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

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3792-3797 ◽  
Author(s):  
Janis Taube ◽  
Nicola McWilliam ◽  
Roger Luddington ◽  
Christopher D. Byrne ◽  
Trevor Baglin
Keyword(s):  
Platelet Activation ◽  
Protein C ◽  
Activated Protein C ◽  
Plasma Lipoproteins ◽  
Platelet Glycoprotein ◽  
Antithrombotic Effect ◽  
Platelet Factor ◽  
Resistance Phenotype ◽  
Apc Resistance ◽  
Factor Va

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.

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

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3792-3797 ◽  
Author(s):  
Janis Taube ◽  
Nicola McWilliam ◽  
Roger Luddington ◽  
Christopher D. Byrne ◽  
Trevor Baglin
Keyword(s):  
Platelet Activation ◽  
Protein C ◽  
Activated Protein C ◽  
Plasma Lipoproteins ◽  
Platelet Glycoprotein ◽  
Antithrombotic Effect ◽  
Platelet Factor ◽  
Resistance Phenotype ◽  
Apc Resistance ◽  
Factor Va

Abstract 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.

scholarly journals A Factor V Genetic Component Differing From Factor V R506Q Contributes to the Activated Protein C Resistance Phenotype

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1552-1557 ◽  
Author(s):  
F. Bernardi ◽  
E.M. Faioni ◽  
E. Castoldi ◽  
B. Lunghi ◽  
G. Castaman ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Protein C ◽  
Activated Protein C ◽  
Functional Assay ◽  
Factor V ◽  
Resistance Phenotype ◽  
Apc Resistance ◽  
Genetic Components ◽  
Factor V Gene ◽  
V Gene

AbstractFactor V gene polymorphisms were investigated to detect components that may contribute to the activated protein C (APC) resistance phenotype in patients with venous thromboembolism. A specific factor V gene haplotype (HR2) was defined by six polymorphisms and its frequency was found to be similar in normal subjects coming from Italy (0.08), India (0.1), and Somalia (0.08), indicating that it was originated by ancestral mutational events. The relationship between the distribution of normalized APC ratios obtained with the functional assay and haplotype frequency was analyzed in patients heterozygous for factor V R506Q (factor V Leiden). The HR2 haplotype was significantly more frequent in patients with ratios below the 15th percentile than in those with higher ratios or in normal controls. Moreover, the study of 10 patients with APC resistance in the absence of the factor V R506Q mutation showed a 50-fold higher frequency of HR2 homozygotes. The HR2 haplotype was associated with significantly lower APC ratios both in patients with venous thromboembolism and in age- and sex-matched controls. However, the two groups showed similar HR2 haplotype frequencies. Plasma mixing experiments showed that an artificially created double heterozygote for the factor V R506Q mutation and the HR2 haplotype had an APC ratio lower than that expected for a simple R506Q heterozygote. Time-course experiments evaluating the decay of factor V in plasma showed the normal stability of the molecule encoded by the factor V gene marked by the HR2 haplotype, which ruled out the presence of a pseudo-homozygous APC resistance mechanism. Our results provide new insights into the presence of factor V genetic components other than the factor V R506Q that are able to contribute to the APC resistance phenotype in patients with venous thromboembolism.

Evaluation of a New Assay Based on a FV-Dependent Prothrombinase Activator for the Detection of Activated Protein C Resistance Phenotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3994-3994
Author(s):  
Jogin Wu ◽  
Peter Quehenberger ◽  
Katherine Foltyn ◽  
Patricia Dillard
Keyword(s):  
Protein C ◽  
Tertiary Care ◽  
False Negative ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Reference Method ◽  
Coagulation Cascade ◽  
Factor V ◽  
Resistance Phenotype ◽  
Apc Resistance

