A single-domain antibody targeting factor XII inhibits both thrombosis and inflammation.

Factor XII (FXII) is the zymogen of the plasma protease FXIIa that activates the intrinsic coagulation pathway and the kallikrein kinin-system. The role of FXII in inflammation has been obscure. Here, we report a single-domain antibody (nanobody, Nb) fused to the Fc region of a human immunoglobulin (Nb-Fc) that recognizes FXII in a conformation-dependent manner and interferes with FXIIa formation. Nb-Fc treatment inhibited arterial thrombosis in male mice without affecting hemostasis. In a mouse model of extracorporeal membrane oxygenation (ECMO), FXII inhibition or knockout reduced thrombus deposition on oxygenator membranes and systemic microvascular thrombi. ECMO increased circulating levels of D-dimer, alkaline phosphatase, creatinine and TNF-α and triggered microvascular neutrophil adherence, platelet aggregation and their interaction, which were substantially attenuated by FXII blockade. Both Nb-Fc treatment and FXII knockout markedly ameliorated immune complex-induced local vasculitis and anti-neutrophil cytoplasmic antibody-induced systemic vasculitis, consistent with selectively suppressed neutrophil migration. In human blood microfluidic analysis, Nb-Fc treatment prevented collagen-induced fibrin deposition and neutrophil adhesion/activation. Thus, FXII is an important mediator of inflammatory responses in vasculitis and ECMO, and Nb-Fc provides a promising approach to alleviate thrombo-inflammatory disorders.

Targeting factor XIIa for therapeutic interference with hereditary angioedema.

Hereditary angioedema (HAE) is a rare, potentially life-threatening genetic disorder characterized by recurrent attacks of swelling. Local vasodilation and vascular leakage are stimulated by the vasoactive peptide bradykinin, which is excessively produced due to dysregulation of the activated factor XII (FXIIa)-driven kallikrein-kinin system. There is a need for novel treatments for HAE that provide greater efficacy, improved quality of life, minimal adverse effects, and reduced treatment burden over current first-line therapies. FXIIa is emerging as an attractive therapeutic target for interference with HAE attacks. In this review, we draw on preclinical, experimental animal, and in vitro studies, providing an overview on targeting FXIIa as the basis for pharmacologic interference in HAE. We highlight that there is a range of FXIIa inhibitors in development for different therapeutic areas. Of these, garadacimab, an FXIIa-targeted inhibitory monoclonal antibody, is the most advanced and has shown potential as a novel long-term prophylactic treatment for patients with HAE in clinical trials. The evidence from these trials is summarized and discussed, and we propose areas for future research where targeting FXIIa may have therapeutic potential beyond HAE.

Structural basis for the inhibition of ßFXIIa by garadacimab.

Activated FXII (FXIIa) is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of hereditary angioedema (HAE). Here, we describe a high-resolution cryoelectron microscopy (cryo-EM) structure of the beta-chain from FXIIa (ßFXIIa) complexed with the Fab fragment of garadacimab. Garadacimab binds to ßFXIIa through an unusually long CDR-H3 that inserts into the S1 pocket in a non-canonical way. This structural mechanism is likely the primary contributor to the inhibition of activated FXIIa proteolytic activity in HAE. Garadacimab Fab-ßFXIIa structure also reveals critical determinants of high-affinity binding of garadacimab to activated FXIIa. Structural analysis with other bona fide FXIIa inhibitors, such as benzamidine and C1-INH, reveals a surprisingly similar mechanism of ßFXIIa inhibition by garadacimab. In summary, the garadacimab Fab-ßFXIIa structure provides crucial insights into its mechanism of action and delineates primary and auxiliary paratopes/epitopes.

Dual Inhibition of Factor XIIa and Factor XIa Produces a Synergistic Anticoagulant Effect.

