Rivaroxaban Causes Missed Diagnosis of Protein S Deficiency but Not of Activated Protein C Resistance (Factor V Leiden).

CONTEXT: - Rivaroxaban causes a false increase in activated protein C resistance (APCR) ratios and protein S activity. OBJECTIVE: - To investigate whether this increase masks a diagnosis of factor V Leiden (FVL) or protein S deficiency in a "real-world" population of patients undergoing rivaroxaban treatment and hypercoagulation testing. DESIGN: - During a 2.5-year period, we compared 4 groups of patients (n = 60): FVL heterozygous (FVL-HET)/taking rivaroxaban, wild-type/taking rivaroxaban, FVL-HET/no rivaroxaban, and normal APCR/no rivaroxaban. Patients taking rivaroxaban were tested for protein S functional activity and free antigen (n = 32). RESULTS: - The FVL-HET patients taking rivaroxaban had lower APCR ratios than wild-type patients ( P < .001). For FVL-HET patients taking rivaroxaban, mean APCR was 1.75 ± 0.12, versus 1.64 ± 0.3 in FVL-HET patients not taking rivaroxaban ( P = .005). Activated protein C resistance in FVL-HET patients fell more than 3 SDs below the cutoff of 2.2 at which the laboratory reflexes FVL DNA testing. No cases of FVL were missed despite rivaroxaban. In contrast, rivaroxaban falsely elevated functional protein S activity, regardless of the presence or absence of FVL ( P < .001). A total of 4 of 32 patients (12.5%) had low free protein S antigen (range, 58%-67%), whereas their functional protein S activity appeared normal (range 75%-130%). Rivaroxaban would have caused a missed diagnosis of all cases of protein S deficiency during the study if testing relied on the protein S activity assay alone. CONCLUSIONS: - Despite rivaroxaban treatment, APCR testing can distinguish FVL-HET from normal patients, rendering indiscriminate FVL DNA testing of all patients on rivaroxaban unnecessary. Free protein S should be tested in patients taking rivaroxaban to exclude hereditary protein S deficiency.

Protein S testing in patients with protein S deficiency, factor V Leiden, and rivaroxaban by North American Specialized Coagulation Laboratories.

In 2010-2012, the North American Specialized Coagulation Laboratory Association (NASCOLA) distributed 12 proficiency testing challenges to evaluate laboratory testing for protein S (PS). Results were analysed to assess the performance of PS activity, PS free antigen, and PS total antigen testing. Statistical analysis was performed on the numeric results and qualitative classification submitted for each method. There were 2,106 total results: 716 results from PS activity assays, 833 results from PS free antigen assays, and 557 results from PS total antigen assays. The three assay types performed well in the classification of five normal samples and nine abnormal samples, although certain PS activity methods were more likely to classify normal samples as abnormal and one PS total antigen assay was more likely to classify abnormal samples as normal. PS activity methods were affected by interfering substances such as heterozygous or homozygous factor V Leiden mutation (underestimation) and the anticoagulant drug rivaroxaban (overestimation). In conclusion, NASCOLA laboratories using a variety of PS assays performed well in the classification of clearly normal and abnormal samples. Laboratories performing PS activity assays should be aware of potential interferences in samples positive for FV Leiden or containing certain anticoagulant medications.

Effect of dietary supplementation with beta-casein A1 or A2 on markers of disease development in individuals at high risk of cardiovascular disease.

The present study is the first to examine the hypothesis that dietary supplementation with beta-casein A1 promotes an increased risk relative to supplementation with beta-casein A2 in patients traditionally at high risk of developing CVD. The study was conducted in fifteen asymptomatic participants (six male; nine female) at high risk of developing CVD. A double-blind cross-over study design was used with a total duration of 24 weeks. Dietary intervention was a daily supplementation (25 g) of either casein A1 or A2 (for 12 weeks each). Surrogate measures of cardioprotection studied included the examination of vascular (endothelium and arterial) function, resting blood pressure, plasma lipids and biochemical markers of inflammation. Total plasma cholesterol levels were significantly lower following 12 weeks of both casein A1 and A2 interventions but the decrease was not different between intervention. Plasma insulin, homocysteine, C-reactive protein, fibrinogen, protein C and S and von Willebrand factor levels were not different between the two casein supplements. Endothelium function, measured as a vascular response using venous occlusion plethysmography to intra-arterial infusions of the endothelium-dependent agonist acetylcholine, were not different between the two casein interventions. Similarly, neither blood pressure nor measures of large artery stiffness were affected by differing the casein variant. We therefore conclude that there is no evidence from the present study that supplementation with casein A1 has any cardiovascular health disadvantage over consumption of casein A2.

