Factor VIIIa is inactivated by a combination of two mechanisms. Activation of factor VIII by thrombin results in a heterotrimeric factor VIIIa that spontaneously inactivates due to dissociation of the A2 subunit. Additionally, factor VIIIa is cleaved by the anticoagulant serine protease, activated protein C, at two cleavage sites, Arg336 in the A1 subunit and Arg562 in the A2 subunit. We previously characterized an engineered variant of factor VIII which contains a disulfide bond between the A2 and the A3 subunits that prevents the spontaneous dissociation of the A2 subunit following thrombin activation. Thus, in the absence of activated protein C, this variant has stable activity following activation by thrombin. To isolate the effects of the individual activated protein C cleavage sites on factor VIIIa, we engineered mutations of the activated protein C cleavage sites into the disulfide bond-cross-linked factor VIII variant. Arg336 cleavage is 6-fold faster than Arg562 cleavage, and the Arg336 cleavage does not fully inactivate factor VIIIa when A2 subunit dissociation is blocked. Protein S enhances both cleavage rates but enhances Arg562 cleavage more than Arg336 cleavage. Factor V also enhances both cleavage rates when protein S is present. Factor V enhances Arg562 cleavage more than Arg336 cleavage as well. As a result, in the presence of both activated protein C cofactors, Arg336 cleavage is only twice as fast as Arg562 cleavage. Therefore, both cleavages contribute significantly to factor VIIIa inactivation. Factor VIIIa is inactivated by a combination of two mechanisms. Activation of factor VIII by thrombin results in a heterotrimeric factor VIIIa that spontaneously inactivates due to dissociation of the A2 subunit. Additionally, factor VIIIa is cleaved by the anticoagulant serine protease, activated protein C, at two cleavage sites, Arg336 in the A1 subunit and Arg562 in the A2 subunit. We previously characterized an engineered variant of factor VIII which contains a disulfide bond between the A2 and the A3 subunits that prevents the spontaneous dissociation of the A2 subunit following thrombin activation. Thus, in the absence of activated protein C, this variant has stable activity following activation by thrombin. To isolate the effects of the individual activated protein C cleavage sites on factor VIIIa, we engineered mutations of the activated protein C cleavage sites into the disulfide bond-cross-linked factor VIII variant. Arg336 cleavage is 6-fold faster than Arg562 cleavage, and the Arg336 cleavage does not fully inactivate factor VIIIa when A2 subunit dissociation is blocked. Protein S enhances both cleavage rates but enhances Arg562 cleavage more than Arg336 cleavage. Factor V also enhances both cleavage rates when protein S is present. Factor V enhances Arg562 cleavage more than Arg336 cleavage as well. As a result, in the presence of both activated protein C cofactors, Arg336 cleavage is only twice as fast as Arg562 cleavage. Therefore, both cleavages contribute significantly to factor VIIIa inactivation. Hemophilia A is an X-linked bleeding disorder, affecting 1 in 5000 males. It is caused by deficiency of blood coagulation factor (F) VIII and can cause severe bleeding after e.g. surgery and trauma or chronic bleeding into muscles and joints (1Lenting P.J. van Mourik J.A. Mertens K. Blood. 1998; 92: 3983-3996Crossref PubMed Google Scholar). Upon initial production of small amounts of thrombin, the intrinsic coagulation pathway is initiated by a positive feedback loop (2Gailani D. Broze Jr., G.J. Science. 1991; 253: 909-912Crossref PubMed Scopus (648) Google Scholar) in which thrombin activates factors V, VIII, and XI. Activated FXI (FXIa) activates FIX to FIXa, which in turn activates FX to FXa. FXa then converts prothrombin to thrombin, leading to fibrin clot formation. FVa and FVIIIa are cofactors for FXa and FIXa, respectively, and enhance the proteolytic function of these enzymes considerably, resulting in rapid production of thrombin. FVIII is a 2332-amino-acid protein with the domain structure A1-A2-B-A3-C1-C2 (3Kane W.H. Davie E.W. Blood. 