[Skin necrosis during long-term fluindione treatment revealing protein C deficiency]. / Nécroses cutanées tardives sous fluindione révélant un déficit en protéine C.

BACKGROUND: Cutaneous necrosis is a rare complication of vitamin K antagonist therapy. It presents as cutaneous hemorrhagic necrosis and usually occurs at the start of treatment. We describe an atypical case of recurrent skin necrosis after two years of treatment with fluindione. CASE REPORT: A 70-year old woman with a history of venous thromboembolism and obesity presented with a large haemorrhagic necrosis of the abdominal wall. She had been treated with fluindione for two years. Genetic protein C deficiency was discovered. Resumption of vitamin K antagonist therapy was followed by recurrence of skin necrosis despite concomitant administration of heparin. Treatment with vitamin K antagonists could not be continued. DISCUSSION: This observation is unusual due to the late onset of skin necrosis. The condition usually begins shortly after initiation of vitamin K antagonist therapy, generally between the third and the sixth day of treatment. It is due to a transient hypercoagulable state in patients with protein C deficiency or, in rare cases, protein S deficiency. This late-onset skin necrosis, occurring many years after initiation of anticoagulant therapy, may be explained by a sudden worsening of pre-existing protein C deficiency due to infectious and iatrogenic factors.

Poor performance of albumin or protein-adjusted plasma calcium to diagnose dyscalcemia in hospitalized patients: A confirmatory study in a general internal medicine department.

INTRODUCTION: Hypo- and hypercalcemia are common and some causes require urgent diagnosis and treatment. Measurement of ionized calcium is the reference test to diagnose calcium disorders but total calcium adjusted for protein or albumin concentration is more often used. METHODS: Patients hospitalised in a general internal medicine department from September 2013 to December 2015 who had a total plasma calcium concentration and a serum albumin or protein concentration measured within 24h of a ionized calcium blood measurement were included. Total calcium was adjusted for protein or albumin concentration using widely used formulas and compared to ionized calcium as the gold standard. RESULTS: Among 210 included patients, 46 (22%) had hypocalcemia, 124 (59%) normocalcemia and 40 (19%) hypercalcemia according to ionized calcium concentration. Total calcium had 50% sensitivity and 95% specificity to diagnose hypocalcemia and a 93% sensitivity and 89% specificity to diagnose hypercalcemia. Adjusting total calcium for protein or albumin concentrations did not increase and sometimes decreased diagnostic accuracy. CONCLUSION: Total calcium, with or without albumin/protein adjustment, is poorly sensitive to screen for hypocalcemia. Unadjusted total calcium is as sensitive as protein- or albumin-adjusted total calcium to screen for hypercalcemia. These data argue against the use of albumin- or protein-adjusted calcium. Ionized calcium measurement should be performed to confirm dyscalcemia in patients with abnormal total calcium concentration and to rule out hypocalcemia in patients with total calcium concentration in the lower range of normal values.

Gross deletions/duplications in PROS1 are relatively common in point mutation-negative hereditary protein S deficiency.

Hereditary protein S (PS) deficiency is an autosomal disorder caused by mutations in the PS gene (PROS1). Conventional PCR-based mutation detection identifies PROS1 point mutations in approximately 50% of the cases. To verify if gross copy number variations (CNVs) are often present in point mutation-negative hereditary PS deficiency we used multiplex ligation-dependent probe amplification (MLPA) as a detection tool in samples from individuals with a high probability of having true PS deficiency. To this end, DNA samples from nine PS deficient probands with family members (seven type I and two type III) and nine isolated probands (three type I and six type III), in whom PROS1 mutations were not found by DNA sequencing, were evaluated. An independent quantitative PCR (qPCR) was performed to confirm the findings of the MLPA assay. Family members were also tested when DNA was available. Gross abnormalities of PROS1 were found in six out of eighteen probands. In three probands complete deletion of the gene was detected. Two probands had a partial deletion involving different parts of the gene (one from exon 4 through 9 and another from exon 9 through 11). One family showed a duplication of part of PROS1. qPCR analysis was in accordance with these results. In conclusion, this study substantiates that gross gene abnormalities in PROS1 are relatively common in hereditary PS deficient patients and that MLPA is a useful tool for direct screening of CNVs in PROS1 point mutation-negative individuals.

