High Molecular Weight Kininogen Research Articles

Increased Prolylcarboxypeptidase Expression Can Serve as a Biomarker of Senescence in Culture.

Prolylcarboxypeptidase (PRCP, PCP, Lysosomal Pro-X-carboxypeptidase, Angiotensinase C) controls angiotensin- and kinin-induced cell signaling. Elevation of PRCP appears to be activated in chronic inflammatory diseases [cardiovascular disease (CVD), diabetes] in proportion to severity. Vascular endothelial cell senescence and mitochondrial dysfunction have consistently been shown in models of CVD in aging. Cellular senescence, a driver of age-related dysfunction, can differentially alter the expression of lysosomal enzymes due to lysosomal membrane permeability. There is a lack of data demonstrating the effect of age-related dysfunction on the expression and function of PRCP. To explore the changes in PRCP, the PRCP-dependent prekallikrein (PK) pathway was characterized in early- and late-passage human pulmonary artery endothelial cells (HPAECs). Detailed kinetic analysis of cells treated with high molecular weight kininogen (HK), a precursor of bradykinin (BK), and PK revealed a mechanism by which senescent HPAECs activate the generation of kallikrein upon the assembly of the HK-PK complex on HPAECs in parallel with an upregulation of PRCP and endothelial nitric oxide (NO) synthase (eNOS) and NO formation. The NO production and expression of both PRCP and eNOS increased in early-passage HPAECs and decreased in late-passage HPAECs. Low activity of PRCP in late-passage HPAECs was associated with rapid decreased telomerase reverse transcriptase mRNA levels. We also found that, with an increase in the passage number of HPAECs, reduced PRCP altered the respiration rate. These results indicated that aging dysregulates PRCP protein expression, and further studies will shed light into the complexity of the PRCP-dependent signaling pathway in aging.

The effect of estrogen-containing birth control pills on the constituents of bradykinin expression in plasma

Hereditary angioedema with C1-inhibitor deficiency (HAE-C1INH) is a rare autosomal disorder presenting with recurrent angioedema. Estrogen-containing medications trigger angioedema in some patients, and conversely, progesterone may decrease attack frequency. The mechanism by which estrogen may exacerbate angioedema in HAE-C1INH is not well characterized. Our aim was to investigate the link between estrogen and bradykinin constituents to better understand the specific underlying triggers that may exacerbate angioedema in patients with HAE-C1INH. As estrogen is contraindicated for patients with HAE-C1INH, females without a history of angioedema were recruited to evaluate whether estrogen-containing oral contraceptive pills (OCPs) alter plasma protein levels of bradykinin, cleaved high-molecular-weight kininogen (cHK), and activated factor XII (FXIIa). Blood (plasma) was collected before initiation of OCP administration and 3 months thereafter. High-molecular-weight kininogen (HK) was measured by ELISA and FXIIa and cHK were analyzed by Western blot analysis. A total of 12 adult females without HAE-CINH (aged <40 years) had a median baseline plasma HK level of 33,976 ng/mL. After 3 months of OCP therapy, their median HK levelincreased to 38,202 ng/mL. With OCPs, there was also a significant increase in level of FXIIa protein (P <.01), as well asan increase in cHK protein level. This preliminary study, performed in females without HAE-C1INH, suggests that estrogen may exacerbateangioedema by increasing the production of cHK and FXIIa.

Oral FXIIa inhibitor KV998086 suppresses FXIIa and single chain FXII mediated kallikrein kinin system activation.

