Free Protein S Antigen Research Articles

Cost-Effective Use of the Protein S Algorithm in Thrombophilia Testing.

One of the most complex risk factors for the laboratory assessment of thrombophilia is Protein S (PS). The testing algorithm for PS employs the plasma-based assays of free PS antigen, total PS antigen, and PS activity creating a complex diagnostic scheme that can lead to misdiagnosis if incorrectly used, and a potential waste of resources and money. This paper compares the recently published evidence-based algorithm from the International Society for Hemostasis and Thrombosis (ISTH) with several commonly performed nonevidence-based testing schemes, to demonstrate the efficiency of the evidence-based algorithm for diagnostic efficiency with improved patient care and increased cost savings for the laboratory. Significant savings (31%-60%) can be realized when the evidence-based algorithm is used in place of other testing modalities of initial PS activity testing (31%) or testing with all 3 assays simultaneously (60%). This study utilizing the PS testing evidence-based algorithm as part of a thrombophilia evaluation demonstrates that the appropriate testing methods can be used to limit wasteful practices while achieving the maximum level of information in this time of limited resources and need for increase monetary savings.

Evaluation of the hemostatic capacity of methylene blue-treated liquid (not frozen) plasma stored up 14 days at 2° to 6°C.

Early plasma transfusion for management of bleeding, particularly trauma, is associated with better outcomes. Improving the availability/safety of plasma transfusion for patients is essential for transfusion services. The aim of this study is to evaluate the hemostatic capacity of methylene-blue (MB) liquid (not frozen) plasma over time. Twenty whole blood-derived plasma units collected from male donors were separated and processed within 18 h of collection. Individual plasmas were treated with MB and stored in liquid status at 2-6°C for 14 days. A range of coagulation assays, including thrombin generation, rotational thromboelastometry (ROTEM), and Thrombodynamics were tested at different time-points, together with bacterial growth. Apart from Factor (F)XII, other coagulation factors (fibrinogen, FV, FVIII, FXI) reduced significantly after MB treatment, with levels remaining stable except for FVIII afterward. By day 14, most clotting factors were >0.7IU/ml, apart from FVIII. There was a disproportionate decrease in Protein S (PS) activity compared to free PS antigen and by day 14 its value was ~50%. There was no significant difference in maximum clot formation (ROTEM) and clot-density (Thrombodynamics) over time. Endogenous thrombin potential (Thrombin-Generation), clot-size, and velocity index (Thrombodynamics) decreased significantly over time consistent with clotting factor reduction. There was no bacterial growth. MB-treated liquid plasma stored at 2-6°C can be used for up to 14 days: the long shelf-life, the liquid status, and the MB treatment will improve its availability for management of bleeding as well as providing a safe component from pathogens.

Effects of low-dose combined oral contraceptives and protein S K196E mutation on anticoagulation factors: a prospective observational study.

The association between low-dose combined oral contraceptives (COCs) and anticoagulation factors in Japanese women has been rarely studied. A total of 394 Japanese women with a new beginning cycle of COC use were enrolled, of whom 335 women visited the clinic within 4weeks after starting the first cycle of COC. Visits occurred in the active phase (272 women) and the placebo phase (63 women). Free protein S (PS) antigen and activity levels and antithrombin activity levels decreased significantly in both the active and placebo phase groups. Protein C (PC) activity levels increased significantly in both groups. Larger reductions in free PS antigen and activity levels occurred with COC comprising either 30µg ethinylestradiol/desogestrel or 20µg ethinylestradiol/drospirenone than that comprising 35µg ethinylestradiol/norethisterone. In four women with the Japanese-specific PS K196E mutation, mean PS activity was 65% before COC use and 57% during COC use, indicating further decrease with COC use. In conclusion, decreased antigen and activity levels of PS and antithrombin and increased activity levels of PC were observed even during the first cycle of low-dose COC use. The effects on PS and PC activities were also observed in the hormone-free interval.