[Multiple osteonecroses and venous thrombosis: one case of patient with a novel mutation of protein C (N102S) and heterozygous for FV Leiden]. / Ostéonécroses et thromboses veineuses multiples : un cas de patient porteur d'une nouvelle mutation de la protéine C (N102S) et hétérozygote pour le facteur V Leiden.

The association of a thrombo-embolic venous disease and multiple osteonecroses occurring in the presence of biological risk factors for thrombosis is rarely described in the literature. We report here the case of a 35-year old patient with such clinical manifestations. This patient is heterozygous for a novel mutation of the protein C gene (N102S) and for FV Leiden polymorphism. The clinical history is characterized by numerous thrombo-embolic venous episodes associated with several episodes of epiphysis osteonecrosis requiring two hip total prostheses and two knee total prostheses. The particular clinical features here are the multiple osteonecroses and the unusual localisation of brain and genital thromboses. The absence of both venous thromboembolic and osteonecrosis events in the relatives presenting the same genetic pattern suggests broad phenotype variations in the clinical expression of these genetic abnormalities. In osteonecrosis associated with thrombophilia, some authors have proposed treatment with stanazolol, which increase circulating protein C concentration. The effectiveness of this drug among such patients should be evaluated by clinical studies.

Met 358 to Arg mutation of alpha 1-antitrypsin associated with protein C deficiency in a patient with mild bleeding tendency.

The molecular defect responsible for a dramatic prolongation of all standard clotting tests discovered in a 15-yr-old boy has been identified. Initial investigations revealed the presence of an activated Factor X (Factor Xa) and thrombin inhibitor which copurified with alpha 1-antitrypsin (alpha 1-AT), thereby suggesting the occurrence of an alpha 1-AT variant similar to alpha 1-AT Pittsburgh. This was confirmed by dot-blot analysis and direct sequencing after amplification by the polymerase chain reaction. A G to T transition at nucleotide 10038 results in the substitution of Met to an Arg, converting alpha 1-AT into an Arg-Ser protease inhibitor (serpin) that inhibited thrombin and Factor Xa more effectively than antithrombin III. Surprisingly, there was no bleeding history in the proband. The common mutation Z, which may explain a reduced expression of the allele bearing the Arg 358 Met mutation, was not observed in the propositus' DNA. To exclude the presence of another mutation, the coding regions and intron/exon junctions were sequenced. No other mutation was found. Recently, the patient experienced his first hemorrhagic episode at the age of 17. The level of the abnormal inhibitor had increased twofold 2 mo before. The large decrease in protein C concentration may account for the mild bleeding tendency in this case, despite the presence of the alpha 1-AT Pittsburgh mutation. An abnormal protein C pattern was observed in patient's plasma, suggesting that the circulating deficiency might be due to a deleterious effect of the abnormal inhibitor on both intracellular processing and catabolism of protein C.

Rat high-molecular-weight kininogen: purification, production of antibodies and demonstration of lack of immunoreactive kininogen in a strain of brown Norway rats.

High-molecular-weight kininogen was purified to apparent homogeneity from Wistar rat plasma by a two-steps chromatographic procedure. 3 mg of kininogen were obtained from 205 ml of plasma. The purified high-Mr kininogen had a bradykinin content of 10.2 micrograms bradykinin equivalents/mg protein. Under denatured and reduced conditions it gave a single band on polyacrylamide gel electrophoresis corresponding to an apparent molecular mass of 110 kDa. Antibodies obtained against rat high-Mr kininogen gave a single precipitation line when tested against rat plasma in double immunodiffusion and in crossed immunoelectrophoresis. Although rat high-Mr kininogen possesses physicochemical properties (molecular mass, kinin content per molecule and amino acid composition) similar to human high-Mr kininogen, its antibodies do not cross-react with human, monkey or rabbit plasma, indicating major interspecies differences in the structure of the molecule. Immunoreactive kininogen of Wistar rats was identical to that of Brown Norway rats from a strain bred in Orleans, France (BN/Orl). However, plasma from a strain of Brown Norway rats bred in Leuven, Belgium (BN/Kat), reported to be deficient in a kinin precursor (Damas, J. and Adam, A. (1980) Experientia 36, 586-587), did not contain immunoreactive material discernible by double immunodiffusion or crossed immunoelectrophoresis.

