FXII Activation Research Articles

Clinical and genetic spectrum of factor XII deficiency in the Han population of East China

BackgroundFactor XII (FXII or F12) deficiency is a rare inherited disorder, typically lacking haemorrhagic symptoms. There is limited literature exists on FXII deficiency and mutations within the Chinese population. This study aimed to characterize the spectrum of F12 gene mutations in a Chinese cohort and to investigate the relationship between FXII mutations and clinical phenotypes.MethodsGenetic and clinical data from 51 unrelated probands with FXII deficiency, along with their families, were meticulously collected and analysed.ResultsGenetic analysis revealed that 94.1% of probands carried genetic defects, with 29 mutations pinpointed in the F12 gene. Of these, 18 mutations were previously reported for the first time by our research group, including c.303_304delCA, c.1078G > A, c.1285 C > T, among others. Of the mutations, 17 are missense, constituting 58.6% of the total. Additionally, 11 are deletions or insertions, of which 8 result in frameshifts, while the remaining one is a nonsense mutation. These mutations were predominantly concentrated in two crucial regions: the catalytic domain and the kringle domain. The most frequently observed mutations were c.1681G > A, closely followed by c.1561G > A and c.1078G > A, indicating a dominance among these mutations. Additionally, a prevalent polymorphism at position 46 was observed in the majority of probands, with 47.1% having the 46T/T genotype and 13.7% having the 46 C/T genotype, which may potentially impact FXII activity. The broad spectrum of asymptomatic FXII deficiency observed within the Han population of East China.ConclusionsWe speculate on the potential impact of recurrent mutations on the efficacy of new drugs being developed to target FXII for thrombosis prevention and treatment. Furthermore, it is important to explore their influence on FXII-related pathways beyond the activation of the contact pathway in the coagulation cascade.

Pharmacokinetics, pharmacodynamics, and safety of asundexian in healthy Chinese and Japanese volunteers, and comparison with Caucasian data.

There is an unmet clinical need for effective anticoagulant therapies for the management of thromboembolic diseases that are not associated with a relevant risk of bleeding. Asundexian (BAY 2433334) is an oral, direct, small-molecule inhibitor of activated factor XI (FXIa). Phase I data from healthy Caucasian male participants indicated predictable pharmacokinetic (PK) and pharmacodynamic (PD) profiles and no clinically relevant bleeding-related adverse events (AEs). Reported here are data from two phase I, randomized, placebo-controlled, single- and multiple-dose escalation studies of asundexian conducted in 60 healthy men: 24 Japanese and 36 Chinese. Baseline characteristics were comparable between the treatment groups. All treatment-emergent AEs were mild, with no serious AEs or AEs of special interest reported. Systemic exposure to asundexian increased dose proportionally after single or multiple dosing, with relatively low accumulation following multiple once-daily dosing in both Chinese and Japanese volunteers. Asundexian induced dose-dependent prolongation of activated partial thromboplastin time and inhibition of FXIa activity, with no effects on prothrombin time or FXI concentration in Japanese participants. There were no clinically relevant interethnic differences in PK profile across the Japanese, Chinese, and Caucasian (data from the previous phase I study) participants and no clinically relevant difference in PD response between Japanese and Caucasian participants.

Real world management of individuals with severe FXI deficiency and its impact on clinical outcomes: Experience from a haemophilia treatment centre.

The management of Factor XI deficiency is challenged by a variable association between FXI level and bleeding phenotype. Additionally, there is scarce data describing management strategies and their outcomes, specifically bleeding, thrombosis, and other complications. To evaluate bleeding, thrombosis, and other complications in individuals with severe FXI deficiency seen in our comprehensive haemophilia treatment centre (HTC). Peri-procedural management strategies and the resulting impact on bleeding and other clinically relevant outcomes were reported. Retrospective review of the electronic medical record of adult patients with severe FXI deficiency (<20% activity) seen at a New York City comprehensive HTC between 2017 and 2022. Procedures, haemostatic management, and outcomes were collected and analysed. We identified 38 individuals (64%) females with severe FXI deficiency. The mean age was 56±21 years (SD). The median FXI activity level was 3% (IQR: 1-8%). The mean BAT score was 3.1±2.4; (52%) individuals did not have a history of bleeding. A total of 256 surgeries and procedures were performed. There was reduced bleeding with preventative or reactive treatment during procedures. Arterial but not venous thrombotic complications were observed. Plasma was mostly used for procedures associated with higher risk of bleeding and antifibrinolytics for procedures at sites of high fibrinolysis. Current management strategies pose a burden of care for these patients and manifested as nonbleeding adverse events and changes in clinical management. These findings highlight the need for novel investigation in predicting and managing bleeding for individuals with severe FXI deficiency.

