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Prepublished online as a Blood First Edition Paper on May 31, 2002; DOI 10.1182/blood-2002-03-0843.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Hematology, Stanford University
School of Medicine, CA.
The coagulation factors V (FV) and VIII (FVIII) are important at
sites of vascular injury for the amplification of the clotting cascade.
Natural variants of these factors frequently lead to severe bleeding
disorders. To understand the mechanisms of activation of FVIII by
thrombin, we used a bank of mutant thrombins to define residues
important for its activation. From the initial screening of 53 mutant
thrombins for the activation of human recombinant FVIII, we mapped
thrombin mutants with 50% or less activity to anion-binding exosite-I
(Lys21Ala, His66Ala, Lys65Ala, Arg68Ala, Arg70Ala, and Tyr71Ala) and
anion-binding exosite-II (Arg98Ala), the Na+-binding site
(Glu229Ala, Arg233Ala, Asp234Ala, and Asp193Ala/Lys196Ala), and the
50-insertion loop (Trp50Ala), which were similar to our results for the
activation of FV. The role of these residues for cleavage at Arg372 and
Arg1689 was investigated using plasma FVIII. Anion-binding exosite-I
appears to be important for cleavage at both sites, whereas the
anion-binding exosite-II residue Arg98Ala is important for cleavage at
Arg372 alone. The Glu229Ala mutant, which contributes to the
Na+-binding site, and the 50-insertion loop mutant W50A
have severely impaired cleavage at Arg372 and Arg1689. This suggests
that the integrity of the active site and the Na+-bound
form of thrombin are important for its procoagulant activity against
FVIII. Detailed mutagenic analysis of thrombin can assist in
understanding the pathogenesis of bleeding disorders and may lead to
the rational design of selective thrombin inhibitors.
(Blood. 2002;100:2820-2826) The serine protease thrombin acts as a positive
feedback amplifier of the coagulation cascade through the specific
cleavage of factor V (FV) and factor VIII (FVIII). Factor Va (FVa) and factor VIIIa (FVIIIa) function as cofactors for prothrombinase and
tenase complexes, respectively, on the surfaces of anionic phospholipids. This leads to the amplification of the coagulation pathway at sites of vascular injury.1 Both factors measure approximately 300 kDa and share a common domain organization, A1-A2-B-A3-C1-C2.2,3
The A and C domains are 40% identical between FV and FVIII, but there
is little sequence homology between the B domains. The A domains are
similar to the A domains of ceruloplasmin,4 and the C
domains are similar to phospholipid-binding proteins. The crystal
structures of the FV and FVIII light-chain C2 domains have
been determined and show hydrophobic and electrostatic
interactions important for anchoring to the phospholipid
membrane.5,6 Several studies have shown that the FVIII
C2 domain also contains the von Willebrand factor
(VWF)-,7,8 thrombin-,9 and factor Xa-binding sites.10
FV circulates as a full-length protein and is activated by thrombin
releasing the B-domain activation products (E fragment and
C1 fragment) by cleavage at the residues Arg709, Arg1018, and Arg1545. FVa is composed of the heavy
(A1-A2 domain, 105 kDa) and light
(A3-C1-C2 domain, 74 kDa) chains
bound by a calcium ion.2,11,12 Specific cleavage of FV
occurs preferentially at Arg709, then at Arg1018, followed by the
rate-limiting cleavage at Arg1545. Cleavage at Arg709 and Arg1545 are
important for full FVa cofactor activity, whereas cleavage of Arg1018
enhances the rate of Arg1545 cleavage.13 In contrast,
FVIII circulates in plasma bound to VWF as a heterogeneous mixture of
heterodimers (Figure 1) because of
variable intracellular processing of the B domain. The variable
heavy-chain fragments (A1-A2-B) range from 90 to 210 kDa, and they are bound to the 80-kDa light chain
(A3-C1-C2). Thrombin activates
FVIII by cleavage at Arg372 between the A1-A2 domain and at Arg740 between the A2 and B domains to
generate the 54-kDa A1 and the 44-kDa A2
domains, and at Arg1689 to release a 40-amino acid acidic
