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Background: Protein S, which circulates in plasma in both free and bound forms, is an anticoagulant protein that stimulates activated protein C and tissue factor pathway inhibitor. Hereditary type I protein S deficiency (low total and low free protein S) is a well-established risk factor for venous thrombosis, whereas the thrombosis risk associated with type III deficiency (normal total and low free protein S) has been questioned., Design and Methods: Kaplan-Meier analysis was performed on 242 individuals from 30 families with protein S deficiency. Subjects were classified as normal, or having type I or type III deficiency according to their total and free protein S levels. Genetic and functional studies were performed in 23 families (132 individuals)., Results: Thrombosis-free survival was not different between type I and type III protein S-deficient individuals. Type III deficient individuals were older and had higher protein S, tissue factor pathway inhibitor and prothrombin levels than type I deficient individuals. Thrombin generation assays sensitive to the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S revealed similar hypercoagulable states in type I and type III protein S-deficient plasma. Twelve PROS1 mutations and two large deletions were identified in the genetically characterized families., Conclusions: Not only type I, but also type III protein S deficiency is associated with a hypercoagulable state and increased risk of thrombosis. These findings may, however, be restricted to type III deficient individuals from families with mixed type I/III protein S deficiency, as these represented 80% of type III deficient individuals in our cohort.

Background: Protein S, which circulates in plasma in a free and bound form, is an anticoagulant protein that stimulates both activated protein C (APC) and tissue factor pathway inhibitor (TFPI). Hereditary type I protein S deficiency (low total and low free protein S) is a well-established risk factor for venous thrombosis, whereas the thrombosis risk associated with type III deficiency (normal total and low free protein S) has been questioned., Design and Methods: Kaplan-Meier analysis was performed on 242 individuals from 30 families with protein S deficiency. Subjects were classified as normal, type I deficient or type III deficient according to their total and free protein S levels. Genetic and functional studies were performed in 23 families (132 individuals)., Results: Thrombosis-free survival was not different between type I and type III protein S-deficient individuals. Type III deficient individuals were older and had higher protein S, TFPI and prothrombin levels than type I deficient individuals. Thrombin generation assays sensitive to the APC- and TFPI-cofactor activities of protein S revealed similar hypercoagulable states in type I and type III protein S-deficient plasma. Twelve PROS1 mutations and 2 large deletions were identified in the genetically characterized families., Conclusions: Not only type I, but also type III protein S deficiency is associated with a hypercoagulable state and increased thrombosis risk. However, these findings may be restricted to type III deficient individuals from families with mixed type I/III protein S deficiency, as these represented 80% of type III deficient individuals in our cohort.

Objective: To determine whether specific subtypes of early-onset hypertensive disorders of pregnancy (haemolysis, elevated liver enzymes, low platelets [HELLP] syndrome; severe preeclampsia; eclampsia; and fetal growth restriction) differ in increased prevalences of thrombophilic disorders., Design: Cohort study., Setting: Two university hospitals in Amsterdam, the Netherlands., Population: 216 patients participating in a randomized clinical trial with severe and early-onset hypertensive disorders of pregnancy., Methods: More than 3 months after delivery, all patients were invited for a thrombophilia screening protocol, including hereditary thrombophilic disorders (Factor II or V-Leiden mutation, APC-resistance, protein S deficiency), antiphospholipid antibodies (anticardiolipin antibodies and lupus anticoagulant activity), and hyperhomocysteinemia (before and after methionin challenge). Disease expression was classified by HELLP syndrome, severe preeclampsia, or neonatal birth weight ratio below the median (0.65). Univariate and multinomial regression analyses examined the association of disease expression with thrombophilic disorders, and other associated factors (chronic hypertension, smoking, body mass index, positive family history of cardiovascular morbidity, and demographic parameters)., Main Outcome Measures: incidence of thrombophilic disorders in different subtypes of disease., Results: Overall prevalence of thrombophilic disorders in 206 (95%) screened women was 36%. Chronic hypertension was present in 32%, and 34% had a positive family history of cardiovascular morbidity. Multinomial regression analysis showed that hereditary thrombophilia was more frequent among women with infants with a birth weight ratio <0.65 than in women with HELLP syndrome or severe preeclampsia (p = 0.01, OR 5.1 (1.5 to 7.3) and OR 3.4 (1.1 to 10.6), respectively). High body mass index was less frequent in women with HELLP syndrome than in those with severe preeclampsia or fetal growth restriction (p = 0.06, OR 0.5 (0.3 to 0.9) and OR 0.4 (0.2 to 1.0), respectively)., Conclusion: In this population, the high prevalence of thrombophilic factors and chronic hypertension was confirmed. There were small differences between groups. Hereditary thrombophilic disorders were associated with fetal growth restriction but not with type of maternal disease, suggesting an effect on placental function. Maternal body mass index was lower in women with HELLP syndrome.