Minimal interference of concizumab with standard clinical coagulation laboratory assays - An in vitro study.

Non-factor replacement therapies are emerging as prophylactic treatment options in haemophilia A or B (HA/HB) with and without inhibitors. Concizumab is an anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody preventing factor (F)Xa inhibition and enhancing thrombin generation. Based on experience with other non-factor therapies and extended half-life products, there is a focus on potential interference with common clinical coagulation assays used to monitor patients treated with concizumab. To evaluate the impact of concizumab on standard clinical coagulation assays. Plasma samples (normal, HA/HB with/without inhibitors) in the presence/absence of added concizumab (250-16,000ng/mL) were analysed in clinical assays including activated partial thromboplastin time (aPTT), prothrombin time (PT), FVIII and FIX one-stage clot and chromogenic substrate assay, assays for detecting FVIII or FIX inhibitors and other assays for coagulation factors. Concizumab did not impact PT assays, but resulted in a small shortening of aPTT (up to 5 s in haemophilia plasma and 0.4 s in normal plasma). Concizumab had no, or only a minor impact on FVIII and FIX activity assays or Bethesda inhibitor assays. FXI and FXII activity in normal plasma, as measured by single factor aPTT-based assay, was significantly increased in the presence of concizumab (+11% each). This was also the case for FVII and FX measured by PT-based assays using plasma with 25% of FVII or FX (+64% and +22%, respectively). The presence of concizumab did not, or only slightly, influence the outcome of standard clinical coagulation assays relevant for HA and HB.

The Clot Thickens: Differential Coagulotoxic and Cardiotoxic Activities of Anguimorpha Lizard Venoms.

Despite their evolutionary novelty, lizard venoms are much less studied in comparison to the intense research on snake venoms. While the venoms of helodermatid lizards have long been assumed to be for defensive purposes, there is increasing evidence of toxic activities more useful for predation than defence (such as paralytic neurotoxicity). This study aimed to ascertain the effects of Heloderma, Lanthanotus, and Varanus lizard venoms on the coagulation and cardiovascular systems. Anticoagulant toxicity was demonstrated for the Varanus species studied, with the venoms prolonging clotting times in human and bird plasma due to the destructive cleavage of fibrinogen. In contrast, thromboelastographic analyses on human and bird plasmas in this study demonstrated a procoagulant bioactivity for Heloderma venoms. A previous study on Heloderma venom using factor-depleted plasmas as a proxy model suggested a procoagulant factor was present that activated either Factor XI or Factor XII, but could not ascertain the precise target. Our activation studies using purified zymogens confirmed FXII activation. Comparisons of neonate and adult H. exasperatum, revealed the neonates to be more potent in the ability to activate FXII, being more similar to the venom of the smaller species H. suspectum than the adult H. exasperatum. This suggests potent FXII activation a basal trait in the genus, present in the small bodied last common ancestor. This also indicates an ontogenetic difference in prey preferences in the larger Heloderma species paralleing the change in venom biochemistry. In addition, as birds lack Factor XII, the ability to clot avian plasma suggested an additional procoagulant site of action, which was revealed to be the activation of Factor VII, with H. horridum being the most potent. This study also examined the effects upon the cardiovascular system, including the liberation of kinins from kininogen, which contributes to hypotension induction. This form of toxicity was previously described for Heloderma venoms, and was revealed in this study was to also be a pathophysiological effect of Lanthanotus and Varanus venoms. This suggests that this toxic activity was present in the venom of the last common ancestor of the anguimorph lizards, which is consistent with kallikrein enzymes being a shared toxin trait. This study therefore uncovered novel actions of anguimorph lizard venoms, not only contributing to the evolutionary biology body of knowledge but also revealing novel activities to mine for drug design lead compounds.