1988; 71: 539-555Crossref PubMed Google Scholar), which is cleaved within the B domain during secretion and circulates in the blood as a heterodimer bound to von Willebrand factor. Thrombin converts FVIII to its activated form (FVIIIa) by cleavages at positions Arg372, Arg740, and Arg1689 (4Amano K. Michnick D.A. Moussalli M. Kaufman R.J. Thromb. Haemostasis. 1998; 79: 557-563Crossref PubMed Scopus (32) Google Scholar). Cleavage at Arg372 and Arg1689 are both required for complete activation of FVIII (5Pittman D.D. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2429-2433Crossref PubMed Scopus (142) Google Scholar, 6O'Brien D.P. Pattinson J.K. Tuddenham E.G. Blood. 1990; 75: 1664-1672Crossref PubMed Google Scholar). The cleavage at Arg740 leads to dissociation of the B domain from the rest of the molecule, but this cleavage is not required for FVIIIa activation (5Pittman D.D. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2429-2433Crossref PubMed Scopus (142) Google Scholar). After cleavage and dissociation of the B domain, the remaining subunits form a non-covalently linked heterotrimer, consisting of the A1 subunit (1–372), the A2 subunit (373–740), and a light chain containing the A3, C1, and C2 domains (1690–2332) (8Lollar P. Knutson G.J. Fass D.N. Biochemistry. 1985; 24: 8056-8064Crossref PubMed Scopus (77) Google Scholar, 9Eaton D. Rodriquez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (397) Google Scholar). In FVIIIa, the A2 subunit spontaneously dissociates, which inactivates FVIIIa with a half-life of about 2 min (10Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Abstract Full Text PDF PubMed Google Scholar, 11Lollar P. Parker E.T. Fay P.J. J. Biol. Chem. 1992; 267: 23652-23657Abstract Full Text PDF PubMed Google Scholar, 12Pipe S.W. Eickhorst A.N. McKinley S.H. Saenko E.L. Kaufman R.J. Blood. 1999; 93: 176-183Crossref PubMed Google Scholar). FVIIIa can also be inactivated through proteolysis by activated protein C (APC), 2The abbreviations used are: APC, activated protein C; MOPS, 4-morpholinepropanesulfonic acid; Bis/Tris, 2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol; APTT, activated partial thromboplastin time. which cleaves FVIIIa at Arg336 in the A1 domain and at Arg562 in the A2 domain. The proteolytic activity of APC is enhanced by its non-enzymatic cofactors protein S (13Koedam J.A. Meijers J.C.M. Sixma J.J. Bouma B.N. J. Clin. Investig. 1988; 82: 1236-1243Crossref PubMed Scopus (192) Google Scholar) and factor V (FV) (14Shen L. Dahlbäck B. J. Biol. Chem. 1994; 269: 18735-18738Abstract Full Text PDF PubMed Google Scholar). Protein S enhances both FVa (15Walker F.J. J. Biol. Chem. 1980; 255: 5521-5524Abstract Full Text PDF PubMed Google Scholar, 16Walker F.J. J. Biol. Chem. 1981; 256: 11128-11131Abstract Full Text PDF PubMed Google Scholar) and FVIIIa (13Koedam J.A. Meijers J.C.M. Sixma J.J. Bouma B.N. J. Clin. Investig. 1988; 82: 1236-1243Crossref PubMed Scopus (192) Google Scholar, 15Walker F.J. J. Biol. Chem. 1980; 255: 5521-5524Abstract Full Text PDF PubMed Google Scholar) inactivation by APC. FV enhances cleavage of FVIIIa by APC in the presence of protein S in purified systems and prolongs clotting time in plasma-based clotting assays measuring FVIIIa activity (14Shen L. Dahlbäck B. J. Biol. Chem. 1994; 269: 18735-18738Abstract Full Text PDF PubMed Google Scholar, 17Lu D. Kalafatis M. Mann K.G. Long G.L. Blood. 1996; 87: 4708-4717Crossref PubMed Google Scholar). This APC cofactor effect of FV was discovered as a result of the thrombosis risk factor FVLeiden (18Dahlbäck B. Carlsson M. Svensson P.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1004-1008Crossref PubMed Scopus (2009) Google Scholar, 19Dahlbäck B. Hildebrand B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1396-1400Crossref PubMed Scopus (341) Google Scholar). FVLeiden has Arg506 mutated to Gln and therefore cannot be cleaved by APC at this position (20Bertina R.M. Koeleman B.P.C. Koster T. Rosendaal F.R. Dirven R.J. de Ronde H. van der Velden P.A. Reitsma P.H. Nature. 1994; 369: 64-67Crossref PubMed Scopus (3819) Google Scholar, 21Greengard J.S. Sun X. Xu X. Fernández J.A. Griffin J.H. Evatt B. Lancet. 1994; 343: 1361-1362Abstract PubMed Scopus (306) Google Scholar). This cleavage is required for APC to fully inactivate FVa (22Nicolaes G.A.F. Tans G. Thomassen M.C.L.G.D. Hemker H.C. Pabinger I. Varadi K. Schwarz H.P. Rosing J. J. Biol. Chem. 1995; 270: 21158-21166Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). However, it is also required for the APC cofactor activity of FV (23Thorelli E. Kaufman R.J. Dahlback B. Blood. 