Single Ventricle Physiology Patients and Coagulation Abnormalities: Is There a Relationship With Hemodynamic Data and Postoperative Course? A Pilot Study.

This study evaluates coagulation profiles of single ventricle (SV) patients in relationship to liver function, hemodynamic variables and outcome. Twenty-six children with SV anatomy were included. Advanced coagulation profiles, invasive preoperative hemodynamic parameters and clinical course were retrospectively analyzed. Median (interquartile range) age and weight at the time of blood sampling was 25.5 (31) months and 10.5 (6.9) kg. Sixteen patients (16/26; 62%) showed decreased antithrombin and/or protein C (PC) and/or free protein S (PS) function and/or free PS antigen. Two patients showed abnormal activated PC resistance ratio due to heterozygous factor V Leiden mutation and 1 heterozygous prothrombin G20210A mutation. Group comparison (abnormal coagulation profile [group 1; n = 16] versus normal coagulation profile [group 2; n = 10]) showed longer postoperative hospitalization time (p = .04), longer postoperative catecholamine support (p = .01), a higher incidence of thromboembolic events (p = .04), and chylothoraxes (p = .007) in group 1. In 5 (31%) of 16 group 1 patients, thromboembolic complications occurred: cerebral stroke (n = 1), intestinal ischemia (n = 2), thrombus formation in inferior caval vein (n = 1), and pulmonary vein (n = 1). Abnormalities in coagulation parameters are common in SV patients. Coagulation abnormalities constitute a preoperative risk factor and affect postoperative course.

Assessment of Hereditary Thrombophilia: Performance of Protein S (PS) Testing.

Protein S (PS) is a Vitamin K-dependent protein that functions as a cofactor for the regulation of the coagulation system. PS works in conjunction with Activated Protein C to inactivate factors V and VIII. PS circulates in plasma either complexed to the complement protein, C4b Binding Protein or unbound. The unbound (or free) component is the functional form for the regulation of the coagulation system. PS can be measured in plasma by functional activity, the free (or unbound form) or both free and bound fractions (Total PS). The test most widely used for clinical evaluations is the Free PS Antigen assay (which is the surrogate of PS anticoagulant activity) and represents the protocol described in this chapter. The Free PS Antigen assay is an immunologic assay which specifically measures the unbound fraction of PS in test plasma. Other methods for assessing PS are also available, including PS activity and total PS Antigen assays, but protocols for these assays are not provided.

Protein S deficiency complicated pregnancy in women with recurrent pregnancy loss

This prospective study aimed to evaluate pregnancy outcome and complications in women with recurrent pregnancy loss (RPL) and protein S (PS) deficiency, who received low dose aspirin (LDA) or LDA plus heparin (LDA/H) therapies. Clinical characteristics, pregnancy outcome and complications of 38 women with two or more RPL and <60% of plasma free PS antigen were compared among three groups: antiphospholipid antibody (aPL)-negative women who received LDA (group A), aPL-negative women who received LDA/H (group B) and aPL-positive women who received LDA/H (group C). Gestational weeks (GW) at delivery in group C (median 32 GW) were earlier than 40 GW in group A and 38.5 GW in group B (p < 0.05). The birth weight in group C (median 1794 g) was less than 2855 g in group B (p < 0.05). The incidences of fetal growth restriction (37.5%), pregnancy-induced hypertension (37.5%), and preterm delivery (62.5%) in group C were higher than those (4.5%, 0%, and 4.5%, respectively) in group B (p<0.05). Women with RPL, PS deficiency, and positive aPL had high risks for adverse pregnancy outcome and complications, even when they received LDA/H therapy. Among women with RPL, PS, and negative aPL, there was no difference in these risks between LDA alone and LDA/H therapies.

