Residues Surrounding Arg372, Arg740, and Arg1689 Contribute to the Rates of Thrombin-Catalyzed Cleavage of Factor VIII.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 847-847
Author(s):  
Jennifer L. Newell ◽  
Amy E. Griffiths ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Cleavage Site ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
Factor Xa ◽  
Wild Type ◽  
Cleavage Rate ◽  
Thrombin Cleavage ◽  
Type Factor ◽  
Bold Type

Abstract Abstract 847 Hemophilia A results from defects or deficiencies in the blood coagulation protein, factor VIII. Factor VIII circulates as an inactive procofactor that must be cleaved by thrombin or factor Xa at Arg740 (A2-B junction), Arg372 (A1-A2 junction), and Arg1689 (a3-A3 junction) to yield the active cofactor, factor VIIIa. Activation of factor VIII by thrombin is exosite-dependent yielding rates of cleavage at Arg740 ∼20-fold faster than Arg372, while cleavage at Arg1689 appears intermediary to Arg740 and Arg372. The contribution of P3-P3' residues flanking each cleavage site to the mechanism of thrombin-catalyzed cleavage of factor VIII has not been extensively studied. The P3-P3' residues for the 372, 1689, and 740 factor VIII sites are 370QIR*↓SVA375, 1687SPR*↓SFQ1692, and 738EPR*↓SFS743, respectively. Residues flanking Arg372 are considered non-optimal for thrombin cleavage with only two residues optimal (in bold type) for cleavage in the P3-P3' sequence, while residues flanking at the two other P1 sites are considered near-optimal with four out of six residues optimal (in bold type). Therefore, we investigated whether the P3-P3'residues surrounding Arg740, Arg372, and Arg1689 affect activation of factor VIII by thrombin. We constructed, stably transfected, and expressed four recombinant P3-P3' factor VIII mutants designated 372(P3-P3')740, 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740/1689(P3-P3')372. For example, the 372(P3-P3')740 variant has replaced the non-optimal P3-P3' residues flanking Arg372 with the near-optimal P3-P3' residues flanking Arg740. The specific activities of the 372(P3-P3')740 and 372(P3-P3')740/740(P3-P3')372 mutants were 98% and 122% the wild-type factor VIII value, respectively. In comparison, the 372(P3-P3')1689 and 372(P3-P3')740/1689(P3-P3')372 showed reductions in specific activity with values that were 14% and 17% of wild-type factor VIII, consistent with possible impaired rates of activation by thrombin. SDS-PAGE and Western blotting of the three variants possessing the 372(P3-P3')740 mutation showed cleavage rates at Arg372 increased 11- to 14-fold compared with wild-type factor VIII as judged by rates of generation of the A1 subunit. Furthermore, these variants revealed 11-21-fold rate increases in the generation of the A2 subunit as compared to wild-type factor VIII. The rates of A1 and A2 subunit generation were moderately increased from 2-3-fold for the 372(P3-P3')1689 mutant. These results indicate that replacing the non-optimal residues flanking Arg372 with near-optimal residues enhances rates of cleavage at this site. Furthermore, since the P2-P2' residues flanking Arg740 and Arg1689 are identical, these results also suggest that the P3 and/or P3' residues from the Arg740 cleavage site make a greater contribution to the enhanced cleavage rate when inserted at Arg372 than the equivalent residues from the Arg1689 site. Thrombin cleavage of light chain showing the largest effect was obtained for the 372(P3-P3')740/1689(P3-P3')372 mutant which yielded a reduced rate of A3-C1-C2 subunit generation by 33-fold. This result suggests that replacing near-optimal P3-P3' residues at Arg1689 with non-optimal residues at Arg372 significantly reduces the rate of thrombin cleavage at Arg1689, an effect that may contribute to its low specific activity. There was no observed defect in Arg1689 cleavage in the 372(P3-P3')740 mutant and moderate 2-3-fold reductions in thrombin-catalyzed cleavage rates at Arg1689 in the 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740 variants. Overall, these results suggest that faster cleavage rates at Arg740 and Arg1689 can be attributed to more optimal residues in the P3-P3' region, while the relatively slower cleavage rate at Arg372 can be accelerated by replacement with more optimal residues for thrombin cleavage. Thus, the P3-P3' residues surrounding Arg740, Arg1689, and Arg372 in factor VIII impact rates of thrombin proteolysis at each site and contribute to the mechanism for thrombin activation of the procofactor. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site

