Association of protein S p.Pro667Pro dimorphism with plasma protein S levels in normal individuals and patients with inherited protein S deficiency.

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.

Type II protein C deficiency: identification and molecular modelling of two natural mutants with low anticoagulant and normal amidolytic activity.

Two mutations in exons 3 and 9 of the protein C gene were identified by amplification and sequencing from symptomatic probands referred for venous thromboembolism and thrombophilia screening. The phenotype associated with the mutations is a type II protein C deficiency with normal amidolytic activity. In one family, the mutation in exon 3 (G3545-->A), which predicts an R9 to H substitution in the Gla domain, was identified. A mutation in exon 9 (G10899-->A), which predicts an R352 to W substitution in the catalytic site, was identified in the second family and has been reported previously in association with type II deficiency with low amidolytic activity. Western blotting of the purified proteins from the probands' plasma did not show any abnormal migratory pattern. Molecular modelling suggested a possible impairment in the recently described Na+ binding pocket for the R352-->W mutant. No conclusions could be drawn relative to the R9-->H mutant.

Activation of the protein C pathway in hereditary thrombophilia.

Levels of free activated protein C are a measure of the activation of the protein C pathway in vivo. The aim of this study was to establish if the protein C pathway is triggered in familial thrombophilia and if activated protein C levels correlate with type of defect or symptoms. We measured activated protein C in 133 patients with a deficiency of antithrombin (n = 31), protein C (n = 24) or protein S (n = 27) or with resistance to activated protein C (n = 51). Levels of activated protein C were evaluated also in 97 healthy individuals. Results indicate that the levels of activated protein C are higher in patients who have experienced a thrombotic event than in patients who have not and that 71% of patients with levels of activated protein C above the normal reference range had had a venous thromboembolic event. We conclude that the protein C pathway is triggered in patients with thrombophilia and that in symptomatic patients, activated protein C levels are increased and may reflect heightened coagulation activation and scavenging through the protein C pathway.

Laboratory screening of thrombophilia. Evaluation of the diagnostic efficacy of a global test to detect congenital deficiencies of the protein C anticoagulant pathway.

Clinical laboratories are at present confronted with increasing demands for thrombophilia work-up, which may seriously overwhelm their capacity. Recently, methods able to investigate the protein C anticoagulant pathway globally have been proposed. In this study we investigated the reliability of one such method for its ability to detect patients with known defects of the pathway by testing plasmas from patients with the FVQ506 mutation, with congenital protein C, protein S or antithrombin deficiencies, and patients with previous history of thrombosis, but no identifiable defects. The results show that the new global test fulfils the requirements for congenital protein C deficiency and activated protein C resistance associated with the FVQ506 mutation, which account for more than half of the congenital defects found in thrombophilia. However, congenital protein S deficiency very often remains undetected by this test. Improvement of sensitivity toward this component of the protein C anticoagulant pathway would enroll the global test as a suitable candidate to explore the pathway. Since antithrombin, which also remains undetected by this test, is an additional important risk factor for venous thrombosis, devoting time and effort to developing global tests able to detect defects in both the antithrombin and protein C pathways is warranted.

Plasma levels of activated protein C in healthy subjects and patients with previous venous thromboembolism: relationships with plasma homocysteine levels.

The proteolytic enzyme activated protein C (APC) is a normal plasma component, indicating that protein C (PC) is continuously activated in vivo. High concentrations of homocysteine (Hcy) inhibit the activation of PC in vitro; this effect may account for the high risk for thrombosis in patients with hyperhomocysteinemia (HyperHcy). We measured the plasma levels of APC in 128 patients with previous venous thromboembolism (VTE) and in 98 age- and sex-matched healthy controls and correlated them with the plasma levels of total Hcy (tHcy) measured before and after an oral methionine loading (PML). Forty-eight patients had HyperHcy and 80 had normal levels of tHcy. No subject was known to have any of the congenital or acquired thrombophilic states at the time of the study. Because the plasma levels of APC and PC were correlated in healthy controls, the APC/PC ratios were also analyzed. Plasma APC levels and APC/PC ratios were significantly higher in VTE patients than in controls (P=0.03 and 0.0004, respectively). Most of the increase in APC levels and APC/PC ratios were attributable to patients with HyperHcy. Patients with normal tHcy had intermediate values, which did not differ significantly from those of healthy controls. There was no correlation between the plasma levels of tHcy or its PML increments and APC or APC/PC ratios in controls. The fasting plasma levels of APC and APC/PC ratios of 10 controls did not increase 4 hours PML, despite a 2-fold increase in tHcy. This study indicates that APC plasma levels are sensitive markers of activation of the hemostatic system in vivo and that Hcy does not interfere with the activation of PC in vivo.

Clinical manifestations in 28 Italian and Iranian patients with severe factor VII deficiency.

There has been wide variation in the reported haemorrhagic manifestations of factor VII deficiency. We examined type and frequency of clinical manifestations in 28 Iranian and Italian patients with severe deficiency (factor VII coagulant activity 2% or less). The most frequent symptoms were epistaxis and menorrhagia, whereas soft tissue bleeding such as haemarthrosis and muscle haematoma was less frequent. Only 5 of 9 patient who underwent surgery without factor VII replacement therapy had postoperative bleeding severe enough to require blood transfusion. No thrombotic manifestation occurred. A factor VII functional assay based on the use of human thromboplastin was a better predictor of the bleeding tendency of these patients than a rabbit thromboplastin-based functional assay or immunoassay. On the whole, this study shows that in severe factor VII deficiency bleeding in mucosal tracts is not uncommon. Surgery can sometimes be performed without replacement therapy and without haemorrhagic complications.

Resistance to activated protein C mimicking dysfunctional protein C: diagnostic approach.

It has been reported that resistance to activated protein C interferes with functional plasma-based coagulation assays of protein C, mimicking a type II deficiency. In this study we confirm and extend these findings. In our laboratory approximately 25% of patients with resistance to activated protein C have an apparent type II protein C deficiency. It is important for rapid and accurate diagnosis to be able to confirm or exclude a dysfunction of protein C associated with resistance. We therefore propose a new coagulation assay that requires first absorption of protein C from plasma, activation with a snake venom and measurement of its anticoagulant activity. This assay is quick, reproducible and can be automated. It is also insensitive to the presence of resistance to activated protein C and allows detection of all types of protein C deficiency. This is important when screening for inherited causes of thrombophilia since more than one defect might be present and interference from resistance to activated protein C is common.

Resistance to activated protein C in nine thrombophilic families: interference in a protein S functional assay.

Nine thrombophilic patients who had had previous diagnoses of functional protein S deficiency were reinvestigated. The functional protein S assays gave dose-response curves that were not parallel to those of the reference plasma. The same pattern was true for approximately half of the first-degree relatives of the propositi. When protein S was extracted from the plasma of the patients by immunoabsorption, it had a normal ratio of functional activity to immunologic concentration. Restriction fragment length polymorphism analysis, informative in one family, showed no linkage between the protein S gene marker and the abnormal behavior of the protein S functional assay. All the propositi and 23/36 first-degree relatives were resistant to the prolongation of activated partial thromboplastin time induced by activated protein C. Furthermore, there was striking concordance in all patients and relatives between the abnormal pattern of the protein S functional assay and resistance to activated protein C. We conclude that a plasma-based functional protein S assay is sensitive to activated protein C resistance and this may lead to spuriously low results in the assay. In agreement with the results of others, this study indicates that resistance to activated protein C is a frequent hemostatic defect in selected thrombophilic populations.