Mechanisms of Plasmin-catalyzed Inactivation of Factor VIII (2006)

Plasmin not only functions as a key enzyme in the fibrinolytic system but also directly inactivates factor VIII and other clotting factors such as factor V. However, the mechanisms of plasmin-catalyzed factor VIII inactivation are poorly understood. In this study, levels of factor VIII activity increased ∼2-fold within 3 min in the presence of plasmin, and subsequently decreased to undetectable levels within 45 min. This time-dependent reaction was not affected by von Willebrand factor and phospholipid. The rate constant of plasmin-catalyzed factor VIIIa inactivation was ∼12- and ∼3.7-fold greater than those mediated by factor Xa and activated protein C, respectively. SDS-PAGE analysis showed that plasmin cleaved the heavy chain of factor VIII into two terminal products, A137–336 and A2 subunits, by limited proteolysis at Lys36, Arg336, Arg372, and Arg740. The 80-kDa light chain was converted into a 67-kDa subunit by cleavage at Arg1689 and Arg1721, identical to the pattern induced by factor Xa. Plasmin-catalyzed cleavage at Arg336 proceeded faster than that at Arg372, in contrast to proteolysis by factor Xa. Furthermore, breakdown was faster than that in the presence of activated protein C, consistent with rapid inactivation of factor VIII. The cleavages at Arg336 and Lys36 occurred rapidly in the presence of A2 and A3-C1-C2 subunits, respectively. These results strongly indicated that cleavage at Arg336 was a central mechanism of plasmin-catalyzed factor VIII inactivation. Furthermore, the cleavages at Arg336 and Lys36 appeared to be selectively regulated by the A2 and A3-C1-C2 domains, respectively, interacting with plasmin. Plasmin not only functions as a key enzyme in the fibrinolytic system but also directly inactivates factor VIII and other clotting factors such as factor V. However, the mechanisms of plasmin-catalyzed factor VIII inactivation are poorly understood. In this study, levels of factor VIII activity increased ∼2-fold within 3 min in the presence of plasmin, and subsequently decreased to undetectable levels within 45 min. This time-dependent reaction was not affected by von Willebrand factor and phospholipid. The rate constant of plasmin-catalyzed factor VIIIa inactivation was ∼12- and ∼3.7-fold greater than those mediated by factor Xa and activated protein C, respectively. SDS-PAGE analysis showed that plasmin cleaved the heavy chain of factor VIII into two terminal products, A137–336 and A2 subunits, by limited proteolysis at Lys36, Arg336, Arg372, and Arg740. The 80-kDa light chain was converted into a 67-kDa subunit by cleavage at Arg1689 and Arg1721, identical to the pattern induced by factor Xa. Plasmin-catalyzed cleavage at Arg336 proceeded faster than that at Arg372, in contrast to proteolysis by factor Xa. Furthermore, breakdown was faster than that in the presence of activated protein C, consistent with rapid inactivation of factor VIII. The cleavages at Arg336 and Lys36 occurred rapidly in the presence of A2 and A3-C1-C2 subunits, respectively. These results strongly indicated that cleavage at Arg336 was a central mechanism of plasmin-catalyzed factor VIII inactivation. Furthermore, the cleavages at Arg336 and Lys36 appeared to be selectively regulated by the A2 and A3-C1-C2 domains, respectively, interacting with plasmin. Factor VIII, a plasma protein deficient or defective in individuals with the severe congenital bleeding disorder hemophilia A, functions as a cofactor in the tenase complex, which is responsible for anionic phospholipid surface-dependent conversion of factor X to Xa by factor IXa (1Mann K.G. Nesheim M.E. Church W.R. Haley P. Krishnaswamy S. Blood. 1990; 76: 1-16Crossref PubMed Google Scholar). Factor VIII circulates as a complex with VWF 2The abbreviations used are: VWF, von Willebrand factor; APC, activated protein C; MES, 4-morpholineethanesulfonic acid; CAPS, 3-(cyclohexylamino) propanesulfonic acid. that protects and stabilizes the cofactor. Factor VIII is synthesized as a single chain molecule consisting of 2,332 amino acid residues with a molecular mass of ∼300 kDa (2Wood W.I. Capon D.J. Simonsen C.C. Eaton D.L. Gitschier J. Keyt B. Seeburg P.H. Smith D.H. Hollingshead P. Wion K.L. Delwart E. Tuddenham E.D.G. Vehar G.A. Lawn R.M. Nature. 1984; 312: 330-337Crossref PubMed Scopus (522) Google Scholar, 3Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Wood W.I. Harkins R.N. Tuddenham E.G.D. Lawn R.M. Capon D.J. Nature. 