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 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.

Activated Protein C-Resistance Induced by a Low Molecular Weight Inhibitor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2136-2136
Author(s):  
Michael Kalafatis ◽  
Rihard E. Chiott ◽  
Richard F. Branda ◽  
Kenneth G. Mann
Keyword(s):  
Molecular Weight ◽  
Protein C ◽  
Activated Protein C ◽  
Low Molecular Weight ◽  
Factor V ◽  
Prolonged Incubation ◽  
Apc Resistance ◽  
Factor Va ◽  
Normal Plasma ◽  
Plasma Factor

Abstract Activated protein C (APC) inactivates factor Va (fVa) following three cleavages in the heavy chain at R506, R306 and R679. Cleavage at R506 precedes cleavage at R306. Cleavage at Arg306 is strictly lipid-dependent and results in total inactivation of the factor Va molecule with dissociation of fragments from the A2 domain from the rest of the molecule. Factor VLeiden is associated with an R→Q substitution at position 506 and is present at approximately 8% of the Caucasian population. The heterozygous presentation of factor VLeiden results in delayed inactivation of factor Va and “APC-resistance” with attendant increased risk of venous thrombosis. However, not all cases of “APC-resistance” are explained by factor VLeiden. We observed “APC-resistance” in a patient displaying heterozygous factor VLeiden, Waldenstrom’s macroglobulinenemia, systemic lupus erythrematosus (anticoagulant) and a history of coronary artery disease. The patient’s plasma resistance to APC inactivation was not repaired by immunodepletion of his factor VLeiden and replacement by normal plasma factor V. Conversely when the patient’s fVa was returned to factor V immunodepleted normal plasma it did not display APC-resistance. Cleavage of the patient’s plasma fVa at R306 was not detected following prolonged incubation of his clotted plasma at 37°C even when 2 nM APC was added following clotting. These data suggested that the APC-resistance observed in the patient was not due to the presence of factor VLeiden, but due to some property which inhibited the lipid dependent cleavage at Arg306. The patient’s plasma was depleted of IgG/IgM and the purified immunoglobulin fraction assessed for inhibition of APC cleavage and inactivation of fVa in a system using purified reagents. The data were compared with the inhibition of fVa inactivation by APC by an IgG/IgM fraction obtained from normal plasma under similar experimental conditions. No inhibition of APC cleavage and inactivation of fVa by the IgM/IgG fraction obtained from either plasma were observed. In addition, the fVa molecule contained in the IgM/IgG-depleted patient plasma was still resistant to cleavage and inactivation by APC. Following dialysis the patient’s plasma lost its ability to inhibit fVa cleavage and inactivation by APC. Overall these studies indicate that inhibition of fVa cleavage and inactivation by APC in the patient’s plasma is caused by a hitherto undescribed metabolite of low molecular weight. The mechanism of action of this metabolite is not yet known, but the evidence suggests that the metabolite interferes with the lipid-dependent cleavage and inactivation of fVa by APC at R306. These data demonstrate the existence of an as yet unknown APC inhibitor of low molecular weight in the plasma of a patient with lupus anticoagulant and severe thrombotic symptoms.

Multicenter Evaluation of a Kit for Activated Protein C Resistance on Various Coagulation Instruments Using Plasmas from Healthy Individuals

1994 ◽  
Vol 72 (02) ◽  
pp. 255-60 ◽  
Author(s):  
S Rosén ◽  
K Johansson ◽  
K Lindberg ◽  
B Dahlbäck ◽  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor V ◽  
Clotting Time ◽  
Apc Resistance ◽  
Normal Plasma ◽  
The Individual ◽  
Anticoagulant Response ◽  
Hemostatic Disorder

