L-asparaginase treatment reduces the anticoagulant potential of the protein C system without affecting vitamin K-dependent carboxylation

The changes in plasma levels of the vitamin K-dependent natural anticoagulants protein C (PC) and protein S (PS) and procoagulant factors II, IX and X were evaluated in 8 adult patients during treatment with L-asparaginase (L-ase i.v. 120,000 U/m² over 10 days). PC anticoagulant activity and factor IX, X and II coagulant activity decreased proportionally to their half-lives to a nadir of 50–60% of pretreatment values after 2–5 L-ase infusions, suggesting that inhibition of protein synthesis rather than consumption is the main mechanism responsible for the observed changes. Free PS antigen levels declined at a rate similar to total PS antigen, reaching a nadir of 56% of pretreatment values after 3 L-ase infusions; however, due to C4b-binding protein levels higher than total PS levels (p <0.05), they were constantly lower than the corresponding total PS antigen levels (0.05 < p <0.001). This implicates that total PS antigen levels cannot be taken as an indicator of PS activity. No differences between the antigenic levels and the anticoagulant activities of PC and free PS could be observed suggesting that L-ase does not affect the mechanisms of vitamin K-dependent carboxylation of Gla-residues. The faster rate of decline of PC and PS activities relative to that of factor II may be responsible for the onset of an hypercoagulable state during the early phase of L-ase treatment.     

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.     

Natural anticoagulants (antithrombin III, protein C, and protein S) in patients with mild to moderate ischemic stroke

BACKGROUND AND PURPOSE: The role of the natural anticoagulants, antithrombin III (AT III), protein C (PC), and protein S (PS), in patients with mild to moderate ischemic stroke remains uncertain. We aimed to find out whether their levels in peripheral blood correlated with the severity of neurological deficit or can predict clinical outcome and recurrence. METHODS: We studied AT III, PC, and free PS levels in 55 consecutive patients likely to survive the study period on admission, 1 week, 1 month and 3 months after a first-ever ischemic stroke. Sex- and age-matched controls were studied once. All patients underwent a full neurological examination and blood sampling at each study time point; comprehensive stroke risk factors were recorded, and the etiology of the ischemic stroke was determined. All patients were contacted 3 years later for possible recurrent ischemic events. RESULTS: AT III level was found to be significantly lower at all time points after stroke; PC level was significantly increased on admission and normal at subsequent measurements, and PS level was normal on admission but significantly decreased later. The levels of the natural anticoagulants did not correlate with the etiology of stroke, any stroke risk factor, or neurological scores, except that the AT III level on admission showed significant correlation with stroke severity and disability at 3 months. Natural anticoagulant levels did not predict recurrence of ischemic stroke. CONCLUSIONS: The measurements of the level of AT III, PC, or PS did not deliver useful information for management of patients with mild or moderate ischemic stroke, expect that AT III level on admission might predict outcome.     

蛋白C、蛋白S与脑血管病关系的研究

目的:探讨蛋白C(PC)、蛋白S(PS)与脑血管病(CVD)的关系。方法:检测94例缺血性脑血管病(ICVD)患者和26例脑出血(CH)患者的血浆蛋白C:抗原(PC:Ag)、总蛋白S(TPS)和游离蛋白S(FPS)。结果:ICVD患者PC: Ag(17％)、TPS(26．6％)、FPS(23．4％)异常降低者多于对照组(2％)(P＜0．01)。CH患者PC:Ag(19．2％)异常降低者多于对照组(P＜0．05)。结论:17％～26．6％ICVD患者存在PC、PS缺陷;19．2％CH患者存在PC缺陷,多数为继发性缺陷,发现这些患者并区别其为原发性或继发性对明确CVD的病因、治疗和预防有重要意义。     

Dysfunctional activated protein C (PC Cádiz) in a patient with thrombotic disease

