Phosphatidylserine Regulation of Coagulation Proteins Factor IXa and Factor VIIIa.

Blood coagulation is an intricate process, and it requires precise control of the activities of pro- and anticoagulant factors and sensitive signaling systems to monitor and respond to blood vessel insults. These requirements are fulfilled by phosphatidylserine, a relatively miniscule-sized lipid molecule amid the myriad of large coagulation proteins. This review limelight the role of platelet membrane phosphatidylserine (PS) in regulating a key enzymatic reaction of blood coagulation; conversion of factor X to factor Xa by the enzyme factor IXa and its cofactor factor VIIIa. PS is normally located on the inner leaflet of the resting platelet membrane but appears on the outer leaflet surface of the membrane surface after an injury happens. Human platelet activation leads to exposure of buried PS molecules on the surface of the platelet-derived membranes and the exposed PS binds to discrete and specific sites on factors IXa and VIIIa. PS binding to these sites allosterically regulates both factors IXa and VIIIa. The exposure of PS and its binding to factors IXa/VIIIa is a vital step during clotting. Insufficient exposure or a defective binding of PS to these clotting proteins is responsible for various hematologic diseases which are discussed in this review.

Single synonymous mutation in factor IX alters protein properties and underlies haemophilia B.

BACKGROUND: Haemophilia B is caused by genetic aberrations in the F9 gene. The majority of these are non-synonymous mutations that alter the primary structure of blood coagulation factor IX (FIX). However, a synonymous mutation c.459G>A (Val107Val) was clinically reported to result in mild haemophilia B (FIX coagulant activity 15%-20% of normal). The F9 mRNA of these patients showed no skipping or retention of introns and/or change in mRNA levels, suggesting that mRNA integrity does not contribute to the origin of the disease in affected individuals. The aim of this study is to elucidate the molecular mechanisms that can explain disease manifestations in patients with this synonymous mutation. METHODS: We analyse the molecular mechanisms underlying the FIX deficiency through in silico analysis and reproducing the c.459G>A (Val107Val) mutation in stable cell lines. Conformation and non-conformation sensitive antibodies, limited trypsin digestion, activity assays for FIX, interaction with other proteins and post-translation modifications were used to evaluate the biophysical and biochemical consequences of the synonymous mutation. RESULTS: The Val107Val synonymous mutation in F9 was found to significantly diminish FIX expression. Our results suggest that this mutation slows FIX translation and affects its conformation resulting in decreased extracellular protein level. The altered conformation did not change the specific activity of the mutated protein. CONCLUSIONS: The pathogenic basis for one synonymous mutation (Val107Val) in the F9 gene associated with haemophilia B was determined. A mechanistic understanding of this synonymous variant yields potential for guiding and developing future therapeutic treatments.

Cluster III of low-density lipoprotein receptor-related protein 1 binds activated blood coagulation factor VIII.

Low-density lipoprotein receptor-related protein 1 (LRP) mediates clearance of blood coagulation factor VIII (FVIII). In LRP, FVIII binds the complement-type repeats (CRs) of clusters II and IV, which also bind a majority of other LRP ligands. No ligand is known for LRP cluster I, and only three ligands, including the LRP chaperone alpha-2 macroglobulin receptor-associated protein (RAP), bind cluster III. Using surface plasmon resonance, we found that in addition to clusters II and IV, activated FVIII (FVIIIa) binds cluster III. The specificity of this interaction was confirmed using an anti-FVIII antibody fragment, which inhibited the binding. Recombinant fragments of cluster III and its site-directed mutagenesis were used to localize the cluster's site for binding FVIIIa to CR.14-19. The interactive site of FVIIIa was localized within its A1/A3'-C1-C2 heterodimer (HDa), which is a major physiological remnant of FVIIIa. In mice, the clearance of HDa was faster than that of FVIII and prolonged in the presence of RAP, which is known to inhibit interactions of LRP with its ligands. In accordance with this, the cluster III site for RAP (CR.15-19) was found to overlap that for FVIIIa. Altogether, our findings support the involvement of LRP in FVIIIa catabolism and suggest a greater significance of the biological role of cluster III compared to that previously known.