The spectrum of factor XI deficiency in Southeast China: four recurrent variants can explain most of the deficiencies

BackgroundFactor XI (FXI) deficiency is an autosomal hemorrhagic disorder characterized by reduced plasma FXI levels. Multiple ancestral variants in the F11 gene have been identified in Ashkenazi Jews and other selected European populations. However, there are few reports of predominant variants in Chinese and/or East Asian populations. The aim of this study is to characterize the genotypes and phenotypes of FXI deficiency and identify the predominant variants.ResultsOf the 41 FXI-deficient patients, 39 exhibited severe FXI defects, considerably more than those with partial defects. The APTT levels showed a negative correlation with FXI activity levels (coefficient=-0.584, P < .001). Only nine patients experienced mild bleeding, including one partially defective patient and eight severely defective patients. The majority of patients were referred for preoperative screenings (n = 22) and checkups (n = 14). Genetic analysis revealed that 90% of the patients had genetic defects, with 2, 16, and 19 cases of heterozygous, homozygous, and compound heterozygous patients, respectively. Seventeen variants were detected in the F11 gene (6 novel), including eleven missense variants, four nonsense variants, and two small deletions scattered throughout the F11. Of the 11 missense variants, six have not yet been studied for in vitro expression. Protein modeling analyses indicated that all of these variants disrupted local structural stability by altering side-chain orientation and hydrogen bonds. Nine variants, consisting of three missense and six null variants, were detected with a frequency of two or more. The highest allele frequency was observed in p.Q281* (21.25%), p.W246* (17.50%), p.Y369* (12.50%), and p.L442Cfs*8 (12.50%). The former two were variants specific to East Asia, while the remaining two were southeast China-specific variants.ConclusionOur population-based cohort demonstrated that no correlation between the level of FXI activity and the bleeding severity in FXI deficiency. Additionally, the prevalence of FXI deficiency may have been underestimated. The nonsense p.Q281* was the most common variant in southeast China, suggesting a possible founder effect.

Abstract 2096: Role Of Divalent Cations In The Enzymatic Activation Of Coagulation Factor XII By Lipopolysaccharides

Background: Lipopolysaccharide (LPS) is the primary pathogenic factor in Gram-negative sepsis. LPS is a polyanionic molecule that may act as a site of initiation of coagulation by activating factor (F)XII. While the physicochemical properties of LPS are known to be sensitive to interactions with divalent cations, it is unknown whether divalent cations modulate the activation of FXII by LPS. Aims: To determine whether divalent cations regulate the kinetics of FXII activation by select LPS chemotypes from E. coli . Methods: Aggregates of E. Coli LPS chemotypes O26:B6 and Rd2 were prepared in ultrapure water in the presence or absence of calcium (Ca 2+ ). The biophysical characterization of LPS chemotypes were determined by dynamic light scattering (DLS) and measurement of zeta potential (ZP). Chromogenic substrate assays were used to study the effects of LPS on FXII activation. Results: The LPS O26:B6 and Rd2 in the absence of Ca 2+ produced aggregates in form of micelles with hydrodynamic diameters of 190±10 and 200±7 nm and ZP of -25±0.8 and -60±2 mV, respectively. The presence of Ca 2+ produced aggregates in the form of vesicles with hydrodynamic diameters of 400±15 and 1300±60 nm and ZP of –11±2 and –3±0.5 mV, respectively. FXII was incubated in HEPES buffer with the distinct LPS morphologies in varying salt concentrations. We found maximal autoactivation of FXII by O26:B6 and Rd2 chemotypes at lower NaCl concentrations. The presence of Ca 2+ , however, selectively reduced the ability of O26:B6 to activate FXII. Strikingly, the neutralization of the surface net charge of the LPS chemotype Rd2 by Ca 2+ ions abrogated the amidolytic activity of Rd2. Conclusions: The presence of calcium ions induces changes in the physicochemical properties of LPS that result in distinct effects on FXII activation in vitro . We are currently studying whether this phenomena is conserved for other divalent cations including Zn, Mg, Cu, and Mn.

