Activated Protein C Sensitivity Ratio Research Articles

Low-dose oral contraceptives and acquired resistance to activated protein C: a randomised cross-over study

We have reported previously that, compared with use of second-generation oral contraceptives, the use of third-generation oral contraceptives is associated with increased resistance to the anticoagulant action of activated protein C (APC). Owing to the cross-sectional design of that study, these observations may have been subject to unknown bias or uncontrolled effects of the menstrual cycle. We aimed to overcome these sources of bias by doing a cycle-controlled randomised cross-over trial. The response to APC in plasma was assessed in 33 women who received two consecutive cycles of a second-generation oral contraceptive (150 microg levonorgestrel and 30 microg ethinyloestradiol) or a third-generation oral contraceptive (150 microg desogestrel and 30 microg ethinyloestradiol), and who switched preparations after two pill-free cycles. Normalised APC sensitivity ratios were calculated by measurement of the effect of APC on thrombin generation in the plasma of these women and in pooled plasma from 90 controls. Of the 33 women, five were excluded because not all required plasma samples were available. In the remaining 28 women, the normalised APC sensitivity ratio increased during treatment with both preparations. Compared with levonorgestrel, desogestrel-containing oral-contraceptive treatment caused a highly significant (p<0.0001) additional increase in normalised APC sensitivity ratio (0.51 [95% CI 0.37-0.66]). Normalised APC sensitivity ratios during oral-contraceptive treatment correlated with the values before oral-contraceptive use. Oral-contraceptive treatment diminishes the efficacy with which APC down-regulates in-vitro thrombin formation. This phenomenon, designated as acquired APC resistance, is more pronounced in women using desogestrel-containing oral contraceptives than in women using levonorgestrel-containing preparations. Whether acquired APC resistance induced by oral contraceptives explains the increased risk of venous thromboembolism in oral-contraceptive users remains to be established.

Detection of decreased response to activated protein C during pregnancy by an endogenous thrombin potential-based assay.

Pregnancy has been widely recognized as a predisposing risk factor for deep vein thrombosis (DVT). However, it still remains unclear why pregnant women without a history of familial thrombophilia or antiphospholipid syndrome (APS) have a higher incidence of DVT and pulmonary embolism (PE) during pregnancy and puerperium. We examined the activated protein C (APC) system in healthy pregnant women and in patients with the onset of DVT during puerperium. Sixty unselected Japanese pregnant women without a past or family history of thrombosis or APS and 3 Japanese women with DVT during puerperium were evaluated. Endogenous thrombin potential-ratio (ETP-r) was measured by determination of thrombin-alpha2-macroglobulin complexes in thromboplastin-activated patient plasma. APC sensitivity ratio (APC-sr) was calculated by the determination of ETP-r in patient plasma in the presence and absence of APC (final concentration [conc.] 5.9 nM) to evaluate the functional APC anticoagulant activity. Mean APC-sr was significantly increased at 30 weeks' gestation (2.35 +/- 0.72) and remained high during puerperium compared with the mean APC-sr in nonpregnant women (1.15 +/- 0.63). Mean APC-sr in patients with DVT at the onset was significantly higher (3.57 +/- 0.54) than mean APC-sr during puerperium was, indicating that the sensitivity to APC was reduced in the ETP-based assay. These data suggest a significant reduction in the functional sensitivity to APC associated with an increased risk of venous thrombosis during pregnancy.