ABSTRACT: Clinical practice shows that a critical unmet need in the field of thrombosis prevention is the availability of anticoagulant therapy without bleeding risk. Inhibitors against FXIa or FXIIa have been extensively studied because of their low bleeding risk. However, whether these compounds produce synergistic effects has not yet been explored. In this study, analyses of activated partial thromboplastin time in combination with the FXIa inhibitor PN2KPI and the FXIIa inhibitor Infestin4 at different proportions were performed using the SynergyFinder tool identifying synergistic anticoagulation effects. Both an FeCl 3 -induced carotid artery thrombosis mouse model and a transient occlusion of the middle cerebral artery mouse model showed that the combination of PN2KPI and Infestin4, which are 28.57% and 6.25% of the effective dose, respectively, significantly prevents coagulation, and furthermore, dual inhibition does not cause bleeding risk.

Discovery of Highly Selective, Potent, Covalent, and Orally Bioavailable Factor XIIa Inhibitors for the Treatment of Thrombo-Inflammation.

Thrombo-inflammation is closely associated with a few severe cardiovascular and infectious diseases. Factor XIIa (FXIIa) in the intrinsic coagulation pathway plays a pivotal role in the development of thrombo-inflammation and its inhibition has emerged as a potential therapeutic approach for thrombo-inflammatory disorders. Nonetheless, as of now, few small-molecule FXIIa inhibitors have demonstrated notable effectiveness against thrombo-inflammation, with none progressing into clinical stages. Herein, we present potent, covalent, reversible, and selective small-molecule FXIIa inhibitors such as 4a and 4j obtained through structure-based drug design. Compounds 4a and 4j showed significant anticoagulation and substantial anti-inflammatory effects in vitro, coupled with exceptional plasma stability. Furthermore, in carrageenan-induced thrombosis models, 4a and 4j demonstrated remarkable dual antithrombotic and anti-inflammatory activity when administered orally. Compound 4j exhibited a favorable safety profile without obvious tissue toxicity in mice, suggesting its potential as an oral therapeutic option for thrombo-inflammation.

Tackling the Root Cause of Surface-Induced Coagulation: Inhibition of FXII Activation to Mitigate Coagulation Propagation and Prevent Clotting.

Factor XII (FXII) is a zymogen present in blood that tends to adsorb onto the surfaces of blood-contacting medical devices. Once adsorbed, it becomes activated, initiating a cascade of enzymatic reactions that lead to surface-induced coagulation. This process is characterized by multiple redundancies, making it extremely challenging to prevent clot formation and preserve the properties of the surface. In this study, a novel modulatory coating system based on C1-esterase inhibitor (C1INH) functionalized polymer brushes, which effectively regulates the activation of FXII is proposed. Using surface plasmon resonance it is demonstrated that this coating system effectively repels blood plasma proteins, including FXII, while exhibiting high activity against activated FXII and plasma kallikrein under physiological conditions. This unique property enables the modulation of FXII activation without interfering with the overall hemostasis process. Furthermore, through dynamic Chandler loop studies, it is shown that this coating significantly improves the hemocompatibility of polymeric surfaces commonly used in medical devices. By addressing the root cause of contact activation, the synergistic interplay between the antifouling polymer brushes and the modulatory C1INH is expected to lay the foundation to enhance the hemocompatibility of medical device surfaces.

Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis.

Background: Dysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. Methods: Citrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. Results: C1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. Conclusion: Citrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.

Activated Coagulation FXII: A Unique Target for In Vivo Molecular Imaging.