Thrombomodulin promoter mutations, venous thrombosis, and varicose veins.

We analyzed the distal promoter region of the thrombomodulin (TM) gene (nucleotides -300 to -2052) in subjects from the Paris Thrombosis Study (PATHROS), a French case-control study of venous thrombosis, to identify polymorphisms that might modify TM gene expression. Eight novel mutations were found in the 40 DNA samples initially screened. Two of these mutations (-1748G/C and -1208/-1209 del TT) were frequent. One rare transition (-1166G/A) might have functional consequences owing to its position. These 3 mutations were screened for in the entire study population of 327 patients and 398 controls. None of the 3 was significantly associated with thrombosis. Interestingly, the -1208/-1209 TT deletion was associated with varicose veins in the patients. This mutation was in tight linkage disequilibrium with the +1418 C/T change in the coding sequence, a known polymorphism that predicts an Ala 455 Val substitution in the sixth epidermal growth factor-like TM module, a domain previously implicated in the proliferative functions of TM. This linkage suggests that the Ala 455 Val mutation may promote changes in these functions and thus be involved in varicose vein formation.

Hypercoagulable states in primary upper-extremity deep vein thrombosis.

BACKGROUND: There are very few data on the prevalence of coagulation abnormalities in primary deep vein thrombosis of the upper limbs. OBJECTIVE: To determine if coagulation abnormalities play a role in effort-related and/or idiopathic (non-effort-related) upper-extremity deep vein thrombosis (UEDVT). METHODS: Fifty-one consecutive patients (21 men and 30 women) who had effort-related (n = 20) or idiopathic (n = 31) UEDVT over an 18-year period (median age at diagnosis, 32 years; age range, 15-86 years) were routinely reexamined. Plasma was screened for antithrombin, protein C, and protein S deficiencies and for antiphospholipid antibodies (lupus anticoagulant and anticardiolipin antibodies). The DNA was screened for factor V Leiden and for prothrombin gene G20210A mutations. RESULTS: The median age (35 vs. 28 years), the proportion of women (81% [25/31] vs. 25% [5/201), the proportion of patients with a personal and/or family history of thromboembolism (42% [13/31] vs. 15% [3/20]), and the proportion of patients with at least 1 coagulation abnormality (42% [13/31] vs. 15% [3/20]) were higher in the idiopathic UEDVT group than in the effort-related UEDVT group. The odds ratio of having a coagulation abnormality was 4.09 (95% confidence interval, 0.99-16.78; P = .06) in the idiopathic UEDVT group compared with the effort-related UEDVT group. CONCLUSION: Hypercoagulable states appear to play a significant role in idiopathic but not in effort-related UEDVT.

Protein C and protein S deficiencies.

The protein C (PC) pathway, with its cofactor protein S (PS), is an important natural antithrombotic mechanism. Both PC and PS deficiencies have been implicated in thrombophilia. The molecular basis for hereditary PC and PS deficiencies is highly heterogeneous, with a large spectrum of mutations that have various effects on the expression of the relevant allele. A small subset of patients who are homozygous or compound heterozygous for a PC gene mutation have severe thrombotic complications at birth, whereas onset occurs later in the other cases. Patients heterozygous for a PC or PS gene abnormality may develop recurrent thrombosis during adulthood, with a probability of remaining free of thrombosis of about 50% at age 45. A PC or PS gene defect is associated with the factor V Arg 506 to Gln mutation in 10% to 30% of symptomatic patients, suggesting that clinical expression is controlled by several genes in heterozygous patients.

Diagnostic score for heparin-induced thrombocytopenia after cardiopulmonary bypass.