peptide from the 80-kDa light chain to generate the 73-kDa
A3-C1-C2 fragment. FVIIIa is a
metal-linked heterotrimer composed of the
A1/A2/A3-C1-C2
domains and lacking the B domain.14,15 Activation of
plasma FVIII/VWF requires cleavages at Arg372 and Arg1689 for cofactor
activity,16,17 whereas cleavage at Arg740 appears not to
be rate limiting.16 Cleavage at Arg1689 releases an acidic
14-kDa fragment that leads to dissociation of VWF, allowing the
association of FVIIIa to the phospholipid membrane.18 In
the absence of VWF, cleavage at Arg372 is sufficient for full cofactor
activity. These results are consistent with the naturally occurring
mutations that give rise to hemophilia A.19
The specificity of thrombin is defined by 2 surface loops, the
50-insertion (Leu45-Asn57, thrombin-numbering system) and autolysis (Leu144-Gly155) loops, which occlude the active site, restricting access, and by 2 ligand-binding sites (exosites). Many thrombin substrates, receptors, and inhibitors overcome the occluded active site
by binding to either of the 2 exosites, which are characterized by a
high density of solvent-exposed basic residues. Anion-binding exosite-I
(ABE-I, fibrinogen-binding exosite) binds fibrinogen,20-22 PAR1,23-25 thrombomodulin,20,26-28 heparin
cofactor II,29-31 and hirudin.32-34
Anion-binding exosite II (ABE-II, heparin-binding exosite) binds
glycosaminoglycan-bound serpins,29,35,36 platelet glycoprotein Ib,37,38 and hemadin.39 Studies
using the inhibitor hirugen (ABE-I specific) and the ABE-II
mutant thrombin RA (Arg89/Arg93/Arg98Ala) implicate ABE-I and
ABE-II for thrombin cleavage and activation of FV and
FVIII.40 Using a bank of 53 mutant thrombins with surface-exposed basic and polar residues substituted with alanine, we
recently defined residues in both exosites, the Na+-binding
site41 and the 50-insertion loop, important for FV activation and for cleavage at Arg709.42 In this study, we
have used the same bank of mutant thrombins to define thrombin residues important in the activation of FVIII. We show that ABE-I and ABE-II are
important for FVIII activation through cleavage of Arg372 but that
ABE-II appears to be less important for cleavage at Arg1689. Recognition and cleavage at both sites requires the
Na+-bound procoagulant form of thrombin41 for
full cofactor activity. These studies are important for understanding
the mechanisms of thrombin activation of FV and FVIII because no
crystal structure data are available for these cofactors in complex
with thrombin. This could lead to coherent strategies designed for the
selective inhibition of thrombin to prevent the activation of
platelets, cleavage of fibrinogen, and dampening of the coagulation
cascade by impairing the formation of the prothrombinase and tenase complexes.
Materials
Activation of human FVIII by WT and mutant thrombins
SDS-PAGE and Western blot analysis of cleavage reactions Cleavage reactions containing 50 nM human plasma-derived FVIII and 1 nM thrombin in assay buffer were incubated from 1 to 120 minutes before they were terminated by the addition of sodium dodecyl sulfate (SDS) loading buffer and boiling for 5 minutes. Cleavage products were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 4% to 20% gradient SDS polyacrylamide gels (Bio-Rad, Hercules, CA) and then were stained with Biosafe Coomassie blue (Bio-Rad). Gels were Western blotted and probed with the monoclonal antibodies OBT0037A (specific for the heavy chain) and FVIIIC (specific for the light chain). Detection was achieved using goat antimouse IgG-horseradish peroxidase (HRP) and enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). The intensity of developed bands was determined by direct scanning of autoradiographs using the UMAX Astra 4000U scanner and Umax VistaScan 3.5.2 software. Scanned images were saved as tagged image file format (TIFF) files at a resolution 600 dots per inch. Pixel densities were calculated for each band from TIFF files imported into Scion Image 1.62c (http://rsb.info.nih.gov/nih-image/).