A3 domain region 1803-1818 contributes to the stability of activated factor VIII and includes a binding site for activated factor IX.

A recent chemical footprinting study in our laboratory suggested that region 1803-1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803-1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803-1810 and 1811-1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811-1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811-1818 on lipospheres. Site-directed mutagenesis revealed that Phe-1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803-1810 and FVIII/FV 1811-1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811-1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811-1818 contributes to FIXa binding. Both regions 1803-1810 and 1811-1818 contribute to FVIIIa stability.

Factor VIIIa A2 subunit shows a high affinity interaction with factor IXa: contribution of A2 subunit residues 707-714 to the interaction with factor IXa.

Factor (F) VIIIa forms a number of contacts with FIXa in assembling the FXase enzyme complex. Surface plasmon resonance was used to examine the interaction between immobilized biotinylated active site-modified FIXa, and FVIII and FVIIIa subunits. The FVIIIa A2 subunit bound FIXa with high affinity (Kd = 3.9 ± 1.6 nm) that was similar to the A3C1C2 subunit (Kd = 3.6 ± 0.6 nm). This approach was used to evaluate a series of baculovirus-expressed, isolated A2 domain (bA2) variants where alanine substitutions were made for individual residues within the sequence 707-714, the C-terminal region of A2 thought to be FIXa interactive. Three of six bA2 variants examined displayed 2- to 4-fold decreased affinity for FIXa as compared with WT bA2. The variant bA2 proteins were also tested in two reconstitution systems to determine activity and affinity parameters in forming FXase and FVIIIa. Vmax values for all variants were similar to the WT values, indicating that these residues do not affect cofactor function. All variants showed substantially greater increases in apparent Kd relative to WT in reconstituting the FXase complex (8- to 26-fold) compared with reconstituting FVIIIa (1.3- to 6-fold) suggesting that the mutations altered interaction with FIXa. bA2 domain variants with Ala replacing Lys(707), Asp(712), and Lys(713) demonstrated the greatest increases in apparent Kd (17- to 26-fold). These results indicate a high affinity interaction between the FVIIIa A2 subunit and FIXa and show a contribution of several residues within the 707-714 sequence to this binding.

Role of hydrophobic mutations on the binding affinity and stability of blood coagulation factor VIIIa: a computational molecular dynamics and free-energy analysis.

Factor VIIIa is a non-covalently bound hetero-trimer among A1, A2 and A3-C1-C2 domains and an essential co-factor for factor IXa enzyme during proteolytic activation of factor X zymogen. The relatively weak interactions between A2 and the interface A1/A3 domains dampen the functional stability of FVIIIa in plasma and results in rapid degradation. We studied the mutational effect of three charged residues (Asp519, Glu665 and Asp666) to several hydrophobic residues by molecular dynamics simulations. Analysis of the binding free energy by MM-PBSA and MM-GBSA methods shows that the mutation of Asp519 and Glu665 residues to either Val or Ala enhance the A2 domain binding affinity in agreement with the experimental site-specific mutagenesis data. Mutation of Asp666 to Val, Tyr, Met and Phe showed largest improvement in the A2-domain binding among the eight hydrophobic mutants studied. Our studies suggest that the enrichment of hydrophobic interactions in the buried surface regions of A2 domain plays crucial role in improving the overall stability of FVIIIa.

Structural insights into the interaction of blood coagulation co-factor VIIIa with factor IXa: a computational protein-protein docking and molecular dynamics refinement study.

