Plasma Kallikrein Research Articles

Structure-based design and synthesis of novel FXIa inhibitors targeting the S2' subsite for enhanced antithrombotic efficacy.

Factor XIa (FXIa), a key component of the intrinsic coagulation pathway, has recently been recognized as a safe and effective target for antithrombotic therapy. Research indicates that FXIa inhibitors can lower bleeding risk compared to novel oral anticoagulants. In this study, we designed and synthesized a series of novel FXIa inhibitors based on the structure of Asundexian, with a particular focus on optimizing the P2' region to enhance binding to the S2' subsite of FXIa. This strategy led to the discovery of compound F47, which demonstrated significantly greater FXIa inhibition (IC50 = 2.0nM) compared to Asundexian (IC50 = 5.0nM). F47 also showed excellent anticoagulant activity in the aPTT assay (EC2x = 0.4μM), with strong efficacy and minimal impact on the extrinsic coagulation pathway. Additionally, F47 exhibited inhibitory activity against plasma kallikrein (PKal), with selectivity comparable to that of Asundexian. The compound also displayed acceptable stability in human liver microsomal stability assays. Molecular modeling revealed that F47 binds tightly to the S1, S1', and S2' pockets of FXIa while maintaining key interactions; notably, its P2' moiety forms two additional π-π stacking interactions with the crucial amino acid TYR143. Further studies demonstrated that F47 exhibits dose-dependent antithrombotic efficacy in a rat FeCl3-induced thrombosis model. Ongoing research aims to further elucidate the potential of compound F47 as a promising lead in antithrombotic therapy.

Novel Plasma Kallikrein Inhibitors for Treating Multiple Diseases

Novel Plasma Kallikrein Inhibitors for Treating Multiple Diseases

CRISPR-Based Therapy for Hereditary Angioedema.

Hereditary angioedema is a rare genetic disease characterized by severe and unpredictable swelling attacks. NTLA-2002 is an in vivo gene-editing therapy that is based on clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9. NTLA-2002 targets the gene encoding kallikrein B1 (KLKB1). A single dose of NTLA-2002 may provide lifelong control of angioedema attacks. In this phase 2 portion of a phase 1-2 trial, we randomly assigned adults with hereditary angioedema in a 2:2:1 ratio to receive NTLA-2002 in a single dose of 25 mg or 50 mg or placebo. The primary end point was the number of angioedema attacks per month (the monthly attack rate) from week 1 through week 16. Secondary end points included safety, pharmacokinetics, and pharmacodynamics (i.e., the change from baseline in total plasma kallikrein protein level); exploratory end points included patient-reported outcomes. Of the 27 patients who underwent randomization, 10 received 25 mg of NTLA-2002, 11 received 50 mg, and 6 received placebo. From week 1 through week 16, the estimated mean monthly attack rate was 0.70 (95% confidence interval [CI], 0.25 to 1.98) with 25 mg of NTLA-2002, 0.65 (95% CI, 0.24 to 1.76) with 50 mg, and 2.82 (95% CI, 0.80 to 9.89) with placebo; the difference in the estimated mean attack rate with NTLA-2002 as compared with placebo was -75% with 25 mg and -77% with 50 mg. Among patients who received NTLA-2002, 4 of the 10 patients who received 25 mg (40%) and 8 of the 11 who received 50 mg (73%) were attack-free with no additional treatment during the period from week 1 through week 16. The most common adverse events among patients who received NTLA-2002 were headache, fatigue, and nasopharyngitis. The mean percent change in total plasma kallikrein protein levels from baseline to week 16 was -55% with 25 mg and -86% with 50 mg; levels remained unchanged with placebo. NTLA-2002 administered in a single dose of 25 mg or 50 mg reduced angioedema attacks and led to robust and sustained reduction in total plasma kallikrein levels in patients with hereditary angioedema. These results support continued investigation in a larger phase 3 trial. (Funded by Intellia Therapeutics; ClinicalTrials.gov number, NCT05120830; EudraCT number, 2021-001693-33.).

Prolonging the circulatory half-life of C1 esterase inhibitor via albumin fusion.

