Interaction of clotting factor V heavy chain with prothrombin and prethrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation

Biochemistry ◽  
1989 ◽  
Vol 28 (5) ◽  
pp. 2348-2354 ◽  
Author(s):  
Elizabeth A. Luckow ◽  
Daryl A. Lyons ◽  
Theresa M. Ridgeway ◽  
Charles T. Esmon ◽  
Thomas M. Laue
Keyword(s):  
Heavy Chain ◽  
Protein C ◽  
Analytical Ultracentrifugation ◽  
Interaction Analysis ◽  
Activated Protein C ◽  
Clotting Factor ◽  
Factor V

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 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 ptFVa ( Pseudonaja Textilis Venom-Derived Factor Va) Retains Structural Integrity Following Proteolysis by Activated Protein C

2021 ◽  
Author(s):  
Mark Schreuder ◽  
Xiaosong Liu ◽  
Ka Lei Cheung ◽  
Pieter H. Reitsma ◽  
Gerry A.F. Nicolaes ◽  
...  
Keyword(s):  
Protein C ◽  
Structural Integrity ◽  
Noncovalent Interactions ◽  
Activated Protein C ◽  
Regulatory Mechanisms ◽  
Factor V ◽  
Factor Va ◽  
Dynamics Simulations ◽  
A2 Domain

Objective: The Australian snake ptFV ( Pseudonaja textilis venom-derived factor V) variant that retains cofactor function despite APC (activated protein C)-dependent proteolysis. Here, we aimed to unravel the mechanistic principles by determining the role of the absent Arg306 cleavage site that is required for the inactivation of Fva (mammalian factor Va). Approach and Results: Our findings show that in contrast to human FVa, APC-catalyzed proteolysis of ptFVa at Arg306 and Lys507 does not abrogate ptFVa cofactor function. Remarkably, the structural integrity of APC-proteolyzed ptFVa is maintained indicating that stable noncovalent interactions prevent A2-domain dissociation. Using Molecular Dynamics simulations, we uncovered key regions located in the A1 and A2 domain that may be at the basis of this remarkable characteristic. Conclusions: Taken together, we report a completely novel role for uniquely adapted regions in ptFVa that prevent A2 domain dissociation. As such, these results challenge our current understanding by which strict regulatory mechanisms control FVa activity.

Mechanism of the Potent Activated Protein C Resistance of Novel Factor V Mutation with W1920R (FV Nara) Relative to R506Q (FV Leiden)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 537-537
Author(s):  
Kenichi Ogiwara ◽  
Keiko Shinozawa ◽  
Keiji Nogami ◽  
Tomoko Matsumoto ◽  
Katsumi Nishiya ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Protein C ◽  
Activated Protein C ◽  
Vena Cava ◽  
Hek293 Cells ◽  
Factor V ◽  
Activated Protein C Resistance ◽  
Sds Page ◽  
Fv Leiden ◽  
Cofactor Activity

Abstract Abstract 537 Introduction: Factor V (FV) functions both as procoagulant and anticoagulant factors. FV R506Q (FV Leiden) showing activated protein C resistance (APCR) is popular among Caucasians, whilst it has not been reported in Asians. Recently, we have reported a Japanese boy with deep venous thrombosis (DVT) who has a novel FV W1920R mutation (FV Nara, ISTH 2009, 2011). Although he showed APCR in activated partial thromboplastin time (APTT)-based assay, low levels of FV activity (10%) and antigen (40%) made it difficult to explain his severe DVT (both lower extremities and inferior vena cava). Here, we show the detailed mechanisms of his thrombotic diathesis via APCR relative to FV Leiden. Methods: We performed the detection of PC pathway inhibition with Thrombopath® (Protac-induced coagulation inhibition %; PiCi%) or the detection of APCR with calibrated automated thrombogram (CAT) in patient's plasma. FV-deficient plasmas containing varying concentrations of FV wild-type (WT) or W1920R were evaluated by an APCR-assay that specifically can measure the APC cofactor activity of FV in activated FVIII (FVIIIa) inactivation and by the APTT-based assay that probes both the susceptibility and APC cofactor components (Castoldi E, 2004). Recombinant FV proteins (FV-WT, -R506Q, and -W1920R) under pMT2/FV vector were expressed in HEK293 cells. In purified assays, activated FV (FVa) was inactivated by APC in the presence of protein S (PS). FVIIIa were also inactivated by APC/PS in the presence of FV. Cleavages of FV heavy chain were observed in SDS-PAGE and Western blotting. Results: PiCi%, a decrease ratio of thrombin generation by the addition of PC activator (protac) was low in patient's plasma (protac absent/present 794/221 mOD/min; PiCi% 72.2%) (v.s. normal plasma absent/present 834/76.1 mOD/min; PiCi% 90.9%). In CAT using platelet-rich plasmas, peak thrombin level of patient was lower than control (169 and 191 nM), but was greater in the presence of 40 nM APC (103 and 71 nM). In FV-deficient plasma containing FV WT or W1920R, W1920R exhibited no cofactor activity in FVIIIa inactivation, likely supporting the contribution to W1920R-associated APCR more significantly rather than poor susceptibility to APC. In purified assays, FVa (8 nM) activity in FV-WT, -R506Q and -W1920R after 5-min reaction with APC (25 pM), PS (30 nM), PL (20 μM) was decreased to 2.6%, 16.7%, and 62.8%, respectively. SDS-PAGE analysis for FVa heavy chain revealed little cleavage of Gln506 in FV-R506Q and markedly delayed cleavage of Arg506 in FV-W1920R. Of surprise, cleavage of Arg306 was little observed in FV-W1920R. In addition, FVIIIa (10 nM) activity after 20-min reaction with APC (0.5 nM), PS (5 nM), PL (20 μM) and FV (0.5 nM) in FV-WT, -R506Q, and -W1920R was diminished to 18.0%, 52.8% and >95% of those without FV, respectively. Although Trp1920 in FV is close to PL-binding site of the C1 domain, the binding ability of FV-W1920R to immobilized PL retained ∼70-80% of that of FV-WT. Conclusion: The novel FV mutant, W1920R possessed the potent APCR relative to FV Leiden, by reducing the susceptibility of FVa to APC-mediated inactivation and/or by interfering with the FV cofactor activity in APC-catalyzed FVIIIa inactivation. This mechanism might be associated with the impaired cleavages of Arg506 and Arg306 due to the conformational change of this FV mutant. Disclosures: Nogami: Bayer Award 2009: Research Funding.