Influence of factor VIII/von Willebrand complex on the activated protein C-resistance phenotype and on the risk for venous thromboembolism in heterozygous carriers of the factor V Leiden mutation

High factor VIII plasma levels have been shown to represent a common increased risk for venous thromboembolism (VTE) and may cause an activated protein C (APC) resistance in the absence of the factor V Leiden mutation, but there are no studies specifically aimed to establish if high factor VIII and von Willebrand factor (vWF) concentrations may influence the APC sensitivity ratio (APC-SR) and increase the risk for VTE in the presence of the factor V Leiden mutation. For this purpose, we performed a retrospective case-control study to investigate the influence of the procoagulant factor VIII (VIII:C) and the antigen of vWF (vWF:Ag) on the normalized APC-SR (n-APC-SR) and on the risk for VTE, in two selected groups of 30 symptomatic (Group I) and 32 asymptomatic (Group II) related heterozygotes for the factor V Leiden mutation. Differences between the two groups (Group I versus Group II) were: n-APC-SR, 0.57+/-0.06 versus 0.63+/-0.08, P = 0.001; factor VIII:C, 1.49+/-0.42 versus 1.13+/-0.28 IU/ml, P<0.001; vWF:Ag, 1.46+/-0.53 versus 1.26+/-0.32 IU/ml, NS. As a whole (Group I + Group II), Pearson correlation coefficients were: n-APC-SR versus factor VIII:C, r = -0.410, P = 0.001; n-APC-SR versus vWF:Ag, r = -0.309, P = 0.01; factor VIII:C versus vWF:Ag, r = +0.640, P<0.0001. The relative risk for VTE in individuals with the factor VIII:C concentration > 1.5 IU/ml was 2.5 (95% confidence interval 1.6-3.9). We concluded that high factor VIII:C levels, probably in the effect of vWF, play a determinant role in worsening the APC-resistance phenotype and represent a common additional risk factor for VTE in heterozygous carriers of the factor V Leiden mutation.

A possible role for activated protein C resistance in patients with first and second trimester pregnancy failure.

Thrombophilia was recently suggested as a possible factor in recurrent pregnancy losses. We studied prospectively 125 patients (mean age 31.4 +/- 5.6 years) with one or more first or second trimester pregnancy losses for the prevalence of activated protein C resistance (APCR). Proteins C and S antigens, antithrombin III, anticardiolipin, and lupus anti-coagulant were also evaluated. Patients with uterine malformations, hormonal abnormalities, chromosomal translocations and infectious causes were excluded. A control group of 125 women with no past fetal loss were matched with the study group. Whenever the APC-sensitivity ratio (APC-SR) was </=2.2, polymerase chain reaction for factor V mutation (Leiden) was performed. Heterozygosity for the mutation was found in 18 patients (14.4%) compared with seven heterozygous among 125 control group (5. 6%; P < 0.05). Acquired APCR (APC-SR 1.8 and Leiden negative) was revealed in seven patients (5.6%) in the study group and in three of the controls (2.4%; not significant). The rate of preclinical pregnancy losses (17/48) and second trimester miscarriages (10/48) in mutation carriers was significantly higher than in patients with no APCR (25/214) and (14/214) respectively (P < 0.001 and P < 0.01 respectively). Live birth rate was not different between the two groups. Occurrence of APCR with any kind of pregnancy loss calculated per patient, in our study group, was approximately 1/7, 1/4 and 1/5 with one, two and three or more pregnancy losses respectively. These findings suggest that assessment of APCR should be considered in a more extended evaluation of such patients.

Activated Protein C Resistance: Effect of Platelet Activation, Platelet-Derived Microparticles, and Atherogenic Lipoproteins

AbstractPlasma and platelet factor Va represent different substrates for activated protein C (APC). In this study, we have measured platelet-dependent APC resistance and the effect of aspirin and a platelet glycoprotein IIbIIIa antagonist (GR144053F) on this phenomenon. In platelet rich plasma (PRP), progressive APC resistance was observed with increasing platelet activation. APC sensitivity ratios of 1.8, 1.7, and 1.4 were observed after platelet activation with thrombin receptor activating peptide (TRAP), collagen, and A23187, respectively. Ultracentrifugation at 77,000g for 1 hour abolished APC resistance indicating that the phenotype is associated exclusively with the platelet membrane. APC resistance was not observed in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles or purified human plasma lipoproteins. APC resistance was observed in the presence of platelet-derived microparticles, but to a lesser degree than that in the presence of activated platelets. The platelet-dependent APC resistance phenotype was also observed when endogenous APC was generated by Protac (American Diagnostica, Inc, Greenwich, CT). In vitro inhibition of platelet activation with aspirin had no effect, but the fibrinogen receptor antagonist, GR144053F, inhibited platelet-dependent APC resistance. These results indicate that platelet activation results in an APC-resistant phenotype comparable to that observed in the plasma of patients with factor V gene mutations affecting critical APC cleavage sites. This suggests that platelet activation at the site of endothelial damage downregulates a critical natural anticoagulant mechanism. The antithrombotic effect of aspirin may be due to an indirect effect on platelet-dependent APC resistance with reduced platelet retention within a developing thrombus. The more potent antithrombotic effect of glycoprotein IIbIIIa antagonists may in addition be the result of reduced platelet factor Va expression and modulation of the platelet-dependent APC resistance phenotype.