Background: The kallikrein kinin system (KKS) is an established pharmacological target for the treatment and prevention of attacks in hereditary angioedema (HAE). Proteolytic activities of FXIIa and single-chain Factor XII (FXII) zymogen contribute to KKS activation and thereby may play roles in both initiating and propagating HAE attacks. In this report, we investigated the effects of potent small molecule FXIIa inhibitors on FXIIa and single chain FXII enzymatic activities, KKS activation, and angioedema in mice. Methods: We examined the effects of 29 structurally distinct FXIIa inhibitors on enzymatic activities of FXIIa and a mutant single chain FXII with R334A, R343A and R353A substitutions (rFXII-T), that does not undergo zymogen conversion to FXIIa, using kinetic fluorogenic substrate assays. We examined the effects of a representative FXIIa inhibitor, KV998086, on KKS activation and both carrageenan- and captopril-induced angioedema in mice. Results: FXIIa inhibitors designed to target its catalytic domain also potently inhibited the enzymatic activity of rFXII-T and the pIC50s of these compounds linearly correlated for rFXIIa and rFXII-T (R 2 = 0.93). KV998086, a potent oral FXIIa inhibitor (IC50 = 7.2nM) inhibited dextran sulfate (DXS)-stimulated generation of plasma kallikrein and FXIIa, and the cleavage of high molecular weight kininogen (HK) in human plasma. KV998086 also inhibited rFXII-T mediated HK cleavage (p < 0.005) in plasma from FXII knockout mice supplemented with rFXII-T and stimulated with polyphosphate or DXS. Orally administered KV998086 protected mice from 1) captopril-induced Evans blue leakage in colon and laryngotracheal tissues and 2) blocked carrageenan-induced plasma HK consumption and paw edema. Conclusion: These findings show that small molecule FXIIa inhibitors, designed to target its active site, also inhibit the enzymatic activity of FXII zymogen. Combined inhibition of FXII zymogen and FXIIa may thereby suppress both the initiation and amplification of KKS activation that contribute to hereditary angioedema attacks and other FXII-mediated diseases.

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.

Predictive Capacity of Plasma and Urinary Biomarkers for Persistent Albuminuria in Patients with Sickle Cell Disease

Introduction: Chronic kidney disease (CKD) is common in sickle cell disease (SCD) and is associated with increased mortality and morbidity. As albuminuria and changes in estimated glomerular filtration rate (eGFR) occur well after substantial structural and functional tissue damage have occurred, novel biomarkers are needed to predict CKD prior to its development. In this multicenter study, we investigated the association of multiple plasma and urine biomarkers with persistent albuminuria (PA) and their potential in predicting PA in patients with SCD. Methods: Adult subjects with HbSS or HbSb 0 thalassemia were enrolled in a prospective, longitudinal cohort study of CKD in SCD. We excluded patients with diabetic nephropathy, cancer, connective tissue disease, other glomerular diseases, end-stage kidney disease on dialysis, known infection with hepatitis (B or C) or HIV, and who had undergone bone marrow transplantation and solid organ transplantation. Participants were enrolled during routine clinical visits in the non-crisis, “steady states.” PA was defined as at least 2 of 3 spot urine albumin-creatinine ratio (ACR) values ≥30 mg/g or a single value ≥100 mg/g. At baseline evaluation, plasma was analyzed for biomarkers of coagulation activation (D-dimer, thrombin antithrombin complex [TAT], ratio of cleaved high molecular weight kininogen-to-intact high molecular weight kininogen [cHK/iHK]), endothelial dysfunction (endothelin-1 [ET1], vascular endothelial growth factor [VEGF], vascular cell adhesion molecule-1 [VCAM-1]), and urine was analyzed for kidney injury molecule 1 (KIM-1), nephrin and angiotensinogen (AGT) using commercially available ELISA. All urinary biomarkers were normalized to urine creatinine. For baseline values, a standard t-test was performed for continuous variables and a chi-square test was performed for categorical variables. Logistic regression analysis was performed to assess univariate predictors of research biomarkers for PA. Results: Of 278 evaluated subjects, 104 (37.4 %) had PA with a median ACR of 194.5 mg/g [IQR: 102.4, 487.1], and 174 subjects (62.6 %) did not have PA (non-PA) with a median ACR of 13.0 mg/g [IQR: 7.4, 22.4]. Subjects with PA were older, had lower hemoglobin and fetal hemoglobin, but higher systolic and diastolic blood pressures than those without PA (Table 1). Plasma VCAM-1, urinary KIM-1, and urinary AGT were significantly associated with PA with odds ratios of 1.001 (AUC: 0.636; 95% CI: 1.00, 1.002), 1.737 (AUC: 0.661; 95% CI: 1.226, 2.575), and 1.053 (AUC: 0.71; 95% CI: 1.029, 1.084), respectively (Table 2). Surprisingly, ET1 and urinary nephrin-creatinine ratio were not significantly associated with PA in this cohort. Conclusion: Urinary AGT exhibited the highest sensitivity for PA, while urinary KIM-1 was the most specific. The relatively modest sensitivity of KIM-1, a marker of renal tubular dysfunction, is likely because albuminuria in SCD results from both glomerular and tubular dysfunction. These results highlight the potential importance of urinary AGT as a key biomarker in identifying individuals at risk for PA. Our ongoing studies will evaluate the predictive capacity of these biomarkers for development of PA in SCD.

