Thrombin activation of the factor XI dimer is a multistaged process for each subunit.

BACKGROUND: Factor (F)XI can be activated by proteases, including thrombin and FXIIa. The interactions of these enzymes with FXI are transient in nature and therefore difficult to study. OBJECTIVES: To identify the binding interface between thrombin and FXI and understand the dynamics underlying FXI activation. METHODS: Crosslinking mass spectrometry was used to localize the binding interface of thrombin on FXI. Molecular dynamics simulations were applied to investigate conformational changes enabling thrombin-mediated FXI activation after binding. The proposed trajectory of activation was examined with nanobody 1C10, which was previously shown to inhibit thrombin-mediated activation of FXI. RESULTS: We identified a binding interface of thrombin located on the light chain of FXI involving residue Pro520. After this initial interaction, FXI undergoes conformational changes driven by binding of thrombin to the apple 1 domain in a secondary step to allow migration toward the FXI cleavage site. The 1C10 binding site on the apple 1 domain supports this proposed trajectory of thrombin. We validated the results with known mutation sites on FXI. As Pro520 is conserved in prekallikrein (PK), we hypothesized and showed that thrombin can bind PK, even though it cannot activate PK. CONCLUSION: Our investigations show that the activation of FXI is a multistaged procedure. Thrombin first binds to Pro520 in FXI; thereafter, it migrates toward the activation site by engaging the apple 1 domain. This detailed analysis of the interaction between thrombin and FXI paves a way for future interventions for bleeding or thrombosis.

Discovery of Teleost Plasma Kallikrein/Coagulation Factor XI-Like Gene from Channel Catfish (Ictalurus punctatus) and the Evidence that the Protein Encoded by it Acts as a Lectin.

Mammalian plasma kallikrein (PK) and coagulation factor XI (fXI) are serine proteases that play in the kinin-kallikrein cascade and in the blood clotting pathway. These proteases share sequence homology and have four apple domains (APDs) and a serine protease domain (SPD) from their N-terminus to C-terminus. No homologs of these proteases are believed to be present in fish species, except for lobe-finned fish. Fish, however, have a unique lectin, named kalliklectin (KL), which is composed of APDs only. In the present study, we found genomic sequences encoding a protein with both APDs and SPD in a few cartilaginous and bony fishes, including the channel catfish Ictalurus punctatus, using bioinformatic analysis. Furthermore, we purified two ~ 70 kDa proteins from the blood plasma of the catfish using mannose-affinity and gel filtration chromatography sequentially. Using de novo sequencing with quadrupole time-of-flight tandem mass spectrometry, several internal amino acid sequences in these proteins were mapped onto possible PK/fXI-like sequences that are thought to be splicing variants. Exploration of APD-containing proteins in the hagfish genome database and phylogenetic analysis suggested that the PK/fXI-like gene originated from hepatocyte growth factor, and that the gene was acquired in a common ancestor of jawed fish. Synteny analysis provided evidence for chromosomal translocation around the PK/fXI-like locus that occurred in the common ancestor of holosteans and teleosts after separation from the lobe-finned fish lineage, or gene duplication into two chromosomes, followed by independent gene losses. This is the first identification of PK/fXI-like proteins in teleosts.

A Common Missense Variant Causing Factor XI Deficiency and Increased Bleeding Tendency in Maine Coon Cats.

Hereditary factor XI (FXI) deficiency is characterized as an autosomal mild to moderate coagulopathy in humans and domestic animals. Coagulation testing revealed FXI deficiency in a core family of Maine Coon cats (MCCs) in the United States. Factor XI-deficient MCCs were homozygous for a guanine to adenine transition resulting in a methionine substitution for the highly conserved valine-516 in the FXI catalytic domain. Immunoblots detected FXI of normal size and quantity in plasmas of MCCs homozygous for V516M. Some FXI-deficient MCCs experienced excessive post-operative/traumatic bleeding. Screening of 263 MCCs in Europe revealed a mutant allele frequency of 0.232 (23.2%). However, V516M was not found among 100 cats of other breeds. Recombinant feline FXI-M516 (fFXI-M516) expressed ~4% of the activity of wild-type fFXI-V516 in plasma clotting assays. Furthermore, fFXIa-M516 cleaved the chromogenic substrate S-2366 with ~4.3-fold lower catalytic efficacy (kcat/Km) than fFXIa-V516, supporting a conformational alteration of the protease active site. The rate of FIX activation by fFXIa-M516 was reduced >3-fold compared with fFXIa-V516. The common missense variant FXI-V516M causes a cross-reactive material positive FXI deficiency in MCCs that is associated with mild-moderate bleeding tendencies. Given the prevalence of the variant in MCCs, genotyping is recommended prior to invasive procedures or breeding.