Protein S deficiency complicated pregnancy in women with recurrent pregnancy loss

This prospective study aimed to evaluate pregnancy outcome and complications in women with recurrent pregnancy loss (RPL) and protein S (PS) deficiency, who received low dose aspirin (LDA) or LDA plus heparin (LDA/H) therapies. Clinical characteristics, pregnancy outcome and complications of 38 women with two or more RPL and <60% of plasma free PS antigen were compared among three groups: antiphospholipid antibody (aPL)-negative women who received LDA (group A), aPL-negative women who received LDA/H (group B) and aPL-positive women who received LDA/H (group C). Gestational weeks (GW) at delivery in group C (median 32 GW) were earlier than 40 GW in group A and 38.5 GW in group B (p < 0.05). The birth weight in group C (median 1794 g) was less than 2855 g in group B (p < 0.05). The incidences of fetal growth restriction (37.5%), pregnancy-induced hypertension (37.5%), and preterm delivery (62.5%) in group C were higher than those (4.5%, 0%, and 4.5%, respectively) in group B (p<0.05). Women with RPL, PS deficiency, and positive aPL had high risks for adverse pregnancy outcome and complications, even when they received LDA/H therapy. Among women with RPL, PS, and negative aPL, there was no difference in these risks between LDA alone and LDA/H therapies.

Determinants of Elevated Levels of Natural Anticoagulants in Healthy Subjects.

The levels of protein C (PC), free protein S (PS) and antithrombin (AT) are evaluated during thrombophilia screening to exclude their deficiencies. The aim of this study was to investigate factors which determine the elevated levels of natural plasma anticoagulants in healthy individuals. The PC activity and antigen, free PS antigen and AT activity together with hematological, biochemical, genetic and immunological laboratory tests were assessed in 130 healthy adults (63 males) aged 20-60 (median 41) years. Individuals with personal or family history of cardiovascular diseases and venous thromboembolism were ineligible. The functionally active PC measured by chromogenic assay (values above 140%) was observed in 14 (11%) of subjects, while the PC antigen determined using the enzyme-linked immunosorbent assay (ELISA) was elevated in 5 (4%) of these patients. Free PS measured by immunoturbidimetry and ELISA was increased in 9 (7%) subjects (values above 139% in men and 114% for women) and in 6 (5%) patients (values above 130% in men and 111% for women), respectively. The AT activity above 118% was found in 5 (4%) subjects measured using chromogenic assay. None of the individuals had any deficiency of natural anticoagulants. Increased C-reactive protein (CRP)>3.0 mg/L was associated with elevated PC activity (odd ratio [OR]: 11.14, 95% confidence interval [CI] 1.67-74.23). Increased free PS assessed by immunoturbidimetric assay and PC activity were associated with hypercholesterolemia (OR: 3.57, 95% CI: 1.09-10.06 and OR: 8.61, 95% CI: 1.07-69.04, respectively). Body mass index≥25 kg/m2 was independently associated with elevated PC activity (OR: 3.42, 95% CI: 1.01-11.52). No risk factors for elevated AT activity were identified. Apart from hypercholesterolemia and overweight, increased serum CRP is associated with elevated PC activity in healthy adults. We confirmed that there are differences in the proportions of subjects with elevated PC and PS depending on the assay used.

Effects of Dabigatran and Rivaroxaban On Routine and Specialized Coagulation Assays: A Study Using Actual Patient Plasma Samples

