The Impacts of G4S Mutation on N-glycosylation and conformation of the Human Coagulation Factor IX GLA domain: In silico and In vitro Analysis

Missense mutations are the most prevalent form of mutation in hemophilia B patients. These alterations may result in the creation of novel and non-native N-glycosylation sites (Asn-X-Ser/Thr) through single amino acid substitutions. The pathogenic mechanisms of N-glycosylation mutations in hemophilia B patients have not been extensively studied yet. By survey among known missense mutations, we found only one N-glycosylation mutation in the γ-carboxyglutamic-rich (GLA) domain of the human coagulation factor IX (hFIX). This mutation that was reported in patients with mild and moderate hemophilia B, is caused by G4S amino acid substitution. To investigate the possibility of glycan attachment to the novel N-glycosylation site in G4S-mutant hFIX and the occurrence of hyperglycosylation, site-directed mutagenesis was applied to introduce the selected mutation into the coding sequence of the hFIX. The nucleotide sequences of the both native and G4S-mutant hFIX were separately cloned into the pcDNA3.1 expression plasmid and transiently expressed in HEK293T cells. Our results from gradient SDS-PAGE and western blotting analysis of the both recombinant native and mutant hFIX demonstrated no glycan attachment to the new N-glycosylation site in the G4S-mutant hFIX. Molecular dynamics (MD) simulation was also conducted to provide atomistic insights into structure and behavior of the native and G4S-mutant GLA domains in the both free and membrane-bound states. The results revealed that the mutation slightly affected the dynamic behavior of the mutant GLA domain. The conformational analysis proved that the native GLA domain had less fluctuation and more stability than the mutant GLA domain. The slight conformational changes may influence the binding capacity and interaction of the mutant GLA domain to phospholipid bilayer which is necessary for coagulation activity of the hFIX. These findings were in accordance with the nature of the G4S mutation which causes mild hemophilia B.

Structure of human factor VIIa–soluble tissue factor with calcium, magnesium and rubidium

Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg2+ ions and four Ca2+ ions in the GLA domain, one Ca2+ ion in the EGF1 domain and one Ca2+ ion in the protease domain. Further, FVIIa contains an Na+ site in the protease domain. Since Na+ and water share the same number of electrons, Na+ sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na+ site in FVIIa, the structure of the FVIIa–soluble tissue factor (TF) complex was solved at 1.8 Å resolution containing Mg2+, Ca2+ and Rb+ ions. In this structure, Rb+ replaced two Ca2+ sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb+ was not detected at the expected Na+ site. In kinetic experiments, Na+ increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ∼20-fold; however, in the presence of Ca2+, Na+ had a negligible effect. Ca2+ increased the hydrolytic activity of FVIIa towards S-2288 by ∼60-fold in the absence of Na+ and by ∼82-fold in the presence of Na+. In molecular-dynamics simulations, Na+ stabilized the two Na+-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163–180. Ca2+ stabilized the Ca2+-binding loop (the 70-loop) and Na+-binding loops but not the TF-binding region. Na+ and Ca2+ together stabilized both the Na+-binding and Ca2+-binding loops and the TF-binding region. Previously, Rb+ has been used to define the Na+ site in thrombin; however, it was unsuccessful in detecting the Na+ site in FVIIa. A conceivable explanation for this observation is provided.

Phosphatidylserine and phosphatidylethanolamine regulate the structure and function of FVIIa and its interaction with soluble tissue factor