Identification of thrombin mutants defective in human FVIII activation We used a site-directed mutagenesis strategy to determine thrombin residues important for human FVIII activation. A bank of 53 mutant thrombins in which solvent-exposed polar and charged residues were substituted with alanine43 was used in a 2-stage chromogenic screening assay with recombinant human FVIII. Recombinant FVIII is devoid of VWF, which usually binds plasma FVIII at a 1:1 molar ratio. For the activation of recombinant FVIII, cleavage at residue Arg372 appears to be sufficient for full cofactor activity of the tenase complex.16-18,40 Hence, decreases in activity from the mutations should reflect impaired cleavage at Arg372. Twelve mutants were identified with 50% or less FVIII activation compared with WT thrombin (Figure 2; Table 1). These residues mapped mainly to ABE-I, ABE-II, the 50-insertion loop, and the Na+-binding site. ABE-I had 3 residues (Lys21, His66, and Arg70) with less than 50% FVIII activation and 3 residues (Lys65, Arg68, Tyr71) with less than 10% activity. Dose-response curves were performed for these mutants with severely diminished activation to ensure that they were within the linear range for cleavage and were found to be comparable to the initial screening (Figure 3; Table 1). Compared to the screening with FV activation, alanine substitution of residues Lys21, His66, Arg68, Arg70, and Tyr71 appears to have similar decreases in FVIII cofactor activation. Among these, Arg68 and Tyr71 are the most important in that alanine substitution leads to a greater than 90% decrease in activation. However, some differences are observed. Thrombin residue Lys65 appears to be more important for FVIII activation than FV (8.5% vs 46.2% of WT activation), whereas the thrombin residues Arg73 and Lys77 appear to be less important for FVIII activation (70.7% and 112.4% vs 37.8% and 36% WT activation, respectively; Table 1).
ABE-II also plays a role in the activation of FVIII, but its importance appears to be less than that for FV activation. The mutant Arg98Ala has 43.3% of WT activation in the initial screen; however, dose-response studies for this mutant show 32.4% WT FVIII activation, which is similar for its activation of FV (Figure 3; Table 1). Two triple mutants, Arg89Ala/Arg93Ala/Glu94Ala and Arg245Ala/Lys248Ala/Gln251Ala, have decreased FV activation but not FVIII activation. The only common ABE-II residue for FV and FVIII activation appears to be Arg98. More than 13 basic residues comprise ABE-II, some of which are not represented in the current collection of alanine-thrombin mutants. Therefore, it is possible that not all electrostatic interactions are represented in this study. All mutant thrombins in ABE-I and ABE-II with impaired FVIII activation have normal catalysis for the chromogenic substrate, S-2238,43 suggesting that impaired binding within the exosites rather than an effect on catalysis is responsible for decreased cofactor activation. The residues Glu229, Arg233, and Asp234, which are important for the integrity of the Na+-binding site,41 have drastically reduced FVIII activation when substituted with alanine (2.9%, 11.5%, and 35.6% of WT FVIII activation, respectively). Dose-response studies for Glu229Ala show a similar decrease in FVIII activation, consistent with the screening (Figure 3; Table 1). These are similar to results observed for FV activation. The double mutant Asp193Ala/Lys196Ala showed 35.6% of WT FVIII activation. Residues lie on an extended insertion loop (residues Tyr190 to Gly198), where both side chains point into the solvent.44 The residue Arg197 on this loop makes a bidentate ion pair with the Na+-binding site residues Asp221 and Asp22241,45; hence, it is possible that either of these 2 mutations could disrupt interactions between the loop and the Na+-binding site, compromising the Na+-bound