SummaryRecently a new hemostatic disorder has been described which appears to be an important risk factor for familial thromboembolism. The disorder is characterized by a poor anticoagulant response to activated Protein C (APC) and has been shown to be due to lack of an APC cofactor activity which is a property of factor V.A kit for determining the response of plasma samples towards addition of APC in an APTT-based assay - COATEST APC Resistance -has been evaluated on 35 coagulation instruments in a multicenter study involving 32 laboratories. A lyophilized normal plasma and identical plasma aliquots from 20 individuals, one of whom had a borderline resistance to APC, were analysed in each laboratory and the sensitivity of each plasma to APC was determined as the ratio between the clotting times obtained in the presence and absence of APC (APC ratio).The plasma from the individual with a borderline resistance to APC activity was correctly classified as the lowest responder in each laboratory, with an APC ratio in the range 1.6-2.4. In comparison, plasmas from individuals with a pronounced response to APC activity resulted in APC ratios above 3.4 in most cases. Interestingly, although the actual APT time for a plasma from a given individual showed a more than 10 s difference due to the type of instrumentation used, the variation in the APC ratio was limited. A similar discrimination was also obtained from evaluation of the actual prolongation of the clotting time in the presence of APC.The intra-laboratory coefficient of variation for the clotting times were on average 2.0% and 3.9% in the absence and presence of APC, respectively, indicating that the precision for the prolonged clotting times obtained in the presence of APC is sufficient to allow a safe assignment of the APC response. The APC ratio for the lyophilized normal plasma was 2.7 ± 0.2 (2 S.D.) illustrating a narrow distribution between instruments which shows the feasibility of including such plasma for assay validation. Altogether, the results indicate that all the coagulation instruments included in the study can be used for detection of individuals with resistance to APC activity through determination of the APC ratio or the prolongation time.

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.

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

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.

Isolation and characterization of an antifactor V antibody causing activated protein C resistance from a patient with severe thrombotic manifestations

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 3985-3992 ◽  
Author(s):  
Michael Kalafatis ◽  
Paolo Simioni ◽  
Daniela Tormene ◽  
Daniel O. Beck ◽  
Sonia Luni ◽  
...  
Keyword(s):  
Antiphospholipid Antibodies ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Factor V ◽  
Apc Resistance ◽  
Factor Va ◽  
Isolation And Characterization ◽  
Sensitivity Ratio ◽  
Total Immunoglobulin

A 44-year-old woman with a history of severe thrombotic manifestations presented with a markedly reduced activated protein C–sensitivity ratio (APC-SR). DNA sequencing of and around the regions encoding the APC cleavage sites in the factor Va molecule excluded the presence of the factor VLeiden mutation and of other known genetic mutations. No antiphospholipid antibodies were present in the patient's plasma and both prothrombin time and activated partial thromboplastin time were normal. The total immunoglobulin fraction was isolated from the patient's plasma and found to induce severe APC resistance when added to normal plasma and to factor V–deficient plasma supplemented with increasing concentrations of factor V. Immunoblotting and immunoprecipitation experiments with the total immunoglobulin fraction purified from the patient's plasma demonstrated that the antibody recognizes factor V, is polyclonal, and has conformational epitopes on the entire factor V molecule (heavy and light chains, and B region). Thus, the immunoglobulin fraction interferes with the anticoagulant pathway involving factor V. The inhibitor was isolated by sequential affinity chromatography on protein G–Sepharose and factor V–Sepharose. The isolated immunoglobulin fraction inhibited factor Va inactivation by APC because of impaired cleavage at Arg306 and Arg506 of the heavy chain of the cofactor. The isolated immunoglobulin fraction was also found to inhibit the cofactor effect of factor V for the inactivation of factor VIII by the APC/protein S complex. Our data provide for the first time the demonstration of an antifactor V antibody not related to the presence of antiphospholipid antibodies, which is responsible for thrombotic rather than hemorrhagic symptoms.