The partial characterization of a dysfunctional protein C (PC), provisionally named "PC Cádiz", in a 45-year-old male patient suffering from recurrent venous thrombosis is described. The only defect found in laboratory assays for haemostasis and hepatic function was a half normal level of both amidolytic and anticoagulant protein C activity, measured by different functional assays that use thrombin-thrombomodulin complex and a snake venom to activate protein C. Protein C antigen was always found to be within normal levels. Two young daughters of the propositus were found to have the same defect. Double-crossed immunoelectrophoresis, performed in the presence and absence of Ca2+ in the first dimension, showed no clear differences between patient and control PC. PC adsorption to barium salts was also found to be normal. Measurement of the PC activation peptide in the barium citrate eluates after PC activation showed no significant differences between patient and 10 normal controls, the concentration of this peptide being very similar to that of PC zymogen in the same eluates before PC activation. These results indicate that this abnormal PC is able to be normally activated by thrombin-thrombomodulin complex but does not exhibit serine protease activity, probably due to a defect in the PC molecule near the active site center.     

Protein S activity in patients with heredofamilial protein S deficiency and in patients with juvenile venous thrombosis. Results of a functional method

Using an ACL 300R coagulometer (Instrumentation Laboratory) we assessed the clinical usefulness of a new method to measure PS activity (PS:Act), based on the prolongation of prothrombin time of a mixture of diluted plasma sample, PS depleted plasma previously incubated with Protac for protein C activation, bovine thromboplastin and calcium ions. The results were compared with those from immunological assays. PS:Act was measured in 42 apparently healthy subjects, in 12 patients with hereditary PS deficiency (HPSD group) diagnosed on the basis of immunologic tests and in 48 patients with episodes of juvenile venous thromboembolism at least three months prior to testing (JVTE group). All the HPSD patients had PS:Act below the normal range (< 62%). In JVTE group 9 patients (18.7%) showed abnormal results for PS:Act, 4 (8.3%) had low levels of free PS:Ag; all patients had normal total PS:Ag levels. Levels of antiphospholipid antibodies (immunologic test) were normal in the 9 JVTE patients with low PS:Act. When all the results were considered together (n=102), the correlation coefficient between PS:Act and free PS:Ag was 0.78 (p<0.01).     

Protein C assays in uremia

Protein C was determined in 42 patients with terminal uremia and 20 healthy controls in three different ways 1) anticoagulant activity 2) amidolytic activity 3) antigen level. Protein C anticoagulant activity was markedly decreased in uremia, but was partly normalized during hemodialysis treatment, whereas the amidolytic activity and antigen level of protein C were normal and without changes during dialysis. The activities and antigen levels of factor II and X were normal before and after hemodialysis. In anticoagulated patients we found a good correlation between prothrombin levels and protein C levels determined with three different assays. We did not find any evidence for a defect carboxylation of protein C as the cause for the defective protein C in uremia. The BaCl2 precipitation in the Protein C anticoagulant assay was incomplete both in uremia and in controls but without differences between the two groups. In vitro addition of urea and creatinine did not decrease protein C activity. The cause of the defective protein C in uremia is still not known but it might contribute to thromboembolic complications.     

Determination of total, free and complexed protein s in plasma by ELISA, and comparison with a standard electroimmunoassay