BACKGROUND: Current clinical imaging of thromboembolic diseases often relies on indirect detection of thrombi, which may delay diagnosis and ultimately the institution of beneficial, potentially lifesaving treatment. Therefore, the development of targeting tools that facilitate the rapid, specific, and direct imaging of thrombi using molecular imaging is highly sought after. One potential molecular target is FXIIa (factor XIIa), which initiates the intrinsic coagulation pathway but also activates the kallikrein-kinin system, thereby initiating coagulation and inflammatory/immune responses. As FXII (factor XII) is dispensable for normal hemostasis, its activated form (FXIIa) represents an ideal molecular target for diagnostic and therapeutic approaches, the latter combining diagnosis/identification of thrombi and effective antithrombotic therapy. METHODS: We conjugated an FXIIa-specific antibody, 3F7, to a near-infrared (NIR) fluorophore and demonstrated binding to FeCl3-induced carotid thrombosis with 3-dimensional fluorescence emission computed tomography/computed tomography and 2-dimensional fluorescence imaging. We further demonstrated ex vivo imaging of thromboplastin-induced pulmonary embolism and detection of FXIIa in human thrombi produced in vitro. RESULTS: We demonstrated imaging of carotid thrombosis by fluorescence emission computed tomography/computed tomography and measured a significant fold increase in signal between healthy and control vessels from mice injected with 3F7-NIR compared with mice injected with nontargeted probe (P=0.002) ex vivo. In a model of pulmonary embolism, we measured increased NIR signal in lungs from mice injected with 3F7-NIR compared with mice injected with nontargeted probe (P=0.0008) and healthy lungs from mice injected with 3F7-NIR (P=0.021). CONCLUSIONS: Overall, we demonstrate that FXIIa targeting is highly suitable for the specific detection of venous and arterial thrombi. This approach will allow direct, specific, and early imaging of thrombosis in preclinical imaging modalities and may facilitate monitoring of antithrombotic treatment in vivo.

Glycated albumin modulates the contact system with implications for the kallikrein-kinin and intrinsic coagulation systems.

BACKGROUND: Human serum albumin (HSA) is the most abundant plasma protein and is sensitive to glycation in vivo. The chronic hyperglycemic conditions in patients with diabetes mellitus (DM) induce a nonenzymatic Maillard reaction that denatures plasma proteins and forms advanced glycation end products (AGEs). HSA-AGE is a prevalent misfolded protein in patients with DM and is associated with factor XII activation and downstream proinflammatory kallikrein-kinin system activity without any associated procoagulant activity of the intrinsic pathway. OBJECTIVES: This study aimed to determine the relevance of HSA-AGE toward diabetic pathophysiology. METHODS: The plasma obtained from patients with DM and euglycemic volunteers was probed for activation of FXII, prekallikrein (PK), and cleaved high-molecular-weight kininogen by immunoblotting. Constitutive plasma kallikrein activity was determined via chromogenic assay. Activation and kinetic modulation of FXII, PK, FXI, FIX, and FX via in vitro-generated HSA-AGE were explored using chromogenic assays, plasma-clotting assays, and an in vitro flow model using whole blood. RESULTS: Plasma obtained from patients with DM contained increased plasma AGEs, activated FXIIa, and resultant cleaved cleaved high-molecular-weight kininogen. Elevated constitutive plasma kallikrein enzymatic activity was identified, which positively correlated with glycated hemoglobin levels, representing the first evidence of this phenomenon. HSA-AGE, generated in vitro, triggered FXIIa-dependent PK activation but limited the intrinsic coagulation pathway activation by inhibiting FXIa and FIXa-dependent FX activation in plasma. CONCLUSION: These data indicate a proinflammatory role of HSA-AGEs in the pathophysiology of DM via FXII and kallikrein-kinin system activation. A procoagulant effect of FXII activation was lost through the inhibition of FXIa and FIXa-dependent FX activation by HSA-AGEs.

A site on factor XII required for productive interactions with polyphosphate.

BACKGROUND: During plasma contact activation, factor XII (FXII) binds to surfaces through its heavy chain and undergoes conversion to the protease FXIIa. FXIIa activates prekallikrein and factor XI (FXI). Recently, we showed that the FXII first epidermal growth factor-1 (EGF1) domain is required for normal activity when polyphosphate is used as a surface. OBJECTIVES: The aim of this study was to identify amino acids in the FXII EGF1 domain required for polyphosphate-dependent FXII functions. METHODS: FXII with alanine substitutions for basic residues in the EGF1 domain were expressed in HEK293 fibroblasts. Wild-type FXII (FXII-WT) and FXII containing the EGF1 domain from the related protein Pro-HGFA (FXII-EGF1) were positive and negative controls. Proteins were tested for their capacity to be activated, and to activate prekallikrein and FXI, with or without polyphosphate, and to replace FXII-WT in plasma clotting assays and a mouse thrombosis model. RESULTS: FXII and all FXII variants were activated similarly by kallikrein in the absence of polyphosphate. However, FXII with alanine replacing Lys73, Lys74, and Lys76 (FXII-Ala73,74,76) or Lys76, His78, and Lys81 (FXII-Ala76,78,81) were activated poorly in the presence of polyphosphate. Both have <5% of normal FXII activity in silica-triggered plasma clotting assays and have reduced binding affinity for polyphosphate. Activated FXIIa-Ala73,74,76 displayed profound defects in surface-dependent FXI activation in purified and plasma systems. FXIIa-Ala73,74,76 reconstituted FXII-deficient mice poorly in an arterial thrombosis model. CONCLUSION: FXII Lys73, Lys74, Lys76, and Lys81 form a binding site for polyanionic substances such as polyphosphate that is required for surface-dependent FXII function.