1999; 93: 2552-2558Crossref PubMed Google Scholar). Cleavage of FVIIIa at Arg336 has been correlated with the inactivation of FVIIIa (9Eaton D. Rodriquez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (397) Google Scholar) and is kinetically favored over cleavage at Arg562 in APC-mediated inactivation of FVIIIa (17Lu D. Kalafatis M. Mann K.G. Long G.L. Blood. 1996; 87: 4708-4717Crossref PubMed Google Scholar, 24Varfaj F. Neuberg J. Jenkins P.V. H. Fay P.J. J. PubMed Scopus Google Scholar). As cleavage at Arg336 also A2 subunit which fully inactivates FVIIIa, this is the inactivation F. Neuberg J. Jenkins P.V. H. Fay P.J. J. PubMed Scopus Google Scholar). The cofactors of APC the cleavage rates of these individual sites M. Fay P.J. Blood. PubMed Google Scholar) FVIIIa proteolysis by APC for the A1 or A2 domains and that protein S enhanced both cleavages but a effect on cleavage at In this we the individual cleavages in FVIIIa by APC that to the inactivation of FVIIIa and the effect of the APC cofactors, protein S and factor V, on cleavage. inactivation of FVIIIa due to dissociation proteolytic inactivation of FVIIIa to This has been in the by of FVIIIa F. Neuberg J. Jenkins P.V. H. Fay P.J. J. PubMed Scopus Google Scholar) or proteolysis of the FVIII than FVIIIa (4Amano K. Michnick D.A. Moussalli M. Kaufman R.J. Thromb. Haemostasis. 1998; 79: 557-563Crossref PubMed Scopus (32) Google Scholar). To this we used a FVIII variant that has a disulfide bond engineered between the A2 and A3 domains This disulfide bond prevents dissociation of the A2 domain and the D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). This to the effects of APC proteolysis on FVIIIa activity of the effects of A2 subunit A disulfide bond in FVa for of individual APC cleavage sites in of A2 subunit dissociation Xu X. Griffin J.H. Protein Sci. PubMed Scopus Google Scholar). After activation by thrombin, FVIIIa has cofactor activity to that of FVIIIa D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). As has been F. Neuberg J. Jenkins P.V. H. Fay P.J. J. PubMed Scopus Google Scholar), cleavage at Arg336 was faster than cleavage at However, we that in the absence of A2 subunit cleavage at Arg336 not fully inactivate FVIIIa, cleavage at Arg562 We also that protein S both FVIIIa cleavages but Arg562 cleavage FV also enhanced cleavage at Arg562 more than cleavage at As a result, in the presence of both protein S and APC cleavage at Arg336 was only faster than APC cleavage at was from FVIII and the from B FVIII was from protein and factor from Activated protein C and factor from was from serine and from was from of FVIII FVIII and as previously D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar, J. Thromb. Haemostasis. PubMed Scopus Google Scholar). The FVIII also in B FVIII that the and mutations the from Arg336 was mutated to Gln by to Arg562 was mutated to Gln by to was after the mutated FVIII into that not through into and for stable with and the FVIII was purified from the and to in the After as J. Thromb. Haemostasis. PubMed Scopus Google Scholar), in FVIII was the factor from and the FVIII from the with FVIII as the FVIII activity was the VIII with FVIII as the as R.M. Griffin J.H. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). FVIII and proteolysis by thrombin and APC with about FVIII was with by with to or with to and and disulfide FVIII at with was with for 1 min by the of to inactivate the thrombin protein S for a at The used the as for the assays that was on a with and with the of FVIIIa and of FVIIIa in assays of the We the of FVIIIa sites for in assays as D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). the FVIIIa for FVIIIa variant in this as with the that FVIII was activated with thrombin for 1 min as as used by to complete inactivation of the thrombin. to a to for of the D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). FX as and to the to and D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). The used was the of the the of this that be on the of the of that FVIIIa variant