Hereditary Angioedema (HAE) is an autosomal dominant disease characterized by episodic swelling, arising from genetic deficiency in C1-esterase inhibitor (C1INH), a regulator of several proteases including activated Plasma kallikrein (Pka). Many existing C1INH treatments exhibit short circulatory half-lives, precluding prophylactic use. Hexahistidine-tagged truncated C1INH (trC1INH lacking residues 1-97) with Mutated N-linked Glycosylation Sites N216Q/N231Q/N330Q (H6-trC1INH(MGS)), its murine serum albumin (MSA) fusion variant (H6-trC1INH(MGS)-MSA), and H6-MSA were expressed in Pichia pastoris and purified via nickel-chelate chromatography. Following intravenous injection in mice, the mean terminal half-life of H6-trC1INH(MGS)-MSA was significantly increased versus that of H6-trC1INH(MGS), by 3-fold, while remaining ~35% less than that of H6-MSA. The extended half-life was achieved with minimal, but significant, reduction in the mean second order rate constant of Pka inhibition of H6-trC1INH(MGS)-MSA by 33% relative to that of H6-trC1INH(MGS). Our results validate albumin fusion as a viable strategy for half-life extension of a natural inhibitor and suggest that H6-trC1INH(MGS)-MSA is worthy of investigation in a murine model of HAE.

Macrocyclic Inhibitors Targeting the Prime Site of the Fibrinolytic Serine Protease Plasmin.

Two series of macrocyclic inhibitors addressing the S1 pocket and the prime site of the fibrinolytic serine protease plasmin have been developed. In the first series, a P1 tranexamoyl residue was coupled to 4-aminophenylalanine in P1' position, which provided moderately potent inhibitors with inhibition constants around 1 μM. In the second series, a substituted biphenylalanine was incorporated as P1' residue leading to approximately 1000-fold stronger plasmin inhibitors, the best compounds possess subnanomolar inhibition constants. The most effective compounds already exhibit a certain selectivity as plasmin inhibitors compared to other trypsin-like serine proteases such as trypsin, plasma kallikrein, thrombin, activated protein Ca, as well as factors XIa and Xa. For inhibitor 28 of the second series, the co-crystal structure in complex with a Ser195Ala microplasmin mutant revealed that the P2' residue adopts multiple conformations. Most polar contacts to plasmin and surrounding water molecules are mediated through the P1 tranexamoyl residue, whereas the bound conformation of the macrocycle is mainly stabilized by two intramolecular hydrogen bonds.

A novel assay of excess plasma kallikrein-kinin system activation in hereditary angioedema.

Cleaved high-molecular-weight kininogen (HKa) is a disease state biomarker of kallikrein-kinin system (KKS) activation in patients with hereditary angioedema due to C1 inhibitor deficiency (HAE-C1INH), the endogenous inhibitor of plasma kallikrein (PKa). Develop an HKa-specific enzyme-linked immunosorbent assay (ELISA) to monitor KKS activation in the plasma of HAE-C1INH patients. A novel HKa-specific antibody was discovered by antibody phage display and used as a capture reagent to develop an HKa-specific ELISA. Specific HKa detection following KKS activation was observed in plasma from healthy controls but not in prekallikrein-, high-molecular-weight kininogen-, or coagulation factor XII (FXII)-deficient plasma. HKa levels in plasma collected from HAE-C1INH patients in a disease quiescent state were higher than in plasma from healthy controls and increased further in HAE-C1INH plasma collected during an angioedema attack. The specificity of the assay for PKa-mediated HKa generation in minimally diluted plasma activated with exogenous FXIIa was demonstrated using a specific monoclonal antibody inhibitor (lanadelumab, IC50 = 0.044 µM). An ELISA was developed for the specific and quantitative detection of HKa in human plasma to support HAE-C1INH drug development. Improved quantification of the HKa biomarker may facilitate further pathophysiologic insight into HAE-C1INH and other diseases mediated by a dysregulated KKS and may enable the design of highly potent inhibitors targeting this pathway.

Advent of oral medications for the treatment of hereditary angioedema.