Abstract 23 Background:To date, all published studies assessing the effects of the new oral anticoagulants dabigatran (dabi) and rivaroxaban (riva) on coagulation assays have used normal pooled plasma spiked with known concentrations of anticoagulant and therefore do not reflect the variability expected from patients taking these medications. Methods:We collected citrated venous blood samples from patients taking either dabi (n=43) or riva (n=10) for management of either atrial fibrillation or acute venous thrombosis. All subjects were required to have taken at least 5 doses of drug in order to reflect steady-state conditions and informed consent was obtained. The interval between last dose and phlebotomy ranged from 1 – 18 hours. Platelet-poor plasma was prepared and using the STA-R.. coagulometer (Diagnostica Stago), the following assays were performed: PT/INR using Neoplastin Plus.. (NP) and Innovin.. (IN), aPTT (Actin.. FS), thrombin time (TT), clot-based protein S (PS) activity, free PS antigen, chromogenic protein C (PC) activity, clot-based antithrombin activity, Clauss fibrinogen, dilute Russell’s viper venom time (DRVVT), and factor VIII (FVIII) activity at several dilutions (1:10 to 1:160). Hemoclot.. dilute thrombin time and STA.. -Rotachrom.. anti-Xa assays were performed for quantitation of dabi and riva drug levels, respectively, using Aniara commercial plasma calibrators. Results:Median INR levels for patients on dabi were 1.1 (IN) and 1.2 (NP) and for riva were 1.15 (IN) and 1.3 (NP). The proportion of subjects with INR levels above 1.3 for dabi were 15% (IN) and 28% (NP) and for riva were 0% (IN) and 40% (NP). Median aPTT levels were 42s dabi and 32.5s riva. Although 73% of patients on dabi had an elevated aPTT (>38s), 60% of these were only mildly elevated (39–45s). Only one subject on riva had an elevated aPTT. The TT was extremely sensitive to the presence of dabi, as 100% of treated subjects had an elevated TT (>20s) and 73% were above the linearity cutoff (>100s). All subjects on riva had normal TT. As shown in Figure 1A, the clot-based PS assay was affected markedly by dabi, with patients showing artificially high PS activity with increasing drug levels, but no such effect was observed for riva (Figure 1B). For both drugs, the PS free antigen and chromogenic PC assays were unaffected. Figure 1C shows dabi also caused false elevations in antithrombin activity, though it did not affect the Clauss fibrinogen assay, which uses a much higher concentration of thrombin. Riva had no discernable effect on the antithrombin and fibrinogen assays (data not shown). Figure 1D shows that increasing levels of both anticoagulants caused artefactual decreases in FVIII activity. Increasing dilutions of patient plasma caused progressive elevation of FVIII activity, with median activity levels nearly doubling between 1:10 and 1:160 dilutions for both dabi (0.65 to 1.28) and riva (0.81 to 1.41). Finally, the DRVVT assay was prolonged (>20% elevated versus normal plasma) for 86% of dabi and 60% of riva patient plasmas. No differences were noted for any of these results between dabi 110 mg (n=14) and 150 mg (n=29) dosing. Conclusions:For the assay platforms assessed in this study, the majority of patients taking dabi and riva had near-normal INR and aPTT levels. This is discrepant with results from studies using spiked plasma samples. Clinicians should be cautioned that normal INR and aPTT results do not rule out the presence of therapeutic anticoagulant levels and the results should not be used to guide clinical decisions. Clotting-based thrombophilia testing should be avoided for patients taking these medications due to assay interferences, particularly with dabigatran. [Display omitted] Disclosures:Teal:Bayer: Consultancy, Honoraria; Boehringer-Ingelheim: Consultancy, Honoraria. Lee:Bayer: Honoraria; Boehringer-Ingelheim: Honoraria.

Protein S abnormalities: A diagnostic nightmare

Heterozygous deficiency of Protein S (PS) increases the risk for developing thrombosis. Many acquired conditions alter plasma PS levels. These complex interactions of PS in plasma make it imperative that clinical PS assay limitations are understood so that the assays are reliable, reproducible and specific to diagnose true genetic abnormalities based on plasma phenotype alone. Unfortunately, the diagnosis of PS deficiency is difficult and complicated. Three basic assays can be utilized for assessing PS in plasma: PS activity assay, Free PS antigen assay, and Total PS antigen assay. This article will review these clinical assays and their associated problems. We also discuss the confounding and interfering factors that make it difficult to obtain an accurate diagnosis of PS deficiency.