Triazol-1-yl Benzamides Promote Anticoagulant Activity via Inhibition of Factor XIIa.

BACKGROUND: Human factor XIIa (FXIIa) is a plasma serine protease that plays a significant role in several physiological and pathological processes. Animal models have revealed an important contribution of FXIIa to thromboembolic diseases. Remarkably, animals and patients with FXII deficiency appear to have normal hemostasis. Thus, FXIIa inhibition may serve as a promising therapeutic strategy to attain safer and more effective anticoagulation. Very few small molecule inhibitors of FXIIa have been reported. We synthesized and investigated a focused library of triazol-1-yl benzamide derivatives for FXIIa inhibition. METHODS: We chemically synthesized, characterized, and investigated a focused library of triazol- 1-yl benzamide derivatives for FXIIa inhibition. Using a standardized chromogenic substrate hydrolysis assay, the derivatives were evaluated for inhibiting human FXIIa. Their selectivity over other clotting factors was also evaluated using the corresponding substrate hydrolysis assays. The best inhibitor affinity to FXIIa was also determined using fluorescence spectroscopy. Effects on the clotting times (prothrombin time (PT) and activated partial thromboplastin time (APTT)) of human plasma were also studied. RESULTS: We identified a specific derivative (1) as the most potent inhibitor in this series. The inhibitor exhibited nanomolar binding affinity to FXIIa. It also exhibited significant selectivity against several serine proteases. It also selectively doubled the activated partial thromboplastin time of human plasma. CONCLUSION: Overall, this work puts forward inhibitor 1 as a potent and selective inhibitor of FXIIa for further development as an anticoagulant.

Factor XII Explored with AlphaFold - Opportunities for Selective Drug Development.

Medical device associated thrombosis is an important clinical problem. This type of thrombosis can result from Factor XII (FXII) binding to non-natural surface materials and subsequent activation of the contact pathway. This drives the development of new therapeutic strategies to block this pathway and information on the structural properties of FXII should catalyse this quest. Presently, there is no publicly available crystal structure of full-length FXII. However, the AlphaFold Protein Structure Database provides a model structure. We here explore this model in combination with previous structure-function studies to identify opportunities for selective pharmacological blockade of the contribution of FXII in medical device associated thrombosis. Previous studies demonstrated that FXII activation is dependent on molecular cleavage after R353. We subsequently proposed that protein conformation protects this cleavage site to ensure zymogen quiescence and prevent inappropriate FXII activation. The AlphaFold model shows that a small loop containing R353 indeed is buried in the globular molecule. This is the result of intra-molecular interactions between the (N-terminal) Fibronectin type II domain, (central) kringle and (C-terminal) protease domain, in a structure that resembles a three-point harness. Furthermore, this interaction pushes the intermediate domains, as well as the flexible proline-rich region (PRR), outward while encapsulating R353 in the molecule. The outward directed positively charged patches are likely to be involved in binding to anionic surfaces. The binding of FXII to surfaces (and several monoclonal antibodies) acccelerates its activation by inducing conformational changes. For prevention of medical device associated thrombosis, it is therefore important to target the surface binding sites of FXII without causing structural changes.

Ayaconin, a novel inhibitor of the plasma contact system from the sand fly Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis.