1984; 312: 337-342Crossref PubMed Scopus (660) Google Scholar). The factor VIII molecule can be divided into three domains arranged in the order of A1-A2-B-A3-C1-C2 according to the amino acid content homology. It is processed into a series of metal ion-dependent heterodimers by cleavage at the B-A3 junction, generating a heavy chain consisting of the A1 and A2 domains, plus heterogeneous fragments of a partially proteolyzed B domain, linked to a light chain consisting of the A3, C1, and C2 domains (2Wood W.I. Capon D.J. Simonsen C.C. Eaton D.L. Gitschier J. Keyt B. Seeburg P.H. Smith D.H. Hollingshead P. Wion K.L. Delwart E. Tuddenham E.D.G. Vehar G.A. Lawn R.M. Nature. 1984; 312: 330-337Crossref PubMed Scopus (522) Google Scholar, 3Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Wood W.I. Harkins R.N. Tuddenham E.G.D. Lawn R.M. Capon D.J. Nature. 1984; 312: 337-342Crossref PubMed Scopus (660) Google Scholar, 4Fay P.J. Anderson M.T. Chavin S.I. Marder V.J. Biochim. Biophys. Acta. 1986; 871: 268-278Crossref PubMed Scopus (124) Google Scholar). The catalytic efficacy of factor VIII in the tenase complex is enhanced over 105 times by conversion into an active form, factor VIIIa, by limited proteolysis by either thrombin or factor Xa (5Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (399) Google Scholar). Both enzymes cleave factor VIII at Arg372 and Arg740 of the heavy chain and produce 50-kDa A1 and 40-kDa A2 subunits. The 80-kDa light chain is also cleaved at Arg1689 generating a 70-kDa A3-C1-C2 subunit. Additionally, factor Xa cleaves at Arg1721 and produces a 67-kDa A3-C1-C2 subunit. Proteolysis at Arg372 and Arg1689 is essential for generating factor VIIIa cofactor activity (6Fay P.J. Blood Rev. 2004; 18: 1-15Crossref PubMed Scopus (161) Google Scholar). Cleavage at the former site exposes a functional factor IXa-interactive site within the A2 domain that is cryptic in the unactivated molecule (7Fay P.J. Mastri M. Koszelak M.E. Wakabayashi H. J. Biol. Chem. 2001; 276: 12434-12439Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Cleavage at the latter site liberates the cofactor from its carrier protein, VWF (8Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar), contributing to the overall specific activity of the cofactor (9Regan L.M. Fay P.J. J. Biol. Chem. 1995; 270: 8546-8552Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 10Donath M.S. Lenting P.J. van Mourik J.A. Mertens K. J. Biol. Chem. 1995; 270: 3648-3655Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Factor VIIIa cofactor activity is down-regulated in the presence of serine proteases such as APC (5Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (399) Google Scholar), factor Xa (5Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (399) Google Scholar), and factor IXa (11Lamphear B.J. Fay P.J. Blood. 1992; 80: 3120-3126Crossref PubMed Google Scholar) by proteolytic inactivation following cleavage at Arg336 within the A1 subunit. This inactivation appears to be the result of altered with the A2 subunit and an increased of the A1 for the factor X M.E. K. J. Mastri M. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Wakabayashi H. K. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the latter reaction of a factor site within residues K. J. Wakabayashi H. Fay P.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Factor Xa and APC also cleave at Lys36 K. Wakabayashi H. K. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and at P.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), respectively. These the of the with the A2 subunit K. Wakabayashi H. K. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and the with factor IXa in the tenase complex L.M. B.J. Fay P.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). is regulated by of by the serine plasmin, at the of the that plasmin also by proteolysis of factor K.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. K.G. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), IXa J.A. Nesheim M.E. Blood. PubMed Google Scholar), X J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and factor VIII R. Full Text PDF PubMed Scopus Google Scholar, M.E. Blood. 