External Quality Assurance of Antithrombin, Protein C, and Protein S Assays: Results of the College of American Pathologists Proficiency Testing Program in Thrombophilia

Hereditary and acquired deficiencies of antithrombin (AT), protein C (PC), and protein S (PS) are risk factors for venous thromboembolism. Proper diagnosis requires high-quality assays for these proteins. To determine the accuracy and interlaboratory precision of AT, PC, and PS assays used by laboratories participating in the United States College of American Pathologists proficiency testing program in thrombophilia and to grade the performance of laboratories. Standardized normal plasma with assigned analyte values was sent in 2 separate challenges to participating laboratories. Participants measured AT, PC, and PS levels using local methods. When compared with the assigned values for the international standard, the order of assay accuracy from highest to lowest was AT activity, PC antigen, AT antigen, total PS antigen, PC activity, PS activity, and free PS antigen (range of assay bias, 2.6%-8.8%). The order of assay precision from highest to lowest was PC activity, AT activity, AT antigen, total PS antigen, PS activity, free PS antigen, and PC antigen (range of assay coefficient of variation, 6.1%-20.0%). Most testing events (87.8%) could be graded as pass or fail using a target range of ±3 standard deviations from the method-specific mean. The pass rate was 98.2% for all AT, PC, and PS testing events combined. Accuracy and precision were higher for AT assays and lower for PC and PS assays. It was feasible to grade individual laboratory performance.

Protein Z, protein S levels are lower in patients with thrombophilia and subsequent pregnancy complications

We posit that low levels of protein S (PS) and protein Z (PZ) contribute to adverse pregnancy outcome (APO). We evaluated 103 women with subsequent normal pregnancy outcome (NPO), 106 women with APO, and 20 women with thrombophilia (TP). We compared first trimester (1st TRI) PZ levels in 103 women with NPO, 106 women with APO, and in 20 women with TP. We compared plasma levels of PZ and free PS antigen during the second (2nd TRI) and third trimesters (3rd TRI) of pregnancy in 51 women with APO and 51 matched women with NPO. The mean 1st TRI PZ level was significantly lower among patients with APO, compared to pregnant controls (1.81 +/- 0.7 vs. 2.21 +/- 0.8 microg mL(-1), respectively, P < 0.001). Of patients with known TP, those with APO had a tendency for lower mean PZ levels compared to those TP women with NPO (1.5 +/- 0.6 vs. 2.3 +/- 0.9 microg mL(-1), respectively, P < 0.0631). There was a significant decrease in the PZ levels in patients with APO compared to NPO (2nd TRI 1.5 +/- 0.4 vs. 2.0 +/- 0.5 microg mL(-1), P < 0.0001; and 3rd TRI 1.6 +/- 0.5 vs. 1.9 +/- 0.5 microg mL(-1), P < 0.0002). Protein S levels were significantly lower in the 2nd and 3rd TRIs among patients with APO compared to patients with NPO (2nd TRI 34.4 +/- 11.8% vs. 38.9 +/- 10.3%, P < 0.05, respectively; and 3rd TRI 27.5 +/- 8.4 vs. 31.2 +/- 7.4, P < 0.025, respectively). We posit that decreased PZ and PS levels are additional risk factors for APO.

Protein S Free Antigen Levels and the Occurrence of IgG and IgM Antibodies to Protein S in Normal Pregnancy.

