Venom-Derived Factor V from the Common Brown Snake P. Textilis Is Expressed as a Constitutively Active Cofactor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1765-1765
Author(s):  
Mettine H.A. Bos ◽  
Michael Boltz ◽  
Liam St. Pierre ◽  
Martin F. Lavin ◽  
Rodney M. Camire
Keyword(s):  
Disulfide Bond ◽  
Factor Xa ◽  
Proteolytic Processing ◽  
Light Chains ◽  
Factor V ◽  
Single Chain ◽  
Processing Step ◽  
Sds Page ◽  
Cofactor Activity ◽  
Constitutively Active

Abstract Factor V (FV) circulates as procofactor with little or no procoagulant activity and is activated upon proteolytic removal of a central B-domain. Recently, we have shown that discrete B-domain sequences stabilize the inactive procofactor state and that their deletion drives the expression of procoagulant function without the need for proteolytic processing (JBC2007;282:15033). While the B-domain length is highly conserved in most mammals (∼800 aa), recent genomic data indicates that in some vertebrates the FV B-domain is dramatically shortened. The most striking example is found in an Australian snake family (P. textilis and O. scutellatus). These snakes, which are among the most venomous in Australia, have a powerful prothrombin activating complex in their venoms consisting of FXa-like and FVa-like components. Remarkably, both plasma FV and venom FV sequences of these snakes predict a B-domain of only 45 and 46 residues, suggesting that snake FV may have lost B-domain sequences that maintain the protein as procofactor. Alternatively, snake FV could use a different strategy to preserve the procofactor state. To gain insight into this, we expressed and purified venom-derived P. textilis FV (pt-FV) in BHK cells. Pt-FV expressed very well (4mg protein/L media), and SDS-PAGE analysis revealed that it migrated as a single-chain protein with a molecular mass of ∼180 kDa. Upon incubation with thrombin (IIa), pt-FV was completely processed to pt-FVa, yielding fragments similar to the human FVa heavy and light chains. Surprisingly, pt-FVa migrated as a single band on a non-reducing gel, indicating that the heavy and light chains are held together by a disulfide bond. Sequence analysis suggests that this disulfide bond is located between the A2 and A3 domains. Functional analysis using a PT-based clotting assay with human FV-deficient plasma demonstrated that pt-FV has low activity compared to human FVa. This is consistent with previous studies which showed that venom FV is a relatively ineffective cofactor when using bovine factor Xa (FXa), suggesting that it needs snake-derived FXa to optimally function. Despite the low reactivity towards human plasma, we were able to demonstrate that processing of pt-FV to pt-FVa did not significantly increase cofactor activity (activation quotient of 2 compared to 10–15 for human FV). This suggests that unlike all nonhuman FV variants characterized so far, pt-FV is expressed as a constitutively active cofactor. Consistent with this, assessment of cofactor activity using a purified prothrombinase assay with either human FXa or transiently expressed O. scutellatus FXa revealed that the initial rates of prothrombin activation were equivalent for pt-FV or pt-FVa. To further confirm these results we expressed and purified pt-FV in which IIa cleavage sites Arg709 and Arg1545 were eliminated (pt-FV-QQ). SDS-PAGE analysis showed that pt-FV-QQ was not cleaved by IIa, yet functional measurements revealed that its activity was similar to pt-FV or pt-FVa. These data indicate that pt-FV has constitutive cofactor activity and thus bypasses the normal proteolytic processing step to function within prothrombinase. This is in agreement with our previous findings that discrete sequences within the FV B-domain are necessary to maintain FV as procofactor. Thus, venom-derived P. textilis FV represents the first example of a naturally occurring FV variant that does not require proteolytic processing to be active.