Effects of long-term administration of high-dose recombinant human antithrombin in immunosuppressed primate recipients of porcine xenografts.

BACKGROUND: Fibrin deposition is central to the acute humoral rejection process occurring in the presence of consumptive coagulopathy when pig organs are transplanted into primates. METHODS: To assess whether strategies aimed at preventing fibrin formation may extend xenograft survival, we administered high daily doses of recombinant human antithrombin (rhAT) (500 U/kg twice daily) to obtain both anticoagulant and anti-inflammatory effects in immunosuppressed primate recipients of porcine kidneys. RESULTS: Some degree of consumptive coagulopathy developed in both rhAT-treated (n=3) and untreated (n=3) primates. No major differences in the coagulation parameters analyzed were observed between the 2 groups. Similarly, no difference in survival was seen between rhAT-treated (20.6+/-4 days; range: 15-23 days) and untreated animals (17.3+/-11.6 days; range: 7-30 days), although the rhAT-treated primates had a higher bleeding tendency. Despite the high daily dose of rhAT, considerable fibrin deposition was observed in the graft as early as 2 weeks after transplantation. CONCLUSIONS: These results suggest that a high daily dose of rhAT fails to influence survival or prevent fibrin formation and deposition in the graft in our pig-to-primate model. However, the potential role of rhAT administered in combination with heparins or other clotting inhibitor concentrates in this model remains to be determined.

Comparison of hemodynamic changes associated with two different polytetraflouroethylene arteriovenous fistulae in hemodialysis patients.

The aim of this prospective study was to identify hemodynamic factors associated with two different types of polytetrafluroethylene (PTFE) AV grafts. The study was conducted on 46 hemodialysis patients over a 3-year period. The subjects were randomly assigned to one of two study groups: Group 1 patients (n = 24) underwent a brachiocephalic loop PTFE fistula; Group 2 patients (n = 22), a brachioaxillary PTFE fistula. Preoperatively, we recorded each individual's subclavian catheter history, hemodialysis frequency, and serum levels of parathormone (PTH), calcium (Ca)-phosphorus (P) product, homocysteine, protein C, and protein S. Doppler ultrasonography was used to evaluate vascular hemodynamic changes in the proximal and distal portions of the AV fistula at 48 hours and 1 week postoperatively. Group 1 showed a significantly greater number of ipsilateral subclavian catheter interventions prior to AV graft surgery than Group 2 (14 versus 7, respectively; P = .05; chi-square). The mean peak systolic velocity in the brachial artery in Group 1 was significantly higher than that in Group 2 at 1-week postoperatively (P = .04, paired t-test). The mean radial artery diameter in Group 1 was greater than that of Group 2 at 1 week postoperatively (P = .05, Student t-test). At 48 hours postoperatively the observed change in cephalic vein diameter in Group 1 was significantly greater than the change in axillary vein diameter in Group 2 (P = .08, paired t-test). Preoperatively, the mean serum protein C and protein S levels in Group 1 were higher than those in Group 2 (P = .03 and P = .04, respectively; Mann-Whitney U test). The total numbers of dialysis sessions per week in each group were significantly different (P = .001, chi-square). Six Group 1 patients exhibited graft thrombosis at 48 hours after AV graft surgery. None of the patients in Group 2 exhibited thrombosis at 48 hours or 1 week postoperatively. The results indicate that patients with brachiocephalic PTFE AV grafts show more significant changes in the cephalic vein and brachial artery than patients with brachioaxillary PTFE AV grafts. The findings also suggest that more ipsilateral subclavian catheter interventions and a higher weekly frequency of hemodialysis prior to AV graft surgery are risk factors for early thrombosis of PTFE AV grafts.

Association of prothrombin and protein S with plasma triglyceride-rich lipoproteins in humans after test meals rich in milk fat or soybean oil.

INTRODUCTION: Prothrombin can associate with rat chylomicrons in vitro. This enhances a platelet factor Xa mediated prothrombin activation when the chylomicron-prothrombin complex is exposed to platelets. Vitamin K-dependent pro- and anti-coagulation proteins are associated with TG-rich lipoproteins obtained from human plasma. In the present study, we examined the effects of saturated and unsaturated fat meals on the association of prothrombin and protein S with TG-rich lipoproteins in vivo. MATERIALS AND METHODS: Human EDTA plasma was separated from normal subjects after overnight fasting and 2.5 h after ingestion of either a saturated fat meal (butter and cream) or an unsaturated fat meal (soybean oil) containing 54-80 g fat (60.2 E%). The prothrombin and protein S in delipidated lipoproteins were determined by SDS-PAGE combined with Western blotting. RESULTS: Both prothrombin and protein S associated with TG-rich lipoproteins in fasting and in postprandial samples. The levels of prothrombin and protein S in postprandial TG-rich lipoproteins, especially after ingestion of a saturated fat meal, were higher than those in fasting TG-rich lipoproteins. CONCLUSIONS: The levels of both prothrombin and protein S in TG-rich lipoproteins in plasma increased after a single fat meal. This association is more pronounced after saturated fat meals and one may hypothesize that it can be linked to atherogenic properties of TG-rich lipoproteins.