First Validated Model of Zymogen Factor XII Provides Insights into Protein Assembly and Activation

Factor XII (FXII), the zymogen of the plasma protease FXIIa, contributes to thrombo-inflammatory processes. As part of the kallikrein-kinin system, FXIIa converts prekallikrein to the protease kallikrein (PKa), which then cleaves high-molecular-weight kininogen (HK) to release the vasoactive peptide bradykinin. In the disorder hereditary angioedema, dysregulation of FXII activation leads to excessive bradykinin production. When blood is exposed to artificial surfaces, such as during cardiopulmonary bypass or ECMO, FXII is rapidly converted to FXIIa by contact activation, and contributes to thrombus formation by activating factor XI. FXII circulates in plasma in a “closed” form that is resistant to activation by PKa and other proteases. Surface-binding “opens” the FXII structure, facilitating activation. Relatively little is known about the structural features that maintain the closed FXII conformation as the full-length protein has been refractory to crystallization. Recently, a crystal structure was reported for part of the non-catalytic portion of FXII. An intriguing feature of the structure is an interaction between the fibronectin type 2 (FN2) and kringle (KNG) domains at opposite ends of the polypeptide that forms a ring-like structure. Functional studies by our group and others indicate these domains are key to maintaining the closed conformation. Here we describe a model for FXII prepared with the program AlphaFold (AF), and its validation using site-directed mutagenesis to disrupt interactions predicted to maintain the closed conformation. The human FXII model in the AF database was modified by removing residues 300-316 from the proline-rich region and by reference to crystal structures of the FXIIa catalytic domain (CD) bound to inhibitors. Molecular graphics and interdomain interaction analyses were performed in ChimeraX. The refined model predicts 15 interdomain bonds involving residues in FN2, KNG, and the CD. Of the 22 amino acids involved in these bonds, 15 are highly conserved in FXII across 25 vertebrate species. We expressed human FXII in which these residues were individually changed to alanine (Table). Alanine replacements for amino acids not predicted to be involved in interdomain interactions were prepared as controls. Four variants (FXII-D253A, FXII-W257A, FXII-W268A and FXII-E502A) were activated in cell culture and one (FXII-R61A) did not express. FXII-R61A, FXII-W257A, and FXII-W268A were not studied further. FXII-D253A was replaced with FXII-D253K. The active site serine of FXII-E502A was replaced with alanine to prevent autocatalysis (FXII-E502A,S544A). In the absence of a surface, FXII-R36A, FXII-E225A, FXII-D253K, and FXII-K346A were activated by PKa (~15 fold) faster than wild type FXII (FXII-WT) in a chromogenic substrate assay, consistent with an open conformation. Using densitometric analyses of stained SDS- polyacrylamide gels, we noted that these four FXII variants and FXII-E502A,S544A were converted to FXIIa by PKa more rapidly than FXII-WT and other variants. FXII-R36A, FXII-E225A, FXII-D253K, and FXII-K346A bound to prekallikrein with higher affinity than FXII-WT in a plate-binding assay, consistent with a change in conformation. When the four variants were added to human plasma in the absence of a surface, they caused a rapid cleavage of HK by accelerating reciprocal activation with prekallikrein. Our functional analyses indicate that non-covalent bonds between R36 on FN2 and E502 on the CD; R36 and E225 on KNG; and D253 on KNG and K346 adjacent to the activation cleavage site are critical for maintaining FXII in a closed conformation. The instability of FXII variants with substitutions for tryptophans at positions 257 and 268 also implicate interactions between these residues and K346. In the AF model these bonds appear to maintain FXII in a conformation in which access to the activation cleavage site is limited. Our binding studies also suggest the closed conformation mask a binding site for prekallikrein and PKa. These bonds are likely disrupted when FXII binds to a surface, as part of the contact activation mechanism that enhances FXII activation.

