Plasminogen activator inhibitor 1 is associated with high-grade serous ovarian cancer metastasis and is reduced in patients who have received neoadjuvant chemotherapy.

Introduction: High-grade serous ovarian cancer (HGSOC) is the most prevalent and deadliest subtype of epithelial ovarian cancer (EOC), killing over 140,000 people annually. Morbidity and mortality are compounded by a lack of screening methods, and recurrence is common. Plasminogen-activator-inhibitor 1 (PAI-1, the protein product of SERPIN E1) is involved in hemostasis, extracellular matrix (ECM) remodeling, and tumor cell migration and invasion. Overexpression is associated with poor prognosis in EOC. Platelets significantly increase PAI-1 in cancer cells in vitro, and may contribute to the hematogenous metastasis of circulating tumor cells (CTCs). CTCs are viable tumor cells that intravasate and travel through the circulation-often aided by platelets - with the potential to form secondary metastases. Here, we provide evidence that PAI-1 is central to the platelet-cancer cell interactome, and plays a role in the metastatic cascade. Methods: SK-OV-3 cells where PAI-1 had been silenced, treated with healthy donor platelets, and treated with platelet-conditioned medium were used as an in vitro model of metastatic EOC. Gene expression analysis was performed using RNA-Seq data from untreated cells and cells treated with PAI-1 siRNA or negative control, each with and without platelets. Four cohorts of banked patient plasma samples (n = 239) were assayed for PAI-1 by ELISA. Treatment-naïve (TN) whole blood (WB) samples were evaluated for CTCs in conjunction with PAI-1 evaluation in matched plasma. Results and discussion: Significant phenotypic changes occurring when PAI-1 was silenced and when platelets were added to cells were reflected by RNA-seq data, with PAI-1 observed to be central to molecular mechanisms of EOC metastasis. Increased proliferation was observed in cells treated with platelets. Plasma PAI-1 significantly correlated with advanced disease in a TN cohort, and was significantly reduced in a neoadjuvant chemotherapy (NACT) cohort. PAI-1 demonstrated a trend towards significance in overall survival (OS) in the late-stage TN cohort, and correlation between PAI-1 and neutrophils in this cohort was significant. 72.7% (16/22) of TN patients with plasma PAI-1 levels higher than OS cutoff were CTC-positive. These data support a central role for PAI-1 in EOC metastasis, and highlight PAI-1's potential as a biomarker, prognostic indicator, or gauge of treatment response in HGSOC.

Procoagulant activity in high grade serous ovarian cancer patients following neoadjuvant chemotherapy-The role of the activated protein C pathway.

INTRODUCTION: Ovarian cancer patients are at high risk of thrombosis particularly during chemotherapy treatment however the mechanism is not understood. The aim of this study is to investigate the role of the activated protein C (aPC) pathway in the procoagulant activity observed in ovarian cancer patients undergoing neoadjuvant chemotherapy. PATIENTS AND METHODS: Thrombin generation was determined before and after addition of thrombomodulin (TM) in high grade serous ovarian cancer (HGSOC) patients treated with neoadjuvant chemotherapy (n = 29) compared with HGSOC patients who were chemo naïve (n = 23) and patients with benign tumours (n = 29). Plasma expression of proteins from the aPC pathway was analysed. mRNA expression was determined in endothelial (EA.hy926) and ovarian (OAW42) cell lines following addition of carboplatin and paclitaxel. RESULTS: Lower levels of ETP (p < 0.007; p < 0.003) and peak thrombin (p < 0.0008; p < 0.0018) were found in the neoadjuvant group compared with both chemo naïve and benign groups. Following addition of TM, ETP (p < 0.0005) and peak thrombin (p < 0.0049) were higher in the neoadjuvant group compared with the benign controls indicating an increase in aPC resistance. Increased TM and lower levels of protein S were found in the neoadjuvant group compared with benign controls (p < 0.05; p < 0.003). Factor V levels were increased in the neoadjuvant group compared with the chemo naïve group (p < 0.05). Carboplatin and paclitaxel altered the expression of EPCR and thrombomodulin in OAW42 cells with a modest effect on EA.hy926 cells. CONCLUSION: Chemotherapy induced procoagulant activity in HGSOC is associated with an alteration in expression of key members of the aPC pathway. This acquired aPC resistance may explain the procoagulant phenotype associated with ovarian cancer patients undergoing chemotherapy.