Coagulation factor X (FX) zymogen activation by factor IXa (FIXa) enzyme plays a critical role in the middle-phase of coagulation cascade. The activation process is catalytically inert and requires FIXa binding and complex formation with co-factor VIIIa (FVIIIa). In order to understand the structural details of the FVIIIa:FIXa complex, we employed knowledge-driven protein-protein docking and aqueous-phase MD refinement methods to develop a stable structural complex between FVIIIa and FIXa. The model shows that all four domains of FIXa wrap across FVIIIa that spans the co-factor binding surface of A2, A3 and C1 domains. The region surrounding the 558-helix of the A2-domain of FVIIIa is predicted to be the key interaction site with the helical segments of Lys293-Lys301 and Asp332-Arg338 residues of the serine-protease domain of FIXa. The hydrophobic helical stack between the GLA and EGF1 domains of FIXa is predicted to be primary interacting region with the A3-C2 domain interface of FVIIIa.

Cyclic peptide analogs of 558-565 epitope of A2 subunit of Factor VIII prolong aPTT. Toward a novel synthesis of anticoagulants.

Novel anticoagulant therapies target specific clotting factors in blood coagulation cascade. Inhibition of the blood coagulation through Factor VIII-Factor IX interaction represents an attractive approach for the treatment and prevention of diseases caused by thrombosis. Our research efforts are continued by the synthesis and biological evaluation of cyclic, head to tail peptides, analogs of the 558-565 sequence of the A2 subunit of FVIII, aiming at the efficient inhibition of Factor VIIIa-Factor IXa interaction. The analogs were synthesized on solid phase using the acid labile 2-chlorotrityl chloride resin, while their anticoagulant activities were examined in vitro by monitoring activated partial thromboplastin time and the inhibition of Factor VIII activity. The results reveal that these peptides provide bases for the development of new anticoagulant agents.

Molecular orientation of factor VIIIa on the phospholipid membrane surface determined by fluorescence resonance energy transfer.

F (Factor) VIIIa binds to phospholipid membranes during formation of the FXase complex. Free thiols from cysteine residues of isolated FVIIIa A1 and A2 subunits and the A3 domain of the A3C1C2 subunit were labelled with PyMPO maleimide {1-(2-maleimidylethyl)-4-[5-(4-methoxyphenyl)-oxazol-2-yl]pyridinium methanesulfonate} or fluorescein (fluorescence donors). Double mutations of the A3 domain (C2000S/T1872C and C2000S/D1828C) were also produced to utilize Cys(1828) and Cys(1872) residues for labelling. Labelled subunits were reacted with complementary non-labelled subunits to reconstitute FVIIIa. Octadecylrhodamine incorporated into phospholipid vesicles was used as an acceptor for distance measurements between FVIII residues and membrane surface by fluorescence resonance energy transfer. The results of the present study indicate that a FVIII axis on a plane that intersects the approximate centre of each domain is orientated with a tilt angle of ~30-50° on the membrane surface. This orientation predicted the existence of contacts mediated by residues 1713-1725 in the A3 domain in addition to a large area of contacts within the C domains. FVIII variants where Arg(1719) or Arg(1721) were mutated to aspartate showed a >40-fold reduction in membrane affinity. These results identify possible orientations for FVIIIa bound to the membrane surface and support a new interaction between the A3 domain and the membrane probably mediated in part by Arg(1719) and Arg(1721).

Variable contributions of basic residues forming an APC exosite in the binding and inactivation of factor VIIIa.

Basic residues contained in the 39-, 60-, and 70-80-loops of activated protein C (APC) comprise an exosite that contributes to the binding and subsequent proteolytic inactivation of factor (F) VIIIa. Surface plasmon resonance (SPR) showed that WT APC bound to FVIII light chain (LC) and the FVIIIa A1/A3C1C2 dimer with equivalent affinity (Kd = 525 and 546 nM, respectively). These affinity values may reflect binding interactions to the acidic residue-rich a1 and a3 segments adjacent to A1 domain in the A1/A3C1C2 and A3 domain in LC, respectively. Results from SPR, using a panel of APC exosite variants where basic residues were mutated, in binding to immobilized FVIIIa A1/A3C1C2 or LC indicated ~4-10-fold increases in the Kd values relative to WT for several of the variants including Lys39Ala, Lys37-Lys38-Lys39/Pro-Gln-Glu, and Arg67Ala. On the other hand, a number of APC variants including Lys38Ala, Lys62Ala, and Lys78Ala showed little if any change in binding affinity to the FVIII substrates. FXa generation assays and Western blotting, used to monitor rates of FVIIIa inactivation and proteolysis at the primary cleavage site in the cofactor (Arg(336)), respectively, showed marked rate reductions relative to WT for the Lys39Ala, Lys37-Lys38-Lys39/Pro-Gln-Glu, Arg67Ala, and Arg74Ala variants. Furthermore, kinetic analysis monitoring FVIIIa inactivation by APC variants at varying FVIIIa substrate concentration showed ~2.6-4.4-fold increases in Km values relative to WT. These results show a variable contribution of basic residues comprising the APC exosite, with significant contributions from Lys39, Arg67, and Arg74 to forming a FVIIIa-interactive site.