Activated Protein C Resistance: Effect of Platelet Activation, Platelet-Derived Microparticles, and Atherogenic Lipoproteins

Plasma and platelet factor Va represent different substrates for activated protein C (APC). In this study, we have measured platelet-dependent APC resistance and the effect of aspirin and a platelet glycoprotein IIbIIIa antagonist (GR144053F) on this phenomenon. In platelet rich plasma (PRP), progressive APC resistance was observed with increasing platelet activation. APC sensitivity ratios of 1.8, 1.7, and 1.4 were observed after platelet activation with thrombin receptor activating peptide (TRAP), collagen, and A23187, respectively. Ultracentrifugation at 77,000g for 1 hour abolished APC resistance indicating that the phenotype is associated exclusively with the platelet membrane. APC resistance was not observed in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles or purified human plasma lipoproteins. APC resistance was observed in the presence of platelet-derived microparticles, but to a lesser degree than that in the presence of activated platelets. The platelet-dependent APC resistance phenotype was also observed when endogenous APC was generated by Protac (American Diagnostica, Inc, Greenwich, CT). In vitro inhibition of platelet activation with aspirin had no effect, but the fibrinogen receptor antagonist, GR144053F, inhibited platelet-dependent APC resistance. These results indicate that platelet activation results in an APC-resistant phenotype comparable to that observed in the plasma of patients with factor V gene mutations affecting critical APC cleavage sites. This suggests that platelet activation at the site of endothelial damage downregulates a critical natural anticoagulant mechanism. The antithrombotic effect of aspirin may be due to an indirect effect on platelet-dependent APC resistance with reduced platelet retention within a developing thrombus. The more potent antithrombotic effect of glycoprotein IIbIIIa antagonists may in addition be the result of reduced platelet factor Va expression and modulation of the platelet-dependent APC resistance phenotype.

A Reduced Sensitivity for Activated Protein C in the Absence of Factor V Leiden Increases the Risk of Venous Thrombosis

AbstractActivated protein C (APC) resistance caused by the factor V Leiden mutation is associated with an increased risk of venous thrombosis. We investigated whether a reduced response to APC, not due to the factor V point mutation, is also a risk factor for venous thrombosis. For this analysis, we used the Leiden Thrombophilia Study (LETS), a case-control study for venous thrombosis including 474 patients with a first deep-vein thrombosis and 474 age- and sex-matched controls. All carriers of the factor V Leiden mutation were excluded. A dose-response relationship was observed between the sensitivity for APC and the risk of thrombosis: the lower the normalized APC sensitivity ratio, the higher the associated risk. The risk for the lowest quartile of normalized APC-SR (<0.92), which included 16.5% of the healthy controls, compared with the highest quartile (normalized APC-SR > 1.05) was greater than fourfold increased (OR = 4.4; 95% confidence interval, 2.9 to 6.6). We adjusted for VIII:C levels, which appeared to affect our APC resistance test. The adjusted (age, sex, FVIII:C) odds ratio for the lowest quartile was 2.5 (95% confidence interval, 1.5 to 4.2). So, after adjustment for factor VIII levels, a reduced response to APC remained a risk factor. Our results show that a reduced sensitivity for APC, not caused by the factor V Leiden mutation, is a risk factor for venous thrombosis.

