The Glasgow Outcome, APCR and Lipid (GOAL) Pregnancy Study: significance of pregnancy associated activated protein C resistance

Activated protein C (APC) resistance secondary to Factor V Leiden (FVL) is associated with pregnancy failure and pre-eclampsia (PET). In non-pregnant subjects, the degree of resistance to APC relates to venous thrombosis risk. In pregnancy, resistance to APC occurs in the absence of FVL. We investigated, in an unselected prospective longitudinal study of 1,671 pregnant, non-FVL subjects, the relationship of the APC sensitivity ratio (APC:SR) with demographic variables and pregnancy outcome. Lower APC:SR values at 7-16 weeks gestation were observed in subjects who subsequently developed PET (median APC:SR 2.55, IQR 2.29-2.70 vs 2.69, IQR 2.48-2.93, Mann-Whitney U-test p = 0.003) in the current pregnancy. An APC:SR < the median (2.69) at 7-16 weeks was associated with a 2.95-fold increased risk (CI95 1.2-7.4) of PET in the current pregnancy. No relationship between the APC:SR, at any gestation, and fetal loss was observed. An inverse correlation between the APC:SR and birth weight was noted. Higher APC:SRs were observed in blood group O subjects and smokers. An inverse relationship of the APC:SR with age, diastolic blood pressure and total serum cholesterol was observed.     

Effects of hereditary and acquired risk factors of venous thrombosis on a thrombin generation-based APC resistance test

BACKGROUND: Several hereditary and acquired risk factors for venous thromboembolism (VTE) are associated with impaired down-regulation of thrombin formation via the protein C pathway. To identify individuals at risk, functional tests are needed that estimate the risk to develop venous thrombosis. METHOD: We determined the effects of hereditary and acquired risk factors of venous thrombosis on an APC resistance test that quantifies the influence of APC on the time integral of thrombin formation (the endogenous thrombin potential, ETP) initiated in plasma via the extrinsic coagulation pathway. APC sensitivity ratios (APCsr) were determined in plasma from carriers of factor V(Leiden) (n = 56) or prothrombin G20210A (n = 18), of individuals deficient in antithrombin (n = 9), protein C (n = 7) or protein S (n = 14) and of women exposed to acquired risk factors such as hormone replacement therapy (n = 49), oral contraceptive use (n = 126) or pregnancy (n = 35). We also analysed combinations of risk factors (n = 60). RESULTS: The thrombin generation-based APC resistance test was sensitive for the factor V(Leiden) and prothrombin G20210A mutation, to protein S deficiency, hormone replacement therapy, oral contraceptive use and pregnancy. The assay was not influenced by antithrombin or protein C deficiency. The presence of more than one risk factor of venous thrombosis resulted in more pronounced APC resistance. The APCsr of individuals with a single or combined risk factors of VTE correlated well with reported risk increases. INTERPRETATION: The thrombin generation-based APC resistance test identifies individuals at risk for venous thrombosis due to acquired risk factors and/or hereditary thrombophilic disorders that affect the protein C pathway.     

TFPI-dependent activities of protein S

Protein S is an essential anticoagulant protein that acts as a cofactor for full length tissue factor pathway inhibitor (TFPI) and activated protein C (APC) in the down regulation of coagulation. Protein S enhances APC-mediated inactivation of the coagulation factors Va and VIIIa, and it stimulates inhibition of factor (F)Xa by TFPI. Because TFPI is a tight binding, but slow inhibitor of FXa, the TFPI/protein S system fails to regulate FXa generation at high tissue factor/FVIIa concentrations. In this review, we explain how TFPI/protein S can regain its activity at high tissue factor concentrations in the presence of APC, resulting in an intertwinement of TFPI- and APC-cofactor activities of protein S, and making TFPI a major determinant of APC-anticoagulant activity in plasma.     