Use of Modified Functional Assays for Activated Protein C Resistance in Patients with Basally Prolonged aPTT

1997 ◽  
Vol 78 (03) ◽  
pp. 1042-1048 ◽  
Author(s):  
Barbara Montaruli ◽  
Piercarla Schinco ◽  
Antonella Pannocchia ◽  
Angelica Giorgianni ◽  
Alessandra Borchiellini ◽  
...  
Keyword(s):  
Protein C ◽  
Dna Analysis ◽  
Activated Protein C ◽  
Laboratory Diagnosis ◽  
Heparin Therapy ◽  
Clotting Factor ◽  
Factor V ◽  
Diagnostic Efficiency ◽  
Prolonged Aptt ◽  
Aptt Reagent

SummaryInherited resistance to activated protein C (APCr) is currently recognized as the most prevalent cause underlying venous thrombophilia, with an estimated prevalence around 20% in thrombotic patients and around 1.8-7% in the general population. A correct laboratory diagnosis of APCr is therefore essential. Two different diagnostic approaches are at present at our disposal: the semi-quantitative plasma test based on the measurement of two aPTTs (in the presence and absence of activated protein C), and the detection of the factor V Arg506 GIn mutation by DNA analysis.In this study we firstly evaluated sensitivity, specificity and diagnostic efficiency of an aPTT-based plasma clotting test (Chromogenix, Sweden) versus DNA analysis; then, since the APC resistance test is invalidated by a basally prolonged aPTT (i.e. during warfarin and heparin therapy or in patients with clotting factor deficiencies or in the presence of a lupus anticoagulant), patient plasmas were conveniently diluted in factor V deficient plasma in order to correct clotting factor abnormalities. Nevertheless, patients with a LA and an aPTT ratio range 1.8-3.17 were still all misclassified. We obtained correct diagnoses in LA positive patients by preincubating plasmas with a mixture of phospholipids; therefore we decided to perform a double modified clotting test adding a mixture of platelet derived phospholipids to samples previously diluted in factor V deficient plasma. The performance characteristics of this novel method with a different aPTT reagent (Behring, Germany) were also evaluated. With this double modified test all patients were correctly classified as negative or positive for factor V mutation in agreement with DNA analysis, irrespectfully of the basal aPTT value and the aPTT reagent employed. We propose this modified version of the APCr clotting test as an easily reproducible, reliable, very sensitive and specific screening test which possibly reduces the need for DNA analysis.

Coagulation Factor V Leiden Mutation Was Detected in the Patients with Activated Protein C Resistance in Thailand

1998 ◽  
Vol 80 (08) ◽  
pp. 344-345 ◽  
Author(s):  
Pasra Arnutti ◽  
Motofumi Hiyoshi ◽  
Wichai Prayoonwiwat ◽  
Oytip Nathalang ◽  
Chamaiporn Suwanasophon ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Coagulation Factor ◽  
Factor V ◽  
Activated Protein C Resistance ◽  
Factor V Leiden Mutation ◽  
Coagulation Factor V

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.

Resistance to Activated Protein C and Factor V Leiden as Risk Factors for Venous Thrombosis

1995 ◽  
Vol 74 (01) ◽  
pp. 449-453 ◽  
Author(s):  
Rogier M Bertina ◽  
Pieter H Reitsma ◽  
Frits R Rosendaal ◽  
Jan P Vandenbroucke
Keyword(s):  
Risk Factors ◽  
Venous Thrombosis ◽  
Protein C ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Factor V

“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.

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