thromboplastin and Activated-Protein-C-Resistance

In normal pregnancy, there is a marked increase in the procoagulant activity in maternal blood characterized by elevation of factors VII, X, VIII, fibrinogen and von Willebrand factor, which is maximal around term. This is associated with an increase in prothrombin fragments (PF1+2) and thrombin-antithrombin complexes. There is a decrease in physiological anticoagulants manifested by a significant reduction in protein S activity and by acquired activated protein C (APC) resistance. The overall fibrinolytic activity is impaired during pregnancy, but returns rapidly to normal following delivery. This is largely due to placental derived plasminogen activator inhibitor type 2 (PAI-2), which is present in substantial quantities during pregnancy. D-dimer, a specific marker of fibrinolysis resulting from breakdown of cross-linked fibrin polymer by plasmin, increases as pregnancy progresses. Overall, there is a 4- to 10-fold increased thrombotic risk throughout gestation and the postpartum period. Local haemostasis at the placental throphoblast level is characterized by increased tissue factor (TF) expression and low expression of the inhibitor TFPI. Microparticles derived from maternal endothelial cells and platelets, and from placental throphoblasts may contribute to the procoagulant effect. Local anticoagulant mechanisms on placental throphoblasts are important for counterbalance of the procoagulant milieu. Disruption of anticoagulant mechanisms, for example, autoantibodies, to annexin V may increase pregnancy complications in patients with antiphospholipid antibodies (APLA).

Topics: Activated Protein C Resistance; Antibodies, Antiphospholipid; Anticoagulants; Blood; Blood Coagulation; Blood Coagulation Factors; Blood Physiological Phenomena; Coagulants; Female; Fibrinolysis; Hemostasis; Humans; Placenta; Plasminogen Activator Inhibitor 2; Pregnancy; Protein S; Thromboplastin; Thrombosis

Topics: Activated Protein C Resistance; Factor V; Humans; Protein S; Prothrombin; Thromboplastin

Activated protein C resistance (APCr) leads to hypercoagulability and is due, often but not exclusively, to Factor V Leiden (FVL). The aim of this study was to assess the ex vivo and in vitro interference of the direct factor Xa inhibitor rivaroxaban (RIV) on a prothrombinase-based assay for APCr detection.. An ex vivo study was performed on fresh plasma samples obtained from 44 subjects with FV wild-type and seven with FVL heterozygous, all treated with RIV. An in vitro study was performed on 15 plasma samples (six from normal subjects, six from heterozygous, and three from homozygous FVL carriers, all frozen specimens) spiked with RIV. RIV concentration was evaluated using a chromogenic assay, and APCr was evaluated by a prothrombinase-based assay.. No significant interference of RIV on APCr results obtained by a prothrombinase-based assay was observed for drug concentrations up to 400 ng/mL in FV wild-type and FVL carriers (homozygous and heterozygous). These results were confirmed both ex vivo and in vitro.. RIV did not significantly interfere with the prothrombinase-based assay used for the assessment of APCr, and this was observed to occur independently of FV status. However, only concentrations up to 400 ng/mL were tested and, therefore, what occurs in the presence of higher doses remains to be investigated.

Topics: Activated Protein C Resistance; Aged; Aged, 80 and over; Blood Coagulation; Blood Coagulation Tests; Drug Monitoring; Factor V; Factor V Deficiency; Female; Heterozygote; Homozygote; Humans; Italy; Male; Rivaroxaban; Thromboplastin