1986; PubMed Google Scholar). These that plasmin tenase activity by factor the mechanism of this inactivation is poorly understood. In this study, the mechanism of plasmin-catalyzed factor VIII inactivation. and SDS-PAGE analysis of the cofactor for the that inactivation was directly with limited proteolysis that to three terminal products, and and subsequently to the cleavage at Cleavage at Arg336 and Lys36 appeared to be selectively following of the with A2 and A3-C1-C2 subunits, respectively. factor VIII from The L.M. Fay P.J. Blood. PubMed Google Scholar) and S. J. 1988; PubMed Scopus Google Scholar) the and of the A1 domain from and respectively. The the A2 domain was from J. R. The the of the domain was as M. M. H. H. J. 1992; PubMed Scopus Google Scholar). VWF was from factor a a and with factor VIII as M. M. H. H. J. 1992; PubMed Scopus Google Scholar). of factor VIII greater than of and J.A. Biochemistry. PubMed Scopus Google Scholar). was from and was to be of factor Xa and Factor APC, and protein from of Factor VIIIa VIII was at in MES, and and light and heavy following and as (7Fay P.J. Mastri M. Koszelak M.E. Wakabayashi H. J. Biol. Chem. 2001; 276: 12434-12439Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). heavy chain was cleaved by and the A2 and A1 by and respectively, as M.E. K. J. Mastri M. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A3-C1-C2 from light chain by as Fay P.J. Biochemistry. 2004; PubMed Scopus Google Scholar). Factor VIIIa was from factor VIII by L.M. Mastri M. Fay P.J. Blood. PubMed Google Scholar). by from A1 and A3-C1-C2 by at in and K. Wakabayashi H. K. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). SDS-PAGE of the by with showed by the of PubMed Scopus Google Scholar). activity was in a clotting factor plasma Scholar). at factor VIII in and plus phospholipid from the at the indicated and plasmin reaction was by the of and The presence of plasmin and in the was not to factor VIII activity in the Cleavage of Factor and by plasmin was to factor and its at a in the presence of phospholipid in and at at the indicated and the and for SDS-PAGE by and for 3 min. and was as by Nature. PubMed Scopus Google Scholar). was a at for following with protein was to a a at for in CAPS, and the indicated VIII by enhanced of of the fragments following plasmin cleavage of factor VIII an The factor VIII fragments and to of Factor VIII and by factor VIII inactivation by plasmin R. Full Text PDF PubMed Scopus Google Scholar, M.E. Blood. 1986; PubMed Google inactivation mechanisms plasmin-catalyzed factor VIII inactivation in the presence of phospholipid and a clotting that the presence of either plasmin or not at of the reaction of factor VIII and plasmin, factor VIIIa activity was 3 min and an This activity was by a to the at and to an undetectable within 45 min. results in the of phospholipid Factor VIII circulates as a complex with VWF, which protects and stabilizes the cofactor from for by APC K. M. K. K. M. H. Blood. PubMed Scopus Google Scholar, P.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In the presence of VWF not the reaction plasmin and factor VIII. of Factor VIII by APC, and Factor serine proteases responsible for factor inactivation are APC and factor Xa. factor VIII inactivation by plasmin, APC, and factor Xa in a clotting was with factor VIII in the presence of phospholipid as showed that the presence of not the APC with protein factor VIII activity in a time-dependent with in activity The activity of factor VIII with factor Xa increased ∼2-fold with that the of plasmin. However, the of factor VIIIa activity in the presence of plasmin was not with factor Xa. In a was for min and was by a in The activity of factor VIIIa at from unactivated activated and activated that following factor VIII subunit the of inactivation of factor VIII by active factor VIIIa as a was with factor VIIIa in the presence of phospholipid. The are in The to a single and showed that the inactivation rate of factor VIIIa activity in the of A2 subunit from factor VIIIa was to P.J. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The rate of inactivation of factor VIIIa activity by plasmin, APC, and factor Xa the and respectively, that the of factor VIIIa inactivation by plasmin was and greater than that with APC and factor respectively. Cleavage of Factor VIII by Plasmin and of Cleavage factor VIII induced by plasmin that by by a rapid in and the in inactivation plasmin, APC, and factor strongly that the mechanism of of plasmin is from those of APC and factor Xa. for this in of factor VIII factor VIII and plasmin in the presence of phospholipid. SDS-PAGE showed that plasmin proteolyzed factor VIII at cleavage The heavy chain fragments consisting of and B domains at min the of plasmin. In the appeared to by limited proteolysis into protein of and The and with The and fragments appeared to as terminal at in the of factor VIII The 80-kDa light chain fragments to 70-kDa and 67-kDa fragments within 45 min. the of fragments to analysis and with the amino acid from factor VIII (2Wood W.I. Capon D.J. Simonsen C.C. Eaton D.L. Gitschier J. Keyt B. Seeburg P.H. Smith D.H. Hollingshead P. Wion K.L. Delwart E. Tuddenham E.D.G. Vehar G.A. Lawn R.M. Nature. 1984; 312: 330-337Crossref PubMed Scopus (522) Google Scholar, 3Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Wood W.I. Harkins R.N. Tuddenham E.G.D. Lawn R.M. Capon D.J. Nature. 1984; 312: 337-342Crossref PubMed Scopus (660) Google Scholar). The results indicated that plasmin cleaved the cofactor at in the heavy Lys36, Arg336, Arg372, and and at two in the light Arg1689 and These appeared to be identical to those cleaved by factor Xa (5Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (399) Google Scholar, K. Wakabayashi H. K. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of factor VIII from cleavage by plasmin from the the molecular for cleavage of plasmin, and in VIII The amino acid of the and fragments from the heavy chain to be residues and respectively, and as and respectively. The amino acid of the and 67-kDa fragments from the light chain residues and respectively, that the light chain was cleaved by plasmin in the order Arg1689 and The fragments is in Cleavage of the Factor VIII by factor VIII activity with the pattern of limited proteolysis of the heavy as by cleavage at Arg372 by thrombin and factor Xa for and cleavage at Arg336 by APC and factor Xa for inactivation. plasmin cleavage of the factor VIII heavy chain of proteolysis two the or of A1 and The cleavage of the heavy chain by plasmin and of factor VIII are in In the presence of factor VIII plasmin and the heavy chain was converted into by cleavage at Arg740 the appeared to be rapidly than the A2 that cleavage at Arg336 occurred than that at the and fragments as These not at the and the and the and the and in respectively, the to and fragments that cleavage at Lys36 and Arg372 by plasmin terminal A137–336 and by cleavage at Arg372 was proteolyzed at Arg336 and Lys36, also in the terminal These results that plasmin proteolyzed the heavy chain into A137–336 and A2 by two cleavage a cleavage at Arg336 and a cleavage at Cleavage of the A1 of Factor by APC, and Factor cleavage within the A1 subunit are Arg336 for APC and Arg372, Arg336, and Lys36 for factor Xa and plasmin. The with the activity of factor VIII with proteases in to this the A1 cleavage pattern mediated by plasmin, APC, and factor Xa was by The of APC with cofactor protein cleaved factor VIII at Arg336 and in the of fragments in a time-dependent Factor Xa cleaved at Arg740 by cleavages at Arg372 and Arg336 consistent with the (5Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (399) Google Scholar). However, plasmin cleaved the A1 subunit at Arg336 rapidly than at Arg372, by cleavage at Lys36 at an These consistent with cleavage of the A1 subunit by plasmin, APC, and factor the cleavage appeared to be or active factor VIIIa, in of factor VIII, to the of A1 was to the proteolysis of the A1 subunit in factor VIIIa and the rate of of subunit The by and the a single The rate of cleavage of A1 in factor VIIIa by plasmin was and was to than greater than that mediated by APC and factor Xa These in with P.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. Wakabayashi H. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that inactivation of factor VIIIa can be to A1 proteolysis at Arg336 and that cleavage plasmin-catalyzed inactivation of factor VIII. of Factor VIIIa in A1 Cleavage by the and mechanisms of subunit in factor for factor fragments used as for plasmin by the the for this the of the A1 fragments but not A137–336 the or of can be to cleavage at Arg336 or Lys36, respectively. The fragments from factor VIII factor VIIIa and heavy chain at an the of plasmin. was and with A1 the was the of plasmin, respectively. Factor and the heavy chain the A2 domain, but this is not in or A1 subunits. results the that cleavage at Arg336 be by the A2 domain the fragments from factor VIII and factor VIIIa within min the of