Protein S levels modulate the activated protein C resistance phenotype induced by elevated prothrombin levels

Elevated plasma prothrombin levels, due to the prothrombin 20210 G/A mutation or to acquired causes, are a risk factor for venous thrombosis, partly because of prothrombin-mediated inhibition of the protein C anticoagulant pathway and consequent activated protein C (APC) resistance. We determined the effect of plasma prothrombin concentration on the APC resistance phenotype and evaluated the role of protein S levels as a modulating variable. The effect of prothrombin and protein S levels on APC resistance was investigated in reconstituted plasma systems and in a population of healthy individuals using both the aPTT-based and the thrombin generation-based APC resistance tests. In reconstituted plasma, APC resistance increased at increasing prothrombin concentration in both assays. Enhanced APC resistance was caused by the effect of prothrombin on the clotting time in the absence of APC in the aPTT-based test, and on thrombin formation in the presence of APC in the thrombin generation-based test. In plasma from healthy individuals prothrombin levels were highly correlated to protein S levels. Since prothrombin and protein S had opposite effects on the APC resistance phenotype, the prothrombin/protein S ratio was a better predictor of APC resistance than the levels of either protein alone. Prothrombin titrations in plasmas containing different amounts of protein S confirmed that protein S levels modulate the ability of prothrombin to induce APC resistance. These findings suggest that carriers of the prothrombin 20210 G/A mutation, who have a high prothrombin/protein S ratio, may experience a higher thrombosis risk than non-carriers with comparable prothrombin levels.     

Circulating levels of inflammatory cytokines (IL-1 beta and TNF-alpha), resistance to activated protein C, thrombin and fibrin generation in uncomplicated pregnancies

We studied 33 women during normal uneventful pregnancies and with no history of previous adverse pregnancy events for markers of activated coagulation and thrombin activity including prothrombin fragment 1.2(PF1.2), thrombin- antithrombin (TAT), and soluble fibrin polymer (SFP). In addition, we measured potential thrombin generation through the addition of thromboplastin to patient plasma in the presence of a thrombin-specific chromogenic substrate determined serially over a period of time--Endogenous Thrombin Potential assay (ETP). This assay was performed with plasma treated and untreated with activated protein C (APC). The fibrinolytic system was assessed by measurement of thrombin activatable fibrinolysis inhibitor (TAFI). These findings were correlated with the levels of pro-inflammatory cytokines, interleukine-1 beta and tumor necrosis factor-alpha. Our data supports previous reports that indicate that resistance to activated protein C and coagulation activation markers are commonly increased in the later 2/3rds of pregnancy. There are no differences in thrombin generation potential, as determined by the ETP assay without the addition of APC, in the three trimesters. However, the thrombin reserve (TR), the ETP result without APC divided by the ETP result with the addition of APC, is increased above the reference range in the 2nd and 3rd trimesters. Patients with increased TR and resistance to APC had increased levels of TNF-alpha. Increased proinflammatory cytokines are reportedly associated with changes in the APC system with a decrease in the ability to generate APC. A sub-group of pregnancies with APC resistance had increased levels of TNF-alpha and may be important in the risk for adverse pregnancy outcomes.     

Modulation of systemic hemostatic parameters by enoxaparin during gestation in women with thrombophilia and pregnancy loss

Recurrent pregnancy loss (PL) is associated with maternal thrombophilia and prophylaxis with low molecular weight heparin (LMWH) can improve pregnancy outcome in this setting.The aim of this study was to investigate the modulation of systemic hemostatic parameters by enoxaparin in women with recurrent PL and to evaluate plasmatic parameters that would potentially enable monitoring LMWH prophylaxis effect during pregnancy. Study group included 87 women with thrombophilia and PL treated with enoxaparin 40 mg daily vs. 40 mg twice daily. The control group comprised 40 women with normal pregnancies. Blood samples have been collected throughout the period starting at 5-10 weeks of gestation until 6-10 weeks postpartum. The determined plasmatic markers included: anti-Xa activity, total and free tissue factor pathway inhibitor (TFPI), D-dimer, prothrombin fragment 1+2 (PT1+2), activated protein C resistance (APC-SR) and free protein S. Successful pregnancy outcome was recorded in 70 (80.5%) women treated with enoxaparin, without correlation to enoxaparin dosage. Seventeen women (19.5%) had pregnancy loss at 16+/-7 (6-32) weeks of gestation. Anti-Xa levels at 10-15 weeks of gestation were higher (0.39+/-0.38 u/ml) in the successful pregnancy outcome group compared to the abortion group (0.22+/-0.2 u/ml). Prophylactic anti-Xa activity levels (0.28+/-0.13 u/ml) were documented from 15 weeks of gestation until delivery in the successful pregnancy outcome group. Significant increase in anti-Xa, total TFPI and free TFPI levels (P<0.001) was achieved after beginning of LMWH prophylaxis in successful pregnancy outcome group but not in the abortion group. D-dimer and PT1+2 levels appeared to be significantly increased while APC-SR and free protein S levels gradually decreased during pregnancy, with no difference between study groups. These results suggest that LMWH prophylaxis during pregnancy enables modulation of systemic hemostatic parameters via inhibition of factor Xa and increase in plasmatic total and free TFPI levels.     

