Marie-Christine Mowinckel

Hormone replacement therapy and acquired resistance to activated protein C: results of a randomized, double-blind, placebo-controlled trial

Recent studies suggest that low‐dose oral contraceptives may cause acquired resistance to activated protein C (APC). The aims of this study were to determine whether hormone replacement therapy (HRT) may also induce acquired APC resistance and to study the effects of APC resistance on the risk of recurrent thrombosis. The patients comprised 140 females with at least one previous venous thromboembolism (VTE), who were randomized to receive continuous treatment with 2 mg 17‐β‐oestradiol and 1 mg norethisterone acetate (n = 71) or placebo (n = 69). Normalized APC sensitivity ratios (nAPCsr) were calculated by measurement of the effect of APC on thrombin generation in plasma collected at baseline and after 3 months of treatment. Of the 140 women, 121 had plasma samples collected both at baseline and after 3 months. The nAPCsr increased significantly (P < 0·001) on HRT (n = 62), both in females not carrying the factor VLeiden mutation [mean change 0·57 (95% CI 0·45–0·70), n = 50] and in females heterozygous for the factor VLeiden mutation [mean change 1·10 (0·71–1·49), n = 12], but remained unchanged on placebo (n = 59). The baseline nAPCsr as well as the increase in nAPCsr associated with HRT use was not higher in the five women who subsequently developed recurrent VTE. Free protein S and free TFPI were both important parameters for the acquired APC resistant phenotype. We conclude that HRT diminishes the efficacy by which APC downregulates in‐vitro thrombin formation in a similar fashion to that observed with low‐dose oral contraceptives, but the increase in nAPCsr alone is not sufficient to explain the increased risk of VTE associated with use of HRT.

Activated protein C resistance determined with a thrombin generation‐based test is associated with thrombotic events in patients with lupus anticoagulants

Summary.  Background: Several studies suggest that antiphospholipid antibodies interfere with the activity of activated protein C (APC). This acquired form of APC resistance has been proposed as a possible pathogenic mechanism underlying hypercoagulability associated with the antiphospholipid syndrome (APS).Objectives: We wanted to investigate the inhibitory effect of recombinant APC (rAPC) on ex vivo thrombin generation in plasma and the modification of this effect by the presence of lupus anticoagulants (LA).Patients/Methods: We analyzed plasmas from 81 patients with LA (52 patients fulfilling the criteria for the APS) and 91 controls. Percent inhibition of the endogenous thrombin potential (ETP) as a parameter of APC sensitivity was determined in plasmas using a thrombin generation‐based APC resistance test probed with rAPC. All results were normalized using pooled normal plasma (PNP) as a reference.Results: Normalized percent inhibition of ETP by APC was lower in patients with LA [61.4%, 95% confidence interval (CI) 45.8–74.5%] compared to controls (107.8%, 95% CI: 107.1–109.3%). In patients with LA and APS, median inhibition was lower than in patients with LA without APS (44.6%, 95% CI: 30.1–55.7% vs. 78.8%, 95% CI: 73.9–95.8%). This difference also persisted when patients on warfarin therapy were excluded from the APS subgroup.Conclusions: APC resistance can be demonstrated with a thrombin generation‐based test in a majority of patients with the LA laboratory phenotype. A history of thrombotic events in patients with LA is associated with a stronger resistance to the anticoagulant effect of APC.

Increased acquired activated protein C resistance in unselected patients with hematological malignancies

Summary.  Background: We have previously found that activation of coagulation in patients with various hematological malignancies was apparently not initiated by tissue factor (TF). Acquired activated protein C (APC) resistance may be another mechanism responsible for such hypercoagulation, and has been demonstrated in patients with solid tumors, but not in patients with hematological malignancy. Objective: To investigate acquired APC resistance in a hypercoagulable cohort of patients with hematological malignancies. Patients/methods: Blood samples from 93 patients with acute myeloid leukemia (AML), chronic lymphatic leukemia, multiple myeloma, or non‐Hodgkin’s lymphoma, were analyzed before start and after completion of cancer therapy. APC resistance was measured using calibrated automated thrombography. The APC sensitivity ratio (APC‐SR) was calculated as the ratio of the endogenous thrombin potential (ETP) determined in plasma probed with either APC or buffer. Results: Untreated patients were found to have higher APC‐SR than healthy controls, and patients with AML had higher APC‐SR as compared to the other diagnoses, both findings being consistent with acquired APC resistance. The acquired APC resistance was partly ameliorated with cancer treatment. Decreased levels of protein S and TF pathway inhibitor were inversely correlated to APC resistance. Conclusions: APC resistance may contribute to the hypercoagulable state in hematological malignancies.