Hereditary angioedema (HAE) is a rare genetic disorder characterized by unpredictable, debilitating episodes of submucosal and/or subcutaneous tissue swelling, which may be life-threatening depending on anatomic location. The two primary management strategies for HAE are ready access to effective on-demand treatment in all patients and the prevention of attacks (short-term prophylaxis [STP] and long-term prophylaxis [LTP]) in appropriate patients. All approved on-demand and most LTP medications require subcutaneous or intravenous administration. Injection-related challenges include trypanophobia (fear of needles), difficulty with self-administration, injection-site reactions (e.g., pain, erythema, bleeding, bruising), and anxiety-all contributing to poor compliance and administration delays. Oral HAE treatments may improve outcomes by reducing treatment barriers. To review oral therapies, approved or in development, for on-demand treatment and/or prevention of HAE attacks. To provide a comprehensive review, data was obtained from publicly available resources through a targeted PubMed literature review and supplemented by information provided on company websites (search cutoff of May 31, 2024). Berotralstat, an oral plasma kallikrein (PKa) inhibitor, is approved for LTP. Sebetralstat, another PKa inhibitor, is the investigational first oral on-demand HAE treatment to complete a phase 3 trial. Deucrictibant, an oral bradykinin B2 receptor antagonist, has completed phase 2 trials for on-demand therapy and LTP. Several other oral PKa inhibitors (ATN249, VE-4666, and VE-4062) are in early development for LTP. Substantial advances have been made in the development of oral treatments for HAE. These treatments have the potential to improve and optimize clinical outcomes, satisfaction, and quality of life among patients with HAE.

Reducing Brain Edema Using Berotralstat, an Inhibitor of Bradykinin, Repurposed as Treatment Adjunct in Glioblastoma

Glioblastomas synthesize, bear receptors for, and respond to bradykinin, triggering migration and proliferation. Since centrifugal migration into uninvolved surrounding brain tissue occurs early in the course of glioblastoma, this attribute defeats local treatment attempts and is the primary reason current treatments almost always fail. Stopping bradykinin-triggered migration would be a step closer to control of this disease. The recent approval and marketing of an oral plasma kallikrein inhibitor, berotralstat (Orladeyo™), and pending FDA approval of a similar drug, sebetralstat, now offers a potential method for reducing local bradykinin production at sites of bradykinin-mediated glioblastoma migration. Both drugs are approved for treating hereditary angioedema. They are ideal for repurposing as a treatment adjunct in glioblastoma. Furthermore, it has been established that peritumoral edema, a common problem during the clinical course of glioblastoma, is generated in large part by locally produced bradykinin via kallikrein action. Both brain edema and the consequent use of corticosteroids both shorten survival in glioblastoma. Therefore, by (i) migration inhibition, (ii) growth inhibition, (iii) edema reduction, and (iv) the potential for less use of corticosteroids, berotralstat may be of service in treatment of glioblastoma, slowing disease progression. This paper recounts the details and past research on bradykinin in glioblastoma and the rationale of treating it with berotralstat.

Delayed plasma kallikrein inhibition fosters post-stroke recovery by reducing thrombo-inflammation

Activation of the kallikrein-kinin system promotes vascular leakage, inflammation, and neurodegeneration in ischemic stroke. Inhibition of plasma kallikrein (PK) – a key component of the KKS – in the acute phase of ischemic stroke has been reported to reduce thrombosis, inflammation, and damage to the blood-brain barrier. However, the role of PK during the recovery phase after cerebral ischemia is unknown. To this end, we evaluated the effect of subacute PK inhibition starting from day 3 on the recovery process after transient middle artery occlusion (tMCAO). Our study demonstrated a protective effect of PK inhibition by reducing infarct volume and improving functional outcome at day 7 after tMCAO. In addition, we observed reduced thrombus formation in cerebral microvessels, fewer infiltrated immune cells, and an improvement in blood-brain barrier integrity. This protective effect was facilitated by promoting tight junction reintegration, reducing detrimental matrix metalloproteinases, and upregulating regenerative angiogenic markers. Our findings suggest that PK inhibition in the subacute phase might be a promising approach to accelerate the post-stroke recovery process.

Unveiling therapeutic frontiers: DON/DRP-104 as innovative Plasma kallikrein inhibitors against carcinoma-associated hereditary angioedema shocks - a comprehensive molecular dynamics exploration

