R. Marino

Influence of factor VIII/von Willebrand complex on the activated protein C-resistance phenotype and on the risk for venous thromboembolism in heterozygous carriers of the factor V Leiden mutation.

High factor VIII plasma levels have been shown to represent a common increased risk for venous thromboembolism (VTE) and may cause an activated protein C (APC) resistance in the absence of the factor V Leiden mutation, but there are no studies specifically aimed to establish if high factor VIII and von Willebrand factor (vWF) concentrations may influence the APC sensitivity ratio (APC-SR) and increase the risk for VTE in the presence of the factor V Leiden mutation. For this purpose, we performed a retrospective case-control study to investigate the influence of the procoagulant factor VIII (VIII:C) and the antigen of vWF (vWF:Ag) on the normalized APC-SR (n-APC-SR) and on the risk for VTE, in two selected groups of 30 symptomatic (Group I) and 32 asymptomatic (Group II) related heterozygotes for the factor V Leiden mutation. Differences between the two groups (Group I versus Group II) were: n-APC-SR, 0.57+/-0.06 versus 0.63+/-0.08, P = 0.001; factor VIII:C, 1.49+/-0.42 versus 1.13+/-0.28 IU/ml, P<0.001; vWF:Ag, 1.46+/-0.53 versus 1.26+/-0.32 IU/ml, NS. As a whole (Group I + Group II), Pearson correlation coefficients were: n-APC-SR versus factor VIII:C, r = -0.410, P = 0.001; n-APC-SR versus vWF:Ag, r = -0.309, P = 0.01; factor VIII:C versus vWF:Ag, r = +0.640, P<0.0001. The relative risk for VTE in individuals with the factor VIII:C concentration > 1.5 IU/ml was 2.5 (95% confidence interval 1.6-3.9). We concluded that high factor VIII:C levels, probably in the effect of vWF, play a determinant role in worsening the APC-resistance phenotype and represent a common additional risk factor for VTE in heterozygous carriers of the factor V Leiden mutation.

Beneficial effects of postmenopausal hormone replacement therapy with transdermal estradiol on sensitivity to activated protein C.

Many hemostatic and fibrinolytic parameters have been evaluated following hormone replacement therapy (HRT) but little is known about its influence on the anticoagulant response to activated protein C (APC-sensitivity). For this purpose, we studied the effect of transdermal 17-beta-estradiol (50 microg/24 h) by a continuous regimen on the APC-sensitivity, in 28 postmenopausal hysterectomized women (mean age, 47 years; range, 44-65 years). We also measured the plasma proteins directly involved in the protein C anticoagulant pathway, such as activities of factor VIII (VIII:C), factor V and free protein S. Von Willebrand factor (vWF) antigen, the carrier protein of factor VIII, was also determined. Blood sampling was done at baseline and after 16-week therapy. A significant increase in the normalized APC-sensitivity ratio (n-APC-SR) values (mean +/- SD: pre-trial, 0.88 +/- 0.14; post-trial, 1.01 +/- 0.12; P < 0.001) and a significant decrease of factor VIII:C plasma levels (pre-trial, 1.13 +/- 0.29 IU/ml; post-trial, 0.98 +/- 0.20 IU/ ml; P = 0.001) were found. No difference was observed in factor V, protein S and vWF plasma levels. Correlation studies demonstrated only a significant negative correlation between the percent change in n-APC-SR and the percent change in factor VIII:C (r = -0.574; P = 0.001). Our findings clearly show that HRT with transdermal estradiol improves the anticoagulant response to APC, probably as a result of a decreased factor VIII:C. We also suggest that a similar but opposite mechanism may occur for perorally administered estrogens used in the HRT. These results may have some clinical implications about the reported increase of the risk for venous thromboembolism following HRT.

Treatment with recombinant activated factor VII in a patient with hemophilia A and an inhibitor: advantages of administration by continuous infusion over bolus intermittent injections.