The aim of this study was to evaluate ex vivo and in vitro interference of a direct factor IIa inhibitor, dabigatran, on a prothrombinase-based assay to detect activated protein C resistance.. An ex vivo study was performed in six heterozygous factor V Leiden carriers and 12 normal subjects without the factor V Leiden mutation who were treated with dabigatran. An in vitro study was also performed considering 12 plasma samples (six from normal subjects and six from heterozygous factor V Leiden carriers) spiked with dabigatran. The dabigatran concentration was evaluated using a diluted thrombin time assay, activated protein C resistance was evaluated using a prothrombinase-based assay.. In both the ex vivo and in vitro studies dabigatran interfered significantly with activated protein C resistance ratios observed in normal subjects and in factor V Leiden heterozygous carriers.. The results reported in this paper seem to confirm that dabigatran is able to interfere with the Penthafarm prothrombinase-based assay used to study activated protein C resistance, significantly increasing observed ratios. This effect appears to be present already at low concentrations of dabigatran (6 ng/mL) and affects both normal subjects and heterozygous carriers of factor V Leiden. In this group of patients, dabigatran, at concentrations in the therapeutic range (100-200 ng/mL), could markedly increase the activated protein C resistance ratio, bringing it up to within the reference range for normal subjects, thus potentially leading to misclassification of patients.

Topics: Activated Protein C Resistance; Antithrombins; Blood Coagulation Tests; Dabigatran; Diagnostic Errors; Factor V; Female; Humans; Male; Mutation; Protein C; Thromboplastin

Essentials Activated protein C (APC) resistance is a prevalent risk factor for venous thrombosis. A novel missense mutation (Ala512Val - FVBonn ) was characterized in vitro and in silico. FVBonn is a new cause of APC resistance and venous thrombosis. FVBonn expresses additionally enhanced procoagulant activity in the absence of APC.. Background Activated protein C (APC) resistance is a prevalent risk factor for venous thrombosis. This phenotype is most commonly associated with the factor V Arg506Gln mutation (FV Leiden), which impairs the APC-mediated inactivation of both activated FV (FVa) and activated FVIII (FVIIIa). Objectives Here, we report the identification and characterization of a novel FV mutation (Ala512Val, FVBonn ) in six patients with APC resistance and venous thrombosis or recurrent abortions. Methods FVBonn was expressed in a recombinant system and compared with recombinant wild-type (WT) FV and FV Leiden in several functional assays. Results FVBonn conferred APC resistance to FV-depleted plasma, both in the activated partial thromboplastin time (APTT)-based test (APC sensitivity ratio [APCsr] of 1.98 for FVBonn versus 4.31 for WT FV and 1.59 for FV Leiden) and in the thrombin generation-based test (normalized APCsr of 5.41 for FVBonn versus 1.00 for WT FV and 8.99 for FV Leiden). The APC-mediated inactivation of FVaBonn was slower than that of WT FVa (mainly because of delayed cleavage at Arg506), but was greatly stimulated by protein S. The APC cofactor activity of FVBonn in FVIIIa inactivation was ~ 24% lower than that of WT FV. In line with these findings, an in silico analysis showed that the Ala512Val mutation is located in the same loop as the Arg506 APC cleavage site and might hamper its interaction with APC. Moreover, FVBonn was more procoagulant than WT FV and FV Leiden in the absence of APC, because of an increased activation rate and, possibly, an enhanced interaction with activated FX. Conclusions FVBonn induces hypercoagulability via a combination of increased activation/procoagulant activity, decreased susceptibility to APC-mediated inactivation, and slightly reduced APC cofactor activity.

Topics: Abortion, Habitual; Activated Protein C Resistance; Adult; Aged; Blood Coagulation; Blood Coagulation Tests; Catalysis; Coagulants; Cohort Studies; Factor V; Factor Va; Factor VIIIa; Female; Humans; Male; Mutation; Mutation, Missense; Partial Thromboplastin Time; Pregnancy; Protein C; Thrombin; Thromboplastin; Venous Thrombosis; Young Adult

Topics: Activated Protein C Resistance; Aged; Aged, 80 and over; Antithrombins; Blood Coagulation; Blood Coagulation Tests; Dabigatran; Factor V; Factor Xa Inhibitors; Female; Heterozygote; Humans; Male; Middle Aged; Partial Thromboplastin Time; Rivaroxaban; Thromboplastin