Dermatan Sulfate and LMW Heparin Enhance the Anticoagulant Action of Activated Protein C

1999 ◽  
Vol 82 (11) ◽  
pp. 1462-1468 ◽  
Author(s):  
José Fernández ◽  
Jari Petäjä ◽  
John Griffin
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Protein C ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor V ◽  
Factor Va ◽  
Anticoagulant Action

SummaryUnfractionated heparin potentiates the anticoagulant action of activated protein C (APC) through several mechanisms, including the recently described enhancement of proteolytic inactivation of factor V. Possible anticoagulant synergism between APC and physiologic glycosaminoglycans, pharmacologic low molecular weight heparins (LMWHs), and other heparin derivatives was studied. Dermatan sulfate showed potent APC-enhancing effect. Commercial LMWHs showed differing abilities to promote APC activity, and the molecular weight of LMWHs correlated with enhancement of APC activity. Degree of sulfation of the glycosaminoglycans influenced APC enhancement. However, because dextran sulfates did not potentiate APC action, the presence of sulfate groups per se on a polysaccharide is not sufficient for APC enhancement. As previously for unfractionated heparin, APC anticoagulant activity was enhanced by glycosaminoglycans when factor V but not factor Va was the substrate. Thus, dermatan sulfate and LMWHs exhibit APC enhancing activity in vitro that could be of physiologic and pharmacologic significance.

“Pseudo Homozygous” Activated Protein C Resistance due to Double Heterozygous Factor V Defects (Factor V Leiden Mutation and Type I Quantitative Factor V Defect) Associated with Thrombosis: Report of Two Cases Belonging to Two Unrelated Kindreds

1996 ◽  
Vol 75 (03) ◽  
pp. 422-426 ◽  
Author(s):  
Paolo Simioni ◽  
Alberta Scudeller ◽  
Paolo Radossi ◽  
Sabrina Gavasso ◽  
Bruno Girolami ◽  
...  
Keyword(s):  
Protein C ◽  
Dna Analysis ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Type I ◽  
Factor V ◽  
Factor V Leiden Mutation ◽  
Thrombotic Risk ◽  
Apc Resistance ◽  
Quantitative Factor

SummaryTwo unrelated patients belonging to two Italian kindreds with a history of thrombotic manifestations were found to have a double heterozygous defect of factor V (F. V), namely type I quantitative F. V defect and F. V Leiden mutation. Although DNA analysis confirmed the presence of a heterozygous F. V Leiden mutation, the measurement of the responsiveness of patients plasma to addition of activated protein C (APC) gave results similar to those found in homozygous defects. It has been recently reported in a preliminary form that the coinheritance of heterozygous F. V Leiden mutation and type I quantitative F. V deficiency in three individuals belonging to the same family resulted in the so-called pseudo homozygous APC resistance with APC sensitivity ratio (APC-SR) typical of homozygous F. V Leiden mutation. In this study we report two new cases of pseudo homozygous APC resistance. Both patients experienced thrombotic manifestations. It is likely that the absence of normal F. V, instead of protecting from thrombotic risk due to heterozygous F. V Leiden mutation, increased the predisposition to thrombosis since the patients became, in fact, pseudo-homozygotes for APC resistance. DNA-analysis is the only way to genotype a patient and is strongly recommended to confirm a diagnosis of homozygous F. V Leiden mutation also in patients with the lowest values of APC-SR. It is to be hoped that no patient gets a diagnosis of homozygous F. V Leiden mutation based on the APC-resi-stance test, especially when the basal clotting tests, i.e., PT and aPTT; are borderline or slightly prolonged.

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

1997 ◽  
Vol 77 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Joan F Guasch ◽  
Ruud P M Lensen ◽  
Rogier M Bertina
Keyword(s):  
Protein C ◽  
Dna Analysis ◽  
Activated Protein C ◽  
Type I ◽  
Factor V ◽  
Sequencing Analysis ◽  
Apc Resistance ◽  
Quantitative Factor ◽  
Factor V Deficiency ◽  
Fv Leiden

SummaryResistance 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.

scholarly journals Factor V Is an Anticoagulant Cofactor for Activated Protein C during Inactivation of Factor Va

2009 ◽  
Vol 37 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Thomas J. Cramer ◽  
John H. Griffin ◽  
Andrew J. Gale
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor V ◽  
Factor Va
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