Evolutionary Adaptation of Factor V from the Venom of the Common Brown Snake into a Potent Procoagulant

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 586-586
Author(s):  
Mettine H.A. Bos ◽  
Michael Boltz ◽  
Liam St. Pierre ◽  
John de Jersey ◽  
Paul P Masci ◽  
...  
Keyword(s):  
Membrane Surface ◽  
Light Chains ◽  
Factor V ◽  
Single Chain ◽  
Prothrombinase Complex ◽  
High Affinity ◽  
The Common ◽  
Cofactor Activity ◽  
Mechanistic Basis ◽  
Normal Requirement

Abstract Some of the most toxic snakes in the world are those from the Australian Elapid family, including the three most venomous land snakes Inland Taipan, Coastal Taipan, and Common Brown snake. Their venom is strongly procoagulant and they are the only species known to have acquired a powerful prothrombin activator in their venom, which consists of a factor Xa (FXa)-like and factor V (FV)-like component. Venom-derived FV (pt-FV) from the Common Brown snake P. textilis shares 44% sequence homology with mammalian FV and has a similar domain organization. Remarkably, the B domain length of pt-FV is dramatically shortened compared to human FV (46 vs. 836 aa). This adaptation provides a unique opportunity to gain new insight into the function of the B domain and to examine the mechanistic basis for the strong procoagulant nature of the venom-derived prothrombinase complex. Pt-FV was expressed in BHK cells, purified, and characterized in functional assays employing FXa purified from P. textilis venom (pt-FXa). SDS-PAGE analysis revealed that pt-FV migrated as a single chain protein (~180 kDa). Thrombin completely processed pt-FV to pt-FVa, yielding the characteristic heavy and light chains. Surprisingly, pt-FVa migrated as a single band on a non-reducing gel, indicating that the heavy and light chains are connected by a unique disulfide bond. Functional analysis of prothrombin and prethrombin-1 conversion using a purified component assay in the presence of pt-FXa and negatively charged phospholipids revealed that pt-FV exhibits kinetic parameters comparable to human prothrombinase. Proteolytic processing of single chain pt-FV to the heterodimer did not significantly increase cofactor activity, indicating that pt-FV is expressed as a constitutively active cofactor that has bypassed the normal requirement for proteolytic activation. These results were confirmed using an uncleavable variant, pt-FV-QQ. We speculate that the mechanistic basis for this constitutive cofactor activity is related to the absence of a key cluster of conserved B domain residues, which we have recently shown to play an important role in maintaining FV as an inactive procofactor (JBC2007;282:15033). Additional experiments revealed that the pt-FV–pt-FXa complex does not require a membrane surface to optimally function, as the kinetics of prethrombin-1 activation were equivalent in the presence or absence of membranes. Binding measurements indicated that this was due to the high affinity interaction (Kd ~8 nM) of pt-FV with pt-FXa in solution. Interestingly, human FVa did not bind soluble pt-FXa with high affinity, suggesting that pt-FXa binding involves unique molecular features on pt-FV. Additional studies revealed that pt-FV does not lose activity following incubation with high concentrations of activated protein C (APC), even though the pt-FV heavy chain was fully proteolyzed. Collectively, our findings provide new insights into FV structure/function as well as a biochemical rationale for the powerful procoagulant nature of the prothrombinase complex from P. textilis venom. Remarkably, pt-FV has acquired at least three gain of function elements: first, it is constitutively active and as such the first example of a naturally occurring active FV variant. Second, pt-FV has a unique conformation as it bypasses the normal requirement for a membrane surface to achieve high affinity FXa binding. Finally, pt-FV is functionally resistant to APC which could be due to its unique disulfide bond. Taken together, venom-derived P. textilis FV represents an exceptional example of a protein that has adapted into a potent biological weapon for host defense and to incapacitate prey. Uncovering the mechanistic details of these gain of function elements will provide a new level of understanding of FV/FVa function.