Abstract 3095: Characterization Of Interactions Between Skeletal Muscle Myosin And Activated Factor XI

Background: We recently demonstrated that the procoagulant activity of skeletal muscle myosin (SkM) involved the binding of coagulation factor (F) XI to enhance FXI activation by thrombin. However, the impact of SkM on the enzymatic activities of activated FXI (FXIa) remains unclear. Aims: Determine the influence of SkM interactions on the expression of FXIa activities. Methods: Binding studies between FXIa and SkM were conducted using biolayer interferometry (BLI). Kallikrein, a FXIa homolog, served as a negative control. The interactions between SkM and FXIa were investigated through fluorescence spectroscopy using dansyl-labeled FXIa. Chromogenic assays for FXIIa were employed to measure FXII activation by FXIa. Tissue factor pathway inhibitor (TFPI) inactivation by FXIa was assessed by determining FXa inhibition by TFPI using FX chromogenic assays. Quantification of complement factor H (CFH) cleavage by FXIa as well as FV activation by FXIa were carried out using SDS PAGE and Coomassie blue staining. Results: BLI data showed that SkM exhibited a high binding affinity for FXIa with a dissociation constant of 1.6 nM, whereas SkM did not bind kallikrein. The addition of SkM induced a 9 nm shift in the emission spectrum of dansyl-labeled FXIa. The presence of SkM increased the rate of feedback FXII activation by FXIa but not by kallikrein. The addition of SkM inhibitors, trifluoperazine and blebbistatin, reduced the enhancement effect of SkM on the ability of FXIa to activate FXII. Furthermore, SkM increased the rate of enzymatic inactivation of TFPI by FXIa, the rate of cleavage of CFH by FXIa, and the rate of activation of FV by FXIa. Conclusions: SkM binds with high affinity to FXIa and serves as a co-factor to enhance multiple different enzymatic activities of FXIa. Future work is needed to examine the multiple procoagulant activities of SkM in the setting of hemostasis and acute trauma.

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

BackgroundFactor (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. ObjectivesTo identify the binding interface between thrombin and FXI and understand the dynamics underlying FXI activation. MethodsCrosslinking 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. ResultsWe 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. ConclusionOur 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.

Forms of Hereditary Angioedema That Are Independent of Kallikrein and Factor XIIa System

Hereditary angioedema (HAE) is an inherited disorder characterized by recurrent episodes of severe soft tissue swelling involving skin and mucous membranes. In most cases, edema is caused by excessive formation of the vasoactive nanopeptide bradykinin (BK) due to dysregulation of the plasma kallikrein-kinin system (KKS). The KKS consists of the zymogens prekallikrein (PK) and factor XII (FXII) and the cofactor/substrate high-molecular-weight kininogen (HK). PK and FXII reciprocally convert each other to the proteases plasma kallikrein (PKa) and FXIIa. PKa cleaves HK after Lys362 and Arg371 to release bradykinin. In most cases of HAE, increased KKS activation is due to low plasma activity of C1-Inhibitor (C1-INH), the main regulator of PKa and FXIIa. However, ~10% of HAE patients have normal C1-INH activity (HAEnC1). A relationship between fibrinolysis and kinin formation was first described in 1971. The fibrinolytic protease plasmin activates FXII and cleaves HK, although it is unclear that this results in significant bradykinin generation. Recently mutations in the PLG gene encoding plasminogen (Lys311Glu) and the KNG1 gene encoding HK and the related protein low-molecular-weight kininogen (LK, Met361Lys) were identified in patients with HAEnC1. Here we describe how the fibrinolytic system contributes to angioedema in patients with these mutations. Human Glu-plasminogen (Plg), HK and LK were expressed in HEK293 cells. Lys311 in plasminogen was changed to glutamic acid, and Met361 in HK and LK were changed to lysine, by site directed mutagenesis. Wild type (Plg-Lys311) and variant (Plg-Glu311) plasminogen were converted to plasmin (Plm-Lys311 and Plm-Glu311) with urokinase. The capacity of plasmin to cleave HK and LK were studied with SDS-PAGE, western blots and kinin ELISA. Cleavage of wild type HK-Met361 and LK-Met361 and variant HK-Lys361 and LK-Lys361 by PKa and plasmin were assessed in a similar manner. The types of kinin released during reactions were determined by mass spectroscopy. Normal plasma was supplemented with Plg-Lys311 or Plg-Glu311, and tPA was added to generate plasmin. Bradykinin release was substantially greater with Plm-Glu311 than Plm-Lys311. This held when reactions were run in plasma lacking FXII or PK, indicating activation of the KKS was not responsible for kinin production. Using plasma-derived proteins, PKa released bradykinin 100-fold faster from HK than LK, and released bradykinin from HK ~50-fold faster than did plasmin. PKa and plasmin released bradykinin at comparable relatively slow rates from LK. Plm-Glu311 released bradykinin from HK 10-fold faster, and from LK 3-fold faster, than in reactions with Plm-Lys311. In experiments in which kininogen deficient plasma was supplemented with either HK or LK to a physiologic concentration, the majority of bradykinin released by Plm-Glu311 was from LK. PKa-catalyzed release of bradykinin from wild type HK-Met361 and variant HK-Lys361 comparably. The same held for LK-Met361 and LK-Lys361. However, plasmin released kinin from HK-Lys361 and LK-Lys361 3.3-fold and 13-fold faster than from HK-Met361 and LK-Met361. While the kinin released from HK-Met361 and LK-Met361 was bradykinin, plasmin released the decapeptide Lys-bradykinin (kallidin) from HK-Lys361 and LK-Lys36. Our studies indicate that angioedema in some patients with HAEnC1 may be due to fibrinolytic activity, and not to KKS activity. Both HK and LK may be substrates in such patients. As the plasma LK concentration is ~4-fold higher than HK, it may be the main pathologic kinin source. The plasmin Lys311Glu substitution converts the protease into a more efficient kininogenase. Fibrinolytic activity is normally brisk in the mouth, perhaps explaining why patients with the Lys311Glu mutation primarily suffer from oral-lingual angioedema. The findings raise the possibility that oral-lingual edema after tPA infusion or ACE inhibitor administration may also be due to plasmin-mediated cleavage of LK and HK. The Met361Lys substitution in HK and LK introduces a novel protease cleavage site immediately upstream from a normal PKa cleavage site. The novel site is preferred over the normal site by plasmin, but not PKa. Several treatments for HAE directed at neutralizing PKa or FXIIa activity have been developed. Our results suggest that such drugs may be less effective in patients with HAEnC1 in which disease mechanisms operate independently of the KKS.

