Inhibitor against coagulation factor V after liver transplantation.

A new case of anti-factor V inhibitor is described in a 46-year-old man, who received a liver transplantation for hepatocellular carcinoma, without exposure to bovine thrombin or fibrin glue during the operative course. The inhibition occurred on the 14th postoperative day, while the patient was being treated with oxacillin, azathioprine, and a new immunosuppressive drug, FK506. The inhibition was of short duration (3 days), and no bleeding complication occurred despite a very low plasmatic level of factor V activity and antigen (<5%). Plasma samples drawn after cessation of FK506 disclosed a dose-dependent inhibitory activity when alcoholic solutions of FK506 were exogeneously added; this suggests a possible role of the FK506 drug in the occurrence of this anti-factor V inhibitor.

Genetic modulation of plasma protein S levels by two frequent dimorphisms in the PROS1 gene.

We studied two polymorphisms located close to or within the 3'-untranslated (3'-UT) region of the PROS1 gene [an A to G transition at nt 2148 (Pro 626) and an A to C substitution at nt 2698] in 110 healthy volunteers. The allele frequency of the nt 2148 G variant was 35%, and that of the nt 2698 A variant was 27%. We found a relationship between the two dimorphisms (both separately and together) and the plasma total protein S antigen (tPS) level. The impact of the neutral Pro 626 dimorphism was more significant than that of nt 2698 C/A (p = 0.0003 and p = 0.013, respectively). The lowest tPS values were observed in subjects with the Pro 626;nt 2698 GG;CC genotype, and the highest values in those with the AA;AA genotype. Both polymorphisms acted independently of sex and age. The mechanisms by which the two polymorphisms regulate tPS synthesis were not revealed by the studies of platelet mRNA. This study provides the first evidence of a genetic modulation of tPS levels, which, in addition to age and sex, contributes to the wide normal range of tPS in plasma. Determination of these two polymorphisms could be a valuable additional tool for studying PS.

The 1691 G-->A mutation in the factor V gene: relationship to activated protein C (APC) resistance and thrombosis in 65 patients.

Four hundred fifty subjects were screened for the 1691 G-->A mutation in the factor V gene. Two hundred ninety-seven patients were referred to us for unexplained thrombosis, 133 were family members of these patients and 20 were normal subjects. We studied the relationships between the mutation, resistance to APC and thrombosis. Among the 450 subjects tested, 65 belonging to 42 families were found to have the 1691 G-->A mutation in one (n = 61) or both alleles (n = 4). The prevalence of the mutation in the thrombotic patients was 13%. Resistance to APC was tested for in 247 subjects not on anticoagulant treatment (4 homozygous and 44 heterozygous for the mutation, and 199 individuals without the mutation). Incomplete cosegregation of heterozygosity for the 1691 G-->A mutation with APC resistance (APC-SR < 2.4 or n-APC-SR < 0.75) was observed, showing that the functional assay alone is insufficient for a firm diagnosis. In patients carrying the mutation, elevated levels of prothrombin fragment 1 + 2 and D-dimers pointed to increased thrombin generation in vivo. Clinical manifestations in the heterozygous subjects were very similar to those reported in heterozygous PC or PS deficiencies, but the first thrombotic event occurred later than in PC- or PS-deficient patients. Homozygosity for the factor V gene mutation appears to be a far more benign thrombotic disorder than homozygous PC and PS deficiencies.

Evaluation of a new chronometric assay for factor V Leiden-dependent APC resistance.