Modification of interdomain interfaces within the A3C1C2 subunit of factor VIII affects its stability and activity.

Factor (F)VIII consists of a heavy chain [A1(a1)A2(a2)B domains] and a light chain [(a3)A3C1C2 domains]. Several reports have shown significant changes in FVIII stability and/or activity following selected mutations at the A1-A2, A1-A3, A2-A3, and A1-C2 domain interfaces. In this study, the remaining inter-FVIII subunit interfaces (A3-C1 and C1-C2) were examined for their contributions to the stability and activity of FVIII and FVIIIa. We prepared FVIII mutants with nascent disulfide bridges between A3 and C1 domains (Gly1750Cys/Arg2116Cys and Ala1866Cys/Ser2119Cys) or C1 and C2 domains (Ser2029Cys/Pro2292Cys). We also prepared mutants via replacement of Arg2116 with hydrophobic residues (Ala and Val) because this C1 domain residue appears to face a pocket of positive electrostatic potential in the A3 domain. Stability was assessed following the rates of loss of FVIII activity at 55 °C and the spontaneous loss of FVIIIa activity from A2 subunit dissociation. FVIII Gly1750Cys/Arg2116Cys showed a marked increase in thermal stability (∼3.7-fold) compared with that of wild-type (WT) FVIII, while the stability of FVIII Ala1866Cys/Ser2119Cys was reduced (∼4.7-fold). Although the Ser2029Cys/Pro2292Cys variant showed a modest loss of FVIII stability, the specific activity and thrombin generation potential of this variant were increased (up to 1.2-fold) compared with those of WT. Furthermore, this variant demonstrated an ∼2-fold reduced Km for FX. Mutation of Arg2116 to hydrophobic residues resulted in variable decreases in stability and thrombin generation parameters, suggesting a role of this Arg residue contributing to FVIII structure. Taken together, selective modification of the contiguous domain interfaces in the FVIII light chain may improve FVIII stability and/or cofactor function.

Mass spectrometry-assisted study reveals that lysine residues 1967 and 1968 have opposite contribution to stability of activated factor VIII.

The A2 domain rapidly dissociates from activated factor VIII (FVIIIa) resulting in a dampening of the activity of the activated factor X-generating complex. The amino acid residues that affect A2 domain dissociation are therefore critical for FVIII cofactor function. We have now employed chemical footprinting in conjunction with mass spectrometry to identify lysine residues that contribute to the stability of activated FVIII. We hypothesized that lysine residues, which are buried in FVIII and surface-exposed in dissociated activated FVIII (dis-FVIIIa), may contribute to interdomain interactions. Mass spectrometry analysis revealed that residues Lys(1967) and Lys(1968) of region Thr(1964)-Tyr(1971) are buried in FVIII and exposed to the surface in dis-FVIIIa. This result, combined with the observation that the FVIII variant K1967I is associated with hemophilia A, suggests that these residues contribute to the stability of activated FVIII. Kinetic analysis revealed that the FVIII variants K1967A and K1967I exhibit an almost normal cofactor activity. However, these variants also showed an increased loss in cofactor activity over time compared with that of FVIII WT. Remarkably, the cofactor activity of a K1968A variant was enhanced and sustained for a prolonged time relative to that of FVIII WT. Surface plasmon resonance analysis demonstrated that A2 domain dissociation from activated FVIII was reduced for K1968A and enhanced for K1967A. In conclusion, mass spectrometry analysis combined with site-directed mutagenesis studies revealed that the lysine couple Lys(1967)-Lys(1968) within region Thr(1964)-Tyr(1971) has an opposite contribution to the stability of FVIIIa.