A mechanism for hereditary angioedema caused by a methionine-379–to–lysine substitution in kininogens

AbstractHereditary angioedema (HAE) is associated with episodic kinin-induced swelling of the skin and mucosal membranes. Most patients with HAE have low plasma C1-inhibitor activity, leading to increased generation of the protease plasma kallikrein (PKa) and excessive release of the nanopeptide bradykinin from high-molecular-weight kininogen (HK). However, disease-causing mutations in at least 10% of patients with HAE appear to involve genes for proteins other than C1-inhibitor. A point mutation in the Kng1 gene encoding HK and low–molecular weight kininogen (LK) was identified recently in a family with HAE. The mutation changes a methionine (Met379) to lysine (Lys379) in both proteins. Met379 is adjacent to the Lys380-Arg381 cleavage site at the N-terminus of the bradykinin peptide. Recombinant wild-type (Met379) and variant (Lys379) versions of HK and LK were expressed in HEK293 cells. PKa-catalyzed kinin release from HK and LK was not affected by the Lys379 substitutions. However, kinin release from HK-Lys379 and LK-Lys379 catalyzed by the fibrinolytic protease plasmin was substantially greater than from wild-type HK-Met379 and LK-Met379. Increased kinin release was evident when fibrinolysis was induced in plasma containing HK-Lys379 or LK-Lys379 compared with plasma containing wild-type HK or LK. Mass spectrometry revealed that the kinin released from wild-type and variant kininogens by PKa is bradykinin. Plasmin also released bradykinin from wild-type kininogens but cleaved HK-Lys379 and LK-Lys379 after Lys379 rather than Lys380, releasing the decapeptide Lys-bradykinin (kallidin). The Met379Lys substitutions make HK and LK better plasmin substrates, reinforcing the relationship between fibrinolysis and kinin generation.

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.

Factor XII promotes the thromboinflammatory response in a rat model of venoarterial extracorporeal membrane oxygenation

BackgroundFactor XII (FXII) is a multifunctional protease capable of activating thrombotic and inflammatory pathways. FXII has been linked to thrombosis in extracorporeal membrane oxygenation (ECMO), but the role of FXII in ECMO-induced inflammatory complications has not been studied. We used novel gene-targeted FXII- deficient rats to evaluate the role of FXII in ECMO-induced thromboinflammation. MethodsFXII-deficient (FXII−/−) Sprague–Dawley rats were generated using CRISPR/Cas9. A minimally invasive venoarterial (VA) ECMO model was used to compare wild-type (WT) and FXII−/− rats in 2 separate experimental cohorts: rats placed on ECMO without pharmacologic anticoagulation and rats anticoagulated with argatroban. Rats were maintained on ECMO for 1 hour or until circuit failure occurred. Comparisons were made with unchallenged rats and rats that underwent a sham surgical procedure without ECMO. ResultsFXII−/− rats were maintained on ECMO without pharmacologic anticoagulation with low resistance throughout the 1-hour experiment. In contrast, WT rats placed on ECMO without anticoagulation developed thrombotic circuit failure within 10 minutes. Argatroban provided a means to maintain WT and FXII−/− rats on ECMO for the 1-hour time frame without thrombotic complications. Analyses of these rats demonstrated that ECMO resulted in increased neutrophil migration into the liver that was significantly blunted by FXII deficiency. ECMO also resulted in increases in high molecular weight kininogen cleavage and complement activation that were abrogated by genetic deletion of FXII. ConclusionsFXII initiates hemostatic system activation and key inflammatory sequelae in ECMO, suggesting that therapies targeting FXII could limit both thromboembolism and inopportune inflammatory complications in this setting.

C1 inhibitor and prolylcarboxypeptidase modulate prekallikrein activation on endothelial cells

We examined how prekallikrein (PK) activation on human microvascular endothelial cells (HMVECs) is regulated by the ambient concentration of C1 inhibitor (C1INH) and prolylcarboxypeptidase (PRCP). We sought to examine the specificity of PK activation on HMVECs by PRCP and the role of C1INH to regulate it, high-molecular-weight kininogen (HK) cleavage, and bradykinin (BK) liberation. Investigations were performed on cultured HMVECs. Immunofluorescence, enzymatic activity assays, immunoblots, small interfering RNA knockdowns, and cell transfections were used to perform these studies. Cultured HMVECs constitutively coexpressed PK, HK, C1INH, and PRCP. PK activation on HMVECs was modulated by the ambient C1INH concentration. In the absence of C1INH, forming PKa on HMVECs cleaved 120-kDa HK completely to a 65-kDa H-chain and a 46-kDa L-chain in 60 minutes. In the presence of 2 μM C1INH, only 50% of the HK became cleaved. C1INH concentrations (0.0-2.5 μM) decreased but did not abolish BK liberated from HK by activated PK. Factor XII did not activate when incubated with HMVECs alone for 1 hour. However, if incubated in the presence of HK and PK, factor XII became activated. The specificity of PK activation on HMVECs by PRCP was shown by several inhibitors to each enzyme. Furthermore, PRCP small interfering RNA knockdowns magnified C1INH inhibitory activity on PK activation, and PRCP transfections reduced C1INH inhibition at any given concentration. These combined studies indicated that on HMVECs, PK activation and HK cleavage to liberate BK weremodulated by the local concentrations of C1INH and PRCP.