Transcriptome analysis of the salivary gland cDNA library from a phlebotomine sand fly, Lutzomyia ayacuchensis, identified a transcript coding for the PpSP15/SL1 family protein as the second most abundant salivary component. In the present study, a recombinant protein of the PpSP15/SL1 family protein, designated ayaconin, was expressed in Escherichia coli, and its biological activity was characterized. The recombinant ayaconin purified from the soluble fraction of E. coli lysate efficiently inhibited the intrinsic but not extrinsic blood coagulation pathway. When the target of ayaconin was evaluated using fluorescent substrates of coagulation factors, ayaconin inhibited factor XIIa (FXIIa) activity more efficiently in a dose-dependent manner, suggesting that FXII is the primary target of ayaconin. In addition, incubation of ayaconin with FXII prior to activation effectively inhibited FXIIa activity, whereas such inhibition was not observed when ayaconin was mixed after the production of FXIIa, indicating that ayaconin inhibits the activation process of FXII to produce FXIIa, but not the enzymatic activity of FXIIa. Moreover, ayaconin was shown to bind to FXII, suggesting that the binding of ayaconin to FXII is involved in the inhibitory mechanism against FXII activation. These results suggest that ayaconin plays an important role in the blood-sucking of Lu. ayacuchensis.

Scaffold stability and P14' residue steric hindrance in the differential inhibition of FXIIa by Aedes aegypti trypsin inhibitor versus Infestin-4.

Kazal-type protease inhibitors strictly regulate Factor XIIa (FXIIa), a blood-clotting serine protease. However, when negatively charged surface of prosthetic device come into contact with FXII, it undergoes conformational change and auto-activation, leading to thrombus formation. Some research suggests that Kazal-type protease inhibitor specificity against FXIIa is governed solely by the reactive-site loop sequence, as this sequence makes most-if not all-of the direct contacts with FXIIa. Here, we sought to compare the inhibitory properties of two Kazal-type inhibitors, Infestin-4 (Inf4), a potent inhibitor of FXIIa, and Aedes aegypti trypsin inhibitor (AaTI), which does not inhibit FXIIa, to better understand Kazal-type protease specificity and determine the structural components responsible for inhibition. There are only three residue differences in the reactive-site loop between AaTI and Inf4. Through site-directed mutagenesis, we show that the reactive-site loop is only partially responsible for the inhibitory specificity of these proteases. The protein scaffold of AaTI is unstable due to an elongated C5C6 region. Through chimeric study, we show that swapping the protease-binding loop and the C5C6 region from Inf4 with that of AaTI can partially enhance the inhibitory activity of the AaTI_Inf4 chimera. Furthermore, the additional substitution of Asn at the P14' position of AaTI with Gly (Gly27 in Inf4) absolves the steric clashing between AaTI and the surface 140-loop of FXIIa, and increases the inhibition of the chimeric AaTI to match that of wild-type Inf4. Our findings suggest that ancillary regions in addition to the reactive-site loop sequence are important factors driving Kazal-type inhibitor specificity.

A mechanism for hereditary angioedema caused by a lysine 311-to-glutamic acid substitution in plasminogen.

Patients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.

Identification of the histidine-rich glycoprotein domains responsible for contact pathway inhibition.

BACKGROUND: Previously, we showed that histidine-rich glycoprotein (HRG) binds factor (F) XIIa with high affinity, inhibits FXII autoactivation and FXIIa-mediated activation of FXI, and attenuates ferric chloride-induced arterial thrombosis in mice. Therefore, HRG downregulates the contact pathway in vitro and in vivo. OBJECTIVE: To identify the domains on HRG responsible for contact pathway inhibition. METHODS: Recombinant HRG domain constructs (N-terminal [N1, N2, and N1N2], proline-rich regions, histidine-rich region [HRR], and C-terminal) were expressed and purified. The affinities of plasma-derived HRG, HRG domain constructs, and synthetic HRR peptides for FXII, FXIIa, ß-FXIIa, and polyphosphate (polyP) were determined using surface plasmon resonance, and their effects on polyP-induced FXII autoactivation, FXIIa-mediated activation of FXI and prekallikrein, the activated partial thromboplastin time (APTT), and thrombin generation were examined. RESULTS: HRG and HRG domain constructs bind FXIIa, but not FXII or ß-FXII. HRR, N1, and N1N2 bind FXIIa with affinities comparable with that of HRG, whereas the remaining domains bind with lower affinity. Synthetic HRR peptides bind FXIIa and polyP with high affinity. HRG and HRR significantly inhibit FXII autoactivation and prolong the APTT. Like HRG, synthetic HRR peptides inhibit FXII autoactivation, attenuate FXIIa-mediated activation of prekallikrein and FXI, prolong the APTT, and attenuate thrombin generation. CONCLUSION: The interaction of HRG with FXIIa and polyP is predominantly mediated by the HRR domain. Like intact HRG, HRR downregulates the contact pathway and contributes to HRG-mediated down regulation of coagulation.

