency is 5-10 times higher in Japanese populations compared with Caucasians. Protein C deficiency is estimated to be 3 times higher in Japanese populations.

The factor V Leiden mutation is common in white populations and is now known to be the result of a founder effect estimated to be 30,000 years old. This mutation is rare and almost never found in Japanese or Asian populations. In general, black Africans and African Americans with VTE have a lower detection of any of the currently recognized thrombophilic disorders, especially factor V Leiden.

Sex

No difference exists in the male-to-female rate of occurrence.

Age

Protein S deficiency is a hereditary disorder, but the age of onset of thrombosis varies by heterozygous versus homozygous state. Most VTE events in heterozygous protein S deficiency occur in persons younger than 40-45 years. The rare homozygous patients have neonatal purpura fulminans, as described above; onset occurs in early infancy. As noted above in the discussion on genetics, age does affect total protein S antigen levels, but not free protein S levels. Older patients deficient in protein S have low free S levels, even if their total protein S level rises into the normal range.