Increasing hydrophobicity or disulfide bridging at the factor VIII A1 and C2 domain interface enhances procofactor stability.

Factor VIII (FVIII) consists of a heavy (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. FVIII x-ray structures show close contacts between A1 and C2 domains. To explore the role of this region in FVIII(a) stability, we generated a variant containing a disulfide bond between A1 and C2 domains by mutating Arg-121 and Leu-2302 to Cys (R121C/L2302C) and a second variant with a bulkier hydrophobic group (A108I) to better occupy a cavity between A1 and C2 domains. Disulfide bonding in the R121C/L2302C variant was >90% efficient as judged by Western blots. Binding affinity between the A108I A1 and A3C1C2 subunits was increased ∼3.7-fold in the variant as compared with WT as judged by changes in fluorescence of acrylodan-labeled A1 subunits. FVIII thermal and chemical stability were monitored following rates of loss of FVIII activity at 57 °C or in guanidinium by factor Xa generation assays. The rate of decay of FVIIIa activity was monitored at 23 °C following activation by thrombin. Both R121C/L2302C and A108I variants showed up to ∼4-fold increases in thermal stability but minimal improvements in chemical stability. The purified A1 subunit of A108I reconstituted with the A3C1C2 subunit showed an ∼4.6-fold increase in thermal stability, whereas reconstitution of the variant A1 with a truncated A3C1 subunit showed similar stability values as compared with WT A1. Together, these results suggest that altering contacts at this A1-C2 junction by covalent modification or increasing hydrophobicity increases inter-chain affinity and functionally enhances FVIII stability.

Reversible activation of cellular factor XIII by calcium.

Factor XIII (FXIII) is a pro-transglutaminase found in the plasma as well as intracellularly in platelets and macrophages. Plasma FXIII is activated by thrombin cleavage (FXIIIa*) and acts in the final stages of blood coagulation cascade. In contrast, the function and activation of cellular FXIII are less characterized. Cellular FXIII relies on a conformational activation of the protein. The nonproteolytic activation of FXIII to FXIIIa° induced by Ca(2+) alone is well known, but up until now it has been discussed under which conditions the process can be induced and whether it can be reversed. Here, we study the nature of the Ca(2+)-induced FXIII activation. Previously used methods to evaluate FXIII activity detect both FXIIIa* and FXIIIa° because they rely on occurrence of enzyme activity or on active site Cys-314 solvent accessibility. Therefore, an analytical HPLC method was developed that separates zymogen recombinant FXIII (rFXIII) from rFXIIIa°. The data demonstrate that nonproteolytic activation and deactivation are highly dependent on Ca(2+) concentration, buffer, and salt components. Moreover, it is established that Ca(2+) activation of rFXIII is fully reversible, and only 2-5 mm CaCl(2) is sufficient to retain full rFXIIIa° activity. However, below 2 mm CaCl(2) the rFXIIIa° molecule deactivates. The deactivated molecule can subsequently undergo a new activation round. Furthermore, it is demonstrated that thermal stress of freeze-dried rFXIII can induce a new predisposed form that activates faster than nonstressed rFXIII.

Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain.