Heparin-induced thrombocytopenia (HIT) occurs in nearly 3% of patients treated with heparin after cardiopulmonary bypass (CPB). HIT carries a risk of severe thrombotic complications, and must be diagnosed rapidly. To identify simple criteria for estimating the probability of HIT after CPB, we retrospectively analyzed the files of 84 patients with suspected HIT after CPB and we analyzed the usefulness of several variables collected at the time of HIT suspicion to estimate HIT probability. HIT was confirmed in 35 cases and ruled out in 49 cases, on the basis of a platelet increment after heparin withdrawal, detection of heparin-dependent antibodies, and absence of other clear cause of thrombocytopenia. A biphasic platelet count from CPB to the first day of suspected HIT, an interval of >/= 5 days from CPB to the first day of suspected HIT, and a CPB duration of

The factor II G20210A gene polymorphism, but not factor V Arg506Gln, is associated with peripheral arterial disease: results of a case-control study.

BACKGROUND: The FIIG20210A polymorphism has been associated with arterial wall thickness and atherothrombotic diseases in selected subgroups. The FVArg506Gln polymorphism does not seem to be associated with arterial diseases. Few data are available on these polymorphisms and the risk of peripheral arterial disease (PAD). OBJECTIVES: To study the association between the FIIG20210A and FVArg506Gln polymorphisms and PAD and its clinical severity. To examine the potential interactions with traditional vascular risk factors. PATIENTS AND METHODS: We studied 184 consecutive male patients under 70 years of age with symptomatic PAD and 330 age-matched male controls free of symptomatic PAD and with no cardiovascular history. We evaluated the FIIG20210A and FVArg506Gln polymorphisms in all subjects. RESULTS: Mean age was 57.1 +/- 7.2 years (cases) and 56.7 +/- 7.6 years (controls). The FII20210A allele was more frequent in PAD patients with odds ratios (OR) of 3.77 (1.39-10.2) in univariate analysis and 4.30 (1.3-14.7) after adjustment for diabetes, smoking, hypertension and hypercholesterolemia. In smokers or past smokers the magnitude of the association was markedly increased but there was no evidence of an interaction between tobacco exposure and FIIG20210A. In case subjects, the FII20210A allele was also associated with critical ischemia [OR = 4.1 (1.1-15.7), P = 0.039 in multivariate analysis]. FVArg506Gln was not associated with PAD [OR = 0.65 (0.27-1.54) and 0.77 (0.28-2.1) in univariate and multivariate analyses, respectively]. CONCLUSIONS: The FIIG20210A gene polymorphism may be a risk factor for PAD and its severity. In contrast, the FVArg506Gln polymorphism is not associated with PAD.

Impact of progestagens on activated protein C (APC) resistance among users of oral contraceptives.

Oral contraceptive (OC) use is associated with an increased risk of venous thromboembolism. Previous data reported higher thrombotic risk in women using third-generation combined OC than in those using second generation OC. The difference could be explained by differential effects of progestagens on plasma sensitivity to activated protein C (APC). The main purpose of this cross-sectional study was to assess the influence of a progestagen-only OC (chlormadinone acetate) as well as the effect of several combined OC with different progestagen components on APC resistance. The effect of APC on endogenous thrombin potential (ETP) was investigated in the plasma of healthy women using either combined OC (n=82) or progestagen-only OC (n=28), and in non-users (n=64). Carriers of factor V Leiden were excluded. Compared with non-users, there was no significant change in APC resistance in women using progestagen-only OC. Women who used combined OC were less sensitive to APC than non-users (P < 0.001) and the difference was significantly more pronounced in women using third-generation OC (n=41) than in those who used second-generation OC containing levonorgestrel (n=22) (P < 0.05). Compared with OC containing levonorgestrel, use of norethisterone-containing OC (n = 9) was associated with an increased resistance to APC (P < 0.05). Women who used cyproterone-containing OC (n = 10) were less sensitive to APC than those using third-generation OC (P < 0.05) or second-generation OC containing levonorgestrel (P < 0.05). Protein S, factor II and FVIII levels explained in part the OC-related changes in APC sensitivity variations. ETP-based APC resistance may contribute to explain why different brands of OC can be associated with different levels of thrombogenicity.