Abstract Activated protein C (APC) resistance is the most frequent hereditary defect associated with deep venous thrombosis. Major cause of APC resistance phenotype is due to a point mutation of factor V (Factor V Leiden). A new clotting assay, Pefakit® APC-R Factor V Leiden (Pentapharm Ltd., Switzerland) for the detection of APC resistance phenotype was evaluated at two tertiary care hospitals, Duke University Medical Center, USA (Duke) and University of Vienna, Austria (Vienna). Samples of 242 subjects from Duke and 187 subjects from Vienna were included in the study among patients who were subjects for thormbophilia screening. The Pefakit® method is based on clotting time measurement triggered by a prothrombin activator added to a mixture of patient plasma diluted with factor V deficient plasma with and without APC. Robustness and specificity of the assay is enhanced by elimination of possible disturbing influence by factors upstream the coagulation cascade and heparin interference is precluded up to heparin level of 2 IU/ml by heparin inhibitor added to the regent. A similar, FDA approved commercial APC-R kit (COATEST of IL) was used for method comparison. Patients with elevated factor VIII (n=11), Coumadin (n=23), Lupus Anticoagulant (n-14), protein C deficient (n=7), protein S deficient (n=9), AT III (n=6) and women with pregnancy (n=9) were included in Duke study and no interference were found in phenotype. Using PCR/FRET DNA method as reference method the Pefakit® method provided 100 % sensitivity and 100 % specificity for the Vienna study and 99.0 % sensitivity and 98.6 % specificity for the Duke study and the COATEST provided 97.1 % specificity and 93.2 % sensitivity with the Duke study. Using two levels of genotype controls both studies showed similar intra and inter-assay precision (less than 6 % for the Vienna study and 9 % for the Duke study) as compared with the gold standard IL APC-R COATEST kit (less than 5 % CV). Of great interest one false positive sample from the Duke study is under investigation due to that the functional detection of the assay is supposed to detect other FV mutations leading to APC-R phenotype as well. Reasons that cause the other two false negative results for the Duke study are still unknown and under investigation. Both studies showed that the Pefakit® is simple and rebust assay. Both wild type and heterozygous groups have much higher ratio as compared with the reference method in differentiating them from homozygous phenotype. Figure Figure

scholarly journals A Factor V Genetic Component Differing From Factor V R506Q Contributes to the Activated Protein C Resistance Phenotype

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1552-1557 ◽  
Author(s):  
F. Bernardi ◽  
E.M. Faioni ◽  
E. Castoldi ◽  
B. Lunghi ◽  
G. Castaman ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Protein C ◽  
Activated Protein C ◽  
Functional Assay ◽  
Factor V ◽  
Resistance Phenotype ◽  
Apc Resistance ◽  
Genetic Components ◽  
Factor V Gene ◽  
V Gene

Factor V gene polymorphisms were investigated to detect components that may contribute to the activated protein C (APC) resistance phenotype in patients with venous thromboembolism. A specific factor V gene haplotype (HR2) was defined by six polymorphisms and its frequency was found to be similar in normal subjects coming from Italy (0.08), India (0.1), and Somalia (0.08), indicating that it was originated by ancestral mutational events. The relationship between the distribution of normalized APC ratios obtained with the functional assay and haplotype frequency was analyzed in patients heterozygous for factor V R506Q (factor V Leiden). The HR2 haplotype was significantly more frequent in patients with ratios below the 15th percentile than in those with higher ratios or in normal controls. Moreover, the study of 10 patients with APC resistance in the absence of the factor V R506Q mutation showed a 50-fold higher frequency of HR2 homozygotes. The HR2 haplotype was associated with significantly lower APC ratios both in patients with venous thromboembolism and in age- and sex-matched controls. However, the two groups showed similar HR2 haplotype frequencies. Plasma mixing experiments showed that an artificially created double heterozygote for the factor V R506Q mutation and the HR2 haplotype had an APC ratio lower than that expected for a simple R506Q heterozygote. Time-course experiments evaluating the decay of factor V in plasma showed the normal stability of the molecule encoded by the factor V gene marked by the HR2 haplotype, which ruled out the presence of a pseudo-homozygous APC resistance mechanism. Our results provide new insights into the presence of factor V genetic components other than the factor V R506Q that are able to contribute to the APC resistance phenotype in patients with venous thromboembolism.