Activation of coagulation FXI promotes endothelial inflammation and amplifies platelet activation in a nonhuman primate model of hyperlipidemia

BackgroundHyperlipidemia is associated with chronic inflammation and thromboinflammation. This is an underlying cause of several cardiovascular diseases, including atherosclerosis. In diseased blood vessels, rampant thrombin generation results in the initiation of the coagulation cascade, activation of platelets, and endothelial cell dysfunction. Coagulation factor (F) XI represents a promising therapeutic target to reduce thromboinflammation, as it is uniquely positioned at an intersection between inflammation and thrombin generation. ObjectivesThis study aimed to investigate the role of FXI in promoting platelet and endothelial cell activation in a model of hyperlipidemia. MethodsNonhuman primates (NHPs) were fed a standard chow diet (lean, n = 6) or a high-fat diet (obese, n = 8) to establish a model of hyperlipidemia. Obese NHPs were intravenously administered a FXI blocking antibody (2 mg/kg) and studied at baseline and at 1, 7, 14, 21, and 28 days after drug administration. Platelet activation and inflammatory markers were measured using fluorescence-activated cell sorting or enzyme-linked immunosorbent assay. Molecular imaging was used to quantify vascular cell adhesion molecule 1 (VCAM-1) expression at the carotid bifurcation. ResultsObese NHPs demonstrated increased sensitivity for platelet P-selectin expression and phosphatidylserine exposure in response to platelet GPVI or PAR agonists compared with lean NHPs. Obese NHPs exhibited elevated levels of C-reactive protein, cathepsin D, and myeloperoxidase compared with lean NHPs. Following pharmacological inhibition of FIX activation by FXIa, platelet priming for activation by GPVI or PAR agonists, C-reactive protein levels, and endothelial VCAM-1 levels were reduced in obese NHPs. ConclusionFXI activation promotes the proinflammatory phenotype of hyperlipidemia by priming platelet activation and inciting endothelial cell dysfunction.

