Physiological fIXa Activation Involves a Cooperative Conformational Rearrangement of the 99-Loop

Sichler, Katrin; Kopetzki, Erhard; Huber, Robert; Bode, Wolfram; Hopfner, Karl‐Peter; Brandstetter, Hans

doi:10.1074/jbc.m210722200

Cited by 43 publications

(43 citation statements)

References 27 publications

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“…All variants had very low amidolytic and proteolytic activity relative to wild-type rFVIIa, except variant T239I, which had an activity comparable with wild-type rFVIIa. Thus, just like FIXa [34], FVIIa is highly sensitive to mutations in this position, indicating that it is important in the substrate and inhibitor recognition by FVIIa.…”

Section: Discussionmentioning

confidence: 99%

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

Larsen

Østergaard

Bjelke

et al. 2007

Biochemical Journal

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The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For FVIIa [activated FVII (Factor VII)], the principal initiator of coagulation via the extrinsic pathway, several exosites have been identified, whereas only little is known about the specificity dictated by the active-site architecture. In the present study, we have profiled the primary P4-P1 substrate specificity of FVIIa using positional scanning substrate combinatorial libraries and evaluated the role of the selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa had P1 specificity exclusively towards arginine and lysine residues. In the S2 pocket, threonine, leucine, phenylalanine and valine residues were the most preferred amino acids. Both S3 and S4 appeared to be rather promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed and, as a consequence, the optimal substrate for FVIIa could not be derived directly from a subsite-directed specificity screen. To evaluate the role of the active-site residues in defining specificity, a series of mutants of FVIIa were prepared at position 239 (position 99 in chymotrypsin), which is considered to be one of the most important residues for determining P2 specificity of the trypsin family members. This was confirmed for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered separately.

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Section: Discussionmentioning

confidence: 99%

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

Larsen

Østergaard

Bjelke

et al. 2007

Biochemical Journal

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“…9,10 One specific feature of FIXa is its minimal activity toward its natural substrate, factor X (FX), and toward small peptide substrates. Only after assembly in the tenase complex, which consists of FIXa, activated FVIII (FVIIIa), and FX, is relevant protease activity achieved, exceeding the activity of FIXa alone by 100 000-to 1 000 000-fold.…”

Section: Introductionmentioning

confidence: 99%

“…Only after assembly in the tenase complex, which consists of FIXa, activated FVIII (FVIIIa), and FX, is relevant protease activity achieved, exceeding the activity of FIXa alone by 100 000-to 1 000 000-fold. [10][11][12] The exchange of amino acid residues specific for FIX with those of FX and the modification of the 99-loop, which is unique to FIX and believed to shield the FIX active site before assembly in the tenase complex, led to FIXa variants with several hundred-to several thousand-fold higher amidolytic activities. 10 We hypothesized that these modifications could mimic the effect of FVIIIa binding to FIXa.…”

Section: Introductionmentioning

confidence: 99%

“…[10][11][12] The exchange of amino acid residues specific for FIX with those of FX and the modification of the 99-loop, which is unique to FIX and believed to shield the FIX active site before assembly in the tenase complex, led to FIXa variants with several hundred-to several thousand-fold higher amidolytic activities. 10 We hypothesized that these modifications could mimic the effect of FVIIIa binding to FIXa. Therefore, we expressed the recombinant full-length variants of FIX and observed measurable clotting activity in FVIII-deficient plasma.…”

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confidence: 99%

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Engineered factor IX variants bypass FVIII and correct hemophilia A phenotype in mice

Milanov

Ivanciu

Abriss

et al. 2012

Blood

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The complex of the serine protease factor IX (FIX) and its cofactor, factor VIII (FVIII), is crucial for propagation of the intrinsic coagulation cascade. Absence of either factor leads to hemophilia, a disabling disorder marked by excessive hemorrhage after minor trauma. FVIII is the more commonly affected protein, either by X-chromosomal gene mutations or in autoimmune-mediated acquired hemophilia. Whereas substitution of FVIII is the mainstay of hemophilia A therapy, treatment of patients with inhibitory Abs remains challenging. In the present study, we report the development of FIX variants that can propagate the intrinsic coagulation cascade in the absence of FVIII. FIX variants were expressed in FVIII-knockout (FVIII-KO) mice using a nonviral genetransfer system. Expression of the variants shortened clotting times, reduced blood loss after tail-clip assay, and reinstalled clot formation, as tested by in vivo imaging of laser-induced vessel injury. In IntroductionThe intrinsic coagulation cascade is a tightly regulated proteaseand cofactor-dependent amplification system that ensures the formation of stable clots after injury. 1 Within this system, deficiencies of the coagulation cofactor, factor VIII (FVIII), or the corresponding coagulation protease, factor IX (FIX), lead to the X-chromosomal inherited bleeding disorders hemophilia A and hemophilia B, respectively. Hemophilia A occurs approximately in 1 of 5000 newborn boys, whereas hemophilia B is less common. 2 Untreated, hemophilia presents with spontaneous bleeding preferentially into large joints and skeletal muscle, and internal and intracranial bleedings can also occur. Substitution of deficient coagulation factors by intravenous infusion of plasma-derived or recombinant coagulation factor concentrates is the therapy of choice. In the last decades, treatment has evolved from so-called on-demand treatment for acute injury/hemorrhage to a secondary preventative approach with regular prophylactic infusions. Prophylactic treatment, securing plasma levels above 1% of normal, already prevents the major long-term consequences of the disease: joint damage and muscular atrophy. 3,4 A major obstacle for protein substitution therapy is the occurrence of neutralizing Abs directed against the FVIII protein. This can either be a result of an immune response after exogenous protein exposure 5 or may appear in adult patients as a spontaneous auto-immune event. 6 In these cases, FVIII infusion is often ineffective and so-called bypassing agents are used. These agents consist of constitutively activated proteases such as activated factor VII (FVIIa), and promote clot formation directly without restoring the intrinsic amplification loop. Although these therapeutics are efficient at stopping acute bleeding, limitations include the relatively short half-lives of activated proteases in the circulation and potential vascular risks in long-term treatment. 7,8 In the present study, we report on the generation of FIX variants with FVIII-independent clotting activity that we...