Cell membranes have important functions in many steps of the blood coagulation cascade, including the activation of factor X (FX) by the factor VIIa (FVIIa)-tissue factor (TF) complex (extrinsic Xase). FVIIa shares structural similarity with factor IXa (FIXa) and FXa. FIXa and FXa are regulated by binding to phosphatidylserine (PS)-containing membranes via their γ-carboxyglutamic acid-rich domain (Gla) and epidermal growth-factor (EGF) domains. Although FVIIa also has a Gla-rich region, its affinity for PS-containing membranes is much lower compared with that of FIXa and FXa. Research suggests that a more common endothelial cell lipid, phosphatidylethanolamine (PE), might augment the contribution of PS in FVIIa membrane-binding and proteolytic activity. We used soluble forms of PS and PE (1,2-dicaproyl-sn-glycero-3-phospho-l-serine (C6PS), 1,2-dicaproyl-sn-glycero-3-phospho-ethanolamine (C6PE)) to test the hypothesis that the two lipids bind to FVIIa jointly to promote FVIIa membrane binding and proteolytic activity. By equilibrium dialysis and tryptophan fluorescence, we found two sites on FVIIa that bound equally to C6PE and C6PS with Kd of ∼ 150–160 μM, however, deletion of Gla domain reduced the binding affinity. Binding of lipids occurred with greater affinity (Kd∼70–80 μM) when monitored by FVIIa proteolytic activity. Global fitting of all datasets indicated independent binding of two molecules of each lipid. The proteolytic activity of FVIIa increased by ∼50–100-fold in the presence of soluble TF (sTF) plus C6PS/C6PE. However, the proteolytic activity of Gla-deleted FVIIa in the presence of sTF was reduced drastically, suggesting the importance of Gla domain to maintain full proteolytic activity.

Procoagulant Activity of Skeletal Muscle Myosin Is Not Caused By Contaminating Phosphatidylserine-Vesicles

Skeletal muscle myosin (SkM) can bind factor (F)Xa and FVa, thereby providing a surface that promotes thrombin generation by the prothrombinase complex (FXa:FVa:Ca++) that cleaves prothrombin. A recent BLOOD paper (Novakovic & Gilbert, 2020) asserted that this activity of SkM preparations is entirely due to contaminating phosphatidylserine (PS)-containing phospholipid vesicles in SkM preparations because annexin V and lactadherin neutralized the ability of SkM to enhance prothrombin activation. However, annexin V and lactadherin are certainly not monospecific for binding PS as they are globular proteins that can bind other lipids and many proteins. Without any PS measurements or use of any reagents specific for PS (e.g., monoclonal (mAb) anti-PS antibodies), that report, in a gross overinterpretation of its incomplete data, dismissed any direct role for myosin for SkM's procoagulant activity. When we previously observed that annexin V is inhibitory of SkM's support for prothrombinase activity, we sent a sample of SkM (Cytoskeletal Inc) to Avanti Polar Lipids for quantitation of the PS content based on liquid chromatography-mass spectrometry. That analysis showed that only a small amount of PS was present in the SkM, approximately 0.90 µmol PS per 40. µmol SkM. This amount of PS present in SkM preparations is not enough to explain SkM's procoagulant activity. For example, standardized purified prothrombinase reaction mixture assays show that 10 nM SkM enables formation of 3 nmol thrombin/min while 0.22 nM PS (in 1.1 nM phosphatidylcholine (PC)(80%)/PS(20%) vesicles) enables formation of only 0.4 nmol thrombin/min (Figure 1A). We directly assessed the role of contaminating PS for SkM's prothrombinase support using the well characterized anti-PS mAb 11.31 (aka mAb PGN632). When the ability of mAb 11.31 to inhibit prothrombinase enhancement by SkM or, in controls, by PC/PS vesicles was determined, the data showed that, in controls, mAb 11.31 at 1.0 nM severely inhibited PC/PS vesicle's enhancement of prothrombinase by > 90% (Figure 1B). The dose-response gave an inhibitory IC50 value of 0.2 nM which is near this mAb's reported Kd of 0.17-0.35 nM for PS, establishing the potent ability of this anti-PS mAb to neutralize PS procoagulant activity. However, there was no substantial inhibition of SkM's enhancement of prothrombinase by the anti-PS mAb 11.31 at up to 1 nM mAb 11.31 (Figure 1B). This indicates that contaminating, PS-containing vesicles are not a significant factor for SkM-dependent enhancement of prothrombin activation by the SkM preparation -- in direct contradiction of the assertion of Novakovic & Gilbert (BLOOD 2020). That recent report was correct that annexin V inhibits the ability of SkM to enhance prothrombinase as well as the ability of PC/PS vesicles to do so (Figure 1B). So it was the overinterpretation of the annexin V and lactadherin data as well as the failure to provide any direct measurement of PS that were problematic in that report. The observation that annexin V and lactadherin inhibit SkM's enhancement of prothrombinase merits further studies to understand what may be their mechanistic influences. Another informative test for an essential role for PS in SkM's enhancement of prothrombinase involves the use of FXa that lacks its gamma-carboxyglutamic acid (Gla) domain because this N-terminal domain of FXa is required for FXa binding to PS-containing phospholipid vesicles. Data in Figure 1A show that SkM, but not PS-containing PL vesicles, supports the prothrombinase activity of des-Gla Domain (DG)-FXa which lacks its Gla domain. Dose-response data show that, in prothrombinase assays, DG-FXa has only 1% activity in the presence of PC/PS vesicles but has 25-35% activity in presence of SkM (Figure 1A). These data indicate that SkM's procoagulant activity does not absolutely require the Gla domain of FXa whereas PS-containing vesicles do require the Gla domain of FXa for significant activity. These data prove that the recent assertion that PS vesicle contamination, not myosin, explains SkM's procoagulant activity represents an overinterpretation of annexin V and lactadherin data and is simply wrong. In conclusion, both new data here, i.e., PS content of SkM preparations and data for effects of anti-PS mAb 11.31 (Figure 1B) and previous data (Deguchi et al, Blood 2016 and J Biol Chem, 2019), affirm that the myosin protein is a key factor for SkM's ability to enhance prothrombin activation. Figure 1 Disclosures No relevant conflicts of interest to declare.