scholarly journals The Carbohydrate Moiety of Factor V Modulates Inactivation by Activated Protein C

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4348-4354 ◽  
Author(s):  
José A. Fernández ◽  
Tilman M. Hackeng ◽  
Kazuhisa Kojima ◽  
John H. Griffin
Keyword(s):  
Risk Factor ◽  
Heavy Chain ◽  
Protein C ◽  
Activated Protein C ◽  
Activated Partial Thromboplastin Time ◽  
Resistance Ratio ◽  
Factor V ◽  
Potential Influence ◽  
Apc Resistance ◽  
Factor Va

AbstractAn important risk factor for thrombosis is the polymorphism R506Q in factor V that causes resistance of factor Va to proteolytic inactivation by activated protein C (APC). To study the potential influence of the carbohydrate moieties of factor Va on its inactivation by APC, factor V was subjected to mild deglycosylation (neuraminidase plus N-glycanase) under nondenaturing conditions. The APC resistance ratio values (ratio of activated partial thromboplastin time [APTT] clotting times with and without APC) of the treated factor V were increased (2.4 to 3.4) as measured in APTT assays. O-glycanase treatment of factor V did not change the APC resistance ratio. The procoagulant activity of factor V as well as its activation by thrombin was not affected by mild deglycosylation. Treatment of factor V with neuraminidase and N-glycanase mainly altered the electrophoretic mobility of the factor Va heavy chain, whereas treatment with O-glycanase changed the mobility of the connecting region. This suggests that the removal of the N-linked carbohydrates from the heavy chain of factor Va, which is the substrate for APC, is responsible for the increase in susceptibility to inactivation by APC. Thus, variability in carbohydrate could account for some of the known variability in APC resistance ratios, including the presence of borderline or low APC resistance ratios among patients who lack the R506Q mutation.

scholarly journals The Mechanism of Inactivation of Human Platelet Factor Va from Normal and Activated Protein C-resistant Individuals

1995 ◽  
Vol 270 (35) ◽  
pp. 20794-20800 ◽  
Author(s):  
Rodney M. Camire ◽  
Michael Kalafatis ◽  
Mary Cushman ◽  
Russell P. Tracy ◽  
Kenneth G. Mann ◽  
...  
Keyword(s):  
Protein C ◽  
Human Platelet ◽  
Activated Protein C ◽  
Platelet Factor ◽  
Factor Va

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

2006 ◽  
Vol 95 (02) ◽  
pp. 236-242 ◽  
Author(s):  
Jeroen Brugge ◽  
Guido Tans ◽  
Jan Rosing ◽  
Elisabetta Castoldi
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Protein S ◽  
Healthy Individuals ◽  
Resistance Phenotype ◽  
Apc Resistance ◽  
Thrombosis Risk ◽  
Highly Correlated

SummaryElevated plasma prothrombin levels, due to the prothrombin 20210 G/A mutation or to acquired causes, area risk factor for venous thrombosis,partly because of prothrombin-mediated inhibition of the protein C anticoagulant pathway and consequent activated proteinC (APC) resistance. We determined the effect of plasma prothrombin concentration on the APC resistance phenotype and evaluated the role of protein S levels asa 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 proteinS 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 proteinS confirmed that proteinS 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.

scholarly journals Regulation of Platelet Factor Va-dependent Thrombin Generation by Activated Protein C at the Surface of Collagen-adherent Platelets