with at that of FVIIIa inactivation by as FVIII at was in with Thrombin was to this at to the and then at 1 was to inactivate the thrombin. was for in the APC protein S FV in over time and to and FX was to the as D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). in the FVIIIa, 2 FIXa, and The was with and FXa was with The variant FVIIIa has two APC cleavage sites, and we that FVIIIa inactivation can two 1 and 2 In the FVIIIa is cleaved at resulting in a with partial activity this cleavage is by cleavage at which results in FVIIIa In the FVIIIa is cleaved at and this cleavage fully inactivates for inactivation can be to a (22Nicolaes G.A.F. Tans G. Thomassen M.C.L.G.D. Hemker H.C. Pabinger I. Varadi K. Schwarz H.P. Rosing J. J. Biol. Chem. 1995; 270: 21158-21166Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, Griffin J.H. Blood. PubMed Google Scholar). However, we that we not in this the of cleavages was Therefore, we not cleavage rates from to inactivation. FVIII and FVIII inactivation are both the result of a inactivation an However, to for the of only the initial of activity was Thus, for FVIII and the initial time that on a to a with the following A is activity of FVIIIa is the initial activity of FVIIIa, is the in and is time in F. Neuberg J. Jenkins P.V. H. Fay P.J. J. PubMed Scopus Google Scholar). The for at to an to a of and we then that was to complete cleavage at in 1 are as of FVIIIa cleavage that of activity is to cleavage of FVIIIa was and APC as in the of inactivation of FVIIIa by APC and protein S or of FVIIIa was over time by FVIIIa with APC. FVIIIa with APC and protein S. FVIIIa with APC and to inactivation was to a and to are as of inactivation of FVIIIa by APC. FVIIIa activity was over time by a purified containing FIXa, and FVIIIa FVIIIa with APC. to inactivation was to a and to are as cleavage rates for cleavages at Arg336 and Arg562 in FVIIIa Arg336 FVIIIa Arg562 in a FVIIIa inactivation by APC and protein S was also with an as that FVIII was activated with for 1 min by J. Thromb. Haemostasis. PubMed Scopus Google Scholar). with the of APC and protein S to of 1 and time was to FVIII activity with a of FVIII for the in the FVIII and of FVIII which contains an engineered disulfide bond between and has been characterized and D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). This variant was in the of B the containing we individual mutations of the APC cleavage sites mutated from to the and The in and purified as D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar, J. Thromb. Haemostasis. PubMed Scopus Google Scholar). FVIII and activity as and and FVIII activity of of FVIII activity of of and FVIII activity of of To the of these and to the presence of the disulfide we of and FVIII protein was in and and form and chain and light chain in the However, a of the FVIII was as a chain and light chain at about at for FVIII and FVIII was we not the of of the of the protein from chain to chain and light FVIII and FVIII to However, FVIII present. In in the was a and the light chain was more than the chain This was the two which that be a protein in FVIII that at about the as the light chain in the This was FVIII was at significantly in the was We not to this in two at this variant. To that mutations at Arg336 and Arg562 cleavage by APC, we FVIIIa in a after proteolysis by thrombin to to FVIIIa and then by APC with protein S Thrombin was in and thrombin with APC and protein S in to from thrombin, APC, and protein S in the FVIII FVIIIa was cleaved at both Arg336 and Arg562 by APC. the A1 subunit was to Arg336 and the A2 subunit was to Arg562 The A1 subunit was by the A1 the A2 subunit The A2 subunit was a that was The resulting be and not in this FVIIIa the A1 and A2 subunit APC cleavage, the A1 was and was by the A1 but the A2 in in Thus, was cleavage at FVIII was not as as the the was not as In in the absence of thrombin or APC the chain and light chain not as was in 1 and As this been due to a This was by the that the not thrombin or APC in and Additionally, and the FVIIIa not as as the FVIIIa However, the of that this not the of this FVIII of the containing variant FVIIIa and after with in a the A1 and A2 subunit be in at and After proteolysis by APC the A1 subunit at