Blood ◽  
2005 ◽  
Vol 105 (11) ◽  
pp. 4362-4368 ◽  
Author(s):  
Keiji Nogami ◽  
Qian Zhou ◽  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Factor Xa ◽  
Maximum Velocity ◽  
Activity Levels ◽  
Wild Type ◽  
Cleavage Rate ◽  
Type Factor ◽  
Reduced Rate

Abstract Thrombin-catalyzed proteolysis at Arg372 of factor VIII is essential for procofactor activation. However, hemophilia A patients with the missense mutation Arg372 to His possess a mild to moderate phenotype yet show no detectable cleavage at this bond. To evaluate this discrepancy, we prepared and stably expressed a recombinant, B-domainless factor VIII mutant (R372H) that possessed approximately 1% the specific activity of wild type. Cleavage at R372H by thrombin occurred with an approximately 80-fold decreased rate compared with wild type. N-terminal sequence analysis of the derived A2 subunit confirmed that cleavage occurred at the His372-Ser373 bond. Factor VIII R372H was activated slowly, attained lower activity levels, and exhibited an apparent reduced inactivation rate compared with factor VIII wild type. These observations were attributed to a reduced cleavage rate at His372. Factor Xa generation assays showed similar Michaelis-Menten constant (Km, apparent) values for thrombin-catalyzed activation for either factor VIII form, but suggested an approximately 70-fold reduced maximum velocity (Vmax) for factor VIII R372H. However, prolonged reaction with thrombin yielded similar activity and stability values for the mutant and wild-type factor VIIIa forms. These results indicate a markedly reduced rate of cleavage following substitution at the P1Arg, and this property likely reflects the severity of the hemophilia A phenotype.

Thrombin-Catalyzed Cleavages of Factor VIII at Arg372 and Arg1689 Are Facilitated by the Acidic Residues Glu720-Asp725.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2686-2686
Author(s):  
Jennifer Newell ◽  
Qian Zhou ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Factor Viiia ◽  
N Terminus ◽  
Acidic Residues ◽  
Type Factor ◽  
A2 Domain

Abstract Factor VIIIa acts as an essential cofactor for the serine protease factor IXa, together forming the Xase complex which catalyzes the conversion of factor X to factor Xa. The procofactor, factor VIII circulates as a heterodimeric protein comprised of a heavy chain (A1–A2-B domains) and a light chain (A3-C1-C2 domains) and is activated by proteolytic cleavage by thrombin at Arg372 (A1–A2 junction), Arg740 (A2-B junction), and Arg1689 (near the N-terminus of A3). The regions adjacent to the A1, A2, and A3 domains contain high concentrations of acidic residues and are designated a1 (residues 337–372), a2 (residues 711–740), and a3 (residues 1649–1689). In addition, the N-terminus of the A2 domain (residues 373–395) is rich in acidic residues, and results from a previous study revealed that this region contributes to the rate of thrombin-catalyzed cleavage at Arg740 (Nogami et. al., J. Biol. Chem. 280:18476, 2005). In this study we reveal a role for the acidic region following the A2 domain (a2, residues 717–725) in thrombin-catalyzed cleavage at both Arg372 and Arg1689. The factor VIII mutations Asp717Ala, Glu720Ala, Asp721Ala, Glu724Ala, Asp725Ala, and the double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala were constructed, expressed, and purified from stably-transfected BHK cells as B-domainless protein. Specific activity values for the variants, relative to the wild type value were reduced to 70% for Asp717Ala; ∼50% for Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala; and ∼30% for Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala. SDS-PAGE and western blotting of reactions containing the factor VIII variants and thrombin showed reductions in the rates of thrombin cleavage at both Arg372 and Arg1689 as compared to wild-type factor VIII. The cleavage rates for the single mutations comprising acidic residues 720–724 of factor VIII were reduced from ∼3-5-fold at Arg372, whereas this rate for the Asp717Ala mutant was similar to the wild-type value. The double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala showed rate reductions of ∼7- and ∼27-fold, respectively at Arg372. While the rate for thrombin-catalyzed cleavage at Arg1689 in the Glu720Ala variant was similar to wild-type, rates for cleavage at this site were reduced ∼30-fold compared to wild-type factor VIII for the Asp721Ala, Glu724Ala, Asp725Ala, and Glu720Ala/Asp721Ala mutants, and ∼50-fold for the Glu724Ala/Asp725Ala variant. Furthermore, the generation of factor VIIIa activity following reaction with thrombin as assayed by factor Xa generation showed that all the mutants possessed peak activity values that were ∼2-3-fold reduced compared to wild type factor VIIIa. Moreover, in all the mutants the characteristic peak of activation was replaced with a slower forming, broad plateau of activity, with the double mutants showing the broadest activation profiles. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 following the A2 domain modulate thrombin interactions with factor VIII facilitating cleavage at Arg372 and Arg1689 during procofactor activation.