Activated partial thromboplastin time (aPTT) is an important routine measure of intrinsic blood coagulation. Addition of activated protein C (APC) to the aPTT test to produce a ratio, provides one measure of APC resistance. The associations of some genetic mutations (eg, factor V Leiden) with these measures are established, but associations of other genetic variations remain to be established. The objective of this work was to test for association between genetic variants and blood coagulation using a high-density genotyping array. Genetic association with aPTT and APC resistance was analysed using a focused genotyping array that tests approximately 50 000 single-nucleotide polymorphisms (SNPs) in nearly 2000 cardiovascular candidate genes, including coagulation pathway genes. Analyses were conducted on 2544 European origin women from the British Women's Heart and Health Study. We confirm associations with aPTT at the coagulation factor XII (F12)/G protein-coupled receptor kinase 6 (GRK6) and kininogen 1 (KNG1)/histidine-rich glycoprotein (HRG) loci, and identify novel SNPs at the ABO locus and novel locus kallikrein B (KLKB1)/F11. In addition, we confirm association between APC resistance and factor V Leiden mutation, and identify novel SNP associations with APC resistance in the HRG and F5/solute carrier family 19 member 2 (SLC19A2) regions. In conclusion, variation at several genetic loci influences intrinsic blood coagulation as measured by both aPTT and APC resistance.

Topics: Activated Protein C Resistance; Blood Coagulation; Blood Coagulation Disorders, Inherited; Factor V; Factor XII; Female; Genetic Association Studies; Genotyping Techniques; Humans; Kallikreins; Oligonucleotide Array Sequence Analysis; Partial Thromboplastin Time; Polymorphism, Single Nucleotide; Protein C; Thromboplastin

Whereas procoagulation abnormalities in acute stress are well established, little is known about the mechanism of hypercoagulation in chronic stress, such as post-traumatic stress disorder (PTSD). This is crucial, given the fact that chronic coagulation disturbances have been associated with increased morbidity and premature mortality due to thromboembolism and cardiovascular disorders, complications recently described in PTSD patients.. To explore the mechanisms of hypercoagulation in chronic PTSD.. Thirty patients diagnosed with chronic PTSD were enrolled and compared with a control group matched for age, gender and ethnicity. Hypercoagulation state was evaluated by levels of fibrinogen, D-dimer, prothrombin fragment F 1+2, von Willebrand factor (vWF) antigen, factor VIII activity, activated protein C resistance, ProC Global assay, and tissue factor antigen. Psychiatric evaluation was performed using the Mini-International Neuropsychiatric Interview and Clinician Administered PTSD Scale (CAPS).. vWF antigen levels were significantly higher in patients with chronic PTSD compared with the controls (121.3 +/- 42 vs. 99.7 +/- 23, respectively, P = 0.034). Higher levels of vWF antigen and factor VIII activity were found in patients with severe chronic PTSD (CAPS > 80), compared to controls and patients with chronic PTSD and less severe symptoms (CAPS < or = 80). However, no differences were observed in any other studied coagulation parameters between patients and controls.. Increased levels of vWF antigen and factor VIII activity were documented in severe chronic PTSD. These findings suggest that the higher risk of arterial and venous thromboembolic events in PTSD patients could be related to endothelial damage or endothelial activation.

Topics: Activated Protein C Resistance; Adult; Biomarkers; Blood Coagulation Factors; Chronic Disease; Factor VIII; Fibrin Fibrinogen Degradation Products; Fibrinogen; Humans; Israel; Male; Peptide Fragments; Protein Precursors; Prothrombin; Stress Disorders, Post-Traumatic; Surveys and Questionnaires; Thrombophilia; Thromboplastin; von Willebrand Factor

Topics: Activated Protein C Resistance; Blood Coagulation; Case-Control Studies; Factor V; Factor VIIa; Factor Xa Inhibitors; Humans; Kinetics; Lipoproteins; Multienzyme Complexes; Protein S; Thromboplastin; Venous Thrombosis