INACTIVATION OF FACTOR Va BY PIASMIN

1987 ◽  
Author(s):  
M N Omar ◽  
C D Lee ◽  
K G Mann
Keyword(s):  
Activated Protein C ◽  
Factor Xa ◽  
Phospholipid Vesicles ◽  
Factor V ◽  
Single Chain ◽  
Clotting Factors ◽  
Activity Data ◽  
Factor Va ◽  
Sds Page ◽  
Active Intermediates

The inactivation of Factor Va by plasmin was studied in the presence and absence of phospholipid vesicles and calcium ions. The action of plasmin resulted in a rapid loss of the ability of Factor Va to serve as a cofactor to Factor Xa , as judged by clotting assays and . direct assays of prothrombin activation using the fluorophore, dansylarginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA) . The rate of Factor Va inactivation catalyzed by plasmin was markedly enhanced by the addition of phospholipid vesicles (PCPS), suggesting that the action of plasmin on Factor Va may be a membrane bound phenomena. Both Factor Xa and prothrombin were capable of protecting Factor Va from inactivation by plasmin. SDS-PAGE was utilized to correlate plasmin catalyzed proteolysis of Factor Va with the concomitant loss of activity. Data obtained with Factor Va and the isolated chains of the cofactor indicated that the light chain (E) was cleaved by plasmin to yield products similar to those obtained with Factor Xa and Activated Protein C (APC). The heavy chain (D) was found to be degraded by plasmin to produce proteolytic fragments distinct from those produced by Factor Xa and APC. The action of plasmin on single chain Factor V was notable for an initial, transient increase in total Factor V activity, followed by subsequent loss of activity, indicating the transient formation of active intermediates. SDS-PAGE analysis revealed the degradation of Factor V by plasmin to final, inactive products via several transient, higher molecular weight intermediates. These findings may be of some significance in pathophysiologic states in which systemic fibrinolysis may occur, possibly contributing to the depletion of clotting factors. The identification of such ongoing processes may eventually be facilitated by the observation that the degradation of Factor Va by plasmin leads to end products which may be unique to this interaction.(Supported by NIH Grants HL-35058 and HL 34575)

Identification of Sulfo-Tyrosines of Factor V.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1731-1731
Author(s):  
Evrim Erdogan ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Factor Xa ◽  
Factor V ◽  
Wild Type ◽  
Single Chain ◽  
Amino Acid Region ◽  
Thrombin Cleavage ◽  
Factor Va ◽  
Cofactor Activity ◽  
Acid Region

Abstract The factor Va molecule is the essential cofactor of the prothrombinase complex. This complex composed of factor Xa and factor Va assembled on a platelet membrane-surface in the presence of Ca2+ ions converts membrane-bound prothrombin to thrombin. Single chain factor V does not bind factor Xa. Single-chain factor V is cleaved by thrombin first at Arg709 followed by cleavages at Arg1018 and Arg1545 to produce the heavy and light chains of the active cofactor (factor Va) and two activation peptides. Efficient thrombin cleavage and activation of factor V is essential for cofactor function and requires tyrosine sulfation. Tyrosine sulfation of factor V also appears to regulate its activity. Seven tyrosine residues in factor V, Tyr665, Tyr696, Tyr698, Tyr1494, Tyr1510, Tyr1515, and Tyr1565 have been identified as potential sites of sulfation. However, which residues are sulfated and their contribution to procofactor activation and cofactor function still remain to be elucidated. Two of the sulfation sites Tyr696 and Tyr698 are located in the acidic amino acid region near to the first required thrombin cleavage site at Arg709. Recent data demonstrated that these residues are essential for factor V activation and cofactor activity. Another acidic amino acid region, 1490–1520 is adjacent to the thrombin cleavage site at Arg1545 required for light chain formation. This region also contains three potential sulfation sites at residues 1494, 1510, and 1515 and was shown to be required for optimum procofactor activation. To ascertain which of these three residues is important for procofactor activation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations 1493DY1494→AF, 1508DDY1510→AAF and 1514DY1515→AF. The clotting and cofactor activity of the 1493DY1494→AF and 1514DY1515→AF mutants was similar to the clotting activity observed with the wild type recombinant factor Va molecule following activation by thrombin or RVV-V activator. In contrast, under similar experimental conditions recombinant factor V with the substitution 1508DDY1510→AAF was deficient in its clotting activity and had impaired cofactor activity. Moreover, following prolonged incubation with thrombin, no light chain formation was observed in the factor V molecule bearing the 1508DDY1510→AAF mutation. Thus, amino acid residues 1508–1510 of factor V are required for thrombin interaction with the procofactor which in turn appears necessary for cleavage at Arg1545. Studies of sulfated proteins have shown that the effect of sulfo-tyrosines on protein structure/function can be preserved by replacing them with glutamic acid. To explicitly identify the sulfated tyrosines on the factor V molecule, we mutated Tyr696, Tyr698 and Tyr1510 to glutamic acid and transfected them into COS-7L cells. Expression was performed in the presence of media containing or devoid of sulfate. In the presence of sulfate, the cofactor and clotting activities of the DY696DY698→DEDE and DDY1510→DDE mutants, separately were similar to the wild type recombinant factor Va molecule. However, in the absence of sulfate, the wild type and the mutant recombinant factor V molecules had both impaired cofactor activity and clotting activity following their activation with thrombin. However, their respective activity was higher than the activity of the factor V molecule bearing the 1508DDY1510→AAF mutation. Our data suggest that residues 696, 698, and 1510 of factor V appear to be sulfated and might be important for procofactor activation and cofactor function.