plasmin. In with the heavy chain and A1 subunit the fragments strongly at the the the the of plasmin, the of It that cleavage at Lys36 in the was with cleavage at The factor molecule and the A3-C1-C2 subunits, but are not of the heavy chain or that cleavage at Lys36 was regulated by the presence of the A3-C1-C2 subunit that specific cleavages at Arg336 and Lys36 by plasmin appeared to be selectively following of plasmin with the A2 and A3-C1-C2 respectively. In is a and or to or to the and inactivation of and fibrinolytic factors Google Scholar, B. Google Scholar). In the of the is by proteolysis of clotting factors D.P. Tuddenham E.G.D. and Scholar). the of proteases are to be active and in plasmin is to be a of the proteolysis Nesheim M.E. H. D.C. 1990; PubMed Scopus Google Scholar, H. Nesheim M.E. R. D.C. Blood. 1992; PubMed Google Scholar). In this the proteolysis of by plasmin and in the D.P. and Scholar). It is that other mechanisms proteases a and that plasmin an by the activity of factor or factor VIII and of the 1995; 80: Full Text PDF PubMed Scopus Google Scholar) that an in the plasma of plasmin with decreased factor VIIIa This was to the factor VIII and plasmin. that plasmin factor VIII activity rapidly an in activity in a This was for factor VIII and active factor VIIIa not However, the of plasmin factor VIII was from that for other clotting the inactivation of factor by plasmin appeared to be K.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the mechanism to be with residues of the factor molecule M. K.G. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). cleavage and inactivation of factor X appeared to be J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Factor VIII and factor are W.R. J. R.M. J. Nesheim M.E. K.G. S. 1984; PubMed Scopus Google Scholar), and in the of plasmin factor VIII and factor V. Factor VIII circulates in a complex with for factor VIII with VWF to the of the domain (8Lollar P. Hill-Eubanks D.C. Parker C.G. J. Biol. Chem. 1988; 263: 10451-10455Abstract Full Text PDF PubMed Google Scholar), to the of the C2 domain M. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and within the domain K. M. K. K. M. H. Blood. PubMed Scopus Google Scholar, S. K. Blood. PubMed Google Scholar), the that the A3-C1-C2 an for with In VWF phospholipid affected plasmin-catalyzed factor VIII inactivation. It appeared that plasmin with factor VIII not VWF and and was not activated unactivated of the cofactor. These indicated that plasmin be a for factor VIII. and inactivation of factor VIII by other APC K. M. K. K. M. H. Blood. PubMed Scopus Google Scholar, P.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and factor Xa J.A. J. 1990; PubMed Scopus Google Scholar, K. M. K. D. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), are affected by the presence of an by and M.E. Blood. 1986; PubMed Google Scholar) that factor VIII and inactivation by plasmin was affected by the presence of but not by phospholipid The for this was not but be that the of factor VIII to be to plasmin with that to phospholipid phospholipid and are in to in reaction phospholipid and of SDS-PAGE and analysis that limited proteolysis of factor VIII occurred at in the heavy Lys36, Arg336, Arg372, and and at two in the light Arg1689 and Arg1721, in the of three terminal products, and within of plasmin. The amino acid of cleavage is in with the of the for either or residues Biochim. Biophys. Acta. PubMed Scopus Google Scholar). The plasmin cleavage those by thrombin and factor Xa that result in of factor VIII and those by APC and factor Xa that result in inactivation. the plasmin cleavage identical to those of factor VIII with factor Xa (5Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Crossref PubMed Scopus (399) Google Scholar, K. Wakabayashi H. K. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), but of the APC cleavage was not affected by plasmin. also the A2 with an The of the identical not this is at the Fay (7Fay P.J. Mastri M. Koszelak M.E. Wakabayashi H. J. Biol. Chem. 2001; 276: 12434-12439Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) that the by the heavy chain cleavage by thrombin and factor Xa be the of the was Proteolysis at Arg372 and Arg1689 is essential for generating factor VIIIa cofactor activity (6Fay P.J. Blood Rev. 2004; 18: 1-15Crossref PubMed Scopus (161) Google Scholar). K. Wakabayashi H. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that of proteolysis at Arg740 in cofactor that cleavage at the be an essential in the of In the of factor VIII by plasmin appeared to be with three cleavage inactivation of factor VIII, and was that cleavage by plasmin at Arg336 within A1 subunit was rapid with that at Arg372 the A1 and A2 This cleavage at Arg336 than at Arg372 was in contrast to the cleavage for factor Xa. Furthermore, the cleavage at Arg336 was rapid with cleavage induced by It is that serine APC and factor factor following cleavage at Arg336 P.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. Wakabayashi H. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of an altered the A2 subunit and the A1 and results in the of a factor site within residues K. J. Wakabayashi H. Fay P.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) and an in the for factor X M.E. K. J. Mastri M. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Wakabayashi H. K. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a and SDS-PAGE the that the of factor VIII inactivation is to be the of unactivated activated and following subunit the rapid the cleavage at Arg336 in the A1 the rapid the inactivation of factor that cleavage at Lys36 altered the of the A1 subunit and limited with the A2 subunit K. Wakabayashi H. K. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). appeared to be the of activity of factor VIIIa with the subunit K. Wakabayashi H. K. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In this study, proteolysis by plasmin at Lys36 occurred rapidly than that by factor Xa. These results that the proteolysis at Lys36 to to the rapid inactivation of factor by plasmin. inactivation of factor VIII by cleavage at Arg1721 in the light chain M.S. Lenting P.J. van Mourik J.A. Mertens K. J. Biol. Chem. 1995; 270: 3648-3655Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In this that cleavage at Arg336 was a mechanism responsible for plasmin-catalyzed inactivation of factor VIII. of the proteolysis of the factor VIII heavy chain by plasmin, APC, and factor Xa is in These mechanisms APC cleaves factor VIII at Arg336 within the A1 domain, by within A2 P.J. L.M. O'Brien L.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Factor Xa cleaves at Arg372 and at Lys36 and Arg336 K. Wakabayashi H. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the terminal from plasmin cleavage are identical to those by factor Xa. plasmin-catalyzed cleavage of the heavy chain the two Arg336 and Arg372, within the A1 domain, cleavage at Arg336 to be the The factor also to the domain K. M. K. K. M. H. Blood. PubMed Scopus Google Scholar, D. Fay P.J. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). In K. Wakabayashi H. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. M. K. D. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that factor at Arg336 and Lys36 are selectively regulated by with the A3-C1-C2 and A1 subunits, respectively. The from this indicated that cleavage at Arg336 was selectively enhanced by plasmin with the A2 and cleavage at Lys36 was regulated by the A3-C1-C2 of the A1 subunit with factor not be this that this mechanism of plasmin activity is from that of factor the cleavage are identical for Plasmin is of a heavy chain domains and a light chain the catalytic It is with by site with D.P. and Scholar). It is to that residues of within A2 and A3-C1-C2 but not the A1 domain (2Wood W.I. Capon D.J. Simonsen C.C. Eaton D.L. Gitschier J. Keyt B. Seeburg P.H. Smith D.H. Hollingshead P. Wion K.L. Delwart E. Tuddenham E.D.G. Vehar G.A. Lawn R.M. Nature. 1984; 312: 330-337Crossref PubMed Scopus (522) Google Scholar, 3Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Wood W.I. Harkins R.N. Tuddenham E.G.D. Lawn R.M. Capon D.J. Nature. 1984; 312: 337-342Crossref PubMed Scopus (660) Google Scholar) the for plasmin in the factor VIII The of plasmin-catalyzed cleavage of factor VIII, in and inactivation of cofactor to be However, of from plasma of be to a catalytic rate and greater than APC and factor respectively. that of plasmin in the fibrinolytic to the and of Furthermore, the plasmin-catalyzed inactivation mechanism of other clotting factors such as factor M. K.G. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) and factor J.A. Nesheim M.E. Blood. PubMed Google Scholar) also In factor to factor VIII and also the presence of a of plasmin proteolytic reaction in the reaction as as fibrinolytic for