Differential impact of conventional and low-dose oral hormone therapy (HT), tibolone and raloxifene on functionality of the activated protein C system

Recent studies have shown that hormone therapy (HT) is associated with an acquired resistance to activated protein C (APC). The aims of the present study were to evaluate a possible dose-response relationship and differential effects of different HT regimens on functionality of the APC system. Two hundred two healthy women were randomly assigned to receive treatment for 12 weeks with tablets containing either low-dose HT containing 1 mg 17ss-oestradiol + 0.5 mg norethisterone acetate (NETA) (n = 50), conventional-dose HT containing 2 mg 17ss-oestradiol and 1 mg NETA (n = 50), 2.5 mg tibolone (n = 51), or 60 mg raloxifene (n = 51). Normalized APC system sensitivity ratios (nAPCsr) were determined in plasma collected at baseline and after 12 weeks using a thrombin generation-based APC resistance test probed with either recombinant APC (rAPC) or thrombomodulin (rTM). NAPCsr increased in both the conventional- and low-dose HT groups, consistent with reduced sensitivity to APC. The increase was slightly more pronounced in the conventional-dose group, but the difference between the two HT groups was not statistically significant. The sensitivity to APC was only marginally altered in those allocated to tibolone. Consequently, tibolone showed a different phenotype as compared with the low-dose HT group. A small increase in nAPCsr with both rAPC and rTM was seen in the raloxifene-group, but the increase was less than in the low-dose HT group. Our findings indicate that oestrogen-progestin therapy induces an APC resistant phenotype, which may be related to dose, whereas tibolone and raloxifene only marginally alter the sensitivity to APC.     

Effects of raloxifene and continuous combined hormone therapy on haemostasis variables: a multicenter, randomized, double-blind study

INTRODUCTION: Hormone replacement therapy is known to increase the risk of thromboembolic events. We compared the effects of HRT and raloxifene on some haemostasis variables. MATERIALS AND METHODS: In a multicenter, double-blind study, 54 healthy postmenopausal women were randomized to receive either continuous treatment with 2 mg 17beta-estradiol plus 1 mg norethisterone acetate (n=30) or 60 mg raloxifene (n=24) daily for 12 months. Blood samples were collected at baseline and at 3, 6 and 12 months to evaluate therapy effects on some haemostasis variables (factor VII, factor VIII, prothrombin fragments 1 and 2, protein C, protein C activity, protein S, thrombin-antithrombin complex, D-dimer, antithrombin, fibrinogen and plasminogen activator inhibitor). RESULTS: Both raloxifene and continuous combined hormone therapy modified the haemostasis variables toward a more prothrombotic profile. Factor VIII (p<0.01) and fibrinogen (p<0.05) plasma levels significantly increased at 6 months, prothrombin fragments 1 and 2 (p<0.05) significantly increased at 12 months, whereas protein C activity (p<0.001) and antithrombin (p<0.01) significantly decreased at 12 months in both groups. CONCLUSIONS: Our results demonstrate that raloxifene and continuous combined hormone therapy exhibit the same prothrombotic profile. Both treatments induced an increase in procoagulant parameters at 6 months and a decrease in anticoagulant parameters at 12 months.     

Fibrinolytic function and atrial fibrillation

Atrial fibrillation (AF) is the commonest sustained cardiac arrhythmia, which is associated with a substantial risk of stroke and thromboembolism. A prothrombotic or hypercoagulable state has been observed in these patients, although previous studies have mainly focused on various clotting factors, endothelial damage or dysfunction markers and platelet activation. However, fibrinolytic function has been less frequently studied, despite the fibrinolytic system playing an important role in preventing intravascular thrombosis. Indeed, increasing evidence suggests that an imbalance between the fibrinolytic function is of great importance in cardiovascular disease. This review will begin by providing a brief approach to fibrinolytic function and examine previous studies about fibrinolytic activity and atrial fibrillation.     

Comparison of soluble thrombomodulin, von Willebrand factor, tPA/PAI-1 complex, and high-sensitivity CRP concentrations in serum, EDTA plasma, citrated plasma, and acidified citrated plasma (Stabilyte) stored at -70 degrees C for 8-11 years