An Ultrapotent and Selective Cyclic Peptide Inhibitor of Human ß-Factor XIIa in a Cyclotide Scaffold.

Cyclotides are plant-derived peptides with complex structures shaped by their head-to-tail cyclic backbone and cystine knot core. These structural features underpin the native bioactivities of cyclotides, as well as their beneficial properties as pharmaceutical leads, including high proteolytic stability and cell permeability. However, their inherent structural complexity presents a challenge for cyclotide engineering, particularly for accessing libraries of sufficient chemical diversity to design potent and selective cyclotide variants. Here, we report a strategy using mRNA display enabling us to select potent cyclotide-based FXIIa inhibitors from a library comprising more than 1012 members based on the cyclotide scaffold of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The most potent and selective inhibitor, cMCoFx1, has a pM inhibitory constant toward FXIIa with greater than three orders of magnitude selectivity over related serine proteases, realizing specific inhibition of the intrinsic coagulation pathway. The cocrystal structure of cMCoFx1 and FXIIa revealed interactions at several positions across the contact interface that conveyed high affinity binding, highlighting that such cyclotides are attractive cystine knot scaffolds for therapeutic development.

A novel anticoagulant peptide discovered from Crassostrea gigas by combining bioinformatics with the enzymolysis strategy: inhibitory kinetics and mechanisms.

A novel anticoagulant peptide (IEELEEELEAER) derived from oyster (Crassostrea gigas) was discovered by combining the emerging bioinformatics with the classical enzymolysis approach. The anticoagulant peptide drastically reduced the extrinsic clotting activity (49% residual PT activity) and impaired the intrinsic clotting activity (77% residual PT activity). Consistent with the clotting data, the thrombin peak height reduced to 88.7 from 123.4 nM, and the thrombin generation time delayed to 5.32 from 4.42 min when an extrinsic trigger was applied. The inhibitory kinetics of FXIa, FIXa, FXa, FIIa, and APC in a purified component system rationally explained the reduction of the extrinsic clotting activity and impairment of thrombin generation. Besides the inhibition of FXa and FIIa activity, the activation processes of FX and FII by an intrinsic/extrinsic tenase complex and prothrombinase were also damaged. The anticoagulant activity in the plasma system was the result of comprehensive inhibition of various factors. The research provided a frame for anticoagulant evaluation and inhibitory mechanism of bioactive peptides from food products.

Identification of the factor XII contact activation site enables sensitive coagulation diagnostics.

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317-Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317-Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317-Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.

Microscale Parallel Synthesis of Acylated Aminotriazoles Enabling the Development of Factor XIIa and Thrombin Inhibitors.

Herein we report a microscale parallel synthetic approach allowing for rapid access to libraries of N-acylated aminotriazoles and screening of their inhibitory activity against factor XIIa (FXIIa) and thrombin, which are targets for antithrombotic drugs. This approach, in combination with post-screening structure optimization, yielded a potent 7 nM inhibitor of FXIIa and a 25 nM thrombin inhibitor; both compounds showed no inhibition of the other tested serine proteases. Selected N-acylated aminotriazoles exhibited anticoagulant properties in vitro influencing the intrinsic blood coagulation pathway, but not extrinsic coagulation. Mechanistic studies of FXIIa inhibition suggested that synthesized N-acylated aminotriazoles are covalent inhibitors of FXIIa. These synthesized compounds may serve as a promising starting point for the development of novel antithrombotic drugs.