Skeletal muscle myosin promotes coagulation by binding factor XI via its A3 domain and enhancing thrombin-induced factor XI activation.

Skeletal muscle myosin (SkM) has been shown to possess procoagulant activity; however, the mechanisms of this coagulation-enhancing activity involving plasma coagulation pathways and factors are incompletely understood. Here, we discovered direct interactions between immobilized SkM and coagulation factor XI (FXI) using biolayer interferometry (Kd = 0.2 nM). In contrast, we show that prekallikrein, a FXI homolog, did not bind to SkM, reflecting the specificity of SkM for FXI binding. We also found that the anti-FXI monoclonal antibody, mAb 1A6, which recognizes the Apple (A) 3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying that SkM binds FXI A3 domain. In addition, we show that SkM enhanced FXI activation by thrombin in a concentration-dependent manner. We further used recombinant FXI chimeric proteins in which each of the four A domains of the heavy chain (designated A1 through A4) was individually replaced with the corresponding A domain from prekallikrein to investigate SkM-mediated enhancement of thrombin-induced FXI activation. These results indicated that activation of two FXI chimeras with substitutions of either the A3 domains or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain did not reduce the thrombin-induced activation compared with wildtype FXI. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains. Thus, this study is the first to reveal and support the novel intrinsic blood coagulation pathway concept that the procoagulant mechanisms of SkM include FXI binding and enhancement of FXI activation by thrombin.

Kallikrein directly interacts with and activates Factor IX, resulting in thrombin generation and fibrin formation independent of Factor XI.

Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa- or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.

Hydrogen-deuterium exchange mass spectrometry highlights conformational changes induced by factor XI activation and binding of factor IX to factor XIa.

BACKGROUND: Factor XI (FXI) is a zymogen in the coagulation pathway that, once activated, promotes haemostasis by activating factor IX (FIX). Substitution studies using apple domains of the homologous protein prekallikrein have identified that FIX binds to the apple 3 domain of FXI. However, the molecular changes upon activation of FXI or binding of FIX to FXIa have remained largely unresolved. OBJECTIVES: This study aimed to gain more insight in the FXI activation mechanism by identifying the molecular differences between FXI and FXIa, and in the conformational changes in FXIa induced by binding of FIX. METHODS: Hydrogen-deuterium exchange mass spectrometry was performed on FXI, FXIa, and FXIa in complex with FIX. RESULTS: Both activation and binding to FIX induced conformational changes at the interface between the catalytic domain and the apple domains of FXI(a)-more specifically at the loops connecting the apple domains. Moreover, introduction of FIX uniquely induced a reduction of deuterium uptake in the beginning of the apple 3 domain. CONCLUSIONS: We propose that the conformational changes of the catalytic domain upon activation increase the accessibility to the apple 3 domain to enable FIX binding. Moreover, our HDX MS results support the location of the proposed FIX binding site at the beginning of the apple 3 domain and suggest a mediating role in FIX binding for both loops adjacent to the apple 3 domain.

A non-circulating pool of factor XI associated with glycosaminoglycans in mice.

BACKGROUND: The homologous plasma proteins prekallikrein and factor XI (FXI) circulate as complexes with high molecular weight kininogen. Although evidence supports an interaction between the prekallikrein-kininogen complexes and vascular endothelium, there is conflicting information regarding FXI binding to endothelium. OBJECTIVE: To study the interaction between FXI and blood vessels in mice. METHODS: C57Bl/6 wild-type or F11-/- mice in which variants of FXI were expressed by hydrodynamic tail vein injection, received intravenous infusions of saline, heparin, polyphosphates, protamine, or enzymes that digest glycosaminoglycans (GAGs). Blood was collected after infusion and plasma was analyzed by western blot for FXI. RESULTS AND CONCLUSIONS: Plasma FXI increased 5- to 10-fold in wild-type mice after infusion of heparin, polyphosphates, protamine, or GAG-digesting enzymes, but not saline. Similar treatments resulted in a much smaller change in plasma FXI levels in rats, and infusions of large boluses of heparin did not change FXI levels appreciably in baboons or humans. The releasable FXI fraction was reconstituted in F11-/- mice by expressing murine FXI, but not human FXI. We identified a cluster of basic residues on the apple 4 domain of mouse FXI that is not present in other species. Replacing the basic residues with alanine prevented the interaction of mouse FXI with blood vessels, whereas introducing the basic residues into human FXI allowed it to bind to blood vessels. Most FXI in mice is noncovalently associated with GAGs on blood vessel endothelium and does not circulate in plasma.