Systemic inflammation activates the tissue factor/factor VIIa complex (TF/FVIIa), leading to a procoagulant state, which may be enhanced by impairment of physiological anticoagulant pathways, such as the protein C system. Besides impaired protein C activation, resistance to activated protein C (APC) may occur. We studied the effect of endotoxemia on APC resistance, analysed its determinants and evaluated the effect of TF/FVIIa inhibition on endotoxin-induced APC resistance. Sixteen healthy male volunteers participated in the study, eight receiving endotoxin alone and eight receiving the combination of endotoxin and recombinant Nematode Anticoagulant Protein c2 (rNAPc2), a potent inhibitor of TF/FVIIa. Parameters of coagulation were subsequently studied. The sensitivity to APC was determined by two tests: a test based on the endogenous thrombin potential and a test based on the activated partial thromboplastin time. In response to endotoxemia, both tests detected a transient APC resistance that was predominantly mediated by an increase in factor VIII and was not influenced by TF/FVIIa inhibition. In vitro tests confirmed that an increase in factor VIII induced APC resistance, as measured by both tests. This finding suggests that APC resistance might play a role in the procoagulant state occurring during human endotoxemia.

Topics: Activated Protein C Resistance; Adolescent; Adult; Blood Coagulation; Blood Coagulation Tests; Endotoxemia; Factor V; Factor VIIa; Factor VIII; Helminth Proteins; Humans; Male; Partial Thromboplastin Time; Protein C; Recombinant Proteins; Risk Factors; Thromboplastin

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.

Topics: Activated Protein C Resistance; Adult; Anticoagulants; Blood Coagulation Tests; Contraceptives, Oral; Dose-Response Relationship, Drug; Factor Xa; Female; Humans; Linear Models; Male; Phospholipids; Protein C; Protein S; Protein Structure, Tertiary; Prothrombin; Risk; Thrombin; Thromboplastin; Thrombosis

Activated protein C resistance (APCR), high tissue factor (TF) expression, and hyper-homocysteinemia are associated with thromboembolic diseases. Thromboembolism is a frequent complication of systemic lupus erythematosus (SLE). In this study, we evaluated the prevalence of APCR, high TF, and homocysteine with correlation of the thrombotic tendency in SLE. Ninety-four SLE patients and 28 normal controls were included. APC ratio and TF antigen were measured using commercial kits. Plasma homocysteine level was measured using HPLC. The prevalence of APCR, high TF antigen level, and hyper-homocysteinemia in our SLE patients were 21.3%, 66.0%, and 23.4%, respectively. The median plasma level of TF antigen in SLE patients was 145.23 pg/mL (range, 31.00-778.50 pg/mL), which was significantly higher than the control value of 39.83 pg/mL (range, 1.55-168.50 pg/mL). The median APC ratio in SLE patients was 2.76 (range, 1.48-13.47), which was significantly lower than the control value of 3.59 (range, 0.26-5.66). The plasma level of homocysteine was not significantly different from that of control. A significant association was observed between the presence of APCR (OR = 8.59, P < 0.0001) but not with the presence of high plasma TF antigen level (OR = 1.24, P = 0.67) and thrombotic complications in SLE patients. In conclusion, APCR and high plasma TF levels are common in SLE, but a significant association was observed only between the presence of APCR and thrombosis in SLE patients.

Topics: Activated Protein C Resistance; Adolescent; Adult; Aged; Aged, 80 and over; Case-Control Studies; Female; Homocysteine; Humans; Hyperhomocysteinemia; Lupus Erythematosus, Systemic; Male; Middle Aged; Prevalence; Retrospective Studies; Thromboembolism; Thromboplastin; Thrombosis