Characterization of missense mutations in the signal peptide and propeptide of FIX in hemophilia B by a cell-based assay

Many mutations in the signal peptide and propeptide of factor IX (FIX) cause hemophilia B. A FIX variants database reports 28 unique missense mutations in these regions that lead to FIX deficiency, but the underlying mechanism is known only for the mutations on R43 that interfere with propeptide cleavage. It remains unclear how other mutations result in FIX deficiency and why patients carrying the same mutation have different bleeding tendencies. Here, we modify a cell-based reporter assay to characterize the missense mutations in the signal peptide and propeptide of FIX. The results show that the level of secreted conformation-specific reporter (SCSR), which has a functional γ-carboxyglutamate (Gla) domain of FIX, decreases significantly in most mutations. The decreased SCSR level is consistent with FIX deficiency in hemophilia B patients. Moreover, we find that the decrease in the SCSR level is caused by several distinct mechanisms, including interfering with cotranslational translocation into the endoplasmic reticulum, protein secretion, γ-carboxylation of the Gla domain, and cleavage of the signal peptide or propeptide. Importantly, our results also show that the SCSR levels of most signal peptide and propeptide mutations increase with vitamin K concentration, suggesting that the heterogeneity of bleeding tendencies may be related to vitamin K levels in the body. Thus, oral administration of vitamin K may alleviate the severity of bleeding tendencies in patients with missense mutations in the FIX signal peptide and propeptide regions.

Prothrombin is a binding partner of the human receptor of advanced glycation end products

The receptor for advanced glycation end products (RAGE) plays a key role in mammal physiology and in the etiology and progression of inflammatory and oxidative stress-based diseases. In adults, RAGE expression is normally high only in the lung where the protein concentrates in the basal membrane of alveolar Type I epithelial cells. In diseases, RAGE levels increase in the affected tissues and sustain chronic inflammation. RAGE exists as a membrane glycoprotein with an ectodomain, a transmembrane helix, and a short carboxyl-terminal tail, or as a soluble ectodomain that acts as a decoy receptor (sRAGE). VC1 domain is responsible for binding to the majority of RAGE ligands including advanced glycation end products (AGEs), S100 proteins, and HMGB1. To ascertain whether other ligands exist, we analyzed by MS the material pulled down by VC1 from human plasma. Twenty of 295 identified proteins were selected and associated to coagulation and complement processes and to extracellular matrix. Four of them contained a γ-carboxyl glutamic acid (Gla) domain, a calcium-binding module, and prothrombin (PT) was the most abundant. Using MicroScale thermophoresis, we quantified the interaction of PT with VC1 and sRAGE in the absence or presence of calcium that acted as a competitor. PT devoid of the Gla domain (PT des-Gla) did not bind to sRAGE, providing further evidence that the Gla domain is critical for the interaction. Finally, the presence of VC1 delayed plasma clotting in a dose-dependent manner. We propose that RAGE is involved in modulating blood coagulation presumably in conditions of lung injury.