scholarly journals Human Neutrophil Elastase Activates Human Factor V but Inactivates Thrombin-Activated Human Factor V

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1065-1074 ◽  
Author(s):  
John A. Samis ◽  
Marilyn Garrett ◽  
Reginald P. Manuel ◽  
Michael E. Nesheim ◽  
Alan R. Giles
Keyword(s):  
Sequence Analysis ◽  
Heavy Chain ◽  
Human Neutrophil ◽  
Human Factor ◽  
Time Dependent ◽  
Human Neutrophil Elastase ◽  
Factor V ◽  
Terminal Sequence ◽  
Sds Page ◽  
Cofactor Activity

The effect of human neutrophil elastase (HNE) on human factor V (F.V) or α-thrombin–activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and NH2 -terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with α-thrombin in presence of Ca2+. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 μmol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using α-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted in the cleavage of both the 96 kD heavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2 -terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2 -terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely α-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with α-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.

scholarly journals Cryo-EM structures of human coagulation factors V and Va

Blood ◽  
2021 ◽  
Author(s):  
Eliza A Ruben ◽  
Michael J Rau ◽  
James Fitzpatrick ◽  
Enrico Di Cera
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Proteolytic Processing ◽  
Coagulation Cascade ◽  
Factor V ◽  
Prothrombinase Complex ◽  
A2 Domain

Coagulation factor V is the precursor of factor Va that, together with factor Xa, Ca2+ and phospholipids, defines the prothrombinase complex and activates prothrombin in the penultimate step of the coagulation cascade. Here we present cryo-EM structures of human factors V and Va at atomic (3.3 Å) and near-atomic (4.4 Å) resolution, respectively. The structure of fV reveals the entire A1-A2-B-A3-C1-C2 assembly but with a surprisingly disordered B domain. The C1 and C2 domains provide a platform for interaction with phospholipid membranes and support the A1 and A3 domains, with the A2 domain sitting on top of them. The B domain is highly dynamic and visible only for short segments connecting to the A2 and A3 domains. The A2 domain reveals all sites of proteolytic processing by thrombin and activated protein C, a partially buried epitope for binding factor Xa and fully exposed epitopes for binding activated protein C and prothrombin. Removal of the B domain and activation to fVa exposes the sites of cleavage by activated protein C at R306 and R506 and produces increased disorder in the A1-A2-A3-C1-C2 assembly, especially in the C-terminal acidic portion of the A2 domain responsible for prothrombin binding. Ordering of this region and full exposure of the factor Xa epitope emerge as a necessary step for the assembly of the prothrombin-prothrombinase complex. These structures offer molecular context for the function of factors V and Va and pioneer the analysis of coagulation factors by cryo-EM.