Acquired protein S deficiency caused by estrogen treatment of tall stature.

OBJECTIVE: To evaluate the potential thrombogenic changes in the coagulation and fibrinolytic system related to treatment with ethinyl estradiol (200 and 300 microg). SUBJECTS AND METHODS: Twenty-five healthy girls with expected final height exceeding 185 cm, as calculated by the method of Bayley and Pinneau, were treated with 200 microg or 300 microg of ethinyl estradiol. Coagulation and fibrinolytic parameters were determined before and during estrogen treatment and 2 and 4 weeks after estrogen withdrawal. RESULTS: No difference in the effects on hemostasis was found between the 2 treatment groups. All 25 patients developed protein S deficiency during estrogen treatment, which in most girls lasted for 4 weeks after cessation of estrogen administration. During therapy, protein C activity increased, whereas antithrombin did not change. Plasminogen and plasmin-alpha(2) antiplasmin complexes significantly increased. Protein S deficiency was accompanied by significantly increased prothrombin fragment 1+2 and fibrinopeptide A. In contrast, thrombin-antithrombin complexes did not change. CONCLUSION: High-dose estrogen treatment to reduce the final height in tall girls is associated with a reversible acquired protein S deficiency with indications of a pre-thrombotic state. Risk of venous thrombo-embolism may be enhanced, especially when additional risk factors for thrombosis are present.

Changes of hemostatic factors in children with acute lymphoblastic leukemia receiving combined chemotherapy including high dose methylprednisolone and L-asparaginase.

In this study, protein C (PC), protein S (PS), heparin cofactor II (HCFII), prothrombin fragment 1+2 (PF 1,2), thrombin-antithrombin III complex (TAT), von Willebrand factor (vWF) and thrombomodulin (TM) were investigated in 19 patients with acute lymphoblastic leukemia, (ALL) receiving combined chemotherapy including L-asparaginase (L-ASP) and high dose methylprednisolone (HDMP). HDMP was administered in doses of 30 mg/kg/day for 7 days, and 20 mg/kg/day for another 7 days. In order to evaluate the effect of HDMP on the hemostatic system, the 8 patients studied here received HDMP (30 mg/kg/day) therapy for 4 days before the combined chemotherapy. These parameters were also studied in 12 healthy children as a control group. PC levels were normal in the patients while PS levels were decreased both before and after combined chemotherapies. Patients with ALL have laboratory signs of coagulation activation such as PF 1,2, TAT prior to initiation of chemotherapy. With combined chemotherapy, TAT levels were found to be normal while PF1,2 were not. TM levels were found to be increased both before and after therapies whereas HCFII and vWF levels were not different from those of the control group. The short course of HDMP therapy did not prominently influence these hemostatic parameters. These results indicate that both the malignant process and the drugs used in combined chemotherapy cause a decrease in natural inhibitors and an increase in procoagulant activity and endothelial injury. These hemostatic changes may contribute to a thrombotic tendency in the patients with ALL.

Cerebral venous thrombosis and activated protein C resistance.

BACKGROUND: Activated protein C resistance (APC-R) due to factor V Leiden has recently been established as an important risk factor for cerebral venous thrombosis (CVT). The clinical significance of abnormal or borderline functional APC-R in the absence of factor V Leiden is uncertain. Our observations suggest that APC-R due to mechanisms other than factor V Leiden may also contribute to the development of CVT. CASE DESCRIPTIONS: We describe three women who had superior sagittal and lateral sinus thrombosis while taking oral contraceptives and had a number of additional risk factors for CVT. Each had APC-R for different reasons. CONCLUSIONS: Inherited thrombophilia, including APC-R, should be looked for in all patients with CVT. Functional APC-R is a highly prevalent coagulopathy, but the reasons for this abnormality are diverse; abnormal and borderline functional APC-R results should be supplemented by DNA analysis for the presence of factor V Leiden.

Does aprotinin influence endothelial-associated coagulation in cardiac surgery?