Since the first description by Dahlbäck et al (1) of a hereditary defect in the plasma of three unrelated thrombophilic patients characterized by a poor anticoagulant response to activated protein C (APC), many reports have shown a high prevalence of the APC resistance in both the general and thrombophilic populations (2-6). In almost all cases APC resistance is due to a single point mutation in the factor V gene, (a G to A substitution at nucleotide position 1691), which predicts the synthesis of a factor V molecule (FVQ506 or FV Leiden) in which the Arg 506 in one of the APC cleavage sites is replaced by Gln (7), rendering factor Va resistant to inactivation by APC. The FVQ506 mutation is the most frequent hereditary risk factor for venous thrombosis (2, 5, 6, 8-10) making an assay for APC resistance with a high predictive value for the mutation highly desirable, especially when molecular diagnosis at the gene level is not possible. The aim of this study was to evaluate the performance of a new factor Xa-based assay for the FV Leiden-dependent APC resistance.

Influence of conditions of blood sampling on coagulation activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin complexes and D-dimers) measurements.

In this study, we evaluated the effects of anticoagulants used in blood sampling on the measurements of coagulation activation markers F1 + 2, TAT, D-Dimers by Elisa methods. The study was carried out on normal subjects and patients with inherited deficiency of coagulation inhibitors, antithrombin III (ATIII) protein C (PC) and protein S (PS). Three different anticoagulant solutions were compared: 1) ACD/EDTA/adenosine/heparin, 2) EDTA/aprotinin/a synthetic thrombin inhibitor and 3) sodium citrate. The results showed that sodium citrate, commonly used in coagulation laboratories, is a suitable anticoagulant for the study of coagulation activation markers. In addition, the type of tubes (plastic tubes vs glass Vacutainer R tubes) used for blood sampling as well as the order of sampling (early or late after the phlebotomy procedure) did not influence the results. We concluded that assays of coagulation activation markers F1 + 2 and D-Dimers can be performed in samples collected routinely by haemostasis laboratory staff using Vacutainer R tubes with sodium citrate. Further investigations are needed to understand why TAT measurements gave a pattern of results quite different from F1 + 2 or D-Di measurements.

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.

Functional assay of protein S in 70 patients with congenital and acquired disorders.

A functional assay for the selective measurement of the active form of protein S in plasma, based on the prolongation of an APTT, was previously developed. This assay is sensitive, reproducible and specific, not affected by other clotting factors including FVIII. This method was applied to the measurement of protein S activity in congenital and acquired disorders. Results of protein S activity were compared to those of total and free antigen measured by ELISA. In 30 controls, there was an excellent correlation between protein S activity and free antigen. In patients with inflammatory disease, protein S activity and free antigen were normal, despite high levels of both C4b-binding protein and total protein S antigen. In dicoumarol-treated patients, protein S activity was lower than free antigen due to the presence of acarboxylated forms. Surprisingly, in liver cirrhosis, free antigen was only slightly decreased whereas protein S activity was significantly reduced. In 23 patients with congenital deficiency, protein S activity was consistently decreased, from less than 5% to 60% and showed good correlation with the free antigen. This functional assay allows the rapid diagnosis of congenital or acquired deficiency of protein S.

[Extracorporeal circulation with danaparoid sodium for valve replacement in thrombocytopenia induced by type II heparin]. / Circulation extracorporelle sous danaparoïde sodique pour remplacement valvulaire au cours d'une thrombopénie induite par l'héparine de type II.

A type II heparin-induced thrombocytopenia (HIT) was diagnosed in a 64-year-old woman at day 20 of intravenous unfractionated heparin (UFH) therapy, given after myocardial infarction treated by angioplasty and intracoronary stent. The infarction was complicated by a mitral insufficiency that led to a mitral valve replacement. Cardiopulmonary bypass was successfully performed with sodium danaparoid (Orgaran), as an alternative to UFH, without thrombotic or haemorrhagic complications and the follow-up was uneventful.

[Thrombosis of a cardiopulmonary bypass circuit despite recommended hypocoagulation with danaparoid during the acute phase of type II heparin-induced thrombocytopenia]. / Thrombose d'un circuit de circulation extracorporelle sous danaparoïde sodique à la phase aiguë d'une thrombopénie induite par l'héparine de type II en dépit d'une hypocoagulation recommandée.