Human plasma kallikrein (PKa) is a member of the serine protease family and serves as a key mediator of the kallikrein-kinin system (KKS), which is known for its regulatory roles in inflammation, vasodilation, blood pressure, and coagulation. Genetic dysregulation of KKS leads to Hereditary Angioedema (HAE), which is characterized by spontaneous, painful swelling in various body regions. Importantly, HAE frequently coexists with various cancers. Despite substantial efforts towards the development of PKa inhibitors for HAE, there remains a need for bifunctional agents addressing both anti-cancer and anti-HAE aspects, especially against carcinoma-associated comorbid HAE conditions. Consequently, we investigated the therapeutic potential of the anti-glutamine prodrug, isopropyl(S)-2-((S)-2-acetamido-3-(1H-indol-3-yl)-propanamido)-6-diazo-5-oxo-hexanoate (DRP-104), and its active form, 6-Diazo-5-oxo-l-norleucine (DON), recognized for their anti-cancer properties, as novel PKa inhibitors. Utilizing structure-based in silico methods, we conducted a comparative analysis with berotralstat, a clinically approved HAE prophylactic, and sebetralstat, an investigational HAE therapeutic agent, in Phase 3 clinical trials. Inhibiting PKa with DON resulted in relatively heightened structural stability, rigidity, restricted protein folding, and solvent-accessible loop exposure, contributing to increased intra-atomic hydrogen bond formation. Conversely, PKa inhibition with DRP-104 induced restricted residue flexibility and significantly disrupted the critical SER195-HIS57 arrangement in the catalytic triad. Both DON and DRP-104, along with the reference drugs, induced strong cooperative intra-residue motion and bidirectional displacement in the PKa architecture. The results revealed favorable binding kinetics of DON/DRP-104, showing thermodynamic profiles that were either superior or comparable to those of the reference drugs. These findings support their consideration for clinical investigations into the management of carcinoma-associated HAE.

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation.

Macrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol-containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol-containing elements and used them for the combinatorial synthesis of a 2,688-member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X-ray structure analysis of macrocycle-target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation

AbstractMacrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol‐containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol‐containing elements and used them for the combinatorial synthesis of a 2,688‐member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X‐ray structure analysis of macrocycle‐target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Abstract 155: Plasma Kallikrein Is A Key Driver Of Basal Activation Of Coagulation Under Conditions Of C1 Inhibitor Deficiency

C1 inhibitor (C1INH) functions as a key endogenous inhibitor of contact pathway coagulation factors XII, XI and plasma kallikrein (PKa). Patients with a congenital deficiency in C1INH suffer from a rare swelling disorder called hereditary angioedema (HAE) caused by excess bradykinin generation. Patients with HAE also have elevated circulating levels of markers of activation of coagulation and an increased risk of venous thromboembolism, phenotypes recapitulated in C1INH-deficient mice. We sought to evaluate the molecular mechanism by which C1INH deficiency leads to increased basal activation of coagulation and venous thrombosis. We hypothesized that PKa serves as a key driver of activation of coagulation in humans and mice with congenital C1INH deficiency. Patients with C1INH deficiency-associated HAE were treated with the PKa inhibitor lanadelumab with plasma samples collected before and after treatment. Plasma levels of coagulation markers and contact pathway-initiated thrombin generation were determined. C1INH-deficient mice were treated with the PKa inhibitor avoralstat, the FXIIa inhibitor garadacimab, FXII small interfering RNAs, FXI antisense oligonucleotides, the anti-tissue factor antibody 1H1 or appropriate controls and plasma coagulation markers were measured. Treatment of HAE patients with the PKa inhibitor lanadelumab significantly reduced plasma levels of prothrombin fragment 1.2, thrombin antithrombin (TAT) complexes and D-dimer at 6 months after treatment initiation (P<0.05). Interestingly, landalumab had no significant effect on contact pathway-initiated thrombin generation. In experiments with C1INH deficient mice, TAT complexes were significantly reduced by PKa inhibition (P<0.01) but not by inhibition of FXIIa or FXIa. Interestingly, tissue factor inhibition also significantly reduced plasma TAT complexes (P<0.01). This data suggests that under conditions of C1INH deficiency PKa potentiates activation of coagulation through a non-canonical pathway involving tissue factor. These findings demonstrate that C1INH is a physiologically relevant anticoagulant with C1INH deficiency resulting in enhanced kallikrein-mediated activation of basal coagulation.

Plasma kallikrein supports FXII-independent thrombin generation in mouse whole blood

AbstractPlasma kallikrein (PKa) is an important activator of factor XII (FXII) of the contact pathway of coagulation. Several studies have shown that PKa also possesses procoagulant activity independent of FXII, likely through its ability to directly activate FIX. We evaluated the procoagulant activity of PKa using a mouse whole blood (WB) thrombin-generation (TG) assay. TG was measured in WB from PKa-deficient mice using contact pathway or extrinsic pathway triggers. PKa-deficient WB had significantly reduced contact pathway–initiated TG compared with that of wild-type controls and was comparable with that observed in FXII-deficient WB. PKa-deficient WB supported equivalent extrinsic pathway–initiated TG compared with wild-type controls. Consistent with the presence of FXII-independent functions of PKa, targeted blockade of PKa with either small molecule or antibody-based inhibitors significantly reduced contact pathway–initiated TG in FXII-deficient WB. Inhibition of activated FXII (FXIIa) using an antibody-based inhibitor significantly reduced TG in PKa-deficient WB, consistent with a PKa-independent function of FXIIa. Experiments using mice expressing low levels of tissue factor demonstrated that persistent TG present in PKa- and FXIIa-inhibited WB was driven primarily by endogenous tissue factor. Our work demonstrates that PKa contributes significantly to contact pathway–initiated TG in the complex milieu of mouse WB, and a component of this contribution occurs in an FXII-independent manner.