Cleavage at Arg740 Appears Essential for Thrombin-Catalyzed Cleavage at Arg372 during the Activation of Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1692-1692
Author(s):  
Jennifer Newell ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Time Course ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Thrombin Cleavage ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Initial Cleavage

Abstract Factor VIIIa serves as an essential cofactor for the factor IXa-catalyzed activation of factor X during the propagation phase of coagulation. The factor VIII procofactor is converted to factor VIIIa by thrombin-catalyzed proteolysis of three P1 positions at Arg372 (A1–A2 junction), Arg740 (A2–B junction), and Arg1689 (a3–A3 junction). Cleavage at Arg372 exposes a cryptic functional factor IXa-interactive site, while cleavage at Arg1689 liberates factor VIII from von Willebrand factor and contributes to factor VIIIa specific activity, thus making both sites essential for procofactor activation. However, cleavage at Arg740, separating the A2–B domainal junction, has not been rigorously studied. To evaluate thrombin cleavage at Arg740, we prepared and stably expressed two recombinant factor VIII mutants, Arg740His and Arg740Gln. Results from a previous study examining proteolysis at Arg372 revealed substantially reduced cleavage rates following substitution of that P1 Arg with His, whereas replacing Arg with Gln at residue 372 yielded an uncleavable bond at that site (Nogami et al., Blood, 2005). Specific activity values for the factor VIII Arg740His and Arg740Gln variants as measured using a one-stage clotting assay were approximately 50% and 18%, respectively, that of the wild type protein. SDS-PAGE and western blotting following a reaction of factor VIII Arg740His with thrombin showed reduced rates of cleavage at His740 as well as at Arg372 relative to the wild type. Alternatively, factor VIII Arg740Gln was resistant to thrombin cleavage at Gln740 and showed little, if any, cleavage at Arg372 over an extended time course. The mutant proteins assayed in a purified system by factor Xa generation showed a slight increase in activity for the Arg740His variant compared with the Arg740Gln variant in both the absence and presence of thrombin, and the activities for both variants were reduced compared with wild type factor VIII. These results suggest that cleavage at residue 740 affects subsequent cleavage at Arg372 and generation of the active cofactor factor VIIIa. Preliminary results obtained evaluating proteolysis of these mutants by factor Xa, which cleaves the same sites in factor VIII as thrombin, also revealed slow proteolysis at the P1 His and no cleavage at the P1 Gln. However, subsequent cleavage at Arg372 exhibited less dependence on initial cleavage at residue 740. These observations may explain the higher than predicted specific activity values obtained for the two variants and suggest a different mechanism of action for the two activating proteinases. Overall, these results support a model whereby cleavage of factor VIII heavy chain by thrombin is an ordered pathway with initial cleavage at Arg740 required to facilitate cleavage at the critical Arg372 site to yield the active cofactor.

DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO

1987 ◽  
Author(s):  
Randal J Kaufman ◽  
Debra D Pittman ◽  
Louise C Wasley ◽  
W Barry Foster ◽  
Godfrey W Amphlett ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Cleavage Site ◽  
Factor Xa ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
Terminal Fragment ◽  
Cofactor Activity

Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.