was present. A at was to the A1 subunit cleaved at However, this was a protein in the of thrombin, APC, and protein S In the A2 subunit at was complete cleavage at that the thrombin, which was in this at about and in in the containing APC and protein these also thrombin and This result was in and a cleavage of thrombin in these However, we not a for this of FVIIIa by APC and Protein the inactivation of the FVIII in a time the clotting to FVIIIa activity FVIII activated with thrombin. In the absence of APC and protein FVIIIa and FVIIIa about and only This was due to the disulfide which prevents A2 subunit dissociation in FVIIIa, as was previously with FVIIIa D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). However, in this FVIIIa was more stable than previously D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). We more thrombin to FVIII than used previously and also a of to inactivation of that this the in these results also We that the FVIII is more fully activated with thrombin. APC and protein S to the FVIIIa FVIIIa was inactivated in the absence of A2 subunit dissociation than activity at APC cleavage inactivated more but to of by APC the of inactivation of FVIIIa and FVIIIa, we inactivation of FVIIIa a with purified In this FVIIIa only about activity after min in the absence of APC However, in the presence of APC inactivated FVIIIa was fully inactivated in the time FVIIIa was inactivated more but activity to the time In to FVIIIa about of its but then the activity The inactivation of was to a However, not in these the of inactivation was not Therefore, we not from the inactivation of and to only the initial time that on a for the two cleavage used to initial cleavage rates for the Arg336 cleavage and the Arg562 cleavage In FVIIIa, APC cleaved Arg336 at a of of APC. APC cleaved Arg562 in FVIIIa at a of of APC. of APC on S and FV are both cofactors for APC in the proteolysis of FVIIIa (14Shen L. Dahlbäck B. J. Biol. Chem. 1994; 269: 18735-18738Abstract Full Text PDF PubMed Google Scholar, 15Walker F.J. J. Biol. Chem. 1980; 255: 5521-5524Abstract Full Text PDF PubMed Google Scholar). APC at a of not fully inactivate the FVIIIa during a time However, protein S significantly enhanced the of inactivation of FVIIIa by APC Protein S the of Arg336 cleavage about in protein S the of Arg562 cleavage in FVIIIa FV APC cofactor effect in the absence of protein S A in cleavage at Arg562 or not be This was with results (14Shen L. Dahlbäck B. J. Biol. Chem. 1994; 269: 18735-18738Abstract Full Text PDF PubMed Google Scholar, 17Lu D. Kalafatis M. Mann K.G. Long G.L. Blood. 1996; 87: 4708-4717Crossref PubMed Google Scholar). However, in the presence of protein FV was a cofactor for APC proteolysis of FVIIIa APC to 1 and with protein FV the cleavage of Arg336 in FVIIIa by over the S and it the cleavage of Arg562 in FVIIIa by over the S Arg336 was cleaved at a of of APC, and Arg562 was cleaved at a of of APC. protein S and FV enhanced both cleavage to the Arg562 cleavage about and to the Arg336 cleavage about that the Arg562 cleavage was to of the Arg336 cleavage in the presence of both Cleavage rates be at two APC for APC and and for APC protein S and not significantly the in 1 over both of APC on of FVIIIa a dissociation for of FVIIIa to with a of into the with FVIIIa and We and for the with a of the into the FVIIIa a for of for FX of and of After complete cleavage by APC, FVIIIa cofactor activity was FVIIIa with a of of and of This variant also activity in the after cleavage by APC In these inactivation time FVIIIa a of about activity after complete cleavage at APC proteolysis at Arg336 the for from to and the for FX activation from to the from to Therefore, cleavage at Arg562 the variant fully inactivated FVIIIa, cleavage at Arg336 the variant only inactivated FVIIIa in the absence of dissociation of the A2 subunit. We previously that FVIII a disulfide bond between in the A2 domain and in the A3 domain J. Thromb. Haemostasis. PubMed Scopus Google Scholar). We that FVIII in the but was not inactivated the of spontaneous dissociation of the A2 subunit following activation by thrombin D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). This that the disulfide bond A2 subunit dissociation as it not the FVIIIa protein in that the that are in the cofactor function of FVIIIa in the Additionally, the that the of this disulfide bond does not the of FVIIIa that inactivation due to A2 subunit dissociation inactivation due to proteolysis be D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). significantly from previously results for FVIIIa D. Griffin J.H. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). This was due to a of we activated FVIII with more thrombin in these than previously and We also to for and factor both of which are for this We that these more the of the However, this does not previously We used a FVa variant to isolate the effects of APC proteolysis from A2 subunit dissociation that APC proteolysis in FVa Xu X. Griffin J.H. Protein Sci. PubMed Scopus Google Scholar). Therefore, this disulfide FVIIIa was a for the effects of the individual APC cleavages on FVIIIa A2 subunit dissociation does not it of the effects of the APC cofactors, protein and factor V at We that APC cleaves Arg336 faster than it cleaves However, the in rates that we cleavage was 6-fold faster than Arg562 was than that in a of APC cleavage a disulfide F. Neuberg J. Jenkins P.V. H. Fay P.J. J. PubMed Scopus Google Scholar) that Arg336 cleavage by APC faster than Arg562 cleavage. This be due to in the of FVIIIa that was used in In results these In the absence of A2 subunit the Arg562 cleavage within the A2 subunit fully inactivates This has been in and Arg562 is in a within F. Neuberg J. Jenkins P.V. H. Fay P.J. J. PubMed Scopus Google Scholar, P.J. T. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). Arg336 cleavage within the A1 subunit does not fully inactivate The partial of activity is to a in for as as a in for FX and a in for FX This partial of activity due to cleavage at Arg336 was also in cleaved or FVIIIa subunits at to activity K. H. K. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The effect of cleavage at Arg336 on is this cleavage which to FX Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and cleavages by FXa at both and Arg336 result in a in K. H. K. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, this is the the complete effects and of cleavage at Arg336 in from cleavages or from subunit the of between FVIII and are in the of inactivation of FVIIIa and FVa as a result of APC As was previously cleavage is faster than Arg562 cleavage F. Neuberg J. Jenkins P.V. H. Fay P.J. J. PubMed Scopus Google Scholar). This is in to FVa APC cleavage at Arg506 to is faster than cleavage at to The APC cofactor protein S FV in the of FVIIIa enhances the cleavage of the APC cleavage more than the faster APC cleavage. Thus, protein S enhances cleavage in FVa J. L. G.A.F. Thomassen M.C.L.G.D. Hemker H.C. Varadi K. Schwarz H.P. Tans G. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), protein S and FV enhance Arg562 cleavage in FVIIIa, in FVIIIa, both cleavage rates are The effect for protein S that we was to of protein S effect M. Fay P.J. Blood. PubMed Google Scholar). Therefore, as is the for in the presence of both cofactors for APC, the APC cleavages at are in the inactivation of FVa and In both FVa and FVIIIa, the cleavages in the A1 domain and result in dissociation the of or enhanced dissociation the of of the A2 which fully inactivates the However, when A2 subunit dissociation is both cofactors In both FVa and FVIIIa, the cleavages in the A2 domain or result in a of activity than does the A1 subunit cleavage when A2 subunit dissociation is In Arg506 cleavage results in a in FXa cleavage only results in a in FXa (22Nicolaes G.A.F. Tans G. Thomassen M.C.L.G.D. Hemker H.C. Pabinger I. Varadi K. Schwarz H.P. Rosing J. J. Biol. Chem. 1995; 270: 21158-21166Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, Xu X. Griffin J.H. Protein Sci. PubMed Scopus Google Scholar). In FVIIIa, Arg562 cleavage fully inactivates FVIIIa of subunit Arg336 cleavage only inactivates FVIIIa of subunit dissociation In these FVIIIa to isolate and therefore the effects of APC cleavage on FVIIIa of FVIIIa In this has a of in of the of the APC cofactors, protein S and in these In the presence of the APC cofactors, which is the in both cleavages a in FVIIIa inactivation.
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