The aim was to define the most suitable specimen collection tubes for measurements of soluble Thrombomodulin (sTM), von Willebrand factor (vWF), and tPA/PAI-1 complex concentrations, and in particular whether the strongly acidic citrate additive in Stabilyte plasma would give significantly improved long-term stability of any of these analytes. We measured these analytes in paired specimens from 34 subjects, sampled 8-11 years before analysis, in serum, EDTA plasma, citrated plasma, and acidified citrated plasma (Stabilyte). Results were evaluated by regression analysis and Bland-Altman plots. All associations were linear across a wide assay range. Soluble TM was found to be highly unstable in serum as well as in EDTA plasma and to some extent even in ordinary citrate plasma: acidified citrate plasma is necessary to preserve sTM immunoreactivity in long-term storage. For hsCRP the slopes were not significantly different from that predicted by the dilution effect (0.83-0.86) of the citrate additive and there was no appreciable intercept. vWF values were comparable in citrate and acidified citrate plasma but serum and EDTA plasma samples yielded lower than expected results. For tPA/PAI-1 complex, Stabilyte tubes gave systematically lower results than the other tubes, with serum and EDTA plasma scoring the highest values, suggesting that in vitro increase in complex levels takes places upon blood collection and/or storage. We conclude that Stabilyte plasma is the specimen collection tube of choice for biobank projects aiming to measure fibrinolytic factors as well as several other analytes in the clotting system, such as soluble thrombomodulin and von Willebrand factor, in addition to the inflammatory marker hs-CRP. Indeed, using acidified Stabilyte plasma as the single medium would substantially simplify sampling for many epidemiological studies.     

Short-term effects of estrogen, tamoxifen and raloxifene on hemostasis: a randomized-controlled study and review of the literature

Introduction

Estrogen therapy (ET), tamoxifen and raloxifene are associated with a two- to three-fold increased risk of venous thrombosis (VT); however, the mechanisms by which each drug increases venous thrombosis propensity are not fully understood. The objectives of this investigation were to compare the effects of these three treatments on hemostasis in a head to head randomized placebo-controlled trial.

Patients/methods

Ninety-four postmenopausal women were assigned to receive oral estrogen (conjugated equine estrogen [CEE] 0.625 mg, n=23), tamoxifen 20 mg (n=24), raloxifene 60 mg (n=24) or placebo (n=23) daily for 6 months. Blood samples were analyzed for procoagulant factors (prothrombin, factors VII [fVII], VIII [fVIII], IX [fIX] and XI [fXI], D-dimer and von Willebrand factor [vWf]), anticoagulant factors (antithrombin [AT], total and free protein S, protein C and activated protein C [APC] resistance) and fibrinolytic factors (thrombin activatable fibrinolysis inhibitor [TAFI] and plasminogen activator inhibitor-1 [PAI-1]), at baseline and at 6 months of treatment.

Results

Estrogen increased factor VII and D-dimer, and decreased antithrombin, total and free protein S and PAI-1. Changes with tamoxifen were distinct from estrogen with increases in factors VIII, IX, vWf and free protein S, and decreases in AT, total protein S, protein C and plasminogen activator inhibitor-1. Raloxifene produced similar effects as tamoxifen, but did not increase factor IX or decrease protein C.

Conclusions

Estrogen, tamoxifen and raloxifene affected hemostasis favoring procoagulation and impairing anticoagulation. The biochemical effects of the selective estrogen receptor modulators (SERMs) were distinct from those of estrogen and differed only subtly from each other.     

Thrombin activatable fibrinolysis inhibitor: at the nexus of fibrinolysis and inflammation

TAFI (thrombin activatable fibrinolysis inhibitor) is the precursor of a basic carboxypeptidase (TAFIa) with strong antifibrinolytic and anti-inflammatory activity. Compelling evidence indicates that thrombin, either alone or in complex with thrombomodulin, is the main physiological activator of TAFI. For this reason derangements of thrombin formation, whatever the cause, may influence the fibrinolytic process too. Experimental models of thrombosis suggest that TAFI may participate in thrombus development and persistence under certain circumstances. In several models of pharmacological thrombolysis, the administration of TAFI inhibitors along with the fibrinolytic agent leads to a marked improvement of thrombus lysis, underscoring the potential of TAFI inhibitors as adjuvants for thrombolytic therapy. The role of TAFI in inflammatory diseases is more complex as it may serve as a defense mechanism, exacerbate the disease, or have no influence, depending on the nature of the model and the role played by the mediators controlled by TAFIa. Finally, the numerous clinical studies in patients with thrombotic disease support the idea that increased levels of TAFI and/or the enhancement of TAFI activation may represent a new risk factor for venous and arterial thrombosis.     