Protein S is a cofactor for tissue factor pathway inhibitor

Protein S is a vitamin K-dependent protein that acts as a cofactor of the anticoagulant protein APC. However, protein S also exhibits anticoagulant activity in the absence of APC. Thrombin generation experiments in normal plasma and in plasma deficient in tissue factor pathway inhibitor (TFPI) and/or protein S demonstrated that protein S stimulates the inhibition of TF by TFPI. Kinetic analysis in model systems containing purified proteins showed that protein S enhances the formation of the binary FXa:TFPI complex by reducing the Ki of TFPI from approximately 4 nM to approximately 0.5 nM. Enhancement of inhibitory activity of TFPI by protein S is only observed with full-length TFPI and in the presence of a negatively charged phospholipid surface. The Ki decrease brings the TFPI concentration necessary for FXa:TFPI complex formation within range of the plasma TFPI concentration which increases FXa:TFPI complex formation and accelerates feedback inhibition of the TF pathway by enhancing the formation of the quaternary TFPI:FXa:TF:FVIIa complex. Thus, protein S is not only a cofactor of APC, but also of TFPI. A reduced TFPI cofactor activity may contribute to the increased risk of venous thrombosis in protein-S deficient individuals. Using calibrated automated thrombography we have developed two assays that enable quantification of the functional activity of the TFPI/protein S system in plasma. These assays show that the activity of the TFPI/protein S system is greatly impaired in oral contraceptive users.     

Increased risk of gestational vascular complications in women with low free tissue factor pathway inhibitor plasma levels out of pregnancy

Tissue factor pathway inhibitor (TFPI) plays a crucial role in haemostasis by regulating TF-induced initiation of coagulation. Since it is expressed by endothelial and trophoblastic cells, TFPI is of particular importance at the placental level and might be involved in the occurrence of gestational vascular complications (GVC). In the present study, we investigated plasma free TFPI antigen in four groups of women: healthy non-pregnant women without history of pregnancy complications; women at the beginning (<12 weeks) and women during the third trimester of a normal pregnancy; women with late pregnancy complications (pre-eclampsia / HELLP syndrome, intra-uterine fetal death, fetal growth retardation) at the time of obstetrical event and/or at distance from pregnancy. In normal pregnancy, TFPI increased between first trimester and delivery (median 5.0 ng/ml vs. 7.1 ng/ml; p<0.0001) but remained lower than in non-pregnant controls (median 8.2 ng/ml; p<0.0001). In patients, when measured concomitantly to the obstetrical event, TFPI showed no difference with normal late pregnancy levels. In contrast, at distance from pregnancy, in the absence of any hormonal influence, TFPI was significantly lower than in non-pregnant controls (median 5.9 vs. 8.2ng/ml, p < 0.0001). After categorisation into quartiles, an inverse dose-effect relationship was demonstrated between TFPI categories recorded apart from pregnancy and GVC risk, with a crude odds ratio of 43.5 (95% confidence interval 8.2-230) for patients with TFPI values in the lowest quartile (< 5.7 ng/ml). In conclusion, low free TFPI at distance from pregnancy appears to be a strong indicator of GVC risk.     

Effect of oral contraceptives on the anticoagulant activity of protein S in plasma