Valvular presence of coagulation factor XI correlates with the severity of aortic stenosis

Abstract Background The role of coagulation system as a factor involved in the progression of aortic stenosis (AS) has been proven. Considering recent studies about oral factor XIa (FXIa) inhibitor – asundexian with antithrombotic efficacy, our aim was to investigate whether FXI is present within stenotic leaflets in patients with severe AS and whether its expression correlates with valvular calcification. Methods We recruited 20 patients aged 66 ±9 years with severe AS defined as mean transvalvular pressure gradient (PGmean) ≥ 40mmHg, peak transvalvular velocity (Vmax) of ≥4.0 m/s and aortic valve area (AVA) &amp;lt;1 cm2. Stenotic aortic valves were obtained during valve replacement surgery. Aortic leaflets obtained from AS patients and from age-matched apparently healthy autopsy donors (n=3) were used to evaluate valvular expression of FXI, FIX, tissue factor (TF) and bone morphogenetic protein 2 (BMP2) by single and double-labeled immunostaining. The immunopositive valve area was computed as the ratio (%) of positively and negatively stained areas. The Ethical Committee approved the study and all participants provided written informed consent. Results AS patients with median PGmean of 54 [46-67] mmHg, Vmax of 4.5 [4.4-5.0] m/s and AVA of 0.8 [0.7-0.9] cm2 were treated with angiotensin converting enzyme inhibitors (80%), beta-blockers (90%), statins (70%) and acetylsalicylic acid (80%). In loco analysis revealed valvular expression of FXI within all studied stenotic valves, but not within control ones (Figure 1 A and B). The FXI-immunopositive valve area constituted 21.4±4.5% of the total leaflet area, while these for FIX and TF were 17.2 ±3.8% and 26.5±5.1%, respectively. The mean BMP2-positive area was 22.9±4.1%. The expression of studied proteins was observed at the aortic side of the stenotic leaflets and presented a condensed pattern of fluorescence. Moreover, the expression of FXI co-expressed with FIX in 76% and TF in 71%, suggesting local activation of FXI (Figure 1 D and E). Moreover, FXI co-expressed with BMP2 in 83% (Figure C), which may indicate that FXI activation occurs at the site of leaflet calcification. Interestingly, valvular amounts of FXI correlated with AS severity measured as PGmean (r=0.49, p=0.03), PGmax (r=0.53, p=0.02), Vmax (r=0.54, p=0.01) and AVA (r=-0.53, p=0.02). Conclusions We are the first to show the valvular presence of FXI, co-expressing with FIX and TF, which indicates FXI in loco activation. A strong co-expression of FXI with BMP2 and associations with disease severity highlight the impact of coagulation on valve calcification. It is tempting to speculate that FXIa inhibition could attenuate not only coagulation activation but also valvular calcification and thus retard AS progression. Clinical relevance of these findings requires further studies. This work was supported by the Polish National Science Centre (UMO-2021/41/N/NZ5/03323).Figure 1_ESC_2023

High molecular weight kininogen interactions with the homologs prekallikrein and factor XI: importance to surface-induced coagulation

BackgroundIn plasma, high molecular weight kininogen (HK) is either free or bound to prekallikrein (PK) or factor (F) XI (FXI). During contact activation, HK is thought to anchor PK and FXI to surfaces, facilitating their conversion to the proteases plasma kallikrein and FXIa. Mice lacking HK have normal hemostasis but are resistant to injury-induced arterial thrombosis. ObjectivesTo identify amino acids on the HK-D6 domain involved in PK and FXI binding and study the importance of the HK–PK and HK–FXI interactions to coagulation. MethodsTwenty-four HK variants with alanine replacements spanning residues 542-613 were tested in PK/FXI binding and activated partial thromboplastin time clotting assays. Surface-induced FXI and PK activation in plasma were studied in the presence or absence of HK. Kng1-/- mice lacking HK were supplemented with human or murine HK and tested in an arterial thrombosis model. ResultsOverlapping binding sites for PK and FXI were identified in the HK-D6 domain. HK variants with defects only in FXI binding corrected the activated partial thromboplastin time of HK-deficient plasma poorly compared to a variant defective only in PK-binding. In plasma, HK deficiency appeared to have a greater deleterious effect on FXI activation than PK activation. Human HK corrected the defect in arterial thrombus formation in HK-deficient mice poorly due to a specific defect in binding to mouse FXI. ConclusionClinical observations indicate FXI is required for hemostasis, while HK is not. Yet, the HK–FXI interaction is required for contact activation-induced clotting in vitro and in vivo suggesting an important role in thrombosis and perhaps other FXI-related activities.

PB0566 Prediction and Validation of the Effects of Targeting either Coagulation FXI Activation or Activity on Thrombin Generation

PB0566 Prediction and Validation of the Effects of Targeting either Coagulation FXI Activation or Activity on Thrombin Generation

Histidine-rich glycoprotein attenuates catheter thrombosis.