An enzyme-linked immunosorbent assay (ELISA) for measuring total, free and complexed protein S in plasma was developed. To assay free protein S, C4b-binding protein-bound protein S (C4b-BP-PS) was extracted by addition of polyethyleneglycol (PEG) 6000 (5%, final concentration) to plasma samples. Microtiter plates were coated with rabbit anti-human protein S, and bound protein S was detected with labelled anti-protein S antibody. Diluted plasma samples were incubated in the plates overnight at 22 °C to permit C4b-BP-PS complexes to dissociate. Mean variation coefficients of 2.1 and 3.2% (intra-assay) and 4.3 and 7.9% (inter-assay) were found for total and free protein S assays, respectively. The ELISA measures free and complexed protein S with equal efficiency as is demonstrated by the fact that the sum of free protein S and C4b-BP-PS complex levels in normal individuals, women in their third trimester of gestation and patients with acute deep vein thrombosis (DVT), equaled the level of total protein S present in the corresponding plasma. Total protein S values obtained with the ELISA, in all groups studied, correlated well with those obtained with a standard electroimmunoassay (EIA) (r=0.93; N=40). However, total protein S levels measured by EIA were lower than those assayed by ELISA in pregnant women and in DVT patients. Furthermore, addition of several amounts of purified C4b-BP to NHP, which reduced the recovery of free protein S, did not influence the total protein S values measured by ELISA but slightly decreased the recovery of total protein S measured by EIA. These results indicate the necessity of using assays which accurately and reliably measure the total amount of protein S antigen. After addition of C4b-BP to NHP, the residual functional protein S level was lower than the residual level of free protein S antigen; this lends support to the idea that C4b-BP-PS complex inhibits the activated protein C cofactor activity of protein S.     

Protein C deficiency in insulin-dependent diabetes: a hyperglycemia-related phenomenon

In 30 insulin-dependent diabetic patients protein C (PC) antigen and PC activity were significantly lower than those of matched control healthy subjects. An inverse correlation between fasting plasma glucose and both PC concentration and activity was present in diabetics, while a direct correlation between PC concentration and PC activity was observed. Induced hyperglycemia in diabetic and normal subjects was able to decrease both PC antigen levels and PC activity, and heparin reversed in part this effect. In diabetic patients euglycemia obtained by insulin infusion restored to normal the depressed PC levels. Heparin did not alter both the basal PC concentration and activity in healthy controls. These data stress the major role of hyperglycemia in determining PC decrease in diabetics, and suggest that PC reduction is probably associated to hyperglycemia-enhanced thrombin formation.     

Functional and immunologic protein S in normal pregnant women and in full-term newborns

Total and free protein S antigen and C4b-binding protein (C4bp) were determined by rocket immuno-electrophoresis, and functional protein S was assayed by a coagulation method, throughout pregnancy and normal puerperium and in a group of normal full-term newborns (FTN). The functional protein S assay is based on a modification of the APTT, using a mixture of test sample, protein S deficient plasma, activated protein C, phospholipids and calcium. This protein S functional assay is specific for protein S since the APTT prolongation by normal plasma was abolished by incubation of plasma with monospecific, rabbit anti-protein S IgG. The ratios of functional protein S/free protein S antigen in healthy men (n = 13) and women (n = 14) were 1.0 +/- 0.13 (mean +/- SD) and 1.03 +/- 0.20, respectively. During pregnancy there is a decrease in functional protein S and a progressive decrease in total and free protein S antigen, with a functional/free protein S ratio of 0.75 +/- 0.28 in the third trimester of pregnancy (n = 16). In early puerperium the functional protein S level was lower than the free protein S antigen level (ratio about 0.5). In the FTN group, the free protein S level was 39% and protein S activity was about 70% that of adults, with a functional/free protein S ratio of 1.84 +/- 0.31. C4bp values were 23.5 +/- 10.3% in the FTN group, and crossed immunoelectrophoresis showed that in this group the major protein S peak corresponded to free protein S.(ABSTRACT TRUNCATED AT 250 WORDS)     

Normal reference ranges of antithrombin,protein C and protein S: Effect of sex,age and hormonal status

Introduction

Antithrombin (AT), protein C (PC) and protein S (PS) deficiencies are risk factors for venous thromboembolism. Overlapping values between heterozygous carriers and normal individuals often make a correct classification of a deficiency difficult. The aim of this study was to investigate the effect of sex, age, menopause and hormone therapy on natural anticoagulant plasma levels in a large group of healthy individuals, and to evaluate the need of separate reference ranges.

Materials and Methods

AT and PC were measured with a chromogenic assay, antigenic free PS with an ELISA test. To evaluate the effect of sex, age, oral contraception, hormonal status (and their interaction) on AT, PC and PS levels, linear regression models were used. Biological relevance and the value of the normal deviate z were chosen as rules to decide for separate reference ranges.