Sebetralstat: A Rapidly Acting Oral Plasma Kallikrein Inhibitor for the On-Demand Treatment of Hereditary Angioedema

Sebetralstat is a novel, potent, and selective oral plasma kallikrein inhibitor drug candidate in clinical development for the on-demand treatment of hereditary angioedema (HAE). Upon binding, sebetralstat induces a conformational change in the active site of plasma kallikrein, which contributes to its high potency (Ki 3 nM) and selectivity (>1500 fold) against other serine proteases. Its physiochemical properties promote both rapid dissolution in the stomach and rapid absorption in the upper intestine that contribute to its fast and efficient absorption. A single oral administration of sebetralstat rapidly provides near-complete inhibition of plasma kallikrein and blockade of high-molecular-weight kininogen cleavage as early as 15 min, which drives its clinical efficacy. In a phase 2 clinical trial, sebetralstat significantly reduced the time to beginning of symptom relief (p < 0.0001) with median times of 1.6 h (95% CI: 1.5–3.0) with sebetralstat versus 9.0 h (4.0–17.2) with placebo. KONFIDENT (NCT05259917) is a phase 3 clinical trial assessing the on-demand use of sebetralstat for HAE. If successful, this trial could support the approval of sebetralstat as the first noninvasive, on-demand treatment option to rapidly halt HAE attacks and provide fast symptom relief.

Discovery of α-Amidobenzylboronates as Highly Potent Covalent Inhibitors of Plasma Kallikrein.

Hereditary angioedema (HAE), a rare genetic disorder, is associated with uncontrolled plasma kallikrein (PKa) enzyme activity leading to the generation of bradykinin swelling in subcutaneous and submucosal membranes in various locations of the body. Herein, we describe a series of potent α-amidobenzylboronates as potential covalent inhibitors of PKa. These compounds exhibited time-dependent inhibition of PKa (compound 20 IC50 66 nM at 1 min, 70 pM at 24 h). Further compound dissociation studies demonstrated that 20 showed no apparent reversibility comparable to d-Phe-Pro-Arg-chloromethylketone (PPACK) (23), a known nonselective covalent PKa inhibitor.

Inhibitory potential of recombinant and native peanut Bowman-Birk inhibitor against proteases of human blood coagulation pathway

Plant proteinase/protease inhibitors (PIs) have therapeutic potential besides their role in regulating endogenous protease activity in several physiological processes. They exert their function by inhibiting the catalytic activity of specific proteases. The use of PIs for inhibiting coagulation factors and slowing down the blood coagulation process has emerged as a critical intervention point for developing antithrombotic agents. The present study has demonstrated the characterization of bacterially expressed recombinant peanut Bowman-Birk inhibitor (rPnBBI) and evaluation of its anticoagulant potential compared to native PnBBI (isoinhibitors pool) purified from peanuts. The full-length BBI gene was cloned, and rPnBBI (7.53 kDa) expressed in the soluble fraction of Escherichia coli BL21 (DE3) strain was isolated by passing through the trypsin affinity column. The purified rPnBBI with β-sheets as major secondary structural elements exhibited inhibitory activity towards bovine trypsin and chymotrypsin at diverse pH and temperatures. Both, rPnBBI and PnBBI showed anticoagulant effects by prolonging the activated partial thromboplastin time (aPTT) and inhibiting the activity of proteases/factors (plasma kallikrein, FXIIa, FXIa, FIXa, and FXa) involved in the blood coagulation pathway. Besides, rPnBBI and PnBBI showed a similar inhibitory tendency towards FXIIa, FXIa, and FIXa, while rPnBBI exhibited a higher inhibition potential than PnBBI towards FXa. Contrarily, PnBBI showed higher inhibitory potential than rPnBBI on plasma kallikrein. Altogether, the bioactive BBI molecules from the natural food source peanuts showed significant anticoagulant potential, which could be exploited in the prophylaxis of thromboembolic disorders associated with various human diseases.