A risk score for prediction of venous thromboembolism in gynecologic cancer: The Thrombogyn score.

BACKGROUND: Gynecologic cancers are associated with high rates of venous thromboembolism (VTE), which is exacerbated by pelvic surgery and chemotherapy. OBJECTIVES: The aim of this study was to develop and validate a risk score for VTE in patients with gynecologic cancer and to test the predictive ability of the score following addition of procoagulant biomarker data. PATIENTS AND METHODS: Clinical and laboratory variables were used to develop a risk score for the prediction of VTE in patients with gynecological cancer (n = 616), which was validated in a separate cohort of patients (n = 406). Endogenous thrombin potential and D-dimer levels were determined in a subset (n = 290) of patients and used to produce an extended score in the validation cohort. RESULTS: Multivariable regression analysis identified BMI >30, hemoglobin <11.5 g/dL and chemotherapy as independent predictors of VTE, which formed the Thrombogyn score. Following competing risk regression analysis, subdistribution hazard ratios (SHRs), adjusted for cancer stage, were 8.16 (95% confidence interval [CI], 1.69-43.77) in the high-risk group (score = 2-3) and 4.12 (95% CI, 0.85-20.15) in the intermediate-risk group (score = 1) compared with the low-risk group (score = 0). SHRs for the validation cohort were 6.26 (95% CI, 1.24-31.39) and 3.00 (95% CI, 0.67-13.32), respectively. Cumulative incidence of VTE in the validation cohort high-risk group was 10.34% (95% CI, 6.51-16.41) per women-years compared with 1.06% (95% CI, 0.26-4.26) in the low-risk group. Using the extended Thrombogyn score, adjusted SHRs were 16.83 (95% CI, 4.20-67.37) in the high-risk group with a cumulative incidence of 21.15% (95% CI, 10.32-45.24). External validation of the score is required. CONCLUSIONS: The Thrombogyn score identifies patients with gynecologic cancer at high and low risk of VTE. Addition of biomarker data improves the predictive power of the score.

Estrogen receptor alpha augments changes in hemostatic gene expression in HepG2 cells treated with estradiol and phytoestrogens.

Phytoestrogens are popular alternatives to estrogen therapy however their effects on hemostasis in post-menopausal women are unknown. The aim of this study was to determine the effect of the phytoestrogens, genistein, daidzein and equol on the expression of key genes from the hemostatic system in human hepatocyte cell models and to determine the role of estrogen receptors in mediating any response seen. HepG2 cells and Hep89 cells (expressing estrogen receptor alpha (ERα)) were incubated for 24 h with 50 nM 17ß-estradiol, genistein, daidzein or equol. Tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), Factor VII, fibrinogen γ, protein C and protein S mRNA expression were determined using TaqMan PCR. Genistein and equol increased tPA and PAI-1 expression in Hep89 cells with fold changes greater than those observed for estradiol. In HepG2 cells (which do not express ERα), PAI-1 and tPA expression were unchanged. Increased expression of Factor VII was observed in phytoestrogen treated Hep89 cells but not in similarly treated HepG2s. Prothrombin gene expression was increased in equol and daidzein treated HepG2 cells in the absence of the classical estrogen receptors. These data suggest that phytoestrogens can regulate the expression of coagulation and fibrinolytic genes in a human hepatocyte cell line; an effect which is augmented by ERα.

Norethisterone acetate alters coagulation gene expression in vitro in human cell culture.