Structures of factor XI and prekallikrein bound to domain 6 of high–molecular weight kininogen reveal alternate domain 6 conformations and exosites

BackgroundHigh–molecular weight kininogen (HK) circulates in plasma as a complex with zymogen prekallikrein (PK). HK is both a substrate and a cofactor for activated plasma kallikrein, and the principal exosite interactions occur between PK N-terminal apple domains and the C-terminal D6 domain of HK. ObjectivesTo determine the structure of the complex formed between PK apple domains and an HKD6 fragment and compare this with the coagulation factor XI (FXI)-HK complex. MethodsWe produced recombinant FXI and PK heavy chains (HCs) spanning all 4 apple domains. We cocrystallized PKHC (and subsequently FXIHC) with a 31-amino acid synthetic peptide spanning HK residues Ser565-Lys595 and determined the crystal structure. We also analyzed the full-length FXI-HK complex in solution using hydrogen deuterium exchange mass spectrometry. ResultsThe 2.3Å PKHC-HK peptide crystal structure revealed that the HKD6 sequence WIPDIQ (Trp569-Gln574) binds to the apple 1 domain and HK FNPISDFPDT (Phe582-Thr591) binds to the apple 2 domain with a flexible intervening sequence resulting in a bent double conformation. A second 3.2Å FXIHC-HK peptide crystal structure revealed a similar interaction with the apple 2 domain but an alternate, straightened conformation of the HK peptide where residues LSFN (Leu579-Asn583) interacts with a unique pocket formed between the apple 2 and 3 domains. HDX-MS of full length FXI-HK complex in solution confirmed interactions with both apple 2 and apple 3. ConclusionsThe alternate conformations and exosite binding of the HKD6 peptide likely reflects the diverging relationship of HK to the functions of PK and FXI.

Mutant plasminogen in hereditary angioedema is bypassing FXII/kallikrein to generate bradykinin.

Hereditary angioedema (HAE) is characterized by recurrent localized edema in various organs, which can be potentially fatal. There are different types of hereditary angioedema, which include genetic deficiency of C1 inhibitor (C1-INH) and hereditary angioedema with normal C1-INH (HAEnCI). In HAEnCI patients mutations have been identified in the F12, PLG, KNG1, ANGPT1, MYOF, and HS3ST6 genes. The release of bradykinin from kininogen via the kallikrein-kinin system (KKS) has been shown to be the main mediator in HAE-FXII, but for HAE-PLG there are only first indications how the PLG mutations can result in bradykinin release. Here we identified in a multi-generation HAE-PLG family an additional F12 mutation, resulting in the loss of one F12 allele. There were no differences in the clinical presentation between HAE-PLG patients with and without the additional F12 mutation, thus we concluded that the kallikrein-kinin system is bypassed in HAE-PLG. Structural modeling and in vitro assays using purified proteins confirmed the PLG mutation c.988A>G; p.K330E to be a gain of function mutation resulting in an increased bradykinin release by direct cleavage of high molecular weight kininogen (HMWK). Thus, we can provide clinical and experimental evidence that mutant plasminogen in HAE-PLG is bypassing FXII/kallikrein to generate bradykinin.