Results

The study population consisted of 1837 healthy adult individuals (741 men, 1096 women), aged 18-85 years (median age: 44 years). In men AT levels decreased after the age of 50 years. Men had higher levels of PS than women, particularly at young ages. In women, after correction for menopause, only PC levels increased with age. Menopause affected AT and PS, but not PC levels. Oral contraceptive intake was associated with a decrease of AT and PS, and an increase of PC levels.

Conclusions

For AT, PC and PS, sex- and age-specific normal reference ranges can be useful, in order to better discriminate true carriers of a natural anticoagulant deficiency.     

Behavior of protein S during long-term oral anticoagulant therapy

It has recently been reported that a natural anticoagulant, protein S (PS), is depressed during oral anticoagulation. Since more detailed information is required from the clinical standpoint, we measured plasma levels of PS [both total and free (not complexed) PS antigen], C4b-binding protein (C4bp) and other vitamin K-dependent proteins (factors II, VII, IX, X and protein C) in 60 plasma samples from patients on long-term oral anticoagulant therapy with warfarin. Together with the reduction of other vitamin K-dependent plasma proteins, PS decreased during warfarin treatment, being dependent on the intensity of the therapy. A considerable variation in plasma PS levels was also observed among individuals with a similar intensity of anticoagulation. Plasma concentration of C4bp was closely correlated with total PS level, and free PS/total PS ratio was independent of thrombotest values. These findings indicate that long-term oral anticoagulant therapy results in the suppression of the synthesis of PS, and that its reduction is on the whole balanced with C4bp and vitamin K-dependent coagulation factors. It was suggested that the metabolism of C4bp might be regulated by the plasma PS level, although this hypothesis needs further exploration.     

Stability of coagulation assays performed in plasma from citrated whole blood transported at ambient temperature

Many preanalytical variables affect the results of coagulation assays. A possible way to control some of them would be to accept blood specimens shipped in the original collection tube. The aim of our study was to investigate the stability of coagulation assays in citrated whole blood transported at ambient temperature for up to two days after specimen collection. Blood samples from 59 patients who attended our haematology outpatient ward for thrombophilia screening were transported at ambient temperature (outdoor during the day, indoor overnight) for following periods of time: <1 hour, 4-6, 8-12, 24-28 and 48-52 hours prior to centrifugation and plasma-freezing. The following coagulation tests were performed: PT, aPTT, fibrinogen, FII:C, FV:C, FVII:C, FVIII:C, FIX:C, FX:C, FXI:C, VWF:RCo, VWF:Ag, AT, PC activity, total and free PS antigen, modified APC-sensitivity-ratio, thrombin-antithrombin-complex and D-dimer. Clinically significant changes, defined as a percentage change of more than 10% from the initial value, were observed for FV:C, FVIII:C and total PS antigen starting at 24-28 hours, and for PT, aPTT and FVII:C at 48-52 hours. No statistically significant differences were seen for fibrinogen, antithrombin, or thrombin-antithrombin complexes (Friedman repeated measures analysis of variance). The present data suggest that the use of whole blood samples transported at ambient temperature may be an acceptable means of delivering specimens for coagulation analysis. With the exception of factor V and VIII coagulant activity, and total PS antigen all investigated parameters can be measured 24-28 hours after specimen collection without observing clinically relevant changes.     