2000 ◽  
Vol 276 (10) ◽  
pp. 7164-7168 ◽  
Author(s):  
Jacob J. Briedé ◽  
Guido Tans ◽  
George M. Willems ◽  
H. Coenraad Hemker ◽  
Theo Lindhout
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Platelet Factor ◽  
Factor Va

scholarly journals Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4695-4707 ◽  
Author(s):  
M Kalafatis ◽  
PE Haley ◽  
D Lu ◽  
RM Bertina ◽  
GL Long ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Protein C ◽  
Activated Protein C ◽  
Clot Formation ◽  
Single Amino Acid ◽  
Factor V ◽  
Apc Resistance ◽  
Factor Va ◽  
Normal Plasma ◽  
Membrane Bound

Human factor V is activated to factor Va by alpha-thrombin after cleavages at Arg709, Arg1018, and Arg1545. Factor Va is inactivated by activated protein C (APC) in the presence of a membrane surface after three sequential cleavages of the heavy chain. Cleavage at Arg506 provides for efficient exposure of the inactivating cleavages at Arg306 and Arg679. Membrane-bound factor V is also inactivated by APC after cleavage at Arg306. Resistance to APC is associated with a single nucleotide change in the factor V gene (G1691-->A) corresponding to a single amino acid substitution in the factor V molecule: Arg506-->Gln (factor V Leiden). The consequence of this mutation is a delay in factor Va inactivation. Thus, the success of the APC-resistance assay is based on the fortuitous activation of factor V during the assay. Plasmas from normal individuals (1691 GG) and individuals homozygous for the factor V mutation (1691 AA) were diluted in a buffer containing 5 mmol/L CaCl2, phospholipid vesicles (10 micromol/L), and APC. APC, at concentrations < or = 5.5 nmol/L, prevented clot formation in normal plasma, whereas under similar conditions, a clot was observed in plasma from APC-resistant individuals. Gel electrophoresis analyses of factor V fragments showed that membrane-bound factor V is primarily cleaved at Arg306 in both plasmas. However, whereas in normal plasma production of factor Va heavy chain is counterbalanced by fast degradation after cleavage at Arg506/Arg306, in the APC-resistant individuals' plasma, early generation and accumulation of the heavy chain portion of factor Va occurs as a consequence of delayed cleavage at Arg306. At elevated APC concentrations (>5.5 nmol/L), no clot formation was observed in either plasma from normal or APC-resistant individuals. Our data show that resistance to APC in patients with the Arg506-->Gln mutation is due to the inefficient degradation (inactivation) of factor Va heavy chain by APC.

scholarly journals Blood coagulation factor Va abnormality associated with resistance to activated protein C in venous thrombophilia

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3120-3125 ◽  
Author(s):  
X Sun ◽  
B Evatt ◽  
JH Griffin
Keyword(s):  
Factor Viii ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Laboratory Finding ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Factor V ◽  
Apc Resistance ◽  
Factor Va ◽  
Common Laboratory

Abstract A coagulation test abnormality, termed activated protein C (APC) resistance, involving poor anticoagulant response to APC is currently the most common laboratory finding among venous thrombophilic patients. Because the anticoagulant activity of APC involves inactivation of factors Va and VIIIa, studies were made to assess the presence of abnormal factors V or VIII. Diluted aliquots of plasma from two unrelated patients with APC resistance and thrombosis were added to either factor VIII-deficient or factor V-deficient plasma and APC resistance assays were performed. The results suggested that patients' factor V but not factor VIII rendered the substrate plasma APC resistant. When factor V that had been partially purified from normal or APC resistant patients' plasmas using immunoaffinity chromatography was added to factor V-deficient plasma, APC resistance assays showed that patients' factor V or factor Va, but not normal factor V, rendered the substrate plasma resistant to APC. Studies of the inactivation of each partially purified thrombin-activated factor Va by APC suggested that half of the patients' factor Va was resistant to APC. These results support the hypothesis that the APC resistance of some venous thrombophilic plasmas is caused by abnormal factor Va.

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