Thrombin-inhibitor complexes in the blood during and after delivery

Activation of coagulation leads to generation of thrombin which in turn is inactivated by the formation of thrombin-antithrombin (TAT) complexes, and thrombin-heparin cofactor complexes (T-HCII). These complexes were measured in plasma by ELISA methods. During normal delivery, the median TAT level in ten women increased from 4.1 to 7.8 times the median normal reference level. There was great individual variation, and levels 42 and 56 times normal median were found in two women shortly after normal delivery. The median T-HCII levels increased only moderately from 2.3 to 3.1 times median normal reference. D-dimer values were elevated in 28 out of the 30 samples. In blood sampled 1-2 days after delivery, the median TAT level was 2.5 times the median normal reference. The median T-HCII level was now 5.6 times the median normal reference value. The values were stable during the first 4 days post partum, and there was little difference between those delivered vaginally or by Caesarean section (C-section). D-dimer values were above normal reference in all women, and higher in women delivered by C-section. In conclusion, increasing TAT levels during labour and delivery indicated generation of thrombin which was mainly inactivated by antithrombin. The T-HCII levels increased less during delivery. In the early post partum period, the T-HCII levels were relatively more increased than the TAT levels. These results suggest that intravascularly generated thrombin is preferably inactivated by antithrombin, even in parturient women. In the post partum period, formation of T-HCII complexes was more evident, possibly reflecting extravascular inactivation of thrombin.

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 17β-oestradiol + 0.5 mg norethisterone acetate (NETA) (n=50), conventional-dose HT containing 2 mg 17β-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.

Decreased anticoagulant response to tissue factor pathway inhibitor type 1 in plasmas from patients with lupus anticoagulants.

Inhibition of tissue factor pathway inhibitor type 1 (TFPI) is one of the mechanisms by which lupus anticoagulants (LA) may upregulate tissue factor (TF) activity. We wanted to examine whether purified immunoglobulin G (IgG) from patients with LA may interfere with the ability of TFPI to inhibit ex vivo TF‐induced thrombin generation. The endogenous thrombin potential (ETP) in pooled normal plasma (PNP) supplemented with IgG from either patients with LA or controls was determined in the absence or presence of recombinant TFPI (rTFPI). In the presence of a heparin neutraliser, the ETP was also determined in plasmas from patients with LA and controls before and after heparin injection in order to quantify the anticoagulant effect of heparin‐releasable TFPI in vivo. Compared with IgG from controls (n = 14), IgG from patients with LA (n = 28) induced a wide range of enhancing or inhibitory effects on the ETP in PNP. The response to rTFPI in PNP with IgG from patients with LA correlated inversely with thrombin generation (rs = 0·637, P = 0·0003). Correspondingly, the relative inhibition of ETP in postheparin plasmas was smaller for patients (n = 11) than for controls (n = 9) (32% vs. 68%, P = 0·007). Our findings support the hypothesis that TFPI anticoagulant activity is inhibited in some patients with LA.

The performance of STA‐Liatest D‐dimer assay in out‐patients with suspected pulmonary embolism

Several studies have shown that d‐dimer can reliably rule out pulmonary embolism (PE) in out‐patients. However, various assays have different sensitivities and specificities to detect thrombosis. Our aim was to evaluate the performance of STA‐Liatest D‐Di in out‐patients referred for suspected PE in a prospective outcome study. 495 consecutive patients referred to Østfold Hospital Trust‐Fredrikstad, Norway for suspected PE between February 2002 and December 2003, were recruited in a study evaluating a decision‐based algorithm combining clinical probability (CP), d‐dimer, and multi‐slice computer tomography (MSCT). d‐dimer was performed as a first step test. No further testing was carried out in patients with d‐dimer ≤0·4 mg/l and low/intermediate CP. The remaining patients proceeded to MSCT. All patients were followed up for 3 months to assess the 3‐month thromboembolic risk. The final cohort consisted of 432 patients. PE was diagnosed in 102 (23%) patients. At a d‐dimer cut‐off point of 0·4 mg/l the tests had the highest sensitivity (100%) and specificity (36%). It safely ruled out PE in 120 (28%) patients. Kappa‐coefficients for comparisons versus VIDAS and Asserachrom showed good concordance. STA‐Liatest is a reliable and effective assay that can safely rule out PE in out‐patients with a performance comparable with that of enzyme‐linked immunosorbent assay‐based d‐dimer levels.