We determined anticoagulant parameters that depend on protein S function in plasma, i.e. the APC-independent anticoagulant activity of protein S (expressed as pSR) and APC resistance determined with thrombin generation-based tests (expressed as APCsr) as well as plasma levels of total and free protein S and prothrombin in men, women not using oral contraceptives (OC), and in women using second or third generation OC. Thrombin generation in the APC resistance assays was initiated either with factor Xa (Xa-APCsr) or tissue factor (TF-APCsr). The APC-independent anticoagulant activity of protein S was highest in men (pSR=1.69) and gradually decreased from women not using OC (pSR=1.49) via women using second generation (pSR=1.35) to women using third generation OC (pSR=1.27). The pSR correlated inversely with nAPCsr determined with the tissue factor-based APC resistance test (TF-APCsr) but not with nAPCsr determined with the factor Xa-based assay (Xa-APCsr). Multiple linear regression analysis in which sex, OC use, and protein S and prothrombin levels were included as independent variables and the pSR, TF-APCsr or Xa-APCsr as dependent variables indicated that plasma protein S levels poorly predict the pSR and the TF-APCsr, but are the main determinant of the Xa-APCsr. This indicates that OC use alters the expression of protein S activity. This phenomenon can be caused by differences in modulation of the activity of protein S by other plasma proteins that change during OC use or by OC-induced changes in the protein S molecule that impair its anticoagulant activity. Functional impairment of protein S as a result of hormonal influence may, at least in part, contribute to the thrombotic risk of OC users.     

Hypercoagulability after trauma: Hemostatic changes and relationship to venous thromboembolism

Background

Major trauma induces a hypercoagulable state, which is frequently complicated by pathological thrombosis. However the sequential changes in coagulation markers and their relationship to clinical thrombosis have been poorly characterized.

Methods

We measured several markers of in vivo coagulation and fibrinolysis and their regulation serially for 2 weeks after multi-system trauma in a prospective cohort of patients who received no anticoagulant prophylaxis. Asymptomatic deep vein thrombosis (DVT) was assessed by routine bilateral venography between day 12 and 14. Clinically suspected DVT and pulmonary embolism (PE) were investigated in a standardized manner.

Results

Among the 135 cohort patients the overall venous thromboembolism (VTE) rate was 59%. Markers of thrombin generation were markedly increased within 24 hours of injury, remained persistently elevated for about 5 days and then decreased by day 14. No early compensatory increase in Tissue Factor Pathway Inhibitor (TFPI) or the complex of Factor Xa and TFPI (FXa-TFPI) was seen; FXa-TFPI remained depressed throughout the study. There was no inverse relationship demonstrated between markers of thrombin generation and thrombin regulation. Acquired APC resistance and hypofibrinolysis did not appear to be important contributors to hypercoagulability after trauma. None of the coagulation markers were independently predictive of VTE. Increasing age was the only significant, independent predictor of VTE.

Conclusion

Major trauma leads to significantly increased and persistent thrombin generation with disruption of its regulation. Coagulation markers do not appear to add independent predictive value in detecting VTE. Increasing age is the most important clinical predictor of VTE after trauma.     

Acute release of tissue factor pathway inhibitor after in vivo thrombin generation in baboons

Tissue factor pathway inhibitor (TFPI), the major downregulator of the procoagulant activity of tissue factor (TF), is synthesised by endothelial cells (EC) and acutely released in vitro after thrombin stimulation. Expression of TF on EC and subsequent thrombin generation occurs in vivo during sepsis or malignancy, inducing disseminated intravascular coagulation (DIC). The present study investigates the changes in plasma TFPI in relation to markers of in vivo thrombin generation induced by injection of factor Xa (FXa)/phospholipids in baboons at dosages leading to partial (48%) or complete fibrinogen depletion. The plasma concentrations of thrombin-antithrombin III (TAT) and fibrinopeptide A (FpA), as markers of in vivo generation of thrombin, were strongly enhanced after injection of FXa/phospholipids. TFPI levels, whether measured as antigen or activity, increased significantly in both treatment groups within few minutes, and were dependent on the dose of FXa/phospholipids. Significant positive correlations between plasma levels of TFPI and of TAT or FpA were observed. Altogether, our results indicate that experimentally induced in vivo generation of thrombin causes the acute release of TFPI, high-lighting a possible novel function of thrombin in downregulation of the coagulation process, potentially relevant for the outcome of DIC.     

Polymorphisms in the endothelial protein C receptor gene and thrombophilia

The protein C anticoagulant pathway plays a crucial role as a regulator of the blood clotting cascade. Protein C is activated on the vascular endothelial cell membrane by the thrombin-thrombomodulin complex. Once formed, activated protein C (APC) down-regulates thrombin formation by inactivating factors (F)Va and FVIIIa. Endothelial protein C receptor (EPCR) is able to bind protein C and increase the rate of protein C activation. Normal APC generation depends on the precise assemblage, on the surface of endothelial cells, of thrombin, thrombomodulin, protein C and EPCR. Therefore, any change in the efficiency of this assemblage may cause reduced/increased APC generation and modify the risk of thrombosis. This review highlights the different mutations/polymorphisms reported in the EPCR gene and their association with the risk of thrombosis.     