Factors V leiden and II 20210A in patients with symptomatic pulmonary embolism and deep vein thrombosis.

PURPOSE: Factor V Leiden and factor II 20210A are inherited disorders of the clotting system that occur frequently in patients with deep vein thrombosis. We conducted this study to determine whether these factors are also common in patients with pulmonary embolism. SUBJECTS AND METHODS: We determined the prevalence of factor V Leiden and factor II 20210A in 773 consecutive patients with objectively documented symptomatic deep vein thrombosis or symptomatic pulmonary embolism, or with a combination of these disorders. RESULTS: Isolated symptomatic deep vein thrombosis occurred in 345 patients; isolated symptomatic pulmonary embolism occurred in 236; and both anomalies occurred in 192. Factor V Leiden was present in 21 (9%) of the patients with isolated symptomatic pulmonary embolism, in 30 (16%) with both manifestations, and in 63 (18%) with isolated symptomatic deep vein thrombosis (P = 0.007). Factor V Leiden was more common among patients with deep vein thrombosis (odds ratio [OR] = 2.1; 95% confidence interval [CI]: 1.2 to 3.7; P = 0.006) or both pulmonary embolism and deep vein thrombosis (OR = 1.8; 95% CI: 1.0 to 3.3; P = 0.07) than among patients with isolated pulmonary embolism. Factor V Leiden was less common in massive pulmonary embolism (5% [7 of 127]) than in submassive pulmonary embolism (13% [21 of 155], P = 0.03). We found no significant difference in the prevalence of factor II 20210A among the three groups. CONCLUSION: Factors V Leiden and II 20210A vary in prevalence among patients with pulmonary embolism and deep vein thrombosis, suggesting that the risk of pulmonary embolization may vary among patients who have different causes of venous thromboses.

Antithrombin III antigen in human platelets.

Immunoreactive AT III was found in human platelets. AT III antigen was quantified in platelets taken from each of 17 healthy donors by a specific competitive enzyme immunoassay using purified AT III and AT III antibodies. AT III antigen levels in extracts of washed platelets disrupted by freezing and thawing ranged from 32 to 140 ng per 10(9) platelets with a mean value of 70.3 +/- 27.3. When stimulated by arachidonic acid, the platelets released AT III antigen together with immunoreactive fibrinogen. These results show that AT III is present in platelets at a level corresponding to approximately 0.01% of total antithrombin in normal blood, and suggest that platelet AT III, like fibrinogen, is contained in the storage granules.

Hormone replacement therapy and circulating ICAM-1 in postmenopausal women--a randomised controlled trial.

Hormone replacement therapy may reduce the risk of coronary heart disease but underlying mechanism has not been adequately explained. Recent data suggest that intercellular adhesion molecule 1 (ICAM-1) plays a critical role in early stage of atherosclerosis and may serve as a molecular marker for the development of arterial disease. We investigated the effects of oral and transdermal cyclic oestradiol combined with progesterone on plasma concentration of soluble ICAM-1 (sICAM-1). Thirty-seven healthy postmenopausal women were randomly assigned to receive either oral estradiol valerate or transdermal estradiol both combined with micronized progesterone or no hormonal treatment. Plasma sICAM-1 was assayed at baseline and after a 6-month period. Oral but not transdermal estradiol regimen significantly decreased mean value of sICAM-1 compared with no treatment. Differences in sICAM-1 levels between active treatments were significant. There were no significant changes in mean values of fibrinogen between the three groups. Our results show a favorable effect of oral estrogen plus progesterone on a soluble marker of vascular inflammation and may provide plausible explanation for a cardioprotective effect of hormone replacement therapy among healthy postmenopausal women.

Compound heterozygosity in a family with protein C deficiency illustrating the complexity of the underlying molecular mechanism.