高通量透析膜对C-反应蛋白及血脂代谢的影响

背景：传统低通量透析不能改善微炎症状态和脂质代谢紊乱,新型的高通量透析则可以很好的改善这种微炎症状态和脂质代谢,有助于提高患者的生活质量和存活率,因此如何改善微炎症状态和脂质代谢成为人们研究的热点。 目的：观察高通量和低通量聚砜膜血液透析器对维持性血液透析患者血浆超敏C-反应蛋白及血脂代谢的影响。 方法：将44例维持性血液透析患者随机分为高通量透析组和低通量透析组,另选22例健康体健者作为正常对照组。高通量透析组使用高通量透析器FX60,低通量透析组使用低通量透析器F6,均每周透析3次,每次透析4 h;治疗1年后,分别比较两组患者治疗前后及与正常对照组的血超敏C-反应蛋白与总胆固醇、三酰甘油及低密度脂蛋白胆固醇等指标的变化。 结果与结论：两组治疗前血浆超敏C-反应蛋白与三酰甘油、总胆固醇及低密度脂蛋白胆固醇水平均高于正常对照组(P < 0.05);治疗后高通量透析组患者的血浆超敏C-反应蛋白与总胆固醇、三酰甘油及低密度脂蛋白胆固醇水平明显下降(P < 0.05),而低通量透析组无明显变化(P > 0.05)。结果提示采用高通量FX60聚砜膜透析器进行透析能改善维持性透析患者的微炎症状态及脂质代谢。 关键词：高通量透析;血液透析膜;血浆超敏C-反应蛋白;血脂;膜材料 doi:10.3969/j.issn.1673-8225.2010.25.032     

Hereditary dysfunctional protein C molecules (type II): assay characterization and proposed classification

Protein C (PC) deficiency is among the increasing number of recognized causes of hereditary thrombotic disease. Two types of PC deficiency have been described: 1) Type I, which is characterized by a concomitant decrease in PC activity and antigen, and 2) Type II, characterized by disproportionately low activity compared to antigen (i.e. a dysfunctional molecule). To date, only a small number of Type II patients have been described. This study was undertaken to evaluate a number of dysfunctional PC molecules by comparing PC clotting and amidolytic activities with antigen levels. For these studies, an automated PTT-based clotting PC assay was developed. This assay was sensitive to 1% of a normal plasma pool, specific, accurate, and reproducible (+/- 12%). A good correlation (r = 0.918) of the clotting activity to antigen was found in normal individuals and Type I heterozygous and homozygous patients. To classify Type II PC deficient patients, the antigen, amidolytic and clotting PC levels were compared in ten affected families. The clotting activities were decreased in all affected members, whereas the antigen levels were within the normal limits. In four of the 10 families, the amidolytic activity was normal and similar to the antigen levels. This suggests that in certain families, defects in the PC molecule occur in regions not associated with amidolytic functions. From these studies, the molecular basis of Type II PC deficiency is varied and complex, involving different functional domains of the PC molecule. Therefore, we have suggested a nomenclature algorithm for Type II PC deficiency based on the location of the defect within the specific domains of the PC molecule.     

Thrombin activatable fibrinolysis inhibitor and its fibrinolytic effect in normal pregnancy

We investigated changes in both thrombin activatable fibrinolysis inhibitor (TAFI) antigen levels and its functional effect on in vitro fibrinolysis in normal pregnancy. 152 pregnant women and 31 women in the immediate postpartum period were studied, with pregnancy divided into 6 windows at 4 weekly intervals. As TAFI influences and is in turn influenced by components of the protein C (PC) pathway, its measurements were correlated with levels of soluble thrombomodulin, PC, protein S (PS) and the overall phenotype of activated PC resistance (APCR). Compared with mean TAFI levels at booking gestation (6.6 +/- 1.2 microg/ml), levels peaked at 35-39 weeks gestation (9.6 +/- 2 microg/ml, p = 0.001), followed by a significant drop within 24 hours of delivery (7.2 +/- 1.1 microg/ml). In functional terms, the mean clot lysis time (CLT) (101 +/- 13 min at booking) also peaked at 35-39 weeks gestation (141 +/- 42 min, p = 0.007) and dropped after delivery (99 +/- 33 min), and was significantly correlated with gestational age (r = 0.410, p = 0.001) and could be abrogated in the presence of an inhibitor to TAFI activation. A significant negative correlation was found between TAFI levels and APCR (r = -0.478, p <0.001), APCRV (r = -0.598; p <0.001), PS (r = -0.490, P <0.001) and PC (r = -0.198, p = 0.02). In summary, there is a significant increase in TAFI levels, which translates into increased CLT during pregnancy. Furthermore, changes in TAFI contribute to the increasing APCR of pregnancy.     