Factor VIII:E113A Represents a High Specific Activity Factor VIII Arising from a Single Point Mutation within a Ca2+ Binding Site.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1725-1725 ◽  
Author(s):  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Wild Type ◽  
Activity Increase ◽  
Wide Range

Abstract We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca2+, an ion necessary for cofactor activity (Wakabayashi et al., J. Biol. Chem.279:12677–12684, 2004). Using Ala-scanning mutagenesis, it was determined that replacement of residue E113 with Ala yielded a factor VIII point mutant that possessed an ~2-fold increased affinity for Ca2+ as compared with wild type, suggesting that this residue did not directly contribute to Ca2+ coordination but rather modulated the affinity of the ion at this site. Furthermore, the E113A factor VIII possessed twice the specific activity of wild type as determined by a one-stage clotting assay. This increased activity was not likely a result of increased affinity for Ca2+, since assays were performed at saturating Ca2+ levels. Saturation mutagenesis at position 113 revealed that substitution at this position with relatively small, nonpolar residues were well-tolerated, whereas replacement with a number of polar or charged residues was detrimental to activity. Ala-substitution yielded the greatest activity increase of ~2-fold and this level was observed over a wide range of factor VIII concentrations. Time course experiments of factor VIII activation following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Interestingly, results from factor Xa generation assays using purified reactants showed the mutant possessed <10% greater specific activity than wild type and yielded similar values for Km for substrate factor X, kcat for factor Xa generation and Kd for factor IXa. Thus the single amino acid substitution minimally altered cofactor structure or inter-molecular interactions relating to its participation in factor Xase. These results indicate that mutations within this Ca2+ coordination site may selectively enhance cofactor specific activity as measured in a plasma-based assay compared to activity determined in a purified system. The enhanced activity observed for E113A factor VIII may derive from a subtle alteration in conformation affecting a yet to be identified functional parameter.

Mechanism of Interaction between the Heavy Chain of Factor VIII and Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1711-1711 ◽  
Author(s):  
Keiji Nogami ◽  
Qian Zhou ◽  
Hironao Wakabayashi ◽  
Timothy Myles ◽  
Lawrence L. Leung ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Active Site ◽  
Specific Activity ◽  
Solid Phase ◽  
Factor Xa ◽  
Site Directed Mutagenesis ◽  
Marginal Effect ◽  
Thrombin Cleavage ◽  
A2 Domain

Abstract Factor VIII is activated by proteolytic cleavages catalyzed by thrombin or factor Xa. An earlier study indicated that thrombin binding within the C2 domain facilitated cleavage at Arg1689 of factor VIII light chain (Nogami et al. (2000) J. Biol. Chem. 275, 25774–25780). However, thrombin-interactive region(s) within the heavy chain involved with cleaving the A1-A2 and A2-B domainal junctions remain to be determined. Several approaches were employed to examine the interactions between factor VIII heavy chain and thrombin. Fluorescence energy transfer using acrylodan-labeled A1 or A2 subunits (fluorescence donors) and a fluorescein-labeled, Phe-Pro-Arg-chloromethyl ketone active site-modified thrombin (Fl-FPR-thrombin; fluorescence acceptor) showed that FPR-thrombin bound to the A2 subunit with an ~6-fold higher affinity (Kd =36.6 nM) compared with the A1 subunit (Kd=234 nM). Solid phase binding assays using immobilized thrombin S205A, where the active-site Ser205 was converted to Ala by site directed mutagenesis, showed that the binding affinity of A2 subunit was ~3-fold greater than that of A1 subunit. Similar solid phase assays indicated that hirudin, a ligand for anion-binding exosite I of thrombin (ABE-I), effectively blocked thrombin interaction with A1 subunit while having little if any effect on its interaction with A2 subunit. Conversely, heparin, which binds ABE-II, blocked thrombin interaction with A2 subunit while showing only a marginal effect on A1 subunit binding. To identify an interactive site for thrombin in the A2 domain, we focused on two regions containing clustered acidic residues (389GluGluGluAspTrpAsp394 and 720GluAspSerTyrGluAsp725), which are localized near the N- and C-termini of the A2 domain, respectively. SDS-PAGE analyses using isolated factor VIII heavy chain as substrate showed peptides with the sequences 373–395 and 719–740 encompassing these acidic regions, blocked thrombin cleavage at both Arg372 (A1–A2 junction) and Arg740 (A2–B junction) while a 373–385 peptide did not block either cleavage. The 373–395 and 719–740 peptides competitively inhibited A2 binding to S205A thrombin in a solid phase assay (Ki=11.5 and 12.4 μM, respectively), and quenched the fluorescence of Fl-FPR-thrombin. These data demonstrate that both A2 terminal regions support interaction with thrombin. Furthermore, a B-domainless, factor VIII double mutant D392A/D394A was constructed and possessed specific activity equivalent to a severe hemophilia phenotype (<1% compared with wild type). This mutant was resistant to cleavage at Arg740 whereas cleavage at Arg372 was not appreciably affected. Thus the low specific activity of this mutant was attributed to small C-terminal extensions on the A2 subunit that were not removed following cleavage at Arg740. However, factor Xa cleavage of the mutant at Arg740 was not affected. These data suggest the acidic region comprising residues 389–394 in factor VIII A2 domain interacts with thrombin via ABE-II of the proteinase facilitating cleavage at Arg740.