A dimorphism in PROS1 gene (c.A2,001G, p.Pro667Pro) has been associated with significantly reduced levels of both free and total protein S in carriers of the GG genotype. It is not known how the GG genotype could influence PS levels in normals, whether it could influence the levels of protein S in carriers of mutations in PROS1 gene and whether this genotype acts as an isolated or additive risk factor for venous thrombosis. With this as background, we evaluated the association of p.Pro667Pro dimorphism with free and total protein S centrally measured in a panel of 119 normal controls, 222 individuals with low protein S and 137 individuals with normal PS levels belonging to 76 families with protein S deficiency enrolled in the ProSIT study. Transient expression of recombinant wild type protein S and p.Pro667Pro protein S was performed to evaluate the role of the A to G transition at position 2001 in vitro. The p.Pro667Pro polymorphism was also expressed together with a p.Glu67Ala variant to assess a possible influence on protein S levels in protein S deficient subjects. Free and total protein S levels were significantly lower in normal women. In normal women only was the GG genotype associated with significantly lower free protein S levels in comparison to AA and AG genotypes (P=0.032). No significant influence of GG genotype was observed in patients, either with known mutations or with low protein S levels. These data were confirmed by in vitro transient expression, showing no difference in secretion levels of the p.Pro667Pro variant (even in association with the p.Glu67Ala mutation), compared to the wild type protein S. The genotype in itself was neither a significant risk factor for venous thrombosis nor a risk modifier in patients with known mutations.

Hereditary protein S (PS) deficiency type I is an established risk factor for venous thromboembolism. Contradictionary data on type III deficiency suggests a difference in risk between both types. We studied 156 first degree relatives (90% of eligible relatives) from type I deficient probands (cohort 1) and 268 (88%) from type III deficient probands (cohort 2) to determine the absolute risk of venous and arterial thromboembolism. Annual incidences of venous thromboembolism were 1.47 and 0.17 per 100 person-years in deficient and non-deficient relatives in cohort 1 [relative risk (RR) 8.9; 95% confidence interval (CI) 2.6-30.0], and 0.27 vs. 0.24 in cohort 2 (RR 0.9; 95% CI 0.4-2.2). Type III deficiency was demonstrated in 20% of non-deficient relatives in cohort 1 and the annual incidence in this subgroup was 0.70 (RR 4.3;0.95-19.0). The cut-off level of free PS to identify subjects at risk was 30%, the lower limit of its normal range (65%). PS deficiency was not a risk factor for arterial thromboembolism. In conclusion, type I deficiency was found to be a strong risk factor for venous thromboembolism, in contrast with type III deficiency. This was because of lower free PS levels in type I deficient subjects and a free PS cut-off level far below the lower limit of its normal range.

This paper presents a critical assessment of protein C (PC) and protein S (PS) functional and immunological approaches with regard to DNA sequencing in a large hospital recruitment for thrombosis exploration in more than 1700 consecutive patients. After examination of clinical status and PC and PS phenotype, a genotypic study was implemented for 17 PC-deficient and 28 PS-deficient patients (activity < 70%). Sixty-five percent of the genotyped PC-deficient patients were found to have heterozygous mutations. Among the < 70% values, decreases in PC activity without gene mutation were always slight (mean value 64 +/- 7%) while patients presenting a PC gene mutation had a mean 50 +/- 17% activity (P < 0.05). Among the eight PC mutations found, only one has previously been described. A novel mutation in the promoter region (-1522), located in the HNF-1 site and associated with the Y226H heterozygous mutation, was found in a 9-month-old girl with 4% PC activity. Determination of PS functional activity was considerably improved by contemporaneous measurement of calibration and samples in a single step. Only 50% of the genotyped PS-deficient patients demonstrated heterozygous alterations of the gene. The benefit of sequencing to identify putative causal mutations was only 39% in PS-deficient women, while it was 90% in men. Among the nine PS mutations found, six have not yet been published. In the present paper, we explain our methodological choices and diagnostic strategy.

While many mutations thought to result in protein S (PS) deficiency are known, there have been few attempts to relate genotype expression with plasma phenotype. We have investigated the nature and consequence of PS gene (PROS1) mutations in 17 PS-deficient families who presented with mixed type I and type III phenotypes. Seven different mutations were found in nine families: delG-34 (STOP codon at -24), Val-24Glu, Arg49Cys, Asn217Ser, Gly295Val, +5 G to A intron j and His623Pro. PS wild type (PSWT) and the five missense mutants were transiently expressed in COS-1 cells. All mutants expressed lower (p<0.05) PS antigen compared to PSWT (100%). The mutants Val-24Glu, Gly295Val and His623Pro expressed very low/undetectable PS levels. The mutant Asn217Ser produced around 30% of PSWT, while the mutant Arg49Cys had the highest PS levels (around 50%). Metabolic labelling and pulse-chase experiments showed that all of the mutants had impaired secretion, but this was of variable severity. Also, enhanced intracellular degradation of unsecreted material was found for all mutants. There was a strong correspondence between plasma free PS levels in carriers of the mutations, secreted PS from transfected COS-1 cells and labelled PS from 24 h conditioned medium in pulse-chase experiment. We conclude that the magnitude of secretion defect depends on the nature of the PROS1 mutation and influences the level of free PS in plasma. It is likely that the severity of the secretion defect will determine the risk for venous thrombosis.