The value of 64-detector row computed tomography for the exclusion of pulmonary embolism

Identification of patients at high risk of recurrence after a first event of venous thromboembolism (VTE) remains difficult. Resistance to activated protein C (APC) is a known risk factor for VTE, but data on the risk of recurrence is controversial. We wanted to investigate whether APC resistance in the absence of factor V Leiden, determined with global coagulation test such as the thrombin generation assay, could be used as a marker for increased risk of recurrent VTE among women 18-65 years old after a first event of VTE. In a cohort of 243 women with a first event of VTE, plasma was collected after discontinuation of anticoagulant treatment and the patients were followed up for 46 months (median). Thrombin generation was measured via calibrated automated thrombography, at 1 pM and 10 pM of tissue factor (TF). In women without factor V Leiden (n=117), samples were analysed in the absence and in the presence of APC. Increase in ETP (endogenous thrombin potential) and peak height analysed in the presence of APC correlated significantly with higher risk of recurrence. At 1 pM, peak height correlated with increased risk of recurrence. In conclusion, high thrombin generation in the presence of APC, in women after a first event of VTE is indicative for an increased risk of a recurrence. We also found that thrombin generation at low TF (1 pM) is correlated with the risk of recurrence. Our data suggest that APC resistance in the absence of factor V Leiden is a risk factor for recurrent VTE.     

Tamoxifen induces resistance to activated protein C

Introduction

The estrogen antagonist tamoxifen (TAM) increases the thrombotic risk similar to estrogen containing oral contraceptives (OC). In OC users this risk is attributed to alterations of hemostasis resulting in acquired resistance to activated protein C (APC). TAM-induced APC resistance has not been reported yet.

Materials and Methods

Blood samples were collected prospectively from women with breast cancer before (n = 25) and monthly after start of adjuvant TAM treatment (n = 75). APC resistance was evaluated on basis of the effect of APC on the endogenous thrombin generation potential. To detect increased in vivo APC generation APC plasma levels were measured using a highly sensitive oligonucleotide-based enzyme capture assay. Routine hemostasis parameters were measured additionally.

Results

APC sensitivity decreased by 41% (p = 0.001) compared to baseline after one month of TAM application and remained significantly decreased during the study period. Free protein S increased (p = 0.008) while other analyzed procoagulant factors, inhibitors, and activation markers of coagulation decreased or did not change significantly. In five patients the APC concentration increased to non-physiological levels but an overall significant increase of APC was not observed.

Conclusions

This is the first study showing acquired APC resistance under TAM therapy. Acquired APC resistance might explain the increased thrombotic risk during TAM treatment. Observed changes of hemostasis parameters suggest different determinants of TAM-induced APC resistance than in OC-induced APC resistance. The presence of acquired APC resistance in TAM patients warrants further evaluation if these patients may benefit from antithrombotic prophylaxis in the presence of additional thrombotic risk factors.     

Calibrated automated thrombography demonstrates hypercoagulability in patients with idiopathic pulmonary arterial hypertension

Introduction

Pathogenesis of idiopathic pulmonary arterial hypertension (iPAH) includes endothelial dysfunction and in situ thrombosis. A hypercoagulable state has also been postulated but never demonstrated. Our objective was to determine whether patients with iPAH had a hypercoagulable state using calibrated automated thrombography (CAT), a new tool to phenotype coagulation in vitro.

Patients and methods

16 patients with iPAH and 29 controls were studied. In vitro platelet dependent coagulation phenotyping by CAT monitored the activity of thrombin generation over time. Plasma levels of soluble thrombomodulin, tissue factor pathway inhibitor (TFPI) and von Willebrand factor (VWF) were measured as endothelial biomarkers.