Exosite Interactions Contribute to the Activated Protein C-Catalyzed Inactivation of Factor VIIIa.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1739-1739
Author(s):  
Fatbardha Varfaj ◽  
Julie Neuberg ◽  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Cleavage Site ◽  
Protein C ◽  
Human Factor ◽  
Activated Protein C ◽  
Q Factor ◽  
Wild Type ◽  
Factor Viiia ◽  
Human Factor Viii ◽  
Type Factor

Abstract Activated Protein C (APC) is an anticoagulant serine protease that proteolytically inactivates cofactors Va and VIIIa. Cleavage of human factor VIIIa occurs at Arg336 and Arg562 located within the A1 and A2 subunits, respectively. While cleavages are not ordered, the former site appears to represent a preferred cleavage site. Efficient catalysis requires binding of APC to a phospholipid surface and to the A3-C1-C2 subunit of factor VIIIa. The latter observation suggests that APC likely binds substrate via an exosite(s) thereby contributing to substrate specificity. A study was undertaken to evaluate contributions of substrate docking at the active site and exosite tethering to the APC catalytic mechanism. Recombinant, human factor VIII mutants where P1 Arg residues at 336 and 562 were substituted with Ala or Gln were constructed and stably expressed. Purified factor VIII was converted to factor VIIIa by thrombin and used as substrate to elucidate the mechanism of cleavages. Proteins mutated at Arg336 were also mutated at Lys338 because the latter residue may serve as an alternative APC cleavage site when residue 336 is mutated. Rates of inactivation of wild type and mutant factor VIIIa molecules and rates of cleavage at Arg336 and Arg562 by APC were monitored in the presence and absence of protein S. The R336A/K338A mutant was inactivated and cleaved at the 336 site approximately 20-fold slower than the wild type, whereas the R336Q/K338Q mutant was completely resistant to cleavage at the 336 site. These results indicate that residues other than Arg may be tolerated at the P1 site, whereas Gln yields a cleavage-resistant substrate. Indeed, the R336Q/K338Q/R562Q (triple Q) mutant was resistant to cleavage at both P1 sites. Furthermore, mutations retarding cleavage at residue 336 showed a dramatic decrease in rates of inactivation suggesting that cleavage at this site correlated with the inactivation of factor VIIIa. The importance of exosite interactions was explored by inhibition experiments examining the inactivation of wild type factor VIIIa in the presence of triple Q mutant factor VIIIa. Wild type factor VIIIa inactivation rates decreased as the proportion of triple Q factor VIIIa increased, indicating that the P1 mutant factor VIIIa effectively sequestered APC from the native substrate. Evaluation of inactivation rates suggested that APC possessed an ~8-fold greater affinity for the triple Q FVIIIa than the wild type factor VIIIa. Consistent with that observation, the Ki for triple Q factor VIIIa (29.5 ± 3.6 nM) was ~5-fold less than the Km for wild type factor VIIIa (133 ± 27 nM). Taken together, these results indicate that mutations in the P1 site that prevent cleavage may also retard dissociation of the enzyme-substrate complex. Overall, results from this study suggest that exosite interactions make a primary contribution to substrate affinity during APC-catalyzed inactivation of factor VIIIa.

scholarly journals Role of the B domain for factor VIII and factor V expression and function

Blood ◽  
1994 ◽  
Vol 84 (12) ◽  
pp. 4214-4225 ◽  
Author(s):  
DD Pittman ◽  
KA Marquette ◽  
RJ Kaufman
Keyword(s):  
Factor Viii ◽  
Mrna Expression ◽  
Procoagulant Activity ◽  
Coagulation Cascade ◽  
Factor V ◽  
Wild Type ◽  
Thrombin Cleavage ◽  
Thrombin Activation ◽  
Type Factor ◽  
And Function