Destabilization of the Factor V B-Domain Results in Procofactor Activation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 198-198
Author(s):  
Rodney M. Camire ◽  
Hua Zhu ◽  
Mettine H.A. Bos ◽  
Raffaella Toso
Keyword(s):  
Amino Acids ◽  
Blood Clot ◽  
Coagulation Factor ◽  
Active Species ◽  
Quiescent State ◽  
Factor V ◽  
Single Chain ◽  
Direct Binding ◽  
Cofactor Activity ◽  
Domain Length

Abstract A hallmark of hemostasis is that proteins involved in the formation of a blood clot remain in a quiescent state and are only activated following an appropriate stimulus. Blood coagulation factor V (FV), which is structurally homologous to FVIII, cannot function in the prothrombinase complex and is thus considered a procofactor. Thrombin catalyzes the conversion of FV to FVa following three cleavages (Arg709, Arg1018, and Arg1545) releasing a large heavily glycosylated central B-domain (836 amino acids). Explanations as to how bond cleavage or B-domain release facilitates the transition to the active species are incomplete. Recent studies using a partial B-domainless form of FV (FV-810 des811–1491) support a model in which removal of B-domain sequences from FV rather than specific proteolysis underlies the mechanism by which cofactor function is realized. This single-chain derivative is functionally equivalent to FVa suggesting that the deleted B-domain sequences somehow suppress cofactor activity. To investigate this further, we have expressed and purified several single-chain derivatives of FV that vary in B-domain length from 155 to 497 residues. Functional activity assays as well as direct binding fluorescent measurements revealed that elimination of most of the C-terminal half of the B-domain (residues 1034–1491; 458 out of 836 a.a. deleted) had no influence on maintaining the procofactor state. However, deletion of sequences from the N-terminal half of the B-domain resulted in derivatives with cofactor-like properties. Using progressively finer deletion variants we were able to demonstrate that either a B-domain length of at least 378 amino acids or specific sequences contained within residues 902–1033 is sufficient to suppress cofactor activity. To examine these possibilities, we constructed additional FV variants in which a B-domain length of 378 amino acids was maintained, but specific portions of 902–1033 were exchanged with FVIII B-domain. Using the FV-1033 derivative (residues 1034–1491 of B-domain deleted) as a scaffold, three constructs were prepared, s-131, s-104, and s-46, representing 131, 104, and 46 amino acids from the FV B-domain exchanged with FVIII B-domain. In activity assays and direct binding measurements, each of these variants had properties consistent with the cofactor-like form indicating that a length of ~375 residues is not sufficient to maintain the procofactor state. These findings demonstrate for the first time that there are indeed specific FV-B domain sequences between 902–1033 that directly or indirectly stabilize the procofactor state. Remarkably, simply replacing these sequences in FV-1033 resulted in activation of the proteins in the absence of proteolysis. These observations change existing ideas about FV activation and provide insight into specific regions of the B-domain that assist in preserving the procofactor state.

PURIFICATION AND CHARACTERIZATION OF THE PROCOAGULANT OF THE VENOM OF TROPIDECHIS CARINATUS

1987 ◽  
Author(s):  
P P Masci ◽  
A N Whitaker ◽  
J J Morrison ◽  
E A Bennett
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Factor Xa ◽  
Factor V ◽  
Crude Venom ◽  
Adult Rats ◽  
Sds Page ◽  
Sepharose 4B ◽  
Blue Sepharose

Tropidechis carinatus is a venomous elapid snake distributed throughout Eastern Queensland. It has been considered as a tropical relative of Notechis scutatus and, similarly, the crude venom contains an indirect prothrombin activator, which will clot plasma provided that Factor V is present. Myotoxins and neurotoxins are also present. Envenomated patients regularly develop disseminated intravascular coagulation. The crude whole venom of T.carinatus was shown to have five major components by gel filtration, SDS PAGE and HPLC, and even more components by isoelectric focusing. The procoagulant eluted with a molecular weight of 55,000, being found in peak II on gel filtration on Sephadex-G150. The procoagulant was purified using a combination of Sephadex-G150 chromatography and ion-exchange on DEAE Sephadex-A50 and shown to migrate as a single band of molecular weight 55.000 by SDS PAGE. On reduction by β -mercaptoethanol this component was resolvec into u heavy chain of molecular weight 30.000 and a light chain of 25,000. The procoagulant was shown to bind to con A-Sepharose 4B and Blue Sepharose 4B. Coagulation studies using this purified procoagulant confirmed a factor Xa-like activity activating prothrombin in the presence of factor V. The purified fraction is unstable in buffer solutions at 4°C, probably because of trypsin - like autodigestion. Ouchterlony studies of the procoagulants of T.carinatus and N.scutatus show both lines of homogeneity and spurring, indicating similarities but also significant differences between the two proteins. The purified procoagulant was lethal to adult rats, an IV injection of 10 μg killing in 1 - 2 minutes. Death was prevented by prior heparinization, suggesting that the procoagulant is toxic in the absence of neurotoxin and other components.