INTRODUCTION: Both oestrogen and progestin and the route of administration have been implicated in cardiovascular and thromboembolic risk in post menopausal hormone users. Transdermal preparations have been reported as safer indicating that liver derived metabolites of oestrogen may be important. The aim of our study was to investigate the in vitro effects of 17ß-estradiol, its metabolites, and norethisterone acetate (NETA) on the expression of coagulation genes in cultured human cells. METHODS: Human hepatocytes and human umbilical vein endothelial cells(HUVECS) were treated with 17ß-estradiol, estrone, 2-hydroxyestradiol (2-OH), NETA and NETA/17ß-estradiol (10nM) for 24hours. Fibrinogen, factor VII, prothrombin and plasminogen activator inhibitor -1 (PAI-1) mRNA expression was determined in hepatocyte cultures using TaqMan PCR. Tissue factor (TF), tissue factor pathway inhibitor (TFPI), tissue plasminogen activator (tPA) and PAI-1 expression was determined in HUVECS. Expression of estrogen receptors was also determined. RESULTS: Fibrinogen and factor VII mRNA expression was upregulated 2-4 fold by estradiol and estrone. Addition of NETA downregulated fibrinogen and prothrombin. PAI-1 expression in hepatocytes was upregulated by estrone, 2-OH, NETA and NETA/17ß-estradiol. In HUVECS, TF, TFPI and PAI-1 expression was upregulated by estrone but not by 17ß-estradiol. NETA upregulated TF, TFPI and tPA expression. Estrogen receptor status was unaffected by the addition of NETA. CONCLUSIONS: This data suggests a role for progestins in modifying the effects of oestrogen and its metabolites on coagulation gene expression which may contribute to the reduced thrombotic risk associated with transdermal preparations.

Genistein alters coagulation gene expression in ovariectomised rats treated with phytoestrogens.

Recent data has shown that hormone therapy (HT) increases the risk of cardiovascular and thromboembolic disease, particularly in users of oral HT. Phytoestrogens are popular alternatives to oestrogen therapy; however, their effects on cardiovascular risk are unknown. We investigated the effect of the phytoestrogen, genistein on the expression of genes and proteins from the haemostatic system in the liver in an ovariectomised rat model. Fifty-nine virgin female Sprague-Dawley rats were fed with soy-free chow supplemented with 17ß estradiol (E2) (daily uptake 0.19 or 0.75 mg/kg body weight), or genistein (daily uptake 6 or 60 mg/kg body weight), for three months and compared to soy-free control rats. Gene expression of prothrombin, factor VII, fibrinogen alpha and fibrinogen beta was increased with E2 and genistein compared to the soy-free control group (p<0.001). Genistein increased factor VII significantly more than E2 (p<0.005). Plasminogen mRNA was increased in both treatment groups compared to the soy-free control, with genistein expression significantly higher than E2 (p<0.001). Tissue plasminogen inhibitor (tPA), plasminogen activator inhibitor-1 (PAI-1) and C-reactive protein (CRP) expression were also increased in both groups relative the soy-free control. Results of protein analysis largely concurred with those of the mRNA. Oestrogen receptor ß (ERß) was undetected while oestrogen receptor α (ERα) was detected in each sample group. Genistein can increase the expression of coagulation and fibrinolytic genes. This effect was similar and in some cases higher than 17ß estradiol. These results suggest that genistein may not be neutral with respect to the haemostatic system.

Inhibitors and activation markers of the haemostatic system during hormone therapy: a comparative study of oral estradiol (2 mg)/ dydrogesterone and estradiol (2 mg)/ trimegestone.

Epidemiological studies have shown that hormone therapy (HT) increases the risk of venous thromboembolism in post menopausal women. The mechanism of this increased risk is unknown; however, activation of the haemostatic system is known to contribute to the pathogenesis of venous thromboembolism. In post-menopausal women the estrogen/progestogen composition of the HT can influence the level of haemostatic activation. It was the objective of this study to compare changes in inhibitors and activation markers of the haemostatic system in healthy post-menopausal women taking estradiol (2 mg) combined with dydrogesterone or a new progestin, trimegestone. A multicentre study of 186 women randomised to six months therapy with either estradiol (2 mg) +trimegestone (0.5 mg) or estradiol (2 mg) +dydrogesterone (10 mg) was performed. Antithrombin and protein S activity was decreased and activated protein C (APC) resistance, D-dimer and prothrombin fragment 1.2, were increased in both groups on treatment. Protein C activity was decreased and plasmin-antiplasmin complex was increased in the trimegestone group only. The increase in plasmin-antiplasmin complex and D-dimer was greater after six cycles of treatment in the trimegestone group compared with the dydrogesterone group. In conclusion, decreased levels of inhibitors of blood coagulation and increased thrombin production were found in both groups however a greater increase in the levels of plasmin-antiplasmin complex and D-dimer was found in the trimegestone group. This suggests an enhanced fibrinolytic response in this group. Further studies are required to determine the significance of this finding with respect to venous thrombosis risk.