Factor XII (FXII) knockdown attenuates catheter thrombosis in rabbits. Because histidine-rich glycoprotein (HRG) modulates FXIIa activity, we hypothesized that HRG depletion would promote catheter thrombosis. To test this, rabbits were given either antisense oligonucleotides (ASOs) against HRG or FXII, a control ASO, or saline. The activated partial thromboplastin time (aPTT), prothrombin time (PT), and catheter-induced thrombin generation were determined in blood collected before and after treatment. Compared with the controls, the HRG- and FXII-directed ASOs reduced hepatic messenger RNA and plasma levels of HRG and FXII, respectively, by >90%. Although HRG knockdown shortened the aPTT by 2.5 fold, FXII knockdown prolonged it by fourfold; neither of the ASOs affected the PT. Catheter segments shortened the lag time and increased peak thrombin in the plasma from control rabbits; effects were significantly enhanced and attenuated in the plasma from rabbits given the HRG- and FXII-directed ASOs, respectively. Catheters were then inserted into the right external jugular vein of the rabbits, and the time for catheter occlusion was determined. The catheter occlusion times with the control ASO or saline were 62± 8minutes and 60± 11minutes, respectively. The occlusion time was significantly reduced to 34± 9minutes, with HRG knockdown and significantly prolonged to 128± 19minutes with FXII knockdown. HRG levels are decreased with sepsis or cancer, and such patients are prone to catheter thrombosis. Because HRG modulates catheter thrombosis, our findings suggest that HRG supplementation may prevent this problem.

FXII contact activation products have an inhibitory effect on αFXIIa.

It is accepted that the contact activation complex of the intrinsic pathway of blood coagulation cascade produces active enzymes that lead to plasma coagulation following biomaterial contact. In this study, FXII was activated through contact with hydrophilic glass beads and hydrophobic octadecyltrichlorosilane-modified glass beads from neat buffer solutions. These FXII contact activation products generated from material interaction were found to suppress the procoagulant activity of exogenous αFXIIa, and this inhibition was dependent on surface wettability and the concentration of exogenous αFXIIa. Higher relative inhibition rates were generally observed at low concentrations of αFXIIa (1-2 μg/mL) while both hydrophobic and hydrophilic materials showed similar inhibition levels (~39%) at high concentrations of αFXIIa (20 μg/mL). The presence of prekallikrein in the activation system increased the amount of FXIIa produced during FXII contact activation, and also suppressed the apparent levels of inhibitors on hydrophilic surfaces, while having no effect on apparent levels of inhibitors on hydrophobic surface. The combination of FXII contact activation products and activator surfaces was found to dramatically increase inhibition of αFXIIa activity compared to the activation products alone, regardless of activator surface wettability and the presence of prekallikrein. This finding of inhibitors in the suite of proteins generated by contact activation provides additional knowledge into the complex series of interactions that occur when plasma comes into contact with material surfaces.

Molecular mechanism by which spider-driving peptide potentiates coagulation factors

Hemostasis is a crucial process that quickly forms clots at injury sites to prevent bleeding and infections. Dysfunctions in this process can lead to hemorrhagic disorders, such as hemophilia and thrombocytopenia purpura. While hemostatic agents are used in clinical treatments, there is still limited knowledge about potentiators targeting coagulation factors. Recently, LCTx-F2, a procoagulant spider-derived peptide, was discovered. This study employed various methods, including chromogenic substrate analysis and dynamic simulation, to investigate how LCTx-F2 enhances the activity of thrombin and FXIIa. Our findings revealed that LCTx-F2 binds to thrombin and FXIIa in a similar manner, with the N-terminal penetrating the active-site cleft of the enzymes and the intermediate section reinforcing the peptide-enzyme connection. Interestingly, the C-terminal remained at a considerable distance from the enzymes, as evidenced by the retention of affinity for both enzymes using truncated peptide T-F2. Furthermore, results indicated differences in the bonding relationship of critical residues between thrombin and FXIIa, with His13 facilitating binding to thrombin and Arg7 being required for binding to FXIIa. Overall, our study sheds light on the molecular mechanism by which LCTx-F2 potentiates coagulation factors, providing valuable insights that may assist in designing drugs targeting procoagulation factors.