Cleavage of Factor Va Heavy Chain at Arg643 by Thrombin Partially Inactivates the Cofactor.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1703-1703 ◽  
Author(s):  
Evrim Erdogan ◽  
Michael A. Bukys ◽  
Thomas Orfeo ◽  
Kenneth G. Mann ◽  
Michael Kalafatis
Keyword(s):  
Endothelial Cells ◽  
Heavy Chain ◽  
Consensus Sequence ◽  
Factor V ◽  
Single Chain ◽  
Chain Fragment ◽  
Thrombin Cleavage ◽  
Factor Va ◽  
Plasma Factor ◽  
Cofactor Activity

Abstract Prothrombinase, the enzyme complex required to activate prothrombin, is composed of the serine protease factor Xa and the cofactor factor Va, associated in 1:1 stoichiometry on a phospholipid surface in the presence of Ca2+. Incorporation of factor Va in prothrombinase is required for any meaningful rate of thrombin generation and the arrest of hemorrhage. Factor Va inactivation down-regulates thrombin production resulting in the termination of the hemostatic response. The principal enzyme involved in this down-regulation is activated protein C (APC). Factor Va is formed following enzymatic cleavage of the single chain procofactor, factor V (Mr 330,000) by thrombin. Thrombin cleaves and activates the procofactor sequentially at Arg709, Arg1018, and Arg1545. The active cofactor, factor Va, is composed of heavy (HC105, Mr 105,000) and light (Mr 74,000) chains non-covalently associated in the presence of divalent ions. Previous studies of factor Va inactivation on human umbilical vein endothelial cells (HUVEC) have shown that thrombin cleaves the heavy chain at the COOH-terminus to produce a Mr 97,000 fragment containing the NH2-terminal portion of the heavy chain and a Mr 8,000 peptide representing the COOH-terminus of the molecule which remains attached to the heavy chain by a disulfide bond. The thrombin cleavage appeared to occur between residues 586 and 654. This region contains a consensus sequence for cleavage by thrombin located between residues 640–643 (S-P-R). To evaluate the functional importance of thrombin cleavage at Arg643 for factor Va inactivation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations R643→Q (factor VR643Q) and R643→A (factor VR643A). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with thrombin. Under similar experimental conditions, cleavage of HC105 and appearance of the Mr 97,000 heavy chain fragment in the wild type molecule correlated with partial loss of cofactor activity, while following incubation of factor VR643Q and factor VR643A with thrombin no cleavage of HC105 at Arg643 was observed and no presence of the Mr 97,000 heavy chain fragment was noticed. Further, no loss in cofactor activity was observed using these mutant recombinant factor Va molecules following extended incubation with thrombin. The endothelial cell surface has been presumed to be the site of PC activation and factor Va inactivation in vivo. The relative phospholipid composition of endothelial membranes has been suggested to be consistent with their ability to support factor Va inactivation in a manner analogous to the commonly used phospholipid system composed of 25% phosphatidylserine and 75% phosphatidylcholine. In the experiments conducted on the HUVEC surface incubation of 20 nM plasma factor V with 0.1 nM thrombin resulted in almost complete cleavage of HC105 over a 60 minute thrombin treatment. In the experiments presented herein much higher concentrations of thrombin were necessary to obtain a similar effect. The combined data suggest the presence of a cofactor for thrombin on the surface of endothelial cells that would facilitate cleavage of factor Va heavy chain at Arg643. Collectively, the data demonstrate that cleavage of HC105 at Arg643 by thrombin results in a partially inactive cofactor molecule and provide for an APC-independent anticoagulant effect of thrombin.