A 36-year-old patient was admitted to our hospital with ischaemic stroke. The initial assessment allowed the diagnosis of an antiphospholipid syndrome (APS) and an intracardiac mass suggestive of a heart tumour. The patient was treated with unfractionated heparin. Type II heparin-induced thrombopenia (HIT) was diagnosed on the 18th day of therapy. Given the risk of stroke recurrence it was decided to remove the cardiac tumour surgically. Cardiopulmonary bypass (CPB) was performed using danaparoid in a state of deep hypothermia, in accordance with the well-established protocol in use in our department. As the CPB and surgical procedure came to an end a massive thrombus began forming in the circuit, requiring immediate displacement of the CPB cannulae. The anti-Xa activity level obtained had been considered effective at an estimated 1.20 IU/ml, although, the level recommended by Magnani is between 1.50 and 2.0 IU/ml. There was no clinical consequence and postoperative recovery was uneventful. The discrepancy between the satisfactory level of anti-Xa activity and the thrombus formation in the CPB circuit raises the issue of the diversity of published anticoagulation protocols, the difficulty to extrapolate within a surgical team, the need for intensive laboratory monitoring within a narrow therapeutic range, as well as the patient profiles variability with concurrent disorders complicating their clinical management.

A mutation of the active protein S gene leading to an EGF1-lacking protein in a family with qualitative (type II) deficiency.

The genomic analysis of a 70-year-old man with recurrent deep venous thrombosis having a protein S (PS)-deficient phenotype corresponding to both type III and type II evidenced two different mutations: a +5 g-->a mutation in the donor splice site of intron e (ivs e) and a ser 460 to Pro mutation. The propositus' son, who had a type II PS deficiency phenotype, only bore the ivs e +5 g-->a mutation. The study of platelet PS mRNA prepared from this subject showed that the ivs e, +5 g-->a mutation led to the generation of two abnormal transcripts, one lacking exon 5 and the other lacking exons 5 and 6. The presence of an additional PS band with a decreased molecular mass on immunoblots performed in reducing conditions suggested the presence of truncated PS lacking EGF1 (encoded by exon 5). Two monoclonal antibodies (MoAbs) were used to further characterize the nonfunctional plasma PS. Comparison of PS levels measured with each of these MoAbs and PS levels in conventional assays was consistent with the presence of an abnormal inactive protein in the plasma of both patients bearing the ivs e, +5 g-->a mutation, suggesting that variant PS lacking EGF1 is secreted but is devoid of activated protein C cofactor activity.

Decreased protein S activity in sickle cell disease.

In sickle cell disease (SCD), vaso-occlusion is a complex process involving cellular, vascular and humoral factors and possibly thrombotic events. We studied three physiological inhibitors of the coagulation system, antithrombin III (AT III), protein C (PC) and protein S (PS), in three groups of subjects: 27 homozygous patients observed either in crisis or in a steady state, 23 heterozygous patients and 30 healthy subjects. PS study included the measurement of total and free PS antigen, PS activity and C4bBP antigen. In heterozygous subjects the results were similar to those of controls, but in homozygous subjects abnormalities of PS and to a lesser extent PC were observed. Values of PC were extremely variable with 10 cases lower than the normal range (2 SD of the mean) and 17 others within this range. In all cases total PS antigen was slightly reduced (77 +/- 18%, M +/- SD) with a more marked decrease of free antigen (59 +/- 17%) and normal values of C4bBP. Levels of PS activity were greatly reduced and lower than those of free antigen with a mean ratio of PS activity to free antigen of 0.6. These abnormalities were associated with significantly high concentrations of fibrinogen D-dimers. PS deficiency in SCD may be at least partly due to adsorption of free PS to aminophospholipids abnormally expressed on sickle cells membranes, microvesicles and activated platelets, while the discrepancy between PS activity and free antigen could reflect proteolytic inactivation of PS by traces of thrombin.