Changes of Resistance to Activated Protein C in the Course of Pregnancy and Prevalence of Factor V Mutation

Summary: The purpose of this study was to examine the changes in activated protein C (APC) anticoagulant activity during pregnancy and determine whether changes in APC could contribute to thrombosis in the placental bed in preeclampsia. We measured APC anticoagulant activity in 150 women with a normal pregnancy and 50 women with preeclampsia. There was a significant reduction in the mean APC sensitivity ratio (APC-SR) during pregnancy (p<0.001). APC resistance in preeclampsia was significantly higher than in normal pregnancy (p<0.01). Amongst women with APC resistance the presence of the factor V Leiden mutation was significantly higher in the preeclampsia group than in the normal pregnancy group (p<0.01). It seems that both factor V Leiden mutation and APC resistance may be associated with the development of preeclampsia. These results suggest that APC resistance may be an important mechanism underlying placental bed pathology in pregnancy and may be associated with an increased tendency to develop preeclampsia in some women. Assay of APC resistance and factor V Leiden mutation should be performed in women with preeclampsia.

Prediction of Deep Vein Thrombosis after Elective Hip Replacement Surgery by Preoperative Clinical and Haemostatic Variables: The ECAT DVT Study

The European Concerted Action on Thrombosis (ECAT) DVT Study was a collaborative study of preoperative haemostatic tests in prediction of DVT (diagnosed by routine bilateral venography) after elective hip replacement. 480 patients were recruited in 11 centres across Europe. Clinical risk factors were assessed, and stored citrated plasma aliquots were centrally assayed for 29 haemostatic factors according to the ECAT methodology. 120 (32%) of 375 evaluable patients had DVT, and 41 (11%) had proximal DVT. Among clinical variables, DVT was significantly associated with increased age, obesity, and possibly non-use of stockings. Of the 29 haemostatic factors, mean preoperative levels were significantly higher in patients with subsequent DVT (on univariate analyses) for factor VIII activity, prothrombin fragment F1+2, thrombin-antithrombin complexes, and fibrin D-dimer; and significantly lower for APTT and APC sensitivity ratio. Factor V Leiden was also associated with DVT. Most of these variables were also associated with age, while D-dimer was higher in patients with varicose veins. On multivariate analyses including clinical variables, only a shorter APTT (locally but not centrally performed) and APC resistance showed a statistically significant association with DVT. We conclude that (a) DVT is common after elective hip replacement despite prophylaxis; (b) the study provides some evidence that DVT is associated with a preoperative hypercoaguable state; and (c) preoperative haemostatic tests do not add significantly to prediction of DVT from clinical variables, with the possible exception of APC resistance.

Changes of resistance to activated protein C in the course of pregnancy and prevalence of factor V mutation.

The purpose of this study was to examine the changes in activated protein C (APC) anticoagulant activity during pregnancy and determine whether changes in APC could contribute to thrombosis in the placental bed in preeclampsia. We measured APC anticoagulant activity in 150 women with a normal pregnancy and 50 women with preeclampsia. There was a significant reduction in the mean APC sensitivity ratio (APC-SR) during pregnancy (p<0.001). APC resistance in preeclampsia was significantly higher than in normal pregnancy (p<0.01). Amongst women with APC resistance the presence of the factor V Leiden mutation was significantly higher in the preeclampsia group than in the normal pregnancy group (p<0.01). It seems that both factor V Leiden mutation and APC resistance may be associated with the development of preeclampsia. These results suggest that APC resistance may be an important mechanism underlying placental bed pathology in pregnancy and may be associated with an increased tendency to develop preeclampsia in some women. Assay of APC resistance and factor V Leiden mutation should be performed in women with preeclampsia.