Two cases of inherited triple deficiency in a large kindred with thrombotic diathesis and deficiencies of antithrombin III, heparin cofactor II, protein C and protein S.

Thirty-three subjects, belonging to a large family with functional antithrombin III (ATIII) deficiency (type IIa) and recurrent thromboembolism, were investigated for ATIII, heparin cofactor II (HCII), protein C (PC) and protein S (PS). We report the exceptional finding of two cases of triple deficiency: ATIII combined with HCII and PC in the first case aged 15 and ATIII combined with HCII and PS in the second case aged 27. Interestingly, both are asymptomatic thus far. Twenty-five other deficient members were found, among which seven are affected with a double deficiency. Totally, the results of our study show 38 deficiencies of four distinct antithrombotic protein: ATIII (n = 9), HCII (n = 9), PC (n = 7) or PS (n = 13). Two types of HCII deficiency were observed and type I PC deficiency was found. Functional PS deficiency was characterized by reduced levels of cofactor activity for activated PC. Our report demonstrates that combined deficiencies should be sought in a family already known to be deficient in one antithrombotic protein.     

Plasma protein S activity measured using Protac, a snake venom derived activator of protein C

Functional activity of protein S, a cofactor of activated protein C-dependent inhibition of blood coagulation, in human plasma was measured by using Protac, a snake venom derived activator of protein C. This assay appeared to be specific for protein S, because 1) the activated partial thromboplastin time of protein S-depleted plasma depended on the purified protein S added in the presence of Protac; and 2) the level of protein C in plasma sample (0 to 10 micrograms/ml) had no influence on the clotting time. The cofactor activity of protein S in the plasma of normal men (n = 16) and women (n = 14) was 99.4 +/- 23.8% and 98.6 +/- 24.5% respectively. The protein S activity in the plasma of pregnant women at pre- and post-partum (n = 14), and that in the plasma of patients under warfarin therapy (n = 20) were 46.2 +/- 18.9%, 45.8 +/- 19.6% and 24.0 +/- 15.7%, respectively. In these plasmas, the levels of protein S activity were lower than those of total protein S antigen, but were similar to those of free protein S antigen. In 16 patients out of two families with congenital protein S deficiency, the protein S activity, the free antigen and the total antigen were 9.4 +/- 6.9%, 13.3 +/- 4.6% and 57.4 +/- 20.7%, respectively. There was no significant relationship between the level of protein S activity and that of a complemental C4b-binding protein antigen in any of these patients.     

High levels of tissue factor pathway inhibitor in patients with nephrotic proteinuria.

Acquired deficiency of naturally occurring anticoagulant proteins, due to loss in the urine, has been proposed as one of the major thrombogenic alterations in nephrotic proteinuria. The aim of this study was to investigate if proteinuria may induce deficiency of tissue factor pathway inhibitor (TFPI). TFPI, protein C (PC) and antithrombin (AT) were measured in 31 patients with nephrotic proteinuria, compared with 62 age- and sex-matched controls. Plasma levels of TFPI activity, total TFPI antigen and free TFPI antigen were significantly higher in patients with nephrotic proteinuria than in controls, and none of the patients had TFPI deficiency. Intravenous injection of 7500 IU unfractionated heparin induced a significant further increase of TFPI in two patients with high pre-heparin levels. Also plasma levels of PC were significantly higher in patients than in controls. Mean AT antigen levels were not significantly different between patients and controls, and AT activity was only marginally increased with borderline significance. Three out of 31 patients had substantial acquired AT deficiency. In conclusion, proteinuria is not associated with TFPI deficiency, but with a marked increase of this anticoagulant protein. The acquired thrombophilic diathesis of patients with nephrotic proteinuria can therefore not be attributed to TFPI deficiency.