Endogenous tissue plasminogen activator in neonatal cerebrospinal fluid

Tissue type plasminogen activator (tPA) plays a role in differentiation of neurones and activity-dependent structural changes in neurones. We hypothesised that tPA would also be present in CSF during fibrinolysis after intraventricular haemorrhage. We measured tPA antigen in CSF from 13 normal newborn infants and 14 infants with posthaemorrhagic ventricular dilation (PHVD). tPA was undetectable or at the limit of detection (1 μg/l) in normal CSF. The CSF tPA concentration ranged from 1.3 to 3.5 μg/l in the infants with PHVD. Serial tapping in one infant showed persistence of tPA in the CSF from 3 to 8 weeks of age. We conclude that endogenous tPA may be part of the physiological response to intraventricular haemorrhage or may be present as a result of passive diffusion into the CSF.

Resistance to activated protein C is a risk factor for pregnancy- related venous thrombosis in the absence of the F5 rs6025 (factor V Leiden) polymorphism

Resistance to activated protein C (aPC) is most commonly due to the F5 rs6025 (factor V Leiden) polymorphism, which increases the risk of venous thrombosis. In the present population‐based study of 313 cases and 353 controls, we investigated whether reduced sensitivity to aPC was associated with a history of pregnancy‐related venous thrombosis. Calibrated automated thrombography was used to determine the sensitivity to aPC, and normalized aPC sensitivity ratio (n‐aPC‐sr) was calculated. Pregnant women and women using oral contraceptives and/or anticoagulants were excluded due to the effect on the n‐aPC‐sr. In women without the F5 rs6025 polymorphism, free tissue factor pathway inhibitor (TFPI), free protein S and protein C activity were associated with n‐aPC‐sr. Unadjusted odds ratio for venous thrombosis for women with n‐aPC‐sr in the 4th quartile as compared with n‐aPC‐sr below the 4th quartile was 2·4 (95% confidence interval 1·7–3·6). Adjusting for free protein S, free TFPI and age did not influence the odds ratios. Also in carriers of the F5 rs6025 polymorphism the risk for venous thrombosis was increased for women with higher n‐aPC‐sr. Our findings substantiate the importance of the aPC resistant phenotype as a risk factor for pregnancy‐related venous thrombosis.

Enhanced thrombin generation and reduced intact protein S in processed solvent detergent plasma

BACKGROUND Standardized solvent/detergent (S/D)-treated plasma has been developed as an improved alternative to fresh frozen-plasma (FFP) in the management of severe bleeds. This study aimed at exploring compositional modifications that may influence the general applicability of S/D-treated plasma. MATERIALS AND METHODS S/D-treated plasma and FFP were compared in procoagulant microparticles and concentration of coagulation factors and inhibitors. Compositional differences were correlated with hemostatic and fibrinolytic characteristics as measured by PT, APTT, thrombin generation and thromboelastography. RESULTS Procoagulant microparticles were absent in S/D-treated plasma. Procoagulant factors were within the normal range. Antithrombin, TFPI and protein S antigen may be normal or slightly reduced depending on the duration of the S/D-treatment, but S/D-treated plasmas had only 12-14% intact functional protein S. Thrombin generation was subsequently increased, especially at low tissue factor concentration (1 pM). Plasma coagulation times in PT and APTT were normal, but 1.5-fold reduced in thromboelastography at low TF (1 pM). α2-antiplasmin was reduced with a concomitant 3-4 fold shortened clot lysis time measured by thromboelastography in the presence of TF (10 pM) and tissue-type plasminogen activator (0.2μg/ml). Enhanced fibrin degradation could be normalised with tranexamic acid. CONCLUSIONS S/D-treatment seems to induce a procoagulant phenotype that results from a strongly reduced level of intact single chain protein S. Whether this may correct the apparent hemostatic imbalance as suggested from the increased fibrinolysis remains to be established. Our findings may bear implications in patients with deficiencies of natural anticoagulants. Co-administration of tranexamic acid appears beneficial to control enhanced fibrinolysis.