Phenprocoumon-Induced Hepatitis Delaying Precise Diagnosis in a Thrombophilic Patient With Activated Protein C Resistance Due to Factor V R506Q Mutation

A 38-year-old woman was admitted to the hospital with signs of deep venous thrombosis in the right calf. Phlebography confirmed the diagnosis. Continuous intravenous heparin was instituted, followed by oral anticoagulation with phenprocoumon. Two months later, symptoms of deep venous thrombosis in the right calf recurred when oral anticoagulation was not in the therapeutic range (thromboplastin time 40% to 50%, INR not available). Four months after having started with phenprocoumon, she developed jaundice and elevated liver enzymes. Alanine aminotransferase (ALT) was 3,330 U/L (normal o37 U/L), aspartate aminotransferase (AST) 2,090 U/ L (normal o36 U/L), alkaline phosphatase (AP) 272 U/L (normal o108 U/L), g-glutamyl-transpeptidase (g-GT) 357 U/L (normal o45 U/L), and bilirubin 526 mmol/L (normal o18.8 mmol/L). Albumin was in the normal range (38 g/L). She had no history of alcohol or drug abuse and had never had blood transfusions. Apart from positive anti-HBc and anti-HBs due to hepatitis B infection 18 years earlier, serological tests revealed no evidence for active hepatitis A, B, or C, or cytomegalovirus or Epstein-Barr virus infection. Anticoagulation was stopped in order to perform a liver biopsy, which was, however, subsequently refused by the patient. Oral anticoagulation was not resumed, and elevated liver enzymes returned to normal values within 3 months. At the age of 43 years she was admitted to the hospital for replacement of her pacemaker, which had been implanted 6 years before because of thirddegree heart block. Two days after hospital discharge she was readmitted with clinical signs of right subclavian vein thrombosis and pulmonary embolism. Duplex sonography and pulmonary perfusion/ ventilation scintigraphy confirmed the diagnosis. Therapy with intravenous heparin was started, accompanied by phenprocoumon. Five weeks later, the patient complained of nausea and fatigue and became jaundiced. Laboratory values were as follows: ALT 1,724 U/L, AST 1,219 U/L, AP 200 U/L, gGT 326 U/L, and bilirubin 128 mmol/L. Again, albumin was in the normal range (37 g/L), and serological tests did not show any signs of recent viral infection. Phenprocoumon was stopped, and oral anticoagulation was continued with acenocoumarol. Three weeks later, the patient had clinically improved, and elevated liver values were decreasing: ALT 672 U/L, AST 591 U/L, AP 196 U/L, g-GT 244 U/L, and bilirubin 88 mmol/L. Acenocoumarol was continued for 2 months. One month after withdrawal of acenocoumarol, the patient was investigated for thrombophilia. At that time, ALT (51 U/L), AST (49 U/L), and bilirubin (28 mmol/L) were still slightly elevated, whereas albumin, AP, and g-GT were in the normal range. Results (Table) showed reduced levels of protein C, protein S, and antithrombin III, as well as a decreased APC sensitivity ratio indicating APC resistance, whereas plasminogen was within normal limits (0.79 U/mL) and anticardiolipin antibodies were not detected. Routine coagulation parameters (thromboplastin time 78%, thrombin time 12 sec, fibrinogen 2.7 g/L, factor V clotting activity [FV:C] 0.88 U/mL, FVII:C 0.75 U/mL, and FX:C 0.70 U/mL) were within the normal range, whereas FII:C (0.53 U/mL) was slightly reduced. During the following months, plasma concentration of FII:C and the coagulation inhibitors returned to normal values (Table). However, the APC sensitivity ratio remained decreased, and genetic analysis revealed heterozygous FV R506Q mutation (factor V Leiden mutation). The patient is now under prophylactic anticoagulation with daily subcutaneous applications of a low molecular weight heparin and has remained well for the last 20 months.