Protein S has an established role in the protein C anticoagulant pathway, where it enhances the factor Va (FVa) and factor VIIIa (FVIIIa) inactivating property of activated protein C (APC). Despite its physiological role and clinical importance, the molecular basis of its action is not fully understood. To clarify the mechanism of the protein S interaction with APC, we have constructed and expressed a library of composite or point variants of human protein S, with residue substitutions introduced into the Gla, thrombin-sensitive region (TSR), epidermal growth factor 1 (EGF1), and EGF2 domains. Cofactor activity for APC was evaluated by calibrated automated thrombography (CAT) using protein S-deficient plasma. Of 27 variants tested initially, only one, protein S D95A (within the EGF1 domain), was largely devoid of functional APC cofactor activity. Protein S D95A was, however, gamma-carboxylated and bound phospholipids with an apparent dissociation constant (Kd(app)) similar to that of wild-type (WT) protein S. In a purified assay using FVa R506Q/R679Q, purified protein S D95A was shown to have greatly reduced ability to enhance APC-induced cleavage of FVa Arg306. It is concluded that residue Asp95 within EGF1 is critical for APC cofactor function of protein S and could define a principal functional interaction site for APC.

Antithrombotic potential of a single-domain antibody enhancing the activated protein C-cofactor activity of protein S.

BACKGROUND: Protein S (PS) is a natural anticoagulant acting as a cofactor for activated protein C (APC) in the proteolytic inactivation of activated factors V (FVa) and VIII (FVIIIa), but also for tissue factor pathway inhibitor α (TFPIα) in the inhibition of activated factor X (FXa). OBJECTIVE: For therapeutic purposes, we aimed at generating single-domain antibodies (sdAbs) that could specifically modulate the APC-cofactor activity of PS in vivo. METHODS: A llama-derived immune library of sdAbs was generated and screened on recombinant human PS by phage display. PS binders were tested in a global activated partial thromboplastin time (APTT)-based APC-cofactor activity assay. RESULTS: A PS-specific sdAb (PS003) was found to enhance the APC-cofactor activity of PS in our APTT-based assay, and this enhancing effect was greater for a bivalent form of PS003 (PS003biv). Further characterization of PS003biv demonstrated that PS003biv also enhanced the APC-cofactor activity of PS in a tissue factor (TF)-induced thrombin generation assay and stimulated APC in the inactivation of FVa, but not FVIIIa, in plasma-based assays. Furthermore, PS003biv was directed against the sex hormone-binding globulin (SHBG)-like domain but did not inhibit the binding of PS to C4b-binding protein (C4BP) and did not interfere with the TFPIα-cofactor activity of PS. In mice, PS003biv exerted an antithrombotic effect in a FeCl3 -induced thrombosis model, while not affecting physiological hemostasis in a tail-clip bleeding model. DISCUSSION: Altogether, these results showed that pharmacological enhancement of the APC-cofactor activity of PS through an original anti-PS sdAb might constitute a promising and safe antithrombotic strategy.

Fucosylated chondroitin sulfate inhibits plasma thrombin generation via targeting of the factor IXa heparin-binding exosite.

Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chondroitin sulfate with antithrombin-independent antithrombotic properties. Heparin cofactor II (HCII)-dependent and -independent mechanisms for DHG inhibition of plasma thrombin generation were evaluated. When thrombin generation was initiated with 0.2 pM tissue factor (TF), the half maximal effective concentration (EC(50)) for DHG inhibition was identical in mock- or HCII-depleted plasma, suggesting a serpin-independent mechanism. In the presence of excess TF, the EC(50) for DHG was increased 13- to 27-fold, suggesting inhibition was dependent on intrinsic tenase (factor IXa-factor VIIIa) components. In factor VIII-deficient plasma supplemented with 700 pM factor VIII or VIIIa, and factor IX-deficient plasma supplemented with plasma-derived factor IX or 100 pM factor IXa, the EC(50) for DHG was similar. Thus, cofactor and zymogen activation did not contribute to DHG inhibition of thrombin generation. Factor IX-deficient plasma supplemented with mutant factor IX(a) proteins demonstrated resistance to DHG inhibition of thrombin generation [factor IX(a) R233A > R170A > WT] that inversely correlated with protease-heparin affinity. These results replicate the effect of these mutations with purified intrinsic tenase components, and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition by DHG. Glycosaminoglycan-mediated intrinsic tenase inhibition is a novel antithrombotic mechanism with physiologic and therapeutic applications.