We measured protein S (PS) activity in a large group of Japanese subjects to study the frequency of PS deficiency. The study group comprised 213 men, ages 18-58 years, and 179 women, ages 18-60 years. PS activity in the total 392 subjects was 58-135% (mean +/- 2 SD), 65-135% in the men and 54-120% in the women. The men showed significantly higher levels of PS activity than the women (p<0.001). We identified 8 subjects (4 men and 4 women) in whom PS activity was lower than the mean-2SD for men and women, respectively. Moreover, we examined the classifications of PS deficiency. The frequency of PS deficiency in this study was 2.04% (Type I: 0.51%, Type II: 1.02%, Type III: 0.51%). Based on our findings, it would appear that the frequency of Type II PS deficiency in the Japanese population is approximately 1%. The screening of PS decrease should be judged by activity.

Congenital protein S (PS) deficiency is associated with increased risk of venous thrombosis. To investigate the possibility of automating PS testing with decreased turnaround time, a clotting-based functional protein S assay was evaluated and compared to an antigenic method. Samples were collected from 126 patients within 5 days of their first acute cerebral infarction, from 62 controls and from 47 consecutive samples for thrombophilia investigation. The normal range for the clotting-based kit, calculated from the results of 20 healthy controls, was 62-136% (mean +/- 2 SD). Intra- and inter-assay co-efficients of variation were < 3.0 and 10.0% respectively. There was no significant correlation between the two methods (r = 0.30, P > 0.05). Two patients had low PS antigen results with normal functional levels. Both techniques were used to compare a further group of 53 patients with defined abnormalities which included nine antigenic protein S deficiencies, five protein C deficient patients, 10 patients with a lupus anticoagulant (LA), 17 Factor V Leiden (FVL) heterozygotes, two FVL homozygotes and 10 patients on therapeutic levels of heparin. In this group we found that four of nine antigenic PS deficient patients had normal functional PS levels. The test was susceptible to the FVL mutation with four of 17 FVL heterozygotes and both of two FVL homozygotes giving low levels. One of five protein C-deficient patients also had a low functional PS result with a normal antigenic level. Normal results were obtained by both methods for all of the LA and patients on therapeutic heparin. We concluded that the automated protein S clotting assay was rapid and simple to perform but appeared to be influenced by factors other than PS deficiency. Results need to be interpreted with caution but may be useful as part of a full thrombophilia investigation.

DNA sequence analysis of the protein S gene (PROS1) in 22 Spanish probands with type I or III PS deficiency, has allowed the identification of 10 different mutations and 2 new sequence variants in 15 probands. Nine of the mutations, 8 of which are novel, cosegregate with type I or quantitative PS deficiency in 12 of the 13 pedigrees analyzed. One of these mutations (Q238X) also cosegregates with both type I and III PS-deficient phenotypes coexisting in a type I/III pedigree. Another mutation identified in a pedigree with these two PS phenotypes is the missense mutation R520G, present in the homozygous form in the type I propositus and in the heterozygous form in his type III relatives. By contrast, no cosegregating PROS1 mutation has been found in any of the six families with only type III phenotypes. Three of these families, as well as the two families with type I and I/III phenotypes where no other PROS1 mutation has been identified, segregate the P allele of the S460P variant, although this allele does not always cosegregate with the deficient phenotype. From these results we conclude that while mutations in PROS1 are the main cause of type I PS deficiency, the molecular basis of the type III phenotype is probably more complex, with many cases not being explained by a PROS1 mutation.

Protein C and S deficiency states predispose affected individuals to thrombosis, especially venous thrombosis of the lower extremities, usually beginning in the teenage years. Treatment of these patients is generally with oral anticoagulation, following initial heparinization. The classification of the deficiency states is dependent upon determination of the quantity and functional activity of either protein.