Laboratory assessment of vitamin K status

Vitamin K is required for the ɣ-carboxylation of specific glutamic acid residues within the Gla domain of the 17 vitamin K-dependent proteins (VKDPs). The timely detection and correction of vitamin K deficiency can protect against bleeding. Vitamin K also plays a role in bone metabolism and vascular calcification. Patients at increased risk of vitamin K deficiency include those with a restricted diet or malnutrition, lipid malabsorption, cancer, renal disease, neonates and the elderly. Coagulation assays such as the prothrombin time have been used erroneously as indicators of vitamin K status, lacking sufficient sensitivity and specificity for this application. The measurement of phylloquinone (K1) in serum is the most commonly used marker of vitamin K status and reflects abundance of the vitamin. Concentrations <0.15 µg/L are indicative of deficiency. Disadvantages of this approach include exclusion of the other vitamin K homologues and interference from recent dietary intake. The cellular utilisation of vitamin K is determined through measurement of the prevalence of undercarboxylated VKDPs. Most commonly, undercarboxylated prothrombin (Protein Induced by Vitamin K Absence/antagonism, PIVKA-II) is used (reference range 17.4–50.9 mAU/mL (Abbott Architect), providing a retrospective indicator of hepatic vitamin K status. Current clinical applications of PIVKA-II include supporting the diagnosis of vitamin K deficiency bleeding of the newborn, monitoring exposure to vitamin K antagonists, and when used in combination with α-fetoprotein, as a diagnostic marker of hepatocellular carcinoma. Using K1 and PIVKA-II in tandem is an approach that can be used successfully for many patient cohorts, providing insight into both abundance and utilisation of the vitamin.