HIGH PREVALENCE OF ANTITHROMBIN III, PROTEIN C AND PROTEIN S DEFICIENCY, BUT NO FACTOR V LEIDEN MUTATION IN VENOUS THROMBOPHILIC CHINESE PATIENTS IN TAIWAN

We studied the prevalence of antithrombin III (AT III), protein C (PC) and protein S (PS) deficiencies and factor V Leiden mutation in thrombophilia in Taiwan. Eighty-five consecutive and unrelated patients with otherwise unexplained venous thrombophilia were studied. Both antigen and activity of inhibitors were determined using commercial kits (Stago), activated PC sensitivity ratio (APC SR) by Coatest (Chromogenix), and factor V mutation by polymerase chain reaction with sequence specific primer. Of 85 patients, 41 were male, 44 female, and mean age 49.4 years (17–82 years). None had factor V mutation, or APC SR of less than 2; 50 (58.8%) showed a deficiency of inhibitor proteins; 34 (68.0%) were hereditary, 16 (32.0%) non-hereditary; 3 had an AT III deficiency, 16 a PC deficiency, 28 a PS deficiency, and 3 a combined deficiency. Thirty-five were non-deficient without a known cause. The average age at the first thrombotic episode was 48.5 years (13–81 years). Thrombosis occurred spontaneously in 39 (78.0%) of 50 deficient patients. In conclusion, a relatively higher prevalence of AT III, PC and PS deficiency (59%), but no factor V Leiden mutation, was found in venous thrombophilic Chinese patients in Taiwan compared to that in western countries. Screening for inhibitor protein deficiency in Chinese thrombophilic patients is highly recommended. © 1997 Elsevier Science Ltd

Effect of activated protein C on thrombin generation and on the thrombin potential in plasma of normal and APC-resistant individuals.

In this paper we describe the effect of activated protein C (APC) on thrombin generation initiated in platelet-poor plasma via the extrinsic or the intrinsic pathway. Thrombin was determined with a specific chromogenic substrate and quantitated by calculating the time integral of the thrombin generation curve, i.e. the endogenous thrombin potential (ETP). Addition of APC to normal plasma after both extrinsic and intrinsic initiation of coagulation resulted in a dose-dependent inhibition of thrombin generation as reflected by the decrease in ETP. Data obtained in intrinsically triggered plasma of normal individuals were subject to large variation. Therefore, the effect of APC on thrombin generation in APC-resistant plasmas was only studied in extrinsically stimulated reaction systems. APC had much less effect on the ETP of plasma from individuals that were heterozygous or homozygous for the mutation Arg506-->Gln506 in factor V (APC resistance). There appears to be a linear relationship between the ETP and the amount of alpha 2-macro-globulin-thrombin complex (alpha 2 M-IIa) that accumulates in plasma during thrombin formation. Since the alpha 2M-IIa complex possesses amidolytic activity, we measured the effect of APC on thrombin generation via the so-called normalized APC sensitivity ratio (APC-sr). The latter was defined as the ratio of the end levels of amidolytic activity of the alpha 2M-IIa complex determined in the presence and absence of 50 nM APC (alpha 2M-IIa + APC/(alpha 2M-IIa - APC) divided by the ratio of a normal plasma pool. Significant differences (P < 0.001) were observed between APC-sr of plasmas from normal individuals (APC-sr: 0.5-1.9, n = 25) and of plasmas from individuals that were heterozygous (APC-sr: 2.1-6.7, n = 17) or homozygous APC resistant (APC-sr: 3.9-5.9, n = 5). There was no overlap between APC-sr of normal plasmas and plasma from individuals, bearing the factor. V mutation. Abnormal APC-sr in certain plasmas (pregnancy, use of oral contraceptives, anticoagulant therapy, protein S deficiency or lupus anticoagulant) were corrected by performing the assay on a plasma sample that was diluted 10-fold in factor V-deficient plasma. Our data show that measurement of the effect of APC on the ETP yields valuable information about the (pro)thrombotic status of plasma (e.g. APC resistance, pregnancy, use of oral contraceptives).

Activated-protein-C resistance in cancer patients.