Plasma kallikrein structure reveals apple domain disc rotated conformation compared to factor XI.

Essentials Zymogen PK is activated to PKa and cleaves substrates kininogen and FXII contributing to bradykinin generation. Monomeric PKa and dimeric homologue FXI utilize the N-terminal apple domains to recruit substrates. A high-resolution 1.3 Å structure of full-length PKa reveals an active conformation of the protease and apple domains. The PKa protease and four-apple domain disc organization is 180° rotated compared to FXI. SUMMARY: Background Plasma prekallikrein (PK) and factor XI (FXI) are apple domain-containing serine proteases that when activated to PKa and FXIa cleave substrates kininogen, factor XII, and factor IX, respectively, directing plasma coagulation, bradykinin release, inflammation, and thrombosis pathways. Objective To investigate the three-dimensional structure of full-length PKa and perform a comparison with FXI. Methods A series of recombinant full-length PKa and FXI constructs and variants were developed and the crystal structures determined. Results and conclusions A 1.3 Å structure of full-length PKa reveals the protease domain positioned above a disc-shaped assemblage of four apple domains in an active conformation. A comparison with the homologous FXI structure reveals the intramolecular disulfide and structural differences in the apple 4 domain that prevents dimer formation in PK as opposed to FXI. Two latchlike loops (LL1 and LL2) extend from the PKa protease domain to form interactions with the apple 1 and apple 3 domains, respectively. A major unexpected difference in the PKa structure compared to FXI is the 180° disc rotation of the apple domains relative to the protease domain. This results in a switched configuration of the latch loops such that LL2 interacts and buries portions of the apple 3 domain in the FXI zymogen whereas in PKa LL2 interacts with the apple 1 domain. Hydrogen-deuterium exchange mass spectrometry on plasma purified human PK and PKa determined that regions of the apple 3 domain have increased surface exposure in PKa compared to the zymogen PK, suggesting conformational change upon activation.

A case of a severe factor XI deficiency in a Chinese woman with heavy menorrhagia.

: The current study was to elucidate the molecular defect in a 32-year-old Chinese woman with heavy menorrhagia and delayed wound healing. The F11 gene was amplified by PCR and screened for mutations. Then identified mutations were analyzed by in-silico programs and molecular modeling analysis. This woman was found to have severely low levels of factor XI (FXI) (FXI:C: 2.0%; FXI:Ag: 5.4%) by surgical screening. Further DNA sequencing of F11 reveled a novel mutation (p.Ser295Ile) in the Ap4 domain and an already known mutation (p.Trp228stop) in the Ap3 domain. Pedigree analysis showed that the new mutation was inherited from her father (FXI:C: 41%), whereas the other was inherited from her mother (FXI:C: 62%). Modeling analysis indicated Ser295Ile mutation probably determining important structural changes in the protein folding. Both of the heterozygous mutation contribute to the severe FXI deficiency by interfering with correct assembly of the region.

Homologous Lympho-Epithelial Kazal-type Inhibitor Domains Delay Blood Coagulation by Inhibiting Factor X and XI with Differential Specificity.

Despite being initially identified in the blood filtrate, LEKTI is a 15-domain Kazal-type inhibitor mostly known in the regulation of skin desquamation. In the current study, screening of serine proteases in blood coagulation cascade showed that LEKTI domain 4 has inhibitory activity toward only FXIa, whereas LEKTI domain 6 inhibits both FXIa and FXaB (bovine FXa). Nuclear magnetic resonance structural and dynamic experiments plus molecular dynamics simulation revealed that LEKTI domain 4 has enhanced backbone flexibility at the reactive-site loop. A model of the LEKTI-protease complex revealed that FXaB has a narrower S4 pocket compared with FXIa and hence prefers only small side-chain residues at the P4 position, such as Ala in LEKTI domain 6. Mutational studies combined with a molecular complex model suggest that both a more flexible reactive-site loop and a bulky residue at the P4 position make LEKTI domain 4 a weaker but highly selective inhibitor of FXIa.

Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI.

Coagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

Is scaffold hopping a reliable indicator for the ability of computational methods to identify structurally diverse active compounds?

Computational scaffold hopping aims to identify core structure replacements in active compounds. To evaluate scaffold hopping potential from a principal point of view, regardless of the computational methods that are applied, a global analysis of conventional scaffolds in analog series from compound activity classes was carried out. The majority of analog series was found to contain multiple scaffolds, thus enabling the detection of intra-series scaffold hops among closely related compounds. More than 1000 activity classes were found to contain increasing proportions of multi-scaffold analog series. Thus, using such activity classes for scaffold hopping analysis is likely to overestimate the scaffold hopping (core structure replacement) potential of computational methods, due to an abundance of artificial scaffold hops that are possible within analog series.

Differential Roles for the Coagulation Factors XI and XII in Regulating the Physical Biology of Fibrin.

In the contact activation pathway of the coagulation, zymogen factor XII (FXII) is converted to FXIIa, which triggers activation of FXI leading to the activation of FIX and subsequent thrombin generation and fibrin formation. Feedback activation of FXI by thrombin has been shown to promote thrombin generation in a FXII-independent manner and FXIIa can bypass FXI to directly activate FX and prothrombin in the presence of highly negatively charged molecules, such as long-chain polyphosphates (LC polyP). We sought to determine whether activation of FXII or FXI differentially regulate the physical biology of fibrin formation. Fibrin formation was initiated with tissue factor, ellagic acid (EA), or LC polyP in the presence of inhibitors of FXI and FXII. Our data demonstrated that inhibition of FXI decreased the rate of fibrin formation and fiber network density, and increased the fibrin network strength and rate of fibrinolysis when gelation was initiated via the contact activation pathway with EA. FXII inhibition decreased the fibrin formation and fibrin density, and increased the fibrinolysis rate only when fibrin formation was initiated via the contact activation pathway with LC polyP. Overall, we demonstrate that inhibition of FXI and FXII distinctly alter the biophysical properties of fibrin.

An allosteric disulfide bond is involved in enhanced activation of factor XI by protein disulfide isomerase.

Essentials Reduction of three disulfide bonds in factor (F) XI enhances chromogenic substrate cleavage. We measured FXI activity upon reduction and identified a bond involved in the enhanced activity. Reduction of FXI augments FIX cleavage, probably by faster conversion of FXI to FXIa. The Cys362-Cys482 disulfide bond is responsible for FXI enhanced activation upon its reduction. SUMMARY: Background Reduction of factor (F) XI by protein disulfide isomerase (PDI) has been shown to enhance the ability of FXI to cleave its chromogenic substrate. Three disulfide bonds in FXI (Cys118-Cys147, Cys362-Cys482, and Cys321-Cys321) are involved in this augmented activation. Objectives To characterize the mechanisms by which PDI enhances FXI activity. Methods FXI activity was measured following PDI reduction. Thiols that were exposed in FXI after PDI reduction were labeled with 3-(N-maleimidopropionyl)-biocytin (MPB) and detected with avidin. The rate of conversion of FXI to activated FXI (FXIa) following thrombin activation was assessed with western blotting. FXI molecules harboring mutations that disrupt the three disulfide bonds (C147S, C321S, and C482S) were expressed in cells. The antigenicity of secreted FXI was measured with ELISA, and its activity was assessed by the use of a chromogenic substrate. The effect of disulfide bond reduction was analyzed by the use of molecular dynamics. Results Reduction of FXI by PDI enhanced cleavage of both its chromogenic substrate, S2366, and its physiologic substrate, FIX, and resulted in opening of the Cys362-Cys482 bond. The rate of conversion of FXI to FXIa was increased following its reduction by PDI. C482S-FXI showed enhanced activity as compared with both wild-type FXI and C321S-FXI. MD showed that disruption of the Cys362-Cys482 bond leads to a broader thrombin-binding site in FXI. Conclusions Reduction of FXI by PDI enhances its ability to cleave FIX, probably by causing faster conversion of FXI to FXIa. The Cys362-Cys482 disulfide bond is involved in enhancing FXI activation following its reduction, possibly by increasing thrombin accessibility to FXI.

The susceptibility of plasma coagulation factor XI to nitration and peroxynitrite action.