The evaluation of thrombophilia (TP) in pregnancy is central to the assessment for the risk for adverse pregnancy outcomes (APO) and thrombosis. Antibodies to protein S (PS) have been described in patients with autoimmune diseases and have also been related to thrombosis and APO in pregnancy. Therefore several major issues exist in the evaluation of PS in pregnancy for TP and the risk for APO and thrombosis. What are the ranges of free PS antigen (FPSAG) levels in normal pregnancy (NP)? Do antibodies to PS occur in so called NP and do they affect the levels of PS? We studied 82 1st trimester (TRI) samples, 64 2nd TRI samples, and 78 3rd TRI samples from normal pregnancies to evaluate the FPSAG levels and antibodies against PS both IgG and IgM. We also tested 50 normal non-pregnant donors for antibodies against PS to establish the non-pregnant reference range. The data are show in table 1. It reveals that even in NP the FPSAG levels can decrease to as low as 28% in 1st and 2nd TRI, and 20% in 3rd TRI. These values are significantly below the non-pregnant values. The differences of decreased of PS between 1st vs 3rd, and 2nd vs 3rd TRI are statistically significant (p<0.05). We have previously reported that PS lower than 30% in the 2nd to 3rd TRI was associated with a higher risk for APO. If additional TP factors are present, PS levels in lower NP reference range may increase the risk for APO and thrombosis. The data also shows that there is an increase in PS IgG and IgM antibodies during gestation compared to non-pregnant controls. The number of patients with IgG and IgM antibodies above the 3SD of control value increase with gestation (3% vs 8% vs 9% for IgG, and 13% vs 26% vs 31% for IgM, [1st TRI, 2nd TRI, 3rd TRI respectively]). The lack of overall correlation of antibodies with the protein level is similar to the finding of Gris et al with protein Z and APO. Do the antibodies affect the function but not the level of the factors responsible for the natural inhibition of the prothrombotic process? Do these antibodies become part of the pathogenesis of APO and thrombosis in pregnancy associated with the anticardiolipin syndrome? These questions need to be addressed by larger studies of normal and adverse pregnancy patients. The increase in the number of NP subjects with high levels of PS-IgG and IgM antibodies with gestation might indicate an enhanced immune response to components of the hemostatic system in some patients who might become more vulnerable to APO. The low values of FPSAG in NP add to the difficulty of assessing for a pre-existing deficiency state. However, if an inherited deficiency is present the FPSAG should be in the lower ranges of those we have established for the three TRI. This need to be defined by the assessment of a larger group of PS deficiency patients during pregnancy.Table 1 shows the mean and standrad deviation of FPSAG levels and the titer of PS IgG and IgM antibodies1st trimester2nd trimester3dr trimesterCTRFPSAG38.8+/−10.5%35.9+/−8.3%27.9+/−7.0%88+/−19%Anti-PS IgG0.29+/−0.35OD0.45+/−0.25OD0.44+/−0.31OD0.27+/−0.20ODAnti-PS IgM0.48+/−0.38OD0.66+/−0.42OD0.61+/−0.36OD0.26+/−0.15ODCTR= non-pregnant control

Laboratory Diagnosis of Hereditary Thrombophilia

Genetic defects of antithrombin (AT) or one of the components of the protein C pathway are associated with hereditary thrombophilia. Laboratory assays are currently available to diagnose and type hereditary thrombophilia due to deficiency or dysfunction of one of the anticoagulant factors antithrombin (AT), protein C (PC) and protein S (PS), and APC resistance without the need of DNA analysis. There are no functional tests for the prothrombin mutant G20210A and thrombomodulin mutations, which can be diagnosed by a PCR-based test or by gene analysis, respectively. Hereditary AT deficiency is classified in a quantitative type I and three functional type II deficiencies affecting the reactive site (RS), heparin binding site (HBS), or pleiomorphic site of the AT protein. All four types of hereditary AT deficiencies can be diagnosed by a heparin cofactor assay and one immune assay in combination with crossed immunoelectrophoresis of the AT protein. The combination of an enzyme-linked immunoadsorbent assay (ELISA) and a functional Protac-APTT-based assay for PC will detect quantitative type I and dysfunctional type II PC deficiencies. There is a significant overlap in PC antigen and functional levels between heterozygotes of PC deficiency and normals leaving a gray zone of uncertainty in differentiating congenital PC deficiency and normal individuals. Accurate diagnosis of hereditary PS deficiency should be a combination of tests aimed to measure free PS activity and antigen and total PS antigen levels. APTT-, Xa-, and RVVT-based APC-resistance tests, when test plasmas are diluted in factor V deficient plasma, have increased in sensitivity and specificity to 100% for the discrimination of normal individuals from heterozygotes and homozygotes for factor V Leiden. The RVVT-based APC-resistance test provides better separation of factor V Leiden and normals in the various clinical settings, lupus anticoagulant in particular. The modified APC-resistance tests also claim a separation between heterozygotes and homozygotes for factor V Leiden in the normal population, asymptomatic subjects, and thrombosis patients. Below a certain cut-off level, a minor overlap of normalized APC ratios between heterozygotes and homozygotes for factor V Leiden of thrombosis patients has been shown in one study, which still points to the need to perform the more time consuming and expensive DNA test to identify heterozygotes from the more clinically significant homozygotes. The prothrombin-based APC-resistance test, which measures thrombin activated factor Va in highly diluted test plasma, appears to be the most sensitive and specific of all APC-resistance tests and separates normal individuals from heterozygotes and heterozygotes from homozygotes for factor V Leiden without the need of confirmation by a DNA test.