The normalized activated protein C sensitivity ratio (nAPC-sr) determined with an assay that quantifies the effect of APC on thrombin formation initiated via the extrinsic coagulation pathway identifies hereditary and acquired defects of the protein C system. We investigated the influence of assay conditions (analytical variables) and plasma handling (pre-analytical variables) on nAPC-sr obtained with this APC resistance test. The effect of the analytical variables (CaCl2, phospholipid and APC concentrations and the concentration and source of tissue factor) was determined in pooled normal plasma. Inhibition of thrombin formation by APC was dependent on the APC concentration and was also affected by the tissue factor, Ca2+ and phospholipid concentrations. Thus, strict standardization of reactant concentrations is required to obtain reproducible nAPC-sr. Three different tissue factor preparations were compared by determining nAPCsr in plasma samples obtained from 90 healthy individuals. nAPC-sr were similar for all three tissue factor preparations although, compared with the noncommercially available tissue factor used in earlier studies, values determined with commercial tissue factor preparations showed larger variation. Pre-analytical variables, investigated in plasma of nine volunteers (3 normal individuals and 6 individuals with an APC-resistant phenotype) were: concentration of anticoagulant (3.2% vs. 3.8% trisodiumcitrate), time before processing of blood (0, 4 and 24 h), centrifugation speed, storage temperature of plasma (-20 degrees C vs. -80 degrees C) and sample thawing. Multiple linear regression analysis showed that only the citrate concentration affected the nAPC-sr, which was higher in samples collected in 3.2% trisodiumcitrate than in samples collected in 3.8% trisodiumcitrate.

Topics: Activated Protein C Resistance; Blood Specimen Collection; Calcium; Clinical Laboratory Techniques; Female; Humans; Male; Middle Aged; Phospholipids; Pregnancy; Protein C; Reproducibility of Results; Thrombin; Thromboplastin

Atherosclerotic cardiovascular disease is the leading cause of the increased morbidity and mortality observed in uremic patients. Thrombosis is an important contributor to the evolution of atherosclerotic lesions. The physiologically-relevant blood clotting depends on binding of activated factor VII (FVIIa) to exposed tissue factor (TF) on activated/damaged cells.. A cross-sectional study was performed on three age- and sex-matched groups of individuals: one group of 50 patients on maintenance hemodialysis (D group), one of 50 patients with a non-dialysed renal insufficiency (ND group) and one of 50 healthy controls (HC group). We studied basal plasma concentrations of FVIIa, factor VII-related antigen (FVIIAg), soluble TF, tissue factor pathway inhibitor (TFPI), TF-dependent circulating monocytes procoagulant activity (TF-dMPA), tissue factor-dependent plasma reactivity to activated protein C (TF-aPC), D-dimers (D-Di), and circulating markers of cellular activation/injury: soluble thrombomodulin (sTM), circulating microparticles (microP), soluble leukocyte, endothelial and platelet selectins (sL-selectin, sE-selectin, sP-selectin), soluble intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 (sICAM-1 and sVCAM-1). Their variations induced, in hemodialysis patients, by a dialysis run were thereafter studied. Values of FVIIa, FVIIa/FVIIAg ratio, sTF, TFPI, TF-dMPA, D-Di, sTM, microP, sL, sE and sP selectins, sICAM-1 and sVCAM-1 increased all along the hierarchy HC group/ND group/D group. Microparticles were mainly of platelet origin, to a lesser extent of monocyte origin. Dialysis induced an increase of FVIIa, sTF, TF-dMPA and circulating markers of cellular activation/injury. Strong correlations were observed between FVIIa/FVIIAg ratio and serum creatinine levels, sTF, TF-dMPA, sTM, sE-selectin, sVCAM-1. The TF-aPC was impaired in the ND and the D group, and the lower values were, in the D group, associated with antecedents of vascular access thrombosis.. Renal insufficiency is associated to an activation of the tissue factor coagulation pathway, to a platelet, monocyte and endothelial activation/injury and to a deficient tissue-factor induced response to activated protein C which culminate in end-stage disease and are increased by hemodialysis runs. This contributes to linked coagulation and cellular conditions for an enhanced atherosclerosis progression. Due to the TF pathway activation, the therapeutic use of recombinant TFPI should be evaluated.

Topics: Activated Protein C Resistance; Adult; Aged; Blood Coagulation; Blood Coagulation Factors; Case-Control Studies; Cell Adhesion Molecules; Cross-Sectional Studies; Female; Humans; Male; Middle Aged; Renal Dialysis; Renal Insufficiency; Thrombophilia; Thromboplastin