Coagulation factor XI is present in blood plasma as the zymogen, like other serine proteases of hemostatic system, but as the only coagulation factor forms 140-160kDa homodimers. Its activation is induced by thrombin, and a positive feedback increases the generation of the extra thrombin. Experimental and clinical observations confirm protective roles of factor XI deficiencies in certain types of thromboembolic disorders. Thromboembolism still causes serious problems for modern civilization. Diseases associated with the blood coagulation system are often associated with inflammation and oxidative stress. Peroxynitrite is produced from nitric oxide and superoxide in inflammatory diseases. The aim of the current study is to evaluate effects of nitrative stress triggered by peroxynitrite on coagulation factor XI in human plasma employing biochemical and bioinformatic methods. The amidolytic assay shows increase in factor XI activity triggered by peroxynitrite. Peroxynitrite interferes factor XI by nitration and fragmentation, which is demonstrated by immunoprecipitation followed by western blotting. Nitrated factor XI is even present in control blood plasma. The results suggest possible modifications of factor XI on the molecular level. Computer simulations show tyrosine residues as targets of peroxynitrite action. The modifications induced by peroxynitrite in factor XI might be important in thrombotic disorders.

A novel DFP tripeptide motif interacts with the coagulation factor XI apple 2 domain.

Factor XI (FXI) is the zymogen of FXIa, which cleaves FIX in the intrinsic pathway of coagulation. FXI is known to exist as a dimer and interacts with multiple proteins via its 4 apple domains in the "saucer section" of the enzyme; however, to date, no complex crystal structure has been described. To investigate protein interactions of FXI, a large random peptide library consisting of 10(6) to 10(7) peptides was screened for FXI binding, which identified a series of FXI binding motifs containing the signature Asp-Phe-Pro (DFP) tripeptide. Motifs containing this core tripeptide were found in diverse proteins, including the known ligand high-molecular-weight kininogen (HK), as well as the extracellular matrix proteins laminin and collagen V. To define the binding site on FXI, we determined the crystal structure of FXI in complex with the HK-derived peptide NPISDFPDT. This revealed the location of the DFP peptide bound to the FXI apple 2 domain, and central to the interaction, the DFP phenylalanine side-chain inserts into a major hydrophobic pocket in the apple 2 domain and the isoleucine occupies a flanking minor pocket. Two further structures of FXI in complex with the laminin-derived peptide EFPDFP and a DFP peptide from the random screen demonstrated binding in the same pocket, although in a slightly different conformation, thus revealing some flexibility in the molecular interactions of the FXI apple 2 domain.

Procoagulant microparticles promote coagulation in a factor XI-dependent manner in human endotoxemia.

UNLABELLED: Essentials The procoagulant effects of microparticles (MPs) on coagulation in endotoxemia are not known. MPs from endotoxemia volunteers were evaluated for procoagulant activity in a plasma milieu. MPs from endotoxemia volunteers shortened clotting times and enhanced thrombin generation. MP procoagulant effects were mediated in a factor XI-dependent manner. SUMMARY: Background Human endotoxemia is characterized by acute inflammation and activation of coagulation, as well as increased numbers of circulating microparticles (MPs). Whether these MPs directly promote coagulation and through which pathway their actions are mediated, however, has not been fully explored. Objectives In this study, we aimed to further characterize endotoxin-induced MPs and their procoagulant properties using several approaches. Methods Enumeration and characterization of MPs were performed using a new-generation flow cytometer. Relative contributions of the extrinsic and intrinsic pathways in MP-mediated procoagulant activity were assessed using plasmas deficient in factor (F) VII or FXI or with blocking antibodies to tissue factor (TF) or FXIa. Results Total MPs and platelet MPs were significantly elevated in plasma at 6 h after infusion of endotoxin in healthy human subjects. MPs isolated from plasma following endotoxin infusion also demonstrated increased TF activity in a reconstituted buffer system. When added to recalcified platelet-poor plasma, these MPs also promoted coagulation, as judged by a decreased clotting time with shortening of the lag time and time to peak thrombin using calibrated automated thrombography (CAT). However, the use of FVII-deficient plasma or blocking antibody to TF did not inhibit these procoagulant effects. In contrast, plasma clotting time was prolonged in FXI-deficient plasma and a blocking antibody to FXIa inhibited all MP-mediated parameters in the CAT assay. Conclusions The initiation of coagulation by cellular TF in endotoxemia is in contrast to (and presumably complemented by) the intrinsic pathway-mediated procoagulant effects of circulating MPs.