scholarly journals A Prothrombinase-based Assay for Detection of Resistance to Activated Protein C

1996 ◽  
Vol 76 (03) ◽  
pp. 404-410 ◽  
Author(s):  
Gerry A F Nicolaes ◽  
M Christella ◽  
L G D Thomassen ◽  
Rene van Oerle ◽  
Karly Hamulyak ◽  
...  
Keyword(s):  
Protein C ◽  
Plasma Sample ◽  
Activated Protein C ◽  
Factor Xa ◽  
Phospholipid Vesicles ◽  
Factor V ◽  
Plasma Samples ◽  
Factor Va ◽  
Plasma Factor ◽  
Cofactor Activity

SummaryIn this paper we present a new method for the detection of resistance to activated protein C (APC) that is based on direct measurement of the effect of APC on the cofactor activity of plasma factor Va. The factor V present in a diluted plasma sample was activated with thrombin and its sensitivity towards APC was subsequently determined by incubation with phospholipids and APC. The loss of factor Va cofactor activity was quantified in a prothrombinase system containing purified prothrombin, factor Xa and phospholipid vesicles and using a chromogen-ic assay for quantitation of thrombin formation. The reaction conditions were optimized in order to distinguish normal, heterozygous and homozygous APC-resistant plasmas. Maximal differences in the response of these plasmas towards APC were observed when factor Va was inactivated by APC in the absence of protein S and when the cofactor activity of factor Va was determined at a low factor Xa concentration (0.3 nM).Addition of 0.2 nM APC and 20 μM phospholipid vesicles to a 1000-fold diluted sample of thrombin-activated normal plasma resulted in loss of more than 85% of the cofactor activity factor Va within 6 rnin. Under the same conditions, APC inactivated ∼ 60% and ∼20% of the factor Va present in plasma samples from APC-resistant individuals that were heterozygous or homozygous for the mutation Arg506⟶Gln in factor V, respectively. Discrimination between the plasma samples from normal and heterozygous and homozygous APC-resistant individuals was facilitated by introduction of the so-called APC-sensitivity ratio (APC-sr). The APC-sr was defined as the ratio of the factor Va cofactor activities determined in thrombin-activated plasma samples after 6 min incubation with or without 0.2 nM APC and was multiplied by 100 to obtain integers (APC-sr = {factor Va+APC/factor Va−APC} × 100). Clear differences were observed between the APC-sr of plasmas from normal healthy volunteers (APC-sr: 8-20, n = 33) and from individuals that were heterozygous (APC-sr: 35-50, n = 17) or homozygous APC resistant (APC-sr: 82-88, n = 7). There was no mutual overlap between the APC-sr of normal plasmas and plasmas from heterozygous or homozygous APC resistant individuals (p <0.0001). In all cases our test gave the same result as the DNA-based assay. Since the test is performed on a highly diluted plasma sample there is no interference by conditions that affect APC resistance tests that are based on clotting time determinations (e.g. coagulation factor deficiencies, oral anticoagulation, heparin treatment, the presence of lupus anticoagulants, pregnancy or the use of oral contraceptives). Furthermore, we show that part of the factor Va assay can be performed on an autoanalyzer which increases the number of plasma samples that can be handled simultaneously.