Cardiac Myosin Acts Is a Potent Procoagulant in Vitro and In Vivo

Thrombin generation and fibrin formation can cause occlusive thrombosis and myocardial infarction is caused by occlusive thrombi. Exposure and release of cardiac myosin (CM) are linked to myocardial infarction, but CM has not been accorded any thrombotic functional significance. Skeletal muscle myosin (SkM), which is structurally similar to CM, was previously shown to exert procoagulant activities (Deguchi H et al, Blood. 2016;128:1870), leading us to undertake new studies of the in vitro and in vivo procoagulant activities of CM. First, the setting of hemophilia A with its remarkable bleeding risk was used to evaluate the procoagulant properties of CM. In studies of human hemophilia plasma and of murine acquired hemophilia A plasma, CM was added to these plasmas and tissue factor (TF)-induced thrombin generation assays were performed. Plasmas included human hemophilia A plasma and C57BL/6J mouse plasma with anti-FVIII antibody (GMA-8015; 5 microgram/mL final). CM showed strong procoagulant effects in human hemophilia A plasma, which is naturally deficient in factor VIII (&lt;1% FVIII). The addition of only CM (12.5-200 nM) greatly increased thrombin generation in a manner comparable to addition of only recombinant FVIII. In the wild-type C57BL/6J mouse plasma, anti-FVIII antibody greatly reduced TF-induced thrombin generation, as reported. When CM (12.5-200 nM) was added to mouse plasma containing anti-FVIII antibodies, TF-induced thrombin generation was concentration-dependently restored. To study the in vivo hemostatic ability of SkM, an acquired hemophilia A mouse model was employed. Intravenous injection of anti-FVIII antibody (GMA-8015; 0.25 mg/kg) or control vehicle was given retro-orbitally to wild type C57BL/6J mice at 2 hours prior to tail cutting. The distal portion of the tail was surgically removed at 1.5 mm tail diameter to induce moderate bleeding. Tails were immersed in 50 mL of saline at 37 degrees. Total blood loss was measured as the blood volume collected during 20 min normalized for mouse weight (microL/g). Mice given only anti-FVIII antibody had more blood loss (median = 6.7 microL/g) compared to control mice (median &lt; 2 microL/g) (Figure). In this mouse model receiving anti-FVIII antibody, CM (5.4 mg/kg) injected at 15 min prior to tail cutting significantly reduced the median blood loss from 6.7 to 2.0 and 3.2 microL/g, respectively (p &lt; 0.001 for each myosin) (Figure). Thus, these studies provide in vivo proof of concept that both CM and SkM can reduce bleeding and are procoagulant in vivo. Second, studies of the effects of CM on thrombogenesis ex vivo using fresh human flowing blood showed that perfusion of blood over CM-coated surfaces at 300 s-1 shear rate caused extensive fibrin deposition. Addition of CM to blood also promoted the thrombotic responses of human blood flowing over collagen-coated surfaces, evidence of CM's thrombogenicity. Further studies showed that CM enhanced thrombin generation in platelet rich plasma and platelet poor plasma, indicating that CM promotes thrombin generation in plasma primarily independently of platelets. To address the mechanistic insights for CM's procoagulant activity, purified coagulation factors were employed. In a purified system composed of factor Xa, factor Va, prothrombin and calcium ions, CM greatly enhanced prothrombinase activity. Experiments using Gla-domainless factor Xa showed that the Gla domain of factor Xa was not required for CM's prothrombinase enhancement in contrast to phospholipid-enhanced prothrombinase activity which requires that Gla domain. Binding studies showed that CM directly binds factor Xa. In summary, here we show that CM is procoagulant due to its ability to bind factor Xa and strongly promote thrombin generation. In summary, CM acts as procoagulant by its ability to bind factor Xa and strongly promote thrombin generation both in vivo an in vitro. These provocative findings raise many questions about whether and how the protective pro-hemostatic properties or the pathogenic prothrombotic properties of CM contribute to pathophysiology in the coronary circulation. This discovery raises many questions about CM and coronary pathophysiology, and future CM research may enable novel translations of new knowledge regarding CM's procoagulant activities for coronary health and disease. Figure Disclosures Mosnier: The Scripps Research Institute: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Ruggeri:MERU-VasImmune Inc.: Equity Ownership, Other: CEO and Founder.

The Endothelial Protein C Receptor (EPCR) Regulates Endogenous Factor VII Levels in Mice