Resistance to activated protein C (aPC) is usually linked to factor V Leiden, but may occur in other disorders associated with hypercoagulability. In this study, we investigated the frequency of resistance to aPC in patients with advanced cancer and examined the relationship of aPC resistance to other markers of coagulation activation. Patients (n = 39) had established diagnosis of advanced cancer; controls (n = 20) were healthy persons. aPC resistance was measured as the ratio of activated partial thromboplastin times with and without aPC (aPC-sensitivity ratio, aPC-SR). The factor V Leiden mutation was detected by a polymerase-chain-reaction based technique. Other assays were performed by standard laboratory methods. Data were analyzed using t tests and the Pearson correlation. aPC-SR was below 2 SD for 5 of the cancer patients (13%), but none of the controls; only 1 of the 5 had the factor V Leiden mutation. aPC-SR was inversely correlated (p < 0.01) with factor VIII and fibrinogen in patients and with prothrombin activation fragment 1.2 (F1.2) in controls. Patient factor VIII, von Willebrand factor, (vWF), fibrinogen, F1.2 and D dimer were all significantly increased (p < 0.01; antithrombin III, protein C and proteins were similar to controls. Factor VIII correlated with vWF (p < 0.001) and F1.2 with d-dimer (p < 0.001). Other associations (p < 0.05) were observed between factor V and protein C, fibrinogen and protein C, factor V and antithrombin III and protein C and antithrombin III. Four cancer patients had a history of thromboembolism; their aPC-SR was similar to that of patients without thrombosis. Of the several coagulation measures examined, only vWF was higher in the patients with thrombosis (p = 0.01). Cancer patients have evidence of intravascular coagulation and increases in procoagulants and may have aPC resistance. The aPC resistance is not due to factor V Leiden, but is rather associated with elevated levels of factor VIII and fibrinogen, and in itself does not predict thrombosis.

Molecular Characterization of a Type I Quantitative Factor V Deficiency in a Thrombosis Patient that Is “Pseudo Homozygous” for Activated Protein C Resistance

Resistance to activated protein C (APC), which is associated with the FV Leiden mutation in the large majority of the cases, is the most common genetic risk factor for thrombosis. Several laboratory tests have been developed to detect the APC-resistance phenotype. The result of the APC-resistance test (APC-sensitivity ratio, APC-SR) usually correlates well with the FV Leiden genotype, but recently some discrepancies have been reported. Some thrombosis patients that are heterozygous for FV Leiden show an APC-SR usually found only in homozygotes for the defect. Some of those patients proved to be compound heterozygotes for the FV Leiden mutation and for a type I quantitative factor V deficiency. We have investigated a thrombosis patient characterized by an APC-SR that would predict homozygosity for FV Leiden. DNA analysis showed that he was heterozygous for the mutation. Sequencing analysis of genomic DNA revealed that the patient also is heterozygous for a G5509-->A substitution in exon 16 of the factor V gene. This mutation interferes with the correct splicing of intron 16 and leads to the presence of a null allele, which corresponds to the "non-FV Leiden" allele. The conjunction of these two defects in the patient apparently leads to the same phenotype as observed in homozygotes for the FV Leiden mutation.

Resistance to activated protein C, associated with oral contraceptives use; Effect of formulations, duration of assumption, and doses of oestro-progestins

Resistance to activated protein C (APC-R) is at present considered the most frequent laboratory abnormality in patients with deep vein thrombosis. An increased risk for venous thrombosis is associated with the use of oral contraceptives (OCs). We recently described a statistically significant association between APC-R status and oral contraceptives use in a healthy group of women. We re-evaluated 50 healthy women taking low-dose combination OCs in order to consider a possible correlation between the APC sensitivity ratio (APC-SR) and different oral contraceptive formulations. Seven women showed an APC ratio ≤2 (APC-resistant). Only one of the seven women was found to be heterozygous for Leiden factor V mutation. We observed no significant differences between normally sensitive and APC-resistant women in terms of duration of OC use, amount of estrogenic or progestogenic dose, or type of formulation. We conclude that APC-resistance associated with oral contraceptives use seems to occur only in predisposed subjects (in our results, about 12% of the healthy population).