Results

Endogenous thrombin potential (ETP) in the absence of activated protein C (APC) tended to be increased in patients compared to controls (1769 versus 1656 nM.min; p = 0.053). ETP was higher in the presence of APC 25 nM (ETP-APC) in patients (781 versus 494 nM.min; p = 0.005). Five patients had ETP-APC higher than the 95th centile of controls. Other CAT parameters (lag time, peak thrombin and time to peak) were all consistent with some degree of hypercoagulability in patients. Regarding endothelial plasma biomarkers sTM was lower (28.4 versus 40.6 μg/l, p = 0.0108) in patients; TFPI antigen and activity (respectively: 14.3 versus 10.5 μg/l, p = 0.0167; 1.155 versus 1.070, p = 0.0021) and VWF (1300 versus 976%, p = 0.0108) were higher in patients.

Conclusion

We have demonstrated that at least some patients with iPAH have a hypercoagulable phenotype.     

Changes in haemostatic parameters during the menstrual cycle and subsequent use of drospirenone-containing oral contraceptives

Introduction

Oral contraceptives (OC) increase the risk of venous thromboembolism that depends on the OC formulation and could at least partially be explained by impaired function of the protein C-system (APC resistance) and the tissue factor pathway inhibitor (TFPI)-system. There is limited information available on the effects of OC, containing a newer progestogen- drospirenone (DRSP-OC) on these two major anticoagulant pathways, thrombin generation, reflecting the overall state of coagulation, and other coagulation parameters.

Methods

In a study population consisting of 14 healthy women (age 21–33 years) we investigated the effect of the menstrual cycle and subsequent use of DRSP-OC on APC resistance, the function of the TFPI-system, thrombin generation and on their major determinants, i.e. prothrombin, antithrombin, FV, FX, FVIII, protein C, protein S_{(total and free)} and TFPI_{(full-length and free)}.

Results

All studied parameters remained unchanged during the menstrual cycle. During DRSP-OC use we observed a significant increase in APC resistance (~ 2.4-fold), thrombin generation measured at low (~ 2.2-fold) and high tissue factor concentrations (~ 1.4-fold), plasma concentrations of prothrombin (19%), FX (31%), FVIII (17%) and protein C (43%). DRSP-OC use impaired the function of the TFPI-system and decreased plasma levels of antithrombin (− 6%), FV (− 22%), protein S_total (− 21%), protein S_free (− 20%), TFPI_full-length (− 36%) and TFPI_free (− 46%).

Conclusions

DRSP-OC caused procoagulant changes in all studied haemostatic parameters. The impairment of the protein C- and TFPI-systems was more pronounced than the impairment of the coagulation pathways and can at least partially account for the increased risk of venous thromboembolism in users of DRSP-OC.     

Activated protein C resistance and the FV:R506Q mutation in a random population sample--associations with cardiovascular risk factors and coagulation variables.

Activated protein C (APC) resistance, defined as a low APC ratio, is associated with the factor V mutation R506Q (factor V Leiden). APC ratio may also be influenced by other clinical and coagulation variables, which we studied in 460 men and 495 women aged 25-74 years, from a random population sample (Glasgow MONICA Survey). APC ratio correlated positively with APTT; and inversely with factor VIIIc, factor IXc, antithrombin activity, prothrombin F1+2 fragment, and thrombin-antithrombin complexes; but not with other coagulation variables. APC ratio decreased with age, but APTT did not. APC ratio and APTT were significantly lower in women versus men, and were significantly lower in users of oral contraceptives or hormone replacement therapy. The FV:R506Q mutation (prevalence 2.5%) was associated with lower APC ratio and protein C and S activities and with higher factor VIIIc levels; but not with increases in F1+2 fragment or thrombin-antithrombin complexes. APC ratio correlated inversely with total cholesterol and diastolic blood pressure; and in women with triglycerides, systolic blood pressure, and body mass index. Obesity was associated with a significantly lower APC ratio. In contrast, smoking markers correlated positively with APC ratio in men. These associations of APC ratio may be relevant to the increased risks of venous thrombosis with age, female sex, oestrogen use, obesity and high factor VIIIc levels. The association of APC resistance with elevated plasma levels of coagulation markers suggests that this phenotype represents an in vivo hypercoagulable state.     

Activation of coagulation in amniotic fluid during normal human pregnancy

Introduction

Amniotic fluid (AF) is an important medium for fetal development which exhibits high procoagulant activities; however, the role of these procoagulants during pregnancy has not been elucidated and might be associated with pregnancy complications. The current study aimed to evaluate factor X (FX) activation and its association with tissue factor (TF), tissue factor pathway inhibitor (TFPI) and coagulation activation markers in AF during normal human pregnancy.