Aprotinin has been reported to reduce bleeding in cardiac surgery patients. Its mechanisms of action on coagulation have not been fully elucidated. In a prospectively randomized study of 40 patients undergoing elective aortocoronary bypass grafting, the influence of high-dose aprotinin (2 million IU of aprotinin before CPB, 500,000 IU/h until the end of operation, 2 million IU added to the prime) (N = 20) on endothelial-related coagulation was compared to a nontreated control group (N = 20). Thrombomodulin (TM), protein C and (free) protein S as well as thrombin/antithrombin-III (TAT) plasma concentrations were measured by enzyme-linked immunosorbent assays (ELISA) before the aprotinin infusion, before cardiopulmonary bypass (CPB), during CPB and after CPB, at the end of surgery, 5 hours after CPB, and on the first postoperative day. All standard coagulation parameters (AT-III and fibrinogen plasma levels, platelet count, partial thromboplastin time) did not differ between the two groups. At baseline, TM plasma levels were within the normal range (< 40 ng/mL) and similar in both groups. During CPB, TM plasma concentrations decreased similarly in both groups (aprotinin: 18 +/- 6 ng/mL, control: 17 +/- 7 ng/mL) followed by a comparable increase in the postbypass period until the first postoperative day (aprotinin: 60 +/- 10 ng/mL, control: 53 +/- 11 ng/mL). Protein C and (free) protein S plasma levels also showed no differences between the two groups. On the first postoperative day, baseline values for protein C and protein S had not yet been reached.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin K-dependent protein S in Leydig cells of human testis.

Protein S is an anticoagulant plasma protein, functioning as a cofactor to activated protein C in the regulation of blood coagulation. In addition, protein S forms a complex with the complement regulatory protein, C4b-binding protein. Protein S is unique among the vitamin K-dependent proteins in being structurally similar to androgen binding proteins. Protein S immunoreactivity was demonstrated in Leydig cells of human testis. In Northern blotting experiments, the presence of protein S mRNA in human testis tissue could be shown. In situ hybridization experiments localized protein S mRNA to the Leydig cells, demonstrating transcription of the protein S gene in these cells. Five protein S clones were isolated from a human testis cDNA library, partially sequenced and characterized by restriction enzyme mapping. Three unique clones contained information for the entire coding sequence and approximately two-thirds of the 5' and 3' non-coding sequences. The results indicate the nucleotide sequences of testis and liver protein S mRNA to be identical. No binding of androgens to protein S could be demonstrated. In conclusion, we demonstrate the presence of protein S immunoreactivity as well as protein S mRNA in the Leydig cells of human testis. These results suggest local synthesis of protein S in Leydig cells of human testis which may be functionally important for local anticoagulation.

Protein S and protein C level changes with adjuvant tamoxifen therapy in postmenopausal women.

Tamoxifen citrate is a synthetic antiestrogen that provides survival benefit when given as adjuvant treatment in postmenopausal women with breast cancer. Venous thrombophlebitis may complicate tamoxifen treatment at a rate of approximately one per 800 treatment-years. To explore the possible procoagulant effects associated with tamoxifen therapy we evaluated changes in protein S and C activity levels in 58 postmenopausal women with surgically resected breast cancer who were disease-free and participating in a double-blind, placebo-controlled, randomized toxicity study of tamoxifen. The changes in protein C activities for the tamoxifen group (mean level = 113%) compared to those in the placebo group (mean level = 115%) were not significant (p = 0.45). Protein S activity levels increased while protein C activity levels decreased from baseline at 24 months in both tamoxifen and placebo groups. We conclude that the possible thrombophlebitis-promoting effect of tamoxifen in postmenopausal women is unlikely to be explained on the basis of protein S and protein C activity level changes.

Vitamin K status in cystic fibrosis.

Appearance of PIVKA-II (protein induced by vitamin K absence-II) in serum is a biochemical sign of insufficient vitamin K-dependent carboxylation of prothrombin. Plasma concentrations of PIVKA-II and vitamin K1 were determined in 24 children with cystic fibrosis. Eight were supplemented with vitamin K1. The purpose of the study was to determine the occurrence of vitamin K deficiency in cystic fibrosis and to evaluate the effect of vitamin K supplementation. PIVKA-II was detectable in only one unsupplemented child. In this patient, the concentration of vitamin K1 was below the limit of detection of 60 ng/l. Vitamin K1 levels in the other unsupplemented children were normal (mean 476 ng/l = 1 mmol/l). The supplemented patients showed extremely high levels of vitamin K1 (mean 22445 ng/l = 50 nmol/l). In conclusion, vitamin K deficiency occurs infrequently in cystic fibrosis. Checking the coagulation system is advised, but routine vitamin K supplementation is not recommended. If additional vitamin K is needed, the starting dose should not exceed 1 mg daily.