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.

Low molecular weight heparin (tinzaparin) therapy for moderate risk thromboprophylaxis during pregnancy. A pharmacokinetic study.

Low molecular weight heparin (LMWH) is used increasingly for prophylaxis and treatment of venous thromboembolism during pregnancy. However, the prophylactic dose for patients at moderate risk varies between centers, and the recommended LMWH dose for the non pregnant patient is frequently used in pregnant women. The aim of this study was to investigate the effects of pregnancy on the pharmacokinetics of anti-Xa levels during moderate risk thromboprophylaxis with the LMWH, tinzaparin. In 24 pregnant women, one of three doses of tinzaparin (50, 75 or 100 IU/kg) were given according to the treating physician's assessment of their risk profile. Four-hour peak anti-Xa levels were measured throughout pregnancy and 24-hour profiles were measured at 28 and 36 weeks gestation. Doses were adjusted when peak anti-Xa levels fell below 0.1 IU/ml and, in some cases, when levels at 10 and 18 hours post injection were undetectable (<0.05 IU/ml). Our results showed that women receiving tinzaparin (50 IU/kg) frequently had peak (4 hour) anti-Xa levels below 0.1IU/ml and that 46% of these patients required dose adjustment. Similarly anti-Xa levels were also found to be low over the 24-hour period. A starting dose of 75 IU/kg, once daily, gave greater anti-Xa cover over the 24-hour period and may avoid the need for dose adjustment. The results suggest that the pharmacokinetics of tinzaparin are affected by pregnancy. Larger studies are required to determine whether an increased tinzaparin dose (75 IU/kg) would be more effective in the prevention of thrombosis during pregnancy than 50 IU/kg.

Tinzaparin sodium for thrombosis treatment and prevention during pregnancy.

OBJECTIVE: This study was undertaken to assess the pharmacodynamic profile, safety, and efficacy of tinzaparin during pregnancy. STUDY DESIGN: Fifty-four pregnant women, 12 for treatment of thrombosis and 42 for thromboprophylaxis, received tinzaparin by once daily injection. Four-hour postdose anti-Xa results were analyzed by use of repeated measures statistical methods. RESULTS: One woman (3.4%) on the 175 anti-Xa U/kg dose and three women (20%) on the 50 anti-Xa U/kg dose required a dose increase during the initial dose titration phase to achieve target anti-Xa activity. No thrombotic events occurred. CONCLUSION: The 175 anti-Xa U/kg dose is appropriate for treatment and for high-risk thromboprophylaxis throughout pregnancy. In pregnant women at moderate risk of thrombosis, a higher tinzaparin dose is required than in the nonpregnant state and 75 anti-Xa U/kg appears to be appropriate. The majority of women do not need a dose increase with advancing gestation.

Blood coagulation.

Blood coagulation can be initiated by two pathways: the extrinsic pathway, which is triggered by release of tissue factor from the site of injury, and the intrinsic system, which is stimulated by contact with a negatively charged surface. Following initial triggering, a series of serine proteases are sequentially activated, culminating in the formation of thrombin, the enzyme responsible for the conversion of soluble fibrinogen to the insoluble fibrin clot. Activation of coagulation is tightly regulated. Initiation by tissue factor is inhibited by tissue factor pathway inhibitor. Antithrombin can inactivate many of the serine proteases, including thrombin, by forming stable complexes which are rapidly cleared from the circulation. Protein C and protein S combine to inactivate coagulation factors V and VIII. The deposition of excess fibrin is prevented by the fibrinolytic system which can lyse fibrin into fibrin degradation products. Both genetic and environmental factors can influence the activation of coagulation and may predispose affected individuals to thrombosis.