Factor V and factor VIII are homologous cofactors in the blood coagulation cascade that have the domain structure A1-A2-B-A3-C1-C2, of which the B domain has extensively diverged. In transfected COS-1 monkey cells, expression of factor VIII is approximately 10-fold less efficient than that of factor V, primarily because of inefficient protein secretion and, to a lesser extent, reduced mRNA expression. To study the functional significance and effect of the B domain on expression and activity, chimeric cDNAs were constructed in which the B domains of factor V and factor VIII were exchanged. Expression of a factor VIII chimera harboring the B-domain of factor V yielded a fully functional factor VIII molecule that was expressed twofold more efficiently than wild-type factor VIII because of increased mRNA expression. Thus, sequences within the factor VIII B domain were not responsible for the inefficient secretion of factor VIII compared with factor V. Expression of a factor V chimera harboring the B domain of factor VIII was slightly reduced compared with wild-type factor V, although the secreted molecule had significantly reduced procoagulant activity correlating with dissociated heavy and light chains and resistance to thrombin activation. Interestingly, the factor V chimera containing the factor VIII B domain was efficiently activated by Russell's viper venum (RVV). A factor V B domain deletion (residues 710- 1545) molecule also exhibited significantly reduced procoagulant activity caused by resistance to thrombin cleavage and activation, although this molecule was activatable by RVV. These results show that, in contrast to factor VIII, thrombin activation of factor V requires sequences within the B domain. In addition, thrombin activation of factor V occurs through a different mechanism than activation by RVV.

The Mutation Leu335Pro in Factor VIII Results in Its Rapid Inactivation by Thrombin-Catalyzed Proteolysis at Arg336.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2690-2690
Author(s):  
Fatbardha Varfaj ◽  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Time Course ◽  
Specific Activity ◽  
Residual Activity ◽  
Factor Xa ◽  
Activity Data ◽  
Activity Increase ◽  
Thrombin Cleavage ◽  
Factor Viiia

Abstract Activation of the heterodimeric factor VIII procofactor is achieved by proteolysis of the heavy chain at residues Arg740 and Arg372 and of the light chain at Arg1689 in reactions catalyzed by thrombin or factor Xa. The active cofactor, factor VIIIa, is a heterotrimer consisting of the A1, A2, and A3-C1-C2 subunits. Factor VIIIa inactivation occurs by two mechanisms, spontaneous dissociation of the A2 subunit from factor VIIIa and proteolytic inactivation catalyzed by activated protein C (APC) as well as factor Xa. APC-catalyzed inactivation of factor VIIIa results from proteolysis at the P1 residues Arg336 and Arg562 within the A1 and A2 subunits, respectively, with cleavage at Arg336 representing the dominant reaction. Factor Xa-catalyzed inactivation of factor VIIIa occurs following cleavage only at the former site. While investigating the role for residues in the P2 position (residue 335) in the proteolytic mechanism for factor VIIIa inactivation, we stably expressed and purified several recombinant B-domainless factor VIII mutants. One variant, Leu335Pro, was based upon the optimal residue (Pro) at the P2 position for thrombin. Specific activity of the Leu335Pro factor VIII was ∼75% that of WT factor VIII. A time course of thrombin-catalyzed activation of the Leu335Pro factor VIII, as monitored by a one-stage clotting assay, yielded a similar activity increase to that of WT, consistent with similar rates of cleavages converting the procofactor to cofactor. However, this activity was highly unstable in the variant and quickly decayed such that at 10 min following thrombin addition, the residual activity of the variant was reduced ∼40-fold compared to WT. To investigate the reason for this rapid rate of inactivation of the newly generated cofactor, we monitored cleavages in the factor VIII heavy chain following reaction with thrombin, as well as APC and factor Xa by Western blot analysis. As predicted from the activity data, thrombin efficiently cleaved both the WT and Leu335Pro at Arg740 and Arg372, generating the A1 and A2 subunits of factor VIIIa. In WT factor VIIIa, we also observed a relatively slow rate of cleavage in the A1 subunit generating a product consistent with proteolysis at the APC-sensitive Arg336 site. This material represented &lt;20% of the total A1 subunit at an extended time point (40 min) in the reaction suggesting that thrombin-cleavage at this site is a minor pathway. However, thrombin cleavage of the A1 subunit derived from Leu335Pro generated the truncated A1 product at a &gt;50-fold increased rate compared with WT. Since Pro represents an optimal residue at the P2 position for thrombin action, these results suggest that the presence of this residue facilitated cleavage at Arg336. While the presence of Pro335 did not appreciably affect cleavage of Arg336 by APC compared to the cleavage rate observed in the WT factor VIII, this rate was ∼5-fold less than thrombin-catalyzed cleavage at Arg336 in the Leu335Pro variant. Furthermore, the rate of factor Xa-catalyzed cleavage at this site in the variant was accelerated ∼10-fold relative to WT. Overall, these data suggest that replacement of Leu335 with Pro markedly increases proteolytic inactivation of factor VIIIa by both thrombin, and to a lesser extent factor Xa by creating a more optimal region for active site engagement at Arg336.