procoagulant form of thrombin. Alternatively, the residues could be involved in making direct interactions. A large decrease in FVIII activation (8.9% WT activation) was noted for the residue Trp50Ala, which forms part of the hydrophobic 50-insertion loop that occludes the active site restricting the specificity of thrombin. Trp50Ala has diminished activity toward the chromogenic substrate S-2238,43 as reflected by an increase in Km, implying the mutation affects substrate binding by altering the topology of the S subsites affecting specific contacts. FVIII activation is more impaired than FV activation, which suggests that Trp50 has a greater contribution to the specificity of cleavage at one or more of the FVIII cleavage sites. Analysis of human FVIII activation by SDS-PAGE To determine the structural requirements of thrombin necessary for the activation of FVIII by specific cleavage at Arg372 and Arg1689, we used Western blot analysis of cleavage reactions containing purified plasma FVIII using monoclonal antibodies specific for the heavy-chain A2 domain and the light-chain acidic N terminus. Cleavage at Arg1689 releases the 14-kDa acidic fragment, leading to a dissociation of VWF that allows FVIIIa to act as a cofactor in the tenase complex. Using the monoclonal antibody OBT0037A (specific for the heavy-chain A2 domain), we monitored the cleavage of the heavy-chain heterodimers by the specific appearance of the 44-kDa A2 domain. Compared with WT, the thrombin mutants Trp50Ala, Arg68Ala, Try71Ala, Arg98Ala, and Glu229Ala had slightly delayed appearance of the 90-kDa A1-A2 heavy chain from the cleavage of the various-sized heavy-chain heterodimers (ranging from 220 to 90 kDa), suggesting cleavages at Arg740 and Arg1313 (located in the B domain) are not severely affected by the mutations. For most mutants, however, the 90-kDa A1-A2 band persisted, and the appearance of the 44-kDa A2 domain was significantly delayed (Figure 4A). Quantitation of the A2 domain generated by the cleavage of FVIII by WT and mutant thrombins at 10 minutes (Figure 4B) showed dramatically reduced band intensities, suggesting that ABE-I, ABE-II residue Arg98, 50-insertion loop residue Trp50, and Na+-binding site are all necessary for cleavage at Arg372.
Cleavage at Arg1689 was monitored by Western blot analysis using the
monoclonal antibody FVIIIC, which appears to recognize an epitope on
the 14-kDa acidic fragment. With the exception of Arg98Ala, all mutant
thrombins showed a delay in cleavage at Arg1689, indicated by the
continued presence of the 80-kDa uncleaved light chain (Figure
5A), consistent with reduced cofactor
activation. Arg98Ala appears to efficiently cleave at Arg1689 with a
band intensity of 75% that of WT (Figure 5B), suggesting that ABE-II may not play an important role in cleavage of Arg1689 at this site in
the presence of VWF. The Arg98Ala mutant has only 32.4% FVIII
activation using recombinant FVIII (Table 1), suggesting that cleavage
at Arg372 is sufficient for full cofactor activity in the absence of
VWF.
The activation of human FV and FVIII by thrombin plays an
essential role in the amplification of the coagulation cascade at sites
of vascular injury. In this report, we have investigated the role of
specific thrombin residues important in the activation of FVIII and
have compared these findings with our recent studies for the
activation of the structurally related FV using a collection of
alanine-scanning, mutagenesis-generated thrombin
mutants.43 The preliminary screen and dose dependence for
human recombinant FVIII activation primarily reflects cleavage at
Arg372-Ser373 because, in the absence of VWF, cleavage at this site is
sufficient for full cofactor activity. The results implicate the role
of ABE-I, ABE-II, the 50-insertion loop residue Trp50, and the
Na+-binding site for recombinant FVIII cofactor activation.