The effects of gynaecological surgery on coagulation activation, fibrinolysis and fibrinolytic inhibitor in patients with and without ketorolac infusion

The effects of gynaecological surgery on the fibrinolytic and inhibitor mechanisms were followed up for 24h post-operatively in patients receiving a single dose of ketorolac infusion (n = 18) as compared with those not receiving ketorolac infusion (n = 11). A pre-operative state of lower mean t-PA activity and higher PAI-1 levels with increased platelet activation than that reported in normal subjects were observed in both groups of patients. Increased t-PA activity upon anaesthetic induction together with a decreased level at 24h post-operation was seen in both groups. However, fibrinolytic ‘shut-down’ was not evident as significant increase in D-dimer levels was observed post-operatively, suggesting an enhanced lytic state concurrent with an enhanced activation of coagulation and diminished platelet activation although β-TG remained above the normal level; plasmin from this enhanced lytic state affects platelet adhesion and cleaves platelet glycoprotein Ib thus inhibit release reaction. Ketorolac infusion elicited a significant response in PAI-1 activity within 24h post-operation and this was not seen in the non-ketorolac group in spite of the rising trend by 24h post-operation which did not achieve statistical significance. There were no statistical signifcant differences in blood loss and duration of surgery between the two groups of patients. Overall, both groups of patients showed similar haemostatic changes post-operatively for 24h, a longer duration of post-operative study would have revealed any subtle changes in the molecular markers of thrombosis which was not the objective of this study.     

The impact of cardiac ischemia and reperfusion on markers of activated haemostasis and fibrinolysis during cardiopulmonary bypass: comparison of plasma levels in arterial and coronary venous blood

INTRODUCTION: Activation of coagulation and fibrinolysis is common among patients undergoing cardiopulmonary bypass (CPB) surgery. Little is known, however, about the impact of myocardial ischemia and reperfusion on coagulation activation and fibrinolysis in this clinical setting. STUDY DESIGN AND METHODS: We determined the levels of coagulation activation and fibrinolysis markers (CAFM) in 19 patients with severe coronary heart disease (CHD) during CPB surgery. FXIIa, tissue factor (TF), FVIIa, tissue plasminogen activator/plasminogen activator inhibitor-1 complexes (tPA/PAI-1), prothrombin fragments 1+2 (F1+2), D-dimers (DD) and plasmin-plasmin inhibitor complexes (PPI) were measured at baseline, prior to and after cardioplegic myocardial ischemia. Simultaneous blood samples were drawn from the aorta and the coronary sinus to evaluate arteriovenous CAFM plasma level gradients. RESULTS: Myocardial ischemia induced significant increases in gradients of FXIIa and F1+2 levels across the coronary circulation without influencing systemic levels of these markers significantly. Systemic levels of FXIIa, tPA/PAI-1, F1+2, DD and PPI increased significantly during CPB operation. There was a significant linear correlation between FXIIa, FVIIa, F1+2, DD and PPI. CONCLUSIONS: Myocardial ischemia induces contact activation and thrombin generation rather than release of tPA and might thus contribute to postoperative thromboembolic complications. Surgery itself and CPB cause activation of coagulation and fibrinolysis as already described. A significant association between FXIIa, FVIIa, F1+2, DD and PPI suggests a relationship between contact activation, thrombin generation, fibrin formation and fibrinolysis.     

Haemostatic activation in post-menopausal women taking low-dose hormone therapy: less effect with transdermal administration?

Hormone therapy (HT) increases the risk of cardiovascular and thromboembolic disease in post-menopausal women. Recent studies have suggested that prothrombotic mechanisms are likely to be involved. Transdermal HT avoids the first-pass effect of oestrogen in the liver and may have a less marked effect on the haemostatic system than equivalent oral preparations. The majority of studies have compared HT preparations that have different formulations as well as routes of administration. We investigated changes in the haemostatic system in post-menopausal women using two pharmacologically similar HT preparations, which differed only in their route of administration. Three hundred forty-four healthy post-menopausal women were randomised to six months treatment with either a transdermal matrix patch containing 25 microg 17beta-estradiol/125 microg norethisterone acetate (NETA) applied every 3-4 days, or an equivalent oral preparation (estradiol 1 mg and NETA 0.5 mg given once daily). Oral treatment significantly reduced fibrinogen (p < 0.003), factor VIIc (FVIIc) (p < 0.00001), and antithrombin (AT) levels (p < 0.005); the effects in the transdermal group were less marked with no reduction in fibrinogen levels and lesser effect on FVIIc (p < 0.03) compared with oral treatment. Treatment type significantly affected fibrinolysis with lower plasmin-anti-plasmin (PAP) levels in the transdermal group (p < 0.003) and lower plasminogen activator inhibitor-1 antigen (PAI-1) (p < 0.012) and tissue plasminogen activator (tPA) antigen levels in the oral group (p < 0.002). Prothrombin fragment 1.2 and activated protein C (APC) resistance were not affected by either treatment. Transdermal HT has a less marked effect on coagulation than an equivalent oral preparation. Randomised trials are required to investigate whether this translates into less risk of cardiovascular and thromboembolic disease.