The endothelial protein C receptor (EPCR) has been demonstrated to bind activated FVII (FVIIa) through the Gla domain with equal affinity to Protein C (PC). Mouse studies suggest that EPCR is involved in the extravasation of infused human FVIIa, leading to an extended extravascular tissue persistence, longer than expected based on its circulating half-life. This provides a plausible explanation for the long-term benefits of hemophilic patients on human FVIIa prophylaxis. Collectively, these data suggest that EPCR sequesters administered FVIIa in tissues where it may have a hemostatic effect. However, the role of the endogenous FVII-EPCR interaction in normal conditions is largely unknown. For this, we have developed a mouse model to better understand this interaction in vivo. Endogenous mouse FVII and FVIIa (mFVII/FVIIa) do not bind mouse EPCR. However, our laboratory has demonstrated that L4F, L8M, and T9R substitutions in the Gla domain of mFVIIa enable its interaction with mouse EPCR while retaining full enzymatic activity in vitro. Based on that data, we utilized CRISPR/Cas9 technology to knock-in L4F, L8M, and T9R into the mFVII Gla domain in the mouse F7 locus (F7FMR), thereby developing mice with a chimeric endogenous FVII capable of binding EPCR. Founder animals were generated and capable of producing offspring, indicating that the gain-of-function in mFVII was compatible with life. Animals were subsequently backcrossed to wildtype C57BL/6 mice in order to remove potential off-target effects of the CRISPR/Cas9. Resultant heterozygous animals (F7FMR/WT) from the final cross were bred to generate F7FMR/FMR, F7FMR/WT, and F7WT/WTlittermates. We generated 59 male and 52 female animals and a binomial distribution test demonstrated that sex is equally distributed in the population. Moreover, the genotypes expected from the heterozygous crosses were inherited in a 1:2:1 ratio, further indicating that the gain-of-function in FVII is not lethal during development. As additional metrics of health, we measured weight longitudinally during weeks 1-10 of life and found no differences between the three genotypes for either gender. Complete blood counts (CBCs) revealed no differences between the F7FMR/FMR, F7FMR/WT, and F7WT/WTgenotypes, with the exception of a mild elevation in F7FMR/WTanimals compared to animals with wildtype FVII. Collectively, we found that the gain-of-function in EPCR binding by endogenous FVII is not detrimental to the overall health of the mice. Subsequently, we determined the mFVII levels in the F7FMR/FMR, F7FMR/WT, and F7WT/WTanimals using an in-house ELISA. We observed that plasmatic mFVII levels were dependent on the EPCR-binding capacity of the endogenous mFVII. Specifically, F7WT/WTmice, whose mFVII does not bind EPCR, had a plasmatic mFVII concentration of ~690 ng/ml. In contrast, F7FMR/FMRhomozygote mice had ~350 ng/ml of mouse FVII, approximately half the plasma levels of the F7WT/WT. Heterozygote animals F7FMR/WThad an intermediate plasmatic mFVII level (~550 ng/ml), suggesting that EPCR may regulate plasmatic FVII levels in vivo. Lastly, we determined the hemostatic response to injury in the F7FMR/FMR, F7FMR/WT, and F7WT/WTanimals. We did this in two ways, by measuring blood loss following tail clip assay and by determining time to vessel occlusion following ferric chloride injury of the carotid artery. We observed no differences between the three genotypes in response to either injury model. In conclusion, we have generated and characterized a novel mouse model in which endogenous FVII is capable of binding EPCR. Using this model, we demonstrated that EPCR can modulate plasmatic FVII levels in vivo but does not appear to affect hemostasis. Since this model mimics the FVII-EPCR interaction in humans, it can now be used to further investigate how this interaction participates in other normal or pathologic states that depend on FVII and/or EPCR. Disclosures Margaritis: Bayer Hemophilia Awards: Research Funding; Bristol-Myers Squibb: Other: Salary (spouse); CSL Behring: Other: Salary (spouse); NovoNordisk A/S: Research Funding.

Compound heterozygosity of the gamma-glutamyl carboxylase mutants V255M and S300F causes pseudoxanthoma elasticum-like disease through impaired processivity

ABSTRACTVitamin K-dependent (VKD) protein activities require carboxylated Glus (Glas) generated by the gamma-glutamyl carboxylase. Some carboxylase mutations cause severe bleeding, while others cause pseudoxanthoma elasticum (PXE)-like associated with excessive calcification. How carboxylase mutations cause PXE-like was unknown. We analyzed two mutants (V255M and S300F) whose compound heterozygosity causes PXE-like. Substrates derived from VKD proteins important to calcification (MGP) or clotting (factor IX) were studied, which contained the Gla domain and exosite-binding domain that mediates carboxylase binding. Surprisingly, the V255M mutant was more active (4-5 fold) than wild type carboxylase, while S300F activity was low. The V255M results suggested faster substrate release, which could impact carboxylase processivity, where the carboxylase remains bound to VKD proteins throughout multiple Glu to Gla conversions. To assess mutant processivity, we performed a novel challenge assay in which MGP-carboxylase and factor IX-carboxylase complexes were reacted in the presence of excess challenge VKD peptide. Tight complexes between VKD proteins and wild type carboxylase excluded access of the challenge peptide during the carboxylation of VKD protein in the complex. In contrast, VKD protein complexes with V255M or S300F allowed promiscuous access of challenge peptide. Both mutants therefore impair processivity. Most of the V255M product was carboxylated challenge peptide, which could explain mild PXE-like observed in the proband’s mother and aunt. Both have wild type and V255M carboxylase alleles; however, higher V255M production of a potentially defective MGP product could account for their phenotype. The results are an important advance in understanding why carboxylase mutations cause the PXE-like disease.