Hydrogen-Deuterium Exchange Mass Spectrometry Identifies Activated Factor IX-Induced molecular Changes in Activated Factor VIII.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to gain insight into the changes in factor VIII (FVIII) that occur upon its activation and assembly with activated factor IX (FIXa) on phospholipid membranes. HDX-MS analysis of thrombin-activated FVIII (FVIIIa) revealed a marked increase in deuterium incorporation of amino acid residues along the A1-A2 and A2-A3 interface. Rapid dissociation of the A2 domain from FVIIIa can explain this observation. In the presence of FIXa, enhanced deuterium incorporation at the interface of FVIIIa was similar to that of FVIII. This is compatible with the previous finding that FIXa contributes to A2 domain retention in FVIIIa. A2 domain region Leu631-Tyr637, which is not part of the interface between the A domains, also showed a marked increase in deuterium incorporation in FVIIIa compared with FVIII. Deuterium uptake of this region was decreased in the presence of FIXa beyond that observed in FVIII. This implies that FIXa alters the conformation or directly interacts with this region in FVIIIa. Replacement of Val634 in FVIII by alanine using site-directed mutagenesis almost completely impaired the ability of the activated cofactor to enhance the activity of FIXa. Surface plasmon resonance analysis revealed that the rates of A2 domain dissociation from FVIIIa and FVIIIa-Val634Ala were indistinguishable. HDX-MS analysis showed, however, that FIXa was unable to retain the A2 domain in FVIIIa-Val634Ala. The combined results of this study suggest that the local structure of Leu631-Tyr637 is altered by FIXa and that this region contributes to the cofactor function of FVIII.

Identification of residues in the 558-loop of factor VIIIa A2 subunit that interact with factor IXa.

Factor VIIIa is comprised of A1, A2, and A3C1C2 subunits. Several lines of evidence have identified the A2 558-loop as interacting with factor IXa. The contributions of individual residues within this region to inter-protein affinity and cofactor activity were assessed following alanine scanning mutagenesis of residues 555-571 that border or are contained within the loop. Variants were expressed as isolated A2 domains in Sf9 cells using a baculovirus construct and purified to >90%. Two reconstitution assays were employed to determine affinity and activity parameters. The first assay reconstituted factor Xase using varying concentrations of A2 mutant and fixed levels of A1/A3C1C2 dimer purified from wild type (WT), baby hamster kidney cell-expressed factor VIII, factor IXa, and phospholipid vesicles to determine the inter-molecular K(d) for A2. The second assay determined the K(d) for A2 in factor VIIIa by reconstituting various A2 and fixed levels of A1/A3C1C2. Parameter values were determined by factor Xa generation assays. WT A2 expressed in insect cells yielded similar K(d) and k(cat) values following reconstitution as WT A2 purified from baby hamster kidney cell-expressed factor VIII. All A2 variants exhibited modest if any increases in K(d) values for factor VIIIa assembly. However, variants S558A, V559A, D560A, G563A, and I566A showed >9-fold increases in K(d) for factor Xase assembly, implicating these residues in stabilizing A2 association with factor IXa. Furthermore, variants Y555A, V559A, D560A, G563A, I566A, and D569A showed >80% reduction in k(cat) for factor Xa generation. These results identify residues in the 558-loop critical to interaction with factor IXa in Xase.

Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study.

BACKGROUND: Human blood coagulation factor VIII (fVIII) is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3) that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa) which serves as a critical co-factor for factor IXa (fIXa) enzyme in the intrinsic coagulation pathway. RESULTS: In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 A X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740) and A3-C1-C2 (Ser1669-Tyr2332) domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein. CONCLUSIONS: MD simulations predicted that the acidic linker peptide (a1) between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569) region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1) undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the large body of experimental data that implicated the critical importance of the cleavage of the peptide bond between the Arg372 and Ser373 residues for the full co-factor activity of fVIII.