Clinical significance of activated protein C resistance as a potential marker for hypercoagulable state

The activated protein C (APC)-resistance test is a simple and reliable method for detecting reduced sensitivity to the anticoagulant action of this protein. We investigated the sensitivity to APC in 180 Japanese controls and in 96 Japanese patients with venous and arterial thrombosis (28 with deep vein thrombosis; 13 with pulmonary thromboembolism; 41 with cerebral infarction; and 14 with coronary artery disease). All of the patient groups showed significantly reduced sensitivity to APC, reflected by the lower normalized APC-sensitivity ratio (n-APC-SR), as compared with healthy control. The APC-sensitivity ratio was negatively correlated with plasma activated factor VII levels. These results suggest that the low n-APC-SR is related to venous or arterial thrombotic disease. The APC resistance may serve as a potential marker for assessing the hypercoagulable state.

Resistance to activated protein C in an unselected population of patients with pulmonary embolism

SummaryBackground Resistance to activated protein C (APC) is the most frequent cause of inherited thrombophilia. This phenomenon has been reported in 10-50% of selected patients with venous thromboembolism, a variation that might result from different degrees of selection in different reports.Methods We measured the APC sensitivity ratio in 494 frozen blood samples from unselected consecutive outpatients suspected of pulmonary embolism and referred over a 30-month period to the emergency ward of the University Hospital of Geneva, the only public primary-tertiary care hospital in the region of Geneva (400 000 inhabitants).Findings Prevalence of resistance to APC was 5·5% (95% Cl 2·4-10·5%) (8/146) in patients with confirmed pulmonary embolism and 4·0% (2·2-6·7%) (14/348) in patients in whom the diagnosis could be ruled out (p=0·66), giving an odds ratio of i·36 (0·56-3·32).Interpretation The very different risks of venous thromboembolism in the presence of resistance to APC that have been reported in trials published so far are probably due to variable recruitment conditions. The lower prevalence observed in our totally unselected population of patients with pulmonary embolism may be more representative of the real risk with which clinicians will be confronted. Therefore, more data are needed from various populations of patients with venous thromboembolism to help decide which patients will benefit from screening for resistance to APC.

Effect of platelet phospholipid exposure on activated protein C resistance: implications for thrombophilia screening.

The effect of platelet contamination and freeze-thawing on the activated protein C sensitivity ratio (APCsr) was determined. With increasing platelet count there was a progressive reduction in the ratio. Filtration of samples through a 0.2 microm filter before or after freeze-thawing abolished the development of resistance to the addition of activated protein C indicating that the phenomenon is due to the presence of a particulate factor. Contamination of normal plasma with platelets from a patient with homozygous factor V (FV) deficiency was also associated with the same development of resistance to activated protein C, indicating that the phenomenon was not due to exposure of platelet-derived factor V that might be inaccessible to APC. 82% (96/117) of FVQ506 and 32% (138/430) of FVR506 individuals had APC resistance on analysis of unfiltered plasma. However, 85% (42/50) of FVQ506 individuals had APC resistance on analysis of filtered plasma, whilst only 1/50 FV R506 individuals had APC resistance after filtration. For the purpose of identifying individuals at increased risk of venous thromboembolism due to the presence of the FVQ506 and associated APC resistance a PCR-based genotypic analysis is recommended.

Activated protein C sensitivity ratio in pregnant women at delivery

We studied activated protein C sensitivity ratio (APC‐SR), factors V and VIII activity and von Willebrand antigen in control women, women using oral contraceptives, and pregnant women at delivery. The mean APC‐SR of 2.4 in pregnant women was significantly lower than the mean APC‐SR value of 3.5 for both the other groups and 45% of pregnant women had a ratio below the 5th percentile of the control group. None of them carried the R506→Q mutation. This decreased ratio at delivery appeared to be connected, at least in part, with increased VIII activity. Thus, APC‐SR at delivery should not be used to detect APC resistance.