Variable interference of activated protein C resistance in the measurement of protein S activity by commercial assays

Congenital and acquired deficiencies of vitamin K-dependent protein S (PS) are an established risk factor for thrombotic disease (1,2). Due to the multiple PS domains involved in the interaction with other plasma proteins and the expression of activated protein C (APC) cofactor activity, the existence of qualitative defects of PS is anticipated (3). Thus, measurement of PS anticoagulant activity should be preferred to PS antigens determination in the identification of both congenital and acquired deficiencies. At present, two commercial assays are available for the measurement of the APC cofactor activity of PS. with one of these assays qualitative protein S deficiencies had been erroneously identified (4), as originally indicated by a multicenter collaborative study comparing different functional PS assays (5). In the latter study, plasma samples obtained from patients reportedly affected by qualitative PS deficiencies showed virtual coincidence of free PS antigen and anticoagulant activity levels only when this was evaluated after immunoadsorption of PS (6). This observation raised the possibility of artifacts related to PS testing in whole plasma (5). Following the description of APC resistance a new marker of congenital thrombophilia characterized by defective clotting time prolongation upon additon of APC to plasma (7) -, the seemingly PS deficient patients have been correctly diagnosed as APC resistant cases, with the conclusion that APC resistance may interfere in PS activity testing by the available commercial methods (4). To explore the extent of such interference, we have conducted a prospective comparison of two commercial functional PS assays and our home-made assay (6) in a series of patients with APC resistance referred to our Institution for the evaluation of a thrombophilic state.

Inherited protein S deficiency: clinical manifestations and laboratory findings in 63 patients.

To further characterize inherited heterozygous protein S (PS) deficiencies, we studied 63 patients belonging to 33 families. Diagnosis of PS deficiency was based on protein S activity (PS Act) and/or free PS antigen (FPS Ag) levels below the lower limit of the normal range in patients not on oral anticoagulation. Depending on the level of total PS antigen (TPS Ag), two subpopulations could be distinguished: in the first one (25 patients belonging to 11 families) level of TPS Ag was reduced whereas in the second one (38 patients belonging to 22 families), TPS Ag was normal. In none of the families studied the two types of PS deficiency coexisted suggesting that they are different entities. In the 63 patients, thromboembolic events occurred in 57% of cases and were recurrent in 36.5% of patients. Age at the time of the first thrombosis ranged from 14 to 74 years, and was below 40 years in 69% of symptomatic cases. Thrombotic events were spontaneous in 64% of cases, and were associated with other risk factors in 36%. There was no apparent relationship between clinical status, symptomatic or asymptomatic, and the type or degree of the PS deficiency. Long-term anticoagulation prevented the recurrence of thrombosis in every case but one, and led to a decrease in circulating levels of C4b-binding protein suggesting the existence of a regulation between C4b-BP and PS concentrations. Together with previous reports, these findings underline the clinical and biological heterogeneity of inherited protein S deficiency.