Recent studies have shown that treatment with a continuous infusion of recombinant activated factor VII (rFVIIa) is far more convenient than administration by bolus intermittent injections and may allow a substantial reduction in the dose. We present the case of a 26-year-old patient with hemophilia A, who had a high-titer inhibitor to both human and porcine factor VIII, and who had recently been admitted to hospital because of a bilateral severe ilio-psoas hematoma. Two subsequent courses of treatment with rFVIIa by bolus intermittent injection showed only a partial efficacy. A further administration of rFVIIa was therefore carried out using a continuous infusion regimen that proved to be fully efficacious. During the continuous infusion course levels of factor VII coagulant activity were in the range 18.2-5.2 U/ml, while the prothrombin time, expressed as an International Normalized Ratio, remained within the range 0.57-0.71. The continuous infusion, compared with the administration of the bolus intermittent infusion, reduced the amount of rFVIIa required by approximately 40-50%. Statistical analysis demonstrated that there was a strong positive correlation between the rate of infusion of rFVIIa and levels of factor VII coagulant activity (r = +0.941; P < 0.001), and a very significant negative correlation between levels of factor VII coagulant activity and prothrombin time values (r = -0.897; P < 0.001). In accordance with previous findings, our experience confirms that, when prolonged therapy is required, treatment with rFVIIa by continuous infusion is more convenient than administration of bolus intermittent injections, and may allow the saving of a large amount of drug. Moreover, we suggest potential additional advantages of the continuous infusion regimen over bolus intermittent injections, such as a better efficacy and a stronger correlation between prothrombin time and factor VII coagulant activity levels.

Assessment of the olfactory function in Italian patients with type 3 von Willebrand disease caused by a homozygous 253 Kb deletion involving VWF and TMEM16B/ANO2.

Type 3 Von Willebrand disease is an autosomal recessive disease caused by the virtual absence of the von Willebrand factor (VWF). A rare 253 kb gene deletion on chromosome 12, identified only in Italian and German families, involves both the VWF gene and the N-terminus of the neighbouring TMEM16B/ANO2 gene, a member of the family named transmembrane 16 (TMEM16) or anoctamin (ANO). TMEM16B is a calcium-activated chloride channel expressed in the olfactory epithelium. As a patient homozygous for the 253 kb deletion has been reported to have an olfactory impairment possibly related to the partial deletion of TMEM16B, we assessed the olfactory function in other patients using the University of Pennsylvania Smell Identification Test (UPSIT). The average UPSIT score of 4 homozygous patients was significantly lower than that of 5 healthy subjects with similar sex, age and education. However, 4 other members of the same family, 3 heterozygous for the deletion and 1 wild type, had a slightly reduced olfactory function indicating that socio-cultural or other factors were likely to be responsible for the observed difference. These results show that the ability to identify odorants of the homozygous patients for the deletion was not significantly different from that of the other members of the family, showing that the 253 kb deletion does not affect the olfactory performance. As other genes may compensate for the lack of TMEM16B, we identified some predicted functional partners from in silico studies of the protein-protein network of TMEM16B. Calculation of diversity for the corresponding genes for individuals of the 1000 Genomes Project showed that TMEM16B has the highest level of diversity among all genes of the network, indicating that TMEM16B may not be under purifying selection and suggesting that other genes in the network could compensate for its function for olfactory ability.

Co-administration of low molecular weight heparin enhances the profibrinolytic effect of warfarin through different mechanisms

BACKGROUND AND OBJECTIVE Treatment with vitamin K antagonists (VKA) reduces fibrinolytic resistance through the inhibition of thrombin-mediated activation of thrombin activatable fibrinolysis inhibitor (TAFI). Because low-molecular weight heparin (LMWH) is co-administered with VKA during initiation of anticoagulant treatment, we evaluated the effect of dual anticoagulation on fibrinolytic resistance. PATIENTS AND METHODS Two groups of patients were studied: 1) patients on stable warfarin; 2) patients starting oral anticoagulant therapy, who were evaluated during dual anticoagulation and after enoxaparin withdrawal. Only samples with an INR between 2 and 3 were compared. The resistance of clots to t-PA-induced fibrinolysis was evaluated in blood and plasma by thromboelastography (TEG) and turbidimetry, respectively. RESULTS In patients on dual anticoagulation, blood fibrinolysis time (TEG) was significantly shorter than in patients on warfarin alone and significantly correlated with LMWH level. The profibrinolytic effect was partly ascribable to a reduction of thrombin-dependent TAFI activation: 1) thrombin and TAFIa generation were significantly reduced by dual anticoagulation; 2) the addition of enoxaparin to warfarin-blood reduced TAFI-mediated fibrinolysis inhibition. Patients on dual anticoagulation also displayed a reduction in clot strength, a phenomenon known to reduce fibrinolytic resistance. The profibrinolytic effect of LMWH co-administration was not seen in plasma, likely because TAFIa generation was below the threshold required to inhibit fibrinolysis. CONCLUSIONS Co-administration of LMWH in patients under VKA reduces the fibrinolytic resistance of blood clots via TAFI-dependent and TAFI-independent mechanisms. Further studies are warranted to assess the clinical implications of these findings.