The association of two missense mutations, a Leu 223 to Phe and an Ile 403 to Met, is described in a family presenting with various protein C deficiency phenotypes. In this family, two subjects were compound heterozygotes with protein C levels of about 25%, the other members being heterozygous for only one of the mutations. The Leu 223 to Phe mutation was also found in 9 members of 3 other families and, in all cases but one, resulted in protein C levels below 60% associated with a high incidence of thrombotic complications. The other mutation, an Ile 403 to Met, was identified in those of the family' members who presented with borderline protein C concentrations. In such a family, the genomic DNA analysis represents the only way to differentiate between the genetic status of each family member. The results highlight the importance of the genotype determination and the poor discriminative power of the plasma assays currently used.

Thirty-three novel mutations in the protein C gene. French INSERM network on molecular abnormalities responsible for protein C and protein S.

We analyzed the protein C gene (PROC) with the denaturing gradient gel electrophoresis (DGGE) scanning strategy in a series of 129 patients with suspected protein C (PC) deficiency (93 with low plasma PC levels and 36 with borderline level). At least one sequence variation was found in 104 of the 129 patients. Thirty-nine sequence variations (found in 72 patients) were already reported detrimental mutations. Thirty-three were novel sequence variations, of which 19 (found in 25 patients) were probably detrimental. Five novel mutations (A1T, R9H, S11R, S12R and K193Q) were associated with qualitative plasma PC deficiency, suggesting or confirming the functional importance of amino acids at these positions. This strategy confirmed the diagnosis of inherited PC deficiency in 79/93 (84.9%) patients with low plasma PC levels and 14/36 (38.8%) patients with borderline values. In order to explain abnormal PC levels observed in patients who did not carry detrimental mutations, screening for the -1654C/T and -1641A/G PROC promoter polymorphisms known to influence plasma PC concentrations was performed. The frequency of the CG allele associated with lower PC concentrations was slightly but not significantly lower in 82 heterozygotes for detrimental PROC gene mutations than in 36 patients with no identified detrimental mutations.

Influence of six mutations of the protein C gene on the Gla domain conformation and calcium affinity.

The protein C Gla domain was studied in six families presenting a type II hereditary deficiency characterized by low activity in a coagulation assay and normal activity in an amidolytic assay. Five of these mutations, previously described by our group, affected Arg-5, Arg-1, Arg 229 and Ser 252. We report here the first natural Glu 7 to Asp mutation in a sixth family. We evaluated the binding of the mutated protein C to H11, a monoclonal antibody (mAb) known to recognize the sequence Phe4 to Arg9 of the Gla domain; the presence of calcium ions suppresses the recognition of this epitope by H11. Mutation of Arg229 to Gln and Ser252 to Asn did not modify the inhibition of protein C binding, whereas the Arg-1 to His mutation resulted in a loss of inhibition in the presence of CaCl2. This suggests that the protein C of this patient shows impaired carboxylation. The protein C from patients bearing the mutations Arg-5 to Trp, Arg-1 to Cys and Glu 7 to Asp bound poorly to H11 mAb, even in the absence of calcium ions. The calcium affinity of the Gla domain was studied by pseudo-affinity chromatography, in which protein C was successively eluted from a Mono Q column by CaCl2 10 mM and NaCl 0.6 M. Protein C from the patient bearing the Arg-5 to Asp mutation had a normal elution profile, suggesting that a modification of the propeptide cleavage site impairs the conformation of the Gla domain but not carboxylation.(ABSTRACT TRUNCATED AT 250 WORDS)

A new assay based on thrombin generation inhibition to detect both protein C and protein S deficiencies in plasma.

Activated protein C reduces thrombin generation by inactivating factors V and VIII in the presence of protein S. This prompted us to develop an assay which would allow specific exploration of this reaction. The total amount of thrombin formed in plasma after activation by tissue factor and phospholipids was reduced by adding thrombomodulin. This addition allowed protein C to be activated by endogenous thrombin. The inhibition of thrombin generation (ITG) due to protein C activation could be measured by comparing thrombin formation in the presence and in the absence of thrombomodulin. ITG increased with both protein C and protein S concentrations. Normal values of ITG expressed as a percentage were between 40 and 65% and were not influenced by age or sex. ITG increased in patients under heparin therapy, decreased in patients under oral anticoagulant therapy and was decreased in women using oral contraceptives. This method could be used for screening patients for protein C and protein S deficiencies.