Protein S Tokushima: An Abnormal Protein S Found in a Japanese Family with Thrombosis

An abnormal protein S (PS) was found in a Japanese family with a high incidence of thrombosis. The proband is a woman who was born in Tokushima Prefecture. She had superior sagittal sinus thrombosis, thrombophlebitis of the left leg, and thrombosis of the placenta. She had a normal plasma level of free PS antigen but decreased PS activity. Her mother and aunt also had thrombophlebitis of the leg, and together with four other family members also showed a normal level but decreased activity of PS. This suggests that hereditary dysfunction of PS is inherited in this family as an autosomal dominant trait. The proband's PS appears to have a slightly higher molecular weight than normal PS both in the intact and modified form, suggesting that it has a molecular defect on the carboxyl-terminal side of the thrombin-sensitive site. This abnormal PS with apparently unique characteristics was named PS Tokushima.

Combined inherited protein s and heparin co-factor II deficiency in a patient with upper limb thrombosis: A family study

A 42-year-old Italian woman presenting with spontaneous deep vein thrombosis of the right arm, was found to have inherited a deficiency of both protein S (PS) and heparin co-factor II (HC II). The two defects seemed to segregate independently, since her son exhibited only a HC II deficiency while one of her sisters manifested only the PS defect. All affected patients appeared heterozygous for one or other or both deficiency states. The proposita and her sister exhibited a congenital PS deficiency consisting of normal or near normal levels of total PS antigen and C4b-binding protein (C4b-BP) but a moderate reduction both of free PS antigen and of PS functional activity. In addition, the proposita and her son had half normal levels of HC II antigen and activity. Except for the proposita,all were asymptomatic. Inherited deficiencies either of PS or of HC II have been associated with thrombotic manifestations. Since the proposita had an inherited combined defect of the two proteins,severe thrombotic events might be expected. However, this was not found to be the case. The role of HC II deficiency in the pathogenesis of thrombosis whether alone or combined remains to be fully investigated.

Functional protein S in women with lupus anticoagulant inhibitor

Protein C (PC) and protein S (PS) are components of a potent, natural anticoagulant system. A deficiency of one of these two inhibitors is associated with thrombotic events in young people. A significant reduction in functional PS activity has been observed during normal pregnancy, and recurrent fetal loss may occur in women with lupus anticoagulant (LA) inhibitor. We measured functional PS activity and free PS antigen in 16 non pregnant patients with LA inhibitor and in 17 normal women as controls. A significant difference was observed between patients and controls in functional PS activity (65±23% vs 87±15%, p=0.02) but not in free PS antigen (88±17% vs 93±17%). Functional PS activity decreased only in six patients (37%). Removal of IgG from plasma reduced the difference in functional PS activity between patients and controls. Immunologic IgG levels did not correlate with antiphospholipid antibodies (APA) activities, activated partial thromboplastin time/kaolin clotting time (aPTT/KCT) data or functional PS activity.

A Collaborative Study on the Measurement of Protein S Antigen in Plasma

A collaborative study on the measurement of protein S (PS) antigen (total and free) in a freeze-dried ampouled test plasma by assay against local house standard plasmas was carried out in eleven laboratories. Potency estimates of total PS showed good agreement between laboratories with a geometric coefficient of variation (gcv) of 5.9% and an overall combined potency of 0.84 units per ml. Potency estimates of free PS antigen in the test sample were associated with increased variability between laboratories resulting from the polyethylene glycol (PEG) precipitation step which is used to separate free PS from PS bound to the C4b binding protein. Free PS in the test could be expressed relative to either total PS in the house standards (e.g. 0.28 units per ml) or relative to free PS in the house standards following PEG precipitation (e.g. 0.71 units free PS per ml).