Thromboembolic disease results from an hypercoagulable state and multifactorial causes may lead to hypercoagulability. Thrombogenic risk factors can be acquired and/or inherited. For each thrombophilic patient, the main clinical features retained are: the patient age, the familial history, the recurrence of thromboembolic events, an unusual site of thrombosis. Anti-phospholipid antibodies, which are considered as acquired thrombogenic risk factors, can be detected with coagulation tests and/or Elisa methods. The association of antiphospholipid antibodies with thrombosis is defined as the anti-phospholipid syndrome. Last decades, genetic risk factors were identified. First of all, antithrombin, protein C and protein S deficiencies were described. These deficiencies are involved in about 10% of patients who develop thrombosis before the age of 50. In 1993, a new genetic risk factor was discovered: activated protein C resistance which is due to the Q506 mutation in factor V. This defect represents the most prevalent abnormality of inherited thrombophilia, affecting 20 to 40% of thrombophilic patients. Interestingly, hyperhomocysteinemia, known as potentially predisposing to arterial disease, was also recognized as a risk factor for venous occlusive disease. Several genes encoding homocystein metabolism enzymes, such as cystathionine beta-synthase or methylenetetrahydrofolate reductase are concerned. Establishment of a causal association between the presence of a biological abnormality and the occurrence of thrombosis may lead to an adapted prophylaxis whatever the risk situation.

Protein S deficiency is a known risk factor for thrombosis. The coexistence of phenotypic type I (reduction in total and free antigen) and type III (reduction in free antigen only) protein S deficiencies in 14 of 18 families was recently reported. We investigated the cause of this phenotypic variation in the largest of these families (122 family members, including 44 affected individuals) using both molecular genetic and phenotypic analysis. We have identified a sole causative mutation (Gly295Val) in three family members representative of the variable phenotype. Complete cosegregation of the mutation with reduced free protein S antigen levels was found, regardless of the total antigen level. Analysis of phenotypic data showed high correlations between total protein S antigen and age in both normal and protein S-deficient family members, irrespective of gender. Free protein S antigen levels were not influenced by age, a finding explained by an association between beta-chain containing C4b-binding protein (C4bBP-beta+) antigen levels and age. We propose that the identified Gly295Val mutation causes quantitative, or type I, protein S deficiency, and that as age increases the total protein S antigen level normalizes with respect to the reference plasma pool, giving rise to a type III protein S-deficient phenotype.

To elucidate the molecular basis of hereditary protein S (PS) deficiency and, in particular, type III or free PS deficiency, the allelic distribution and segregation patterns of the PS gene (PROS1) polymorphisms P626A/G and S460P (PS Heerlen) have been analyzed in a group of 45 proposita suffering from type I or type III PS deficiency. No differences between patients and controls were found in the frequency of the P626A/G alleles. By contrast, the frequency of the PS Heerlen allele in the group of patients with type III PS deficiency (9 of 46 chromosomes, P = .196) was significantly higher (P < .001) than in the control group (1 of 300 chromosomes, P = .003). The A allele of P626A/G was always associated with the P allele of S460P. However, this haplotype did not co-segregate with the type III PS-deficient phenotype in 3 of the families. Furthermore, multipoint linkage analysis excluded the whole PROS1 gene in 1 of these families, which is in agreement with the absence of mutations in the PROS1 gene, as determined by sequence analysis. Finally, linkage analysis with 4 microsatellite markers linked to the C4BPB and C4BPA loci also excluded these two genes. From these results we conclude that, at least in some families, the molecular basis of type III PS deficiency is not due to the Mendelian inheritance of a single defect in the PROS1 or in the C4BP genes.

We identified potentially causative mutations in the active protein S gene (PROS 1) by direct sequencing of PROS 1-specific polymerase chain reaction (PRC) products of all 15 exons, including exon-intron boundaries in 10 families with hereditary protein S deficiency type I. Seven different mutations were found in 9 of 10 families, including one frame shift mutation, a previously published splice site mutation (both occurring in two unrelated families), four missense mutations, and a stop codon at the beginning of exon 12. In family studies, cosegregation of the mutation with the disease could be demonstrated for five mutations; for two missense mutations, this was not possible due to limited family data. All seven mutations were the only abnormalities identified in the respective index patients and were absent in 44 to 62 normal individuals. Therefore, they most likely represent the causal gene defects. For five mutations, analysis of ectopic RNA could be performed. Mutant transcripts were present in the case of the frame shift and three of the missense mutations, while no mutant RNA could be detected in the case of the stop codon.

A protein S gene polymorphism, detectable by restriction analysis (BstXI) of amplified exonic sequences (exon 15), was studied in seven Italian families with protein S deficiency. In the 17 individuals heterozygous for the polymorphism the study was extended to platelet mRNA through reverse transcription, amplification and densitometric analysis. mRNA produced by the putative defective protein S genes was absent in three families and reduced to a different extent (as expressed by altered allelic ratios) in four families. The allelic ratios helped to distinguish total protein S deficiency (type I) for free protein S deficiency (type IIa) in families with equivocal phenotypes. This study indicates that the study of platelet mRNA, in association with phenotypic analysis based upon protein S assays in plasma, helps to classify patients with protein S deficiency.