Severe high-molecular-weight kininogen deficiency: clinical characteristics, deficiency–causing KNG1 variants, and estimated prevalence

BackgroundSevere high-molecular-weight kininogen (HK) deficiency is a poorly studied autosomal recessive contact system defect caused by pathogenic, biallelic KNG1 variants. AimWe performed the first comprehensive analysis of diagnostic, clinical, genetic, and epidemiological aspects of HK deficiency. MethodsWe collected clinical information and blood samples from a newly detected HK-deficient individual and from published cases identified by a systematic literature review. Activity and antigen levels of coagulation factors were determined. Genetic analyses of KNG1 and KLKB1 were performed by Sanger sequencing. The frequency of HK deficiency was estimated considering truncating KNG1 variants from GnomAD. ResultsWe identified 48 cases of severe HK deficiency (41 families), of these 47 have been previously published (n = 19 from gray literature). We genotyped 3 cases and critically appraised 10 studies with genetic data. Ten HK deficiency–causing variants (one new) were identified. All of them were truncating mutations, whereas the only known HK amino acid substitution with a relevant phenotype instead causes hereditary angioedema. Conservative estimates suggest an overall prevalence of severe HK deficiency of approximately one case per 8 million population, slightly higher in Africans. Individuals with HK deficiency appeared asymptomatic and had decreased levels of prekallikrein and factor XI, which could lead to misdiagnosis. ConclusionHK deficiency is a rare condition with only few known pathogenic variants. It has an apparently good prognosis but is prone to misdiagnosis. Our understanding of its clinical implications is still limited, and an international prekallikrein and HK deficiency registry is being established to fill this knowledge gap.

Titanium is a potent inducer of contact activation: implications for intravascular devices

BackgroundTitanium (Ti) and its alloys are widely used in manufacturing medical devices because of their strength and resistance to corrosion. Although Ti compounds are considered compatible with blood, they appear to support plasma contact activation and may be thrombogenic. ObjectivesThe objective of this study was to compare Ti and titanium nitride (TiN) with known activators of contact activation (kaolin and silica) in plasma-clotting assays and to assess binding and activation of factor XII, (FXII), factor XI (FXI), prekallikrein, and high-molecular-weight kininogen (HK) with Ti/TiN. MethodsTi-based nanospheres and foils were compared with kaolin, silica, and aluminum in plasma-clotting assays. Binding and activation of FXII, prekallikrein, HK, and FXI to surfaces was assessed with western blots and chromogenic assays. ResultsUsing equivalent surface amounts, Ti and TiN were comparable with kaolin and superior to silica, for inducing coagulation and FXII autoactivation. Similar to many inducers of contact activation, Ti and TiN are negatively charged; however, their effects on FXII are not neutralized by the polycation polybrene. Antibodies to FXII, prekallikrein, or FXI or coating Ti with poly-L-arginine blocked Ti-induced coagulation. An antibody to FXII reduced FXII and PK binding to Ti, kallikrein generation, and HK cleavage. ConclusionTitanium compounds induce contact activation with a potency comparable with that of kaolin. Binding of FXII with Ti shares some features with FXII binding to soluble polyanions but may have unique features. Inhibitors targeting FXII or FXI may be useful in mitigating Ti-induced contact activation in patients with titanium-based implants that are exposed to blood.

Nanobodies against factor XI apple 3 domain inhibit binding of factor IX and reveal a novel binding site for high molecular weight kininogen

BackgroundFactor XI (FXI) is a promising target for novel anticoagulants because it shows a strong relation to thromboembolic diseases, while fulfilling a mostly supportive role in hemostasis. Anticoagulants targeting FXI could therefore reduce the risk for thrombosis, without increasing the chance of bleeding side effects. ObjectivesTo generate nanobodies that can interfere with FXIa mediated activation of factor IX (FIX). MethodsNanobodies were selected for binding to the apple 3 domain of FXI and their effects on FXI and coagulation were measured in purified protein systems as well as in plasma‐based coagulation assays. Additionally, the binding epitope of selected nanobodies was assessed by hydrogen–deuterium exchange mass spectrometry. ResultsWe have identified five nanobodies that inhibit FIX activation by FXI by competing with the FIX binding site on FXI. Interestingly, a sixth nanobody was found to target a different binding epitope in the apple 3 domain, resulting in competition with the FXI–high molecular weight kininogen (HK) interaction. ConclusionsWe have characterized a nanobody targeting the FXI apple 3 domain that elucidates the binding orientation of HK on FXI. Moreover, we have produced five nanobodies that can inhibit the FXI–FIX interaction.