Thrombin Cleavage at Arg1689 Influences Subsequent Cleavage at Arg372 during Activation of Factor VIII

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1021-1021
Author(s):  
Jennifer L. Newell ◽  
Philip Fay
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Specific Activity ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Protein Factor ◽  
Thrombin Cleavage ◽  
Fold Reduction ◽  
Sds Page ◽  
Thrombin Activation

Abstract Hemophilia A is the most common of the severe inherited bleeding disorders and is caused by defects or deficiencies in the blood coagulation protein, factor VIII. Factor VIII circulates as an inactive procofactor and is converted to factor VIIIa by thrombin or factor Xa-catalyzed cleavage at Arg740 (A2-B junction), Arg372 (A1-A2 junction), and Arg1689 (a3-A3 junction). Factor VIII cleavage by thrombin is suggested to be an ordered mechanism, with initial cleavage at Arg740 facilitating cleavages at Arg372 and Arg1689 (Newell and Fay, J. Biol. Chem.282: 25376, 2007). The catalytic efficiency for thrombin cleavage at Arg740 is greater than at either Arg1689 or Arg372, while cleavage at Arg372 appears rate-limiting. For this reason, we investigated whether cleavage at Arg1689 influenced catalysis at the Arg372 site. We constructed, stably transfected, and expressed two recombinant factor VIII mutants, Arg1689His and Arg1689Gln, to slow and eliminate, respectively, cleavage at this site. The specific activity of the Arg1689His and Arg1689Gln factor VIII variants were reduced to 47% and 9% the WT factor VIII value, respectively. This result was consistent with impaired cleavage rates of the light chain impacting this parameter. SDS-PAGE and Western blotting analysis of the Arg1689His variant showed a ~150-fold reduction in the rate of cleavage at Arg1689, while the Arg1689Gln variant was resistant to cleavage at this site. Furthermore, the Arg1689His and Arg1689Gln variants showed 2-fold and 7-fold rate reductions, respectively, in the generation of the A2 subunit following thrombin-catalyzed cleavage at Arg372 as compared to WT factor VIII. This result indicated a linkage between cleavage of the light chain and the primary activating cleavage in the factor VIII heavy chain. In monitoring thrombin activation time courses, we observed a 2-fold reduction in the peak activation for the Arg1689His variant and a 12-fold reduction for Arg1689Gln variant compared to wild type factor VIII. To investigate thrombin binding to Arg1689Gln factor VIII, this variant was used as a competitor of thrombin activation of WT factor VIII. The apparent Ki for Arg1689Gln factor VIII (59 nM) was similar to the Km (apparent) of WT factor VIII (55 nM), suggesting that the Arg1689Gln mutation does not impair binding of thrombin to factor VIII. Taken together, these results suggest thrombin cleavage of factor VIII Arg1689 is preferred before cleavage at Arg372 but not required. Since factor Xa also cleaves factor VIII at the same sites as thrombin, we evaluated factor Xa cleavage of factor VIII using SDS-PAGE and Western blot analysis. These results showed that factor Xa cleavage at Arg372, as judged by A2 subunit generation, for both Arg1689 variants was ~2-fold slower than that for WT factor VIII. Moreover, factor Xa cleavage of light chain at the Arg1721 site was reduced by ~2–3 fold for the variants suggesting that factor Xa cleavage at the Arg1689 site is preferred over the Arg1721 site. Together, these results suggest a less selective mechanism for cleavage order using factor Xa as compared with thrombin.

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