Cleavage At Arg1018 During Thrombin-Mediated Activation of Coagulation Factor V Is Dispensable

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1186-1186 ◽  
Author(s):  
Mahesheema Na ◽  
Joesph R Wiencek ◽  
Jamila Hirbawi ◽  
Michael Kalafatis
Keyword(s):  
Conflicts Of Interest ◽  
Membrane Surface ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Factor V ◽  
Cleavage Sites ◽  
Two Stage ◽  
Cofactor Activity

Abstract Abstract 1186 Blood clotting results in the proteolytic conversion of prothrombin (Pro) to thrombin which in turn will produce the fibrin clot. The proteolytic conversion of Pro to thrombin is catalyzed by the prothrombinase complex which is composed of the enzyme, factor Xa (FXa), the cofactor, factor Va (FVa), assembled on a membrane surface in the presence of divalent metal ions. Factor V (FV), is a multidomain protein (A1-A2-B-A3-C1-C2) with nominal procoagulant activity and is activated by thrombin to FVa through three sequential proteolytic cleavages at Arg709, Arg1018 and Arg1545. To understand the significance of each cleavage for active cofactor formation and prothrombinase function, recombinant factor V molecules were created by site-directed mutagenesis with two out of three cleavage sites mutated simultaneously (to glutamine). We have generated a FV molecule mutated at the Arg709/1018 cleavage sites (FVQQR), a FV molecule mutated at the Arg709/1545 cleavage sites (FVQRQ), a FV molecule mutated at the Arg1018/1545 cleavage sites (FVRQQ), and a FV molecule that is mutated at all three cleavage sites (FVQQQ). These recombinant FV molecules along with wild type FV (FVWT) were transiently expressed in COS7L cells, purified to homogeneity and assessed for their capability to interact with factor Xa following activation by thrombin, and participate in prothrombinase. Pro activation by prothrombinase assembled with the mutant molecules was evaluated by SDS-PAGE and the kinetic parameters of the reactions in the presence of saturating concentrations of FXa were determined. Two-stage clotting assays revealed that while FVQQQ was devoid of clotting activity following incubation with thrombin, FVaQQR, FVaQRQ and FVaRQQ all had impaired clotting activities compared to FVaWT and plasma derived FVa (FVaPLASMA). Kinetic analyses demonstrated that FVaWT had a Kd of 0.25nM for FXa while all other mutant molecules had impaired binding capabilities for FXa. FVaQQQ was severely impaired in its ability to interact with FXa. The kcat value for prothrombinase assembled with FVaQQR was approximately 50% lower than the kcat obtained with prothrombinase assembled with FVaWT, while prothrombinase assembled with FVaQRQ and FVaRQQ had approximately 3-fold reduced catalytic efficiency when compared to the values obtained with prothrombinase assembled with FVaWT. Following incubation with thrombin prothrombinase assembled with FVaQQQ had no cofactor activity. To determine the importance of the cleavage site at Arg1018 for procofactor activation and the function of amino acid region 1000–1008 during proteolysis, several other recombinant molecules were generated. FVRQR is a FV molecule with the mutation Arg1018→Gln, and FVΔ1000-1008 is a mutant FV molecule with region 1000–1008 deleted. We have also generated FVΔ1000-1008/RQR and FVΔ1000-1008/QRQ. Two-stage clotting assays revealed that FVaRQR and FVaΔ1000-1008/RQR have similar clotting activities as FVaWT, whereas FVaQRQ, FVaΔ1000-1008/QRQ are impaired in their clotting activities. Kinetic analyses demonstrated that FVaRQR and FVaΔ1000-1008/RQR have similar affinity for FXa as FVa WT while FVaQRQ and FVaΔ1000-1008/QRQ were impaired in their interaction with factor Xa. The kcat values for prothrombinase assembled with FVaRQR and FVaΔ1000-1008/RQR were similar to the kcat obtained with prothrombinase assembled with FVa WT, while prothrombinase assembled with FVaQRQ and FVaΔ1000-1008/QRQ had 2-fold and 7-fold reduced catalytic efficiency respectively, when compared to the kcat values obtained with prothrombinase assembled with FVaWT. Overall, the data demonstrate that cleavage at both Arg709 and Arg1545 are a prerequisite for expression of optimum cofactor activity. Our data also suggests that cleavage at Arg1018 is redundant for cofactor activity. The role of cleavage at this site by thrombin during procofactor activation remains to be determined. Disclosures: No relevant conflicts of interest to declare.

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