Anders Dahm

Determinants of the APTT- and ETP-based APC sensitivity tests.

Summary.  Background: A reduced sensitivity for activated protein C (APC) is associated with an increased risk of venous thrombosis even in the absence of the factor (F)V Leiden mutation. This risk has been demonstrated with two APC sensitivity tests, which quantify the effects of APC on the activated partial thromboplastin time (APTT) and the endogenous thrombin potential (ETP), respectively. Objectives: We examined determinants of both APC sensitivity tests in the control group of the Leiden Thrombophilia Study (LETS). Methods: Multiple linear regression analysis was performed with normalized APC‐SRAPTT or APC‐SRETP as dependent variable and putative determinants [levels of FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII A subunit, FXIII B subunit, protein S total, protein S free, protein C, tissue factor pathway inhibitor (TFPI) total, TFPI free, antithrombin and fibrinogen] as independent variables. Results and conclusions: The major determinant of the APTT‐based test was FVIII level, followed by FII level. The ETP‐based test was influenced most by free protein S and free TFPI levels. In both tests FXa formation plays a major role, as the effect of FVIII and TFPI on the tests seems to be executed via FXa. The ETP‐based test was also strongly influenced by oral contraceptive use, even when we adjusted for all the clotting factors listed above. This means that the effect of oral contraceptives on the ETP‐based test is not fully explained by the changes of coagulation factor levels investigated in this study, and that the molecular basis of acquired APC resistance during use of oral contraceptives remains to be established.

Different effects of oral contraceptives containing different progestogens on protein S and tissue factor pathway inhibitor

Summary.  Background: Oral contraceptives (OC) containing different types of progestogens induce different sensitivities to activated protein C (APC) measured with the thrombin generation‐based APC‐resistance test. These differences in APC resistance may be the biological explanation for the differences in thrombotic risk of the various pills. The mechanistic basis of APC resistance observed in OC users is unknown. Our objective was to study the effect of OC on the two main determinants of the APC‐resistance test, free protein S and free tissue factor pathway inhibitor (TFPI). Patients/methods: We measured free protein S and free TFPI in 156 users of various types of OC. Results: Users of desogestrel‐containing OC, known to double the risk of thrombosis compared with levonorgestrel‐containing OC, had lower free protein S (24 vs. 33 U dL−1) and TFPI free antigen (2.9 vs. 3.6 ng mL−1) levels than users of OC containing levonorgestrel. Women using cyproterone acetate‐containing OC, known to confer a high thrombotic risk, had the lowest free protein S (19 U dL−1) and free TPFI antigen (2.5 ng mL−1) levels. Users of OC containing drospirenone had lower free protein S (23 U dL−1) and TFPI antigen levels (3.2 ng mL−1) than users of levonorgestrel‐containing OC. Low free protein S and low free TFPI antigen levels were associated with an increased resistance to APC, an established risk factor for thrombosis. Conclusions: This study observed that the differences in APC resistance induced by OC containing different progestogens can at least in part be explained by different effects of OC on free protein S and TFPI.

An Exploratory Trial of Cyclooxygenase Type 2 Inhibitor in HIV-1 Infection: Downregulated Immune Activation and Improved T Cell-Dependent Vaccine Responses

ABSTRACT Chronic HIV infection is characterized by chronic immune activation and dysfunctional T cells with elevated intracellular cyclic AMP (cAMP), which inhibits the T cell activation capability. cAMP may be induced by prostaglandin E2 following lipopolysaccharide (LPS)-induced upregulation of cyclooxygenase type 2 (COX-2) in monocytes due to the elevated LPS levels in patients with chronic HIV infection. This hypothesis was tested using celecoxib, a COX-2 inhibitor, for 12 weeks in HIV-infected patients without antiretroviral treatment in a prospective, open, randomized exploratory trial. Thirty-one patients were randomized in the trial; 27 completed the study, including 13 patients on celecoxib. Celecoxib reduced chronic immune activation in terms of CD38 density on CD8+ T cells (−24%; P = 0.04), IgA levels (P = 0.04), and a combined score for inflammatory markers (P < 0.05). Celecoxib further reduced the inhibitory surface receptor programmed death 1 (PD-1) on CD8+ T cells (P = 0.01), including PD-1 on the HIV Gag-specific subset (P = 0.02), enhanced the number of CD3+ CD4+ CD25+ CD127lo/− Treg or activated cells (P = 0.02), and improved humoral memory recall responses to a T cell-dependent vaccine (P = 0.04). HIV RNA (P = 0.06) and D dimers (P = 0.07) tended to increase in the controls, whereas interleukin-6 (IL-6) possibly decreased in the treatment arm (P = 0.10). In conclusion, celecoxib downmodulated the immune activation related to clinical progression of chronic HIV infection and improved T cell-dependent functions in vivo.