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“…A FIXa variant with triple mutation (Y259F/K265T/Y345T) exhibited several 1000-fold increases in amidolytic activity toward synthetic peptide substrates and altered substrate specificity [56]. Based on these outcomes [56,57], Milanov et al explored whether introduction of the mutations Y259F, K265T, and Y345T into the full length FIX protein would give rise to cofactor independent activity under normal physiological conditions by determining its clotting activities in human FIX-, FVIII-and FX-deficient plasma [58]. As a result, the triple variant exposed almost the same FIX clotting activity as wt-FIX and no shift toward FX substrate affinity was observed.…”

Section: Engineering Fix Variants To Bypass Fviiimentioning

confidence: 99%

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

2012

J Genet Syndr Gene Ther

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FVII/FVIIaPatients with hemophilia A and B have deficient protein concentrations of FVIII and FIX, respectively and therefore are treated by protein substitution with plasma-derived or recombinant factor FVIII or FIX concentrates. In about 20-30% of the patients with hemophilia A and in 3-5% of those with hemophilia B this can lead to the development of neutralizing antibodies against the infused proteins which makes therapy inefficient [1,2]. Recombinant activated factor VII (rFVIIa) protein is currently an alternative therapy available for inhibitor patients to treat excessive bleeding. However, supraphysiological concentrations and repeated administration are required to induce hemostasis [3].Consequently, FVIIa analogs were created for future protein replacement therapy or gene delivery approaches based on crystalline structure analysis of free and TF-bound FVIIa or on sequence homology of other more potent proteases [4,5].Like the other vitamin K dependent coagulation factors, FVII is physiologically synthesized in the liver. FVII is secreted into the circulation as a 406-residue single-chain polypeptide which contains an N-terminal γ-carboxyglutamic acid (Gla) domain in which all ten Glu residues are post-translationally carboxylated followed by two regions homologous to epidermal growth factor domains and a serine protease domain [3,6,7]. Upon vascular injury, the cofactor tissue factor (TF) is exposed to FVII and triggers the extrinsic pathway of the coagulation cascade by activation of FIX, factor X and FVII auto-activation on the TF-bearing cells resulting in large-scale thrombin generation and a fibrin clot. FVII is activated to FVIIa by internal proteolysis due to a cleavage of a single Arg152-Ile153 peptide bond. The physiological plasma concentration of FVII is around 10 nM, however, only about 1% of FVII is circulating in its free and active form. FVIIa has a plasma halflife of approximately 2.5 hours [8]. FVII activation results in connected FVII light and heavy chains which form a one-to-one complex with TF in the presence of Ca 2+ ions. The complex formation is an essential process in which TF allosterically leads to a fully active FVIIa [9]. The cleavage alone leaves FVIIa in a zymogen-like conformation of quite low specific activity [3,10]. Several residues in the first EGF-like domain of FVII and in the protease domain, especially methionine at position 306, seem to be pivotal for the cofactor-mediated allosteric stimulation [11]. TF stabilizes the active conformation of FVIIa for accelerated activation of its substrates FIX and FX [12,13] by inducing a conformational change and establishing a stable salt bridge between the residues Ile153 and Asp343 [14] and stabilization of the interaction between the residues Leu305 and Phe374 which in turn stabilizes S 1 and S 3 substrate pocket and the activation pocket [15]. These findings contributed to the development of FVIIa variants with enhanced TFindependent (intrinsic) specific activity by mimicking the effect of TF binding [16]. Additional fin...

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Physiological fIXa Activation Involves a Cooperative Conformational Rearrangement of the 99-Loop

Cited by 43 publications

References 27 publications

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

Engineered factor IX variants bypass FVIII and correct hemophilia A phenotype in mice

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

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