Hemostatic Effects of Raloxifene in Ovariectomized Rats.

This study aimed to explore the effects of raloxifene (Rx) and estradiol (E2) on prothrombin time (PT), partial thromboplastin time (APTT), coagulation factors (VII, X, XI), and fibrinogen concentrations in rats. Female rats were ovariectomized 11 days prior to starting the treatment. Afterward, they received Rx or E2 (1, 10, 100, and 1000 µg/kg) or propylene glycol (0.3 mL; vehicle, V) subcutaneously for 3 consecutive days. Plasma was collected to measure the hemostatic parameters. Rx significantly increased PT (8%, at 1000 µg/kg; p < 0.05) and APTT at all doses evaluated (32, 70, 67, 30%; p < 0.05, respectively). Rx (1, 10, 100, and 1000 µg/kg) decreased the activity of factor VII by -20, -40, -37, and -17% (p < 0.05), respectively, and E2 increased it by 9, 34, 52, and 29%. Rx reduced factor X activity at 10 and 100 µg/kg doses (-30, and -30% p < 0.05), and E2 showed an increment of 24% with 1000 µg/kg dose only. Additionally, Rx (1, 10, 100 µg/kg) diminished FXI activity (-71, -62, -66; p < 0.05), E2 (1 and 10 µg/kg) in -60 and -38, respectively (p < 0.05), and Rx (1000 µg/kg) produced an increment of 29% (p < 0.05) in fibrinogen concentration, but not E2. Our findings suggest that raloxifene has a protective effect on hemostasis in rats.

Isolation and Characterization of Poeciguamerin, a Peptide with Dual Analgesic and Anti-Thrombotic Activity from the Poecilobdella manillensis Leech

When Poecilobdella manillensis attacks its prey, the prey bleeds profusely but feels little pain. We and other research teams have identified several anticoagulant molecules in the saliva of P. manillensis, but the substance that produces the paralyzing effect in P. manillensis is not known. In this study, we successfully isolated, purified, and identified a serine protease inhibitor containing an antistasin-like domain from the salivary secretions of P. manillensis. This peptide (named poeciguamerin) significantly inhibited elastase activity and slightly inhibited FXIIa and kallikrein activity, but had no effect on FXa, trypsin, or thrombin activity. Furthermore, poeciguamerin exhibited analgesic activity in the foot-licking and tail-withdrawal mouse models and anticoagulant activity in the FeCl3-induced carotid artery thrombosis mouse model. In this study, poeciguamerin was found to be a promising elastase inhibitor with potent analgesic and antithrombotic activity for the inhibition of pain and thrombosis after surgery or in inflammatory conditions.

Determination of the Potential Clinical Benefits of Small Molecule Factor XIa Inhibitors in Arterial Thrombosis.

Anticoagulants are the mainstay for the prevention and treatment of thrombosis. However, bleeding complications remain a primary concern. Recent advances in understanding the contribution of activated factor XI (FXIa) in arterial thrombosis with a limited impact on hemostasis have led to the development of several FXIa-targeting modalities. Injectable agents including monoclonal antibodies and antisense oligonucleotides against FXIa have been primarily studied in venous thrombosis. The orally active small molecules that specifically inhibit the active site of FXIa are currently being investigated for their antithrombotic activity in both arteries and veins. This review focuses on a discussion of the potential clinical benefits of small molecule FXIa inhibitors, mainly asundexian and milvexian, in arterial thrombosis based on their pharmacological profiles and the compelling results of phase 2 clinical studies. The preclinical and epidemiological basis for the impact of FXIa in hemostasis and arterial thrombosis is also addressed. In recent clinical study results, asundexian appears to reduce ischemic events in patients with myocardial infarction and minor-to-moderate stroke, whereas milvexian possibly provides benefits in patients with minor stroke or high-risk transient ischemic attack (TIA). In addition, asundexian and milvexian had a minor impact on hemostasis even in combination with dual-antiplatelet therapy. Other orally active FXIa inhibitors also produce antithrombotic activity in vivo with low bleeding risk. Therefore, FXIa inhibitors might represent a new class of direct-acting oral anticoagulants (DOACs) for the treatment of thrombosis, although the explicit clinical positions of asundexian and milvexian in patients with ischemic stroke, high-risk TIA, and coronary artery disease require confirmation from the outcomes of ongoing phase 3 trials.