Mechanisms of thrombosis related to hormone therapy

Combined oral contraceptives and combined oral postmenopausal hormone therapy are associated with a weak, but clinically significant risk of arterial and venous thrombosis (VT). The effects are related to dose of estrogen and type of progestin. The main effects are increase in markers of activated coagulation, reduction in coagulation inhibitors, and acquired activated protein C resistance. Reduction in tissue factor pathway inhibitor (TFPI) is probably an important mechanism, which predicts activation of coagulation and acquired resistance to activated protein C. Coagulation markers should be used as intermediate or surrogate markers in early pharmacodynamic studies to evaluate the risk associated with new formulations.

Risk of venous thrombosis in pregnancy among carriers of the factor V Leiden and the prothrombin gene G20210A polymorphisms.

Background:  Pregnancy is associated with a 10‐fold increased risk of venous thrombosis (VT), with different risk profiles for the antenatal and postnatal periods. The purpose of this study was to assess the risk of pregnancy‐related VT associated with the factor (F)V Leiden and prothrombin gene G20210A polymorphisms.

A novel anticoagulant activity assay of tissue factor pathway inhibitor I (TFPI)

Summary.  Tissue factor (TF) pathway inhibitor I (TFPI) is the physiological inhibitor of TF‐induced blood coagulation. Circulating blood contains full‐length TFPI and TFPI truncated at the C‐terminal end. Previous studies have shown that full‐length TFPI exerts a stronger anticoagulant effect on diluted prothrombin time (DPT) than truncated TFPI, and it has been suggested that full‐length TFPI is biologically more important in vivo. The objective of this study was to develop and validate an assay of TFPI anticoagulant activity. TFPI anticoagulant activity was assayed using a modified DPT assay. Plasmas were incubated in the absence and the presence of TFPI‐blocking antibodies. Results were expressed as a ratio with the clotting time in the presence of anti‐TFPI as the denominator. The ratio was normalized against a ratio obtained with a reference plasma. The assay was compared with assays of TFPI free antigen, total antigen, and bound TFPI, and TFPI chromogenic substrate activity. We performed all tests in 436 healthy individuals. The normalized TFPI anticoagulant ratio was strongly associated with TFPI free antigen (r = 0.73) but was weakly associated with TFPI chromogenic substrate activity (r = 0.46), TFPI total antigen (r = 0.48), and bound TFPI (r = 0.30). TFPI chromogenic substrate activity was strongly associated with TFPI total antigen (r = 0.73). We have developed a novel assay of TFPI anticoagulant activity in plasma, which may be considered a functional assay of full‐length TFPI. Further studies are needed to establish the role of TFPI anticoagulant activity for thrombotic disorders.

The association between protein S levels and anticoagulant activity of tissue factor pathway inhibitor type 1

Protein S (PS) is a vitamin K-dependent coagulation inhibitor of which 40% circulates in a free form and 60% circulates bound to C4b-binding protein (C4BP). PS exerts its anticoagulant activitymainly by being a cofactor for activated protein C (APC) in the inactivation of factor (F) Va and FVIIIa [1]. Tissue factor (TF) pathway inhibitor type 1 (TFPI) is the physiological inhibitor of the TF pathway of blood coagulation. Approximately 80% of circulating TFPI in blood is Cterminal truncated and bound to lipoproteins, while 20% is carrier free full-length TFPI [2]. The TFPI-mediated inhibition of the TF/FVIIa complex occurs in two steps. Firstly, TFPI binds and inhibits FXa. Secondly, the TFPI/FXa complex binds to the complex of TF and FVIIa to form an inactive quaternary TFPI/FXa/TF/FVIIa complex:

The effect of different hormonal contraceptives on plasma levels of free protein S and free TFPI

Use of combined oral contraceptives is associated with a three- to six-fold increased risk of venous thrombosis. Hormonal contraceptives induce acquired resistance to activated protein C (APC), which predicts the risk of venous thrombosis. The biological basis of the acquired APC resistance is unknown. Free protein S (PS) and free tissue factor pathway inhibitor (TFPI) are the two main determinants of APC. Our objective was to assess the effect of both hormonal and non-hormonal contraceptives with different routes of administration on free TFPI and free PS levels. We conducted an observational study in 243 users of different contraceptives and measured APC sensitivity ratios (nAPCsr), free TFPI and free PS levels. Users of contraceptives with the highest risk of venous thrombosis as reported in recent literature, had the lowest free TFPI and free PS levels, and vice versa, women who used contraceptives with the lowest risk of venous thrombosis had the highest free TFPI and free PS levels. An association was observed between levels of free TFPI and nAPCsr, and between free PS and nAPCsr. The effect of oral contraceptives on TFPI and PS is a possible explanation for the increased risk of venous thrombosis associated with oral contraceptives.

Increased coagulation activity and genetic polymorphisms in the F5, F10 and EPCR genes are associated with breast cancer: a case-control study.

BackgroundThe procoagulant state in cancer increases the thrombotic risk, but also supports tumor progression. To investigate the molecular mechanisms controlling cancer and hemostasis, we conducted a case-control study of genotypic and phenotypic variables of the tissue factor (TF) pathway of coagulation in breast cancer.Methods366 breast cancer patients and 307 controls were genotyped for SNPs (n = 41) in the F2, F3 (TF), F5, F7, F10, TFPI and EPCR genes, and assayed for plasma coagulation markers (thrombin generation, activated protein C (APC) resistance, D-dimer, antithrombin, protein C, protein S, and TF pathway inhibitor (TFPI)). Associations with breast cancer were evaluated using logistic regression to obtain odds ratios (ORs) and 95% confidence intervals (CIs), or the chi-square test.ResultsFour SNPs in F5 (rs12120605, rs6427202, rs9332542 and rs6427199), one in F10 (rs3093261), and one in EPCR (rs2069948) were associated with breast cancer. EPCR rs2069948 was associated with estrogen receptor (ER) and progesterone receptor (PR) positivity, while the SNPs in F5 appeared to follow hormone receptor negative and triple negative patients. The prothrombotic polymorphisms factor V Leiden (rs6025) and prothrombin G20210A (rs1799963) were not associated with breast cancer. High APC resistance was associated with breast cancer in both factor V Leiden non-carriers (OR 6.5, 95% CI 4.1-10.4) and carriers (OR 38.3, 95% CI 6.2-236.6). The thrombin parameters short lag times (OR 5.8, 95% CI 3.7-9.2), short times to peak thrombin (OR 7.1, 95% CI 4.4-11.3), and high thrombin peak (OR 6.1, 95% CI 3.9-9.5) predicted presence of breast cancer, and high D-dimer also associated with breast cancer (OR 2.0, 95% CI 1.3-3.3). Among the coagulation inhibitors, low levels of antithrombin associated with breast cancer (OR 5.7, 95% CI 3.6-9.0). The increased coagulability was not explained by the breast cancer associated SNPs, and was unaffected by ER, PR and triple negative status.ConclusionsA procoagulant phenotype was found in the breast cancer patients. Novel associations with SNPs in F5, F10 and EPCR to breast cancer susceptibility were demonstrated, and the SNPs in F5 were confined to hormone receptor negative and triple negative patients. The study supports the importance of developing new therapeutic strategies targeting coagulation processes in cancer.

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.