Methods

Activation of FX and concentration of TF, free TFPI, D-dimer and prothrombin fragments (F1 + 2) were evaluated in AF samples obtained for chromosome analysis from 91 women with normal pregnancy: 65 samples were taken from patients at 16-20 weeks of gestation, 9 samples were drawn at 21-30 weeks and 17 samples−after 30 weeks of gestation.

Results

Activation of FX in AF significantly increased during normal pregnancy (from 65 ± 41 to 205 ± 80 equivalent RVV ng/mg total protein, P < 0.0001). TF and TFPI levels in AF also rose with gestational age. In contrast, the AF concentration of D-dimer and F1 + 2, markers of coagulation activation significantly decreased when expressed per mg total protein. Levels of free TFPI correlated with TF (r = 0.5, P < 0.001), and were 8-fold higher than those of TF during pregnancy.

Conclusion

High levels of TFPI might be associated with the inhibition of procoagulant activity in amniotic fluid during normal pregnancy, which may account for the rarity of clinical amniotic fluid embolism.     

Calibrated automated thrombin generation in normal uncomplicated pregnancy

Pregnancy is associated with substantial changes in the haemostatic system and a six-fold higher incidence of venous thromboembolism. Conventional global tests, such as prothrombin time and activated partial thromboplastin time, do not definitely detect this hypercoagulable condition. We investigated whether the changes in haemostatic system during pregnancy are reflected in the calibrated automated thrombography (CAT). Thrombin generation was measured in platelet-poor plasma (PPP) of 150 healthy pregnant women without any pregnancy associated diseases by means of CAT. In addition, prothrombin (FII), antithrombin (AT), protein S, protein C, tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex (TAT), and prothrombin fragments 1+2 (F1+2) were measured. Endogenous thrombin potential (ETP) and peak of thrombin generation increased significantly with gestational weeks, while lag time and time to peak remained unchanged. A significant increase of PAI-1, TFPI, F1+2 and TAT as well as a significant decrease of free protein S, protein S antigen, and protein S activity was observed. Levels of AT and protein C remained stable during pregnancy. Division of population in trimester of pregnancy and analysis of differences between the trimesters showed rather similar results. Our study shows that endogenous thrombin potential does increase with duration of normal uncomplicated pregnancy. Whether parameters of continuous thrombin generation will correlate with thrombembolic disease remains to be shown.     

L-Asparaginase and the effect of age on coagulation and fibrinolysis in childhood acute lymphoblastic leukemia

Alterations in haemostasis are frequently observed in children with acute lymphoblastic leukemia (ALL). It was the objective of this study to analyse age-related disturbances in coagulation and fibrinolysis parameters during the induction phase of the antileukemic treatment. Sixty-four children were classified by age into three groups (1-5, 6-10, 11-16 years), and studied during induction treatment of ALL including four weeks of dexamethasone, followed by two weeks tapering of dexamethasone during which 6,000 IU/m(2) native L-Asparaginase (total 4 doses) was administered intravenously twice weekly. Blood samples were collected immediately before each L-Asparaginase infusion to analyze procoagulant (fibrinogen, factor [F] II, FV, FVII, F IX, F X) and anticoagulant factors (antithrombin [AT], protein C, protein S), parameters of thrombin generation (F1+2, TAT) and fibrinolysis (alpha2-antiplasmin, plasminogen, PAP, D-dimer). Children were in a hypercoagulable state after four weeks of dexamethasone due to upregulation of coagulation parameters. Upregulation was highest in the two youngest age groups. During L-Asparaginase treatment the 11- to 16-year-olds showed lower values in procoagulant and, even more, in anticoagulant factor levels compared to the younger children. Activation markers of thrombin generation and fibrinolysis did not change over time during the study period. Decreased synthesis of alpha2-antiplasmin and plasminogen during L-Asparaginase treatment resulted in less potential of clot lysis by plasmin in children older than 11 years of age. In conclusion, a more severe decline of anticoagulant and fibrinolytic parameters in children between 11 and 16 years of age underline that these children are at higher risk of thrombosis during ALL induction treatment.