HK is the apple of FXI's eye

HK is the apple of FXI's eye

Hereditary angioedema is associated with an increased risk of venous thromboembolism

Hereditary angioedema is associated with an increased risk of venous thromboembolism

Properdin (factor P) as a new target cleaved by factor XIa: Intrinsic coagulation at the crossroads with inflammation

The complement system and blood coagulation cascade, in particular, the contact activation pathway, are functionally related and exhibit crossover activities. Deficiency or altered function of complement proteins are associated with thrombo‐inflammatory diseases. Factor XI (FXI), as part of the intrinsic pathway, is the zymogen, which can be activated to the plasma protease FXIa by FXIIa with the help of high molecular weight kininogen, and then continue to the downstream cascade of proteases that ultimately triggers the production of thrombin (FIIa).

Blocking domain 6 of high molecular weight kininogen to understand intrinsic clotting mechanisms

BackgroundThe contact system is initiated by factor (F) XII activation and the assembly of high molecular weight kininogen (HK) with either FXI or prekallikrein (PK) on a negatively charged surface. Overactivation ofsystem contributes to thrombosis and inflammation in numerous diseases. To develop effective therapeutics for contact system disorders, a detailed understanding ofpathway is needed. MethodsWe performed coagulation assays in normal human plasma and various factor‐deficient plasmas. To evaluate how HK‐mediated PK and FXI activation contributes to coagulation, we used an anti‐HK antibody to block access to domain 6 of HK, the region required for efficient activation of PK and FXI. ResultsFXI's binding to HK and its subsequent activation by activated FXII contributes to coagulation. We found that the 3E8 anti‐HK antibody can inhibit the binding of FXI or PK to HK, delaying clot formation in human plasma. Our data show that in the absence of FXI, however, PK can substitute for FXI inprocess. Addition of activated FXI (FXIa) or activated PK (PKa) abolished the inhibitory effect of 3E8. Moreover, the requirement of HK in intrinsic coagulation can be largely bypassed by adding FXIa. Like FXIa, exogenous PKa shortened the clotting time in HK‐deficient plasma, which was not due to feedback activation of FXII. Conclusionsstudy improves our understanding of HK‐mediated coagulation and provides an explanation for the absence of bleeding in HK‐deficient individuals. 3E8 specifically prevented HK‐mediated FXI activation; therefore, it could be used to prevent contact activation‐mediated thrombosis without altering hemostasis.

Treatment of hereditary angioedema-single or multiple pathways to the rescue.

Hereditary angioedema (HAE) is a rare disease caused by mutations in the SERPING1 gene. This results in deficient or dysfunctional C1 esterase inhibitor (C1-INH) and affects multiple proteases involved in the complement, contact-system, coagulation, and fibrinolytic pathways. Current options for the treatment and prevention of HAE attacks include treating all affected pathways via direct C1-INH replacement therapy; or specifically targeting components of the contact activation system, in particular by blocking the bradykinin B2 receptor (B2R) or inhibiting plasma kallikrein, to prevent bradykinin generation. Intravenously administered plasma-derived C1-INH (pdC1-INH) and recombinant human C1-INH have demonstrated efficacy and safety for treatment of HAE attacks, although time to onset of symptom relief varied among trials, specific agents, and dosing regimens. Data from retrospective and observational analyses support that short-term prophylaxis with intravenous C1-INH products can help prevent HAE attacks in patients undergoing medical or dental procedures. Long-term prophylaxis with intravenous or subcutaneous pdC1-INH significantly decreased the HAE attack rate vs. placebo, although breakthrough attacks were observed. Pathway-specific therapies for the management of HAE include the B2R antagonist icatibant and plasma kallikrein inhibitors ecallantide, lanadelumab, and berotralstat. Icatibant, administered for treatment of angioedema attacks, reduced B2R-mediated vascular permeability and, compared with placebo, reduced the time to initial symptom improvement. Plasma kallikrein inhibitors, such as ecallantide, block the binding site of kallikrein to prevent cleavage of high molecular weight kininogen and subsequent bradykinin generation. Ecallantide was shown to be efficacious for HAE attacks and is licensed for this indication in the United States, but the labeling recommends that only health care providers administer treatment because of the risk of anaphylaxis. In addition to C1-INH replacement therapy, the plasma kallikrein inhibitors lanadelumab and berotralstat are recommended as first-line options for long-term prophylaxis and have demonstrated marked reductions in HAE attack rates. Investigational therapies, including the activated factor XII inhibitor garadacimab and an antisense oligonucleotide targeting plasma prekallikrein messenger RNA (donidalorsen), have shown promise as long-term prophylaxis. Given the requirement of lifelong management for HAE, further research is needed to determine how best to individualize optimal treatments for each patient.