This shows a similar pattern for FV activation though there are subtle differences (Table 1; Figure 6). Thrombin
uses ABE-I residues Lys21, Lys65, His66, Arg68, Arg70, and Tyr71 for
the activation of recombinant FVIII (presumably at Arg372-Ser373). By
comparison, more residues in ABE-I are required for the activation of
FV (Lys21, Lys65, His66, Arg68, Arg70, Tyr71, Arg73, and Lys77). The
role of these residues could be direct interaction with factor FVIII, or, alternatively, substitution of these residues may cause a conformational change affecting other contacts. ABE-I extends away from
the thrombin active site and is characterized by a high density of
surface-exposed basic and hydrophobic amino acids. Crystal structures
of thrombin bound to PAR1,24
thrombomodulin,46 and the inhibitors
hirudin,33 triabin,47
ornithodorin,48 and rhodniin49 have shown the
importance of one or more of the basic residues (Lys21, Arg68, Arg70,
Arg73, and Lys77) in ABE-I for direct ion pair interactions. They have
also shown the role of Tyr71 on the surface-exposed patch formed with
Phe34, Ile82, Met84, and Leu65 for hydrophobic interactions. Mutant
thrombins Lys65Ala, Arg68Ala, and Tyr71Ala
ABE-II does not appear to be as important for the activation of FVIII as for the activation of FV using our collection of mutant thrombins. Only residue Arg98 appears to have an effect on the activation of FVIII at the Arg372-Ser373 cleavage site. Esmon and Lollar40 suggest ABE-II is more important for the activation of FVIII using the triple mutant, RA (Arg89Ala/Arg93Ala/Arg98Ala). The thrombin mutant collection does not encompass all the possible residues within ABE-II; therefore, it is possible that ABE-II may still have an important role in the activation of FVIII at both cleavage sites. The loss of activity caused by the Arg98Ala substitution could implicate this residue in direct interactions with FVIII. For example, Arg98 has been shown to be involved in ion-pair interactions from the crystal structures of thrombin bound to the leech inhibitor, heamadin,39 and the prothrombin F2 domain.51 For cleavage at Arg372, both ABE-I and ABE-II are required for hydrolysis, suggesting binding to distinct FVIII domains. The A2 and C2 domains have been implicated in binding thrombin,9 and a hirudinlike sequence N-terminal to the Arg372-Ser373 bond (residues 330-363) in the A1 domain could also play a role. Consistent with the mapping studies of FV is the importance of Glu229,
Arg233, and Asp234 at the Na+-binding
site.41,45 The Na+-binding site is defined by
a cavity formed by 3 antiparallel B-chain The thrombin residue Trp50 forms part of the 50-insertion loop that occludes the active site, restricting the specificity of thrombin. Trp50 plays an important role in defining the apolar S2 subsite and has been shown to make substantial contacts in the thrombin complex with hirudin,33 hemadin,39 rhodniin,49 fibrinopeptide A,56 and PPACK44 complexes. Consistent with these crystal structures is the dramatic effect of the W50A mutation for cleavage at FV residue Arg709 and FVIII residues Arg372 and Arg1689, which can be explained as the loss of important contacts or disruption of the S2 subsite. Evidence exists that the mutation can have long-range effects on the Na+-binding site.57 To date, no crystal structures exist to define the precise structural requirements of thrombin in the recognition and cleavage of FV and FVIII. Saturated alanine-scanning mutagenesis of thrombin has proved valuable in defining thrombin residues important in the recognition and cleavage of FV and FVIII that should aid in the rational design of thrombin inhibitors.
Submitted March 19, 2002; accepted May 2, 2002.
Prepublished online as Blood First Edition Paper, May 31, 2002; DOI 10.1182/blood-2002-03-0843.
Supported by National Institutes of Health grant R01 HL57530 and the Cheong Har Family Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Timothy Myles, Lawrence L. K. Leung, Division of Hematology, CCSR Rm 1155, Stanford University School of Medicine, Stanford, CA 94305-5156; e-mail: tmyles{at}stanford.edu, lawrence.leung{at}stanford.edu.
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© 2002 by The American Society of Hematology.
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  J. L. Newell and P. J. Fay Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII J. Biol. Chem., April 24, 2009; 284(17): 11080 - 11089. [Abstract] [Full Text] [PDF]
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T. Myles and L. L. K. Leung Thrombin Hydrolysis of Human Osteopontin Is Dependent on Thrombin Anion-binding Exosites J. Biol. Chem., June 27, 2008; 283(26): 17789 - 17796. [Abstract] [Full Text] [PDF]
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J. L. Newell and P. J. Fay Proteolysis at Arg740 Facilitates Subsequent Bond Cleavages during Thrombin-catalyzed Activation of Factor VIII J. Biol. Chem., August 31, 2007; 282(35): 25367 - 25375. [Abstract] [Full Text] [PDF]
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 T. E. Adams and J. A. Huntington Thrombin-Cofactor Interactions: Structural Insights Into Regulatory Mechanisms Arterioscler Thromb Vasc Biol, August 1, 2006; 26(8): 1738 - 1745. [Abstract] [Full Text] [PDF]
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M. A. Bukys, T. Orban, P. Y. Kim, D. O. Beck, M. E. Nesheim, and M. Kalafatis The Structural Integrity of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII J. Biol. Chem., July 7, 2006; 281(27): 18569 - 18580. [Abstract] [Full Text] [PDF]
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K. Nogami, Q. Zhou, H. Wakabayashi, and P. J. Fay Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site Blood, June 1, 2005; 105(11): 4362 - 4368. [Abstract] [Full Text] [PDF]
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K. Nogami, Q. Zhou, T. Myles, L. L. K. Leung, H. Wakabayashi, and P. J. Fay Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain J. Biol. Chem., May 6, 2005; 280(18): 18476 - 18487. [Abstract] [Full Text] [PDF]
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Y. M. Fortenberry, H. C. Whinna, H. R. Gentry, T. Myles, L. L. K. Leung, and F. C. Church Molecular Mapping of the Thrombin-Heparin Cofactor II Complex J. Biol. Chem., October 8, 2004; 279(41): 43237 - 43244. [Abstract] [Full Text] [PDF]
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T. H. Yun, F. A. Baglia, T. Myles, D. Navaneetham, J. A. Lopez, P. N. Walsh, and L. L. K. Leung Thrombin Activation of Factor XI on Activated Platelets Requires the Interaction of Factor XI and Platelet Glycoprotein Ib{alpha} with Thrombin Anion-binding Exosites I and II, Respectively J. Biol. Chem., November 28, 2003; 278(48): 48112 - 48119. [Abstract] [Full Text] [PDF]
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L. Yang, C. Manithody, T. D. Walston, S. T. Cooper, and A. R. Rezaie Thrombomodulin Enhances the Reactivity of Thrombin with Protein C Inhibitor by Providing Both a Binding Site for the Serpin and Allosterically Modulating the Activity of Thrombin J. Biol. Chem., September 26, 2003; 278(39): 37465 - 37470. [Abstract] [Full Text] [PDF]
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H. Philippou, J. Rance, T. Myles, S. W. Hall, R. A. Ariens, P. J. Grant, L. Leung, and D. A. Lane Roles of Low Specificity and Cofactor Interaction Sites on Thrombin during Factor XIII Activation: COMPETITION FOR COFACTOR SITES ON THROMBIN DETERMINES ITS FATE J. Biol. Chem., August 22, 2003; 278(34): 32020 - 32026. [Abstract] [Full Text] [PDF]
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L. Chen, L. Yang, and A. R. Rezaie Proexosite-1 on Prothrombin Is a Factor Va-dependent Recognition Site for the Prothrombinase Complex J. Biol. Chem., July 18, 2003; 278(30): 27564 - 27569. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
), the ABE-I
mutants Lys65Ala (
), Arg68Ala (
), and Tyr71Ala (
), the ABE-II
mutant Arg98Ala (
), the 50-insertion loop mutant Trp50Ala (
), and
the Na+ loop mutant Glu229Ala (
). All points had
standard deviations of 15% or less from at least 2 separate
experiments performed in duplicate.
all of which had less than
10% WT FVIII activation, were chosen to explore the role of ABE-I for
the recognition and cleavage of plasma-derived FVIII bound with VWF.
Activation of FVIII/VWF occurs through cleavage at Arg372 and Arg1689
for full cofactor activity. Cleavage at Arg1689 releases an acidic
14-kDa N-terminal fragment that allows the dissociation of VWF. The
binding sites on FVIII for VWF
-strands (Met185-Gly189,
Val225-Cys231, and Tyr237-Tyr240) and the loops Tyr190-Gly198 and
Cys231-Tyr237.
-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry and structure function relationships.
Protein Sci.
1992;1:426-471