A tick saliva serpin, IxsS17 inhibits host innate immune system proteases and enhances host colonization by Lyme disease agent.

Lyme disease (LD) caused by Borrelia burgdorferi is among the most important human vector borne diseases for which there is no effective prevention method. Identification of tick saliva transmission factors of the LD agent is needed before the highly advocated tick antigen-based vaccine could be developed. We previously reported the highly conserved Ixodes scapularis (Ixs) tick saliva serpin (S) 17 (IxsS17) was highly secreted by B. burgdorferi infected nymphs. Here, we show that IxsS17 promote tick feeding and enhances B. burgdorferi colonization of the host. We show that IxsS17 is not part of a redundant system, and its functional domain reactive center loop (RCL) is 100% conserved in all tick species. Yeast expressed recombinant (r) IxsS17 inhibits effector proteases of inflammation, blood clotting, and complement innate immune systems. Interestingly, differential precipitation analysis revealed novel functional insights that IxsS17 interacts with both effector proteases and regulatory protease inhibitors. For instance, rIxsS17 interacted with blood clotting proteases, fXII, fX, fXII, plasmin, and plasma kallikrein alongside blood clotting regulatory serpins (antithrombin III and heparin cofactor II). Similarly, rIxsS17 interacted with both complement system serine proteases, C1s, C2, and factor I and the regulatory serpin, plasma protease C1 inhibitor. Consistently, we validated that rIxsS17 dose dependently blocked deposition of the complement membrane attack complex via the lectin complement pathway and protected complement sensitive B. burgdorferi from complement-mediated killing. Likewise, co-inoculating C3H/HeN mice with rIxsS17 and B. burgdorferi significantly enhanced colonization of mouse heart and skin organs in a reverse dose dependent manner. Taken together, our data suggests an important role for IxsS17 in tick feeding and B. burgdorferi colonization of the host.

Biomarkers associated with cardiovascular disease in women with spontaneous preterm birth: A case-control study.

Women with spontaneous preterm birth have an increased risk of cardiovascular disease later in life. Studies suggest potential pathophysiological mechanisms in common, but whether these could be identified by measurement of soluble circulating protein biomarkers in women with spontaneous preterm birth is unknown. The aim of this study was to determine if protein biomarkers associated with cardiovascular disease distinguish women with spontaneous preterm birth from healthy controls, both at pregnancy and at follow up. Study participants were identified in the population-based Uppsala biobank of pregnant women in Sweden, where plasma samples were collected in mid-pregnancy. In a first screening phase, we identified participants who subsequently experienced spontaneous preterm birth (<37 weeks) in the index pregnancy (N = 13) and controls (N = 6). In these samples, differences in protein expression were examined by comparative mass spectrometry. In a second validation phase, we invited 100 cases with previous spontaneous preterm birth in the index pregnancy and 100 controls (matched for age, body mass index, and year of delivery) from the same source population, to a follow-up visit 4-15 years after pregnancy. At follow up, we collected plasma samples and data on cardiovascular risk factors. We measured concentrations of selected biomarkers identified in the screening phase, as well as lipid profiles in samples both from pregnancy (biobank) and follow up. gov registration NCT05693285. In the screening phase, fibrinogen, cadherin-5, complement C5, factor XII, plasma kallikrein, apolipoprotein M, and vitamin D-binding protein differed significantly at pregnancy. In the validation phase, 65 women agreed to participate (35 cases and 30 controls), with a median follow-up time of 11.8 years since pregnancy. The concentration of fibrinogen (p = 0.02) and triglycerides (p = 0.03) were slightly higher in cases compared with matched controls at follow up. Compared with women without preterm birth, those with spontaneous preterm birth had slightly higher concentrations of fibrinogen, both at mid-pregnancy and a decade after pregnancy. Additionally, we found slightly higher concentration of triglycerides at follow up in women with previous spontaneous preterm birth. The relevance of this finding is uncertain but might indicate potential pathophysiological mechanisms in common between spontaneous preterm birth and cardiovascular disease.

CRISPR-Cas9 In Vivo Gene Editing of KLKB1 for Hereditary Angioedema

BackgroundHereditary angioedema is a rare genetic disease that leads to severe and unpredictable swelling attacks. NTLA-2002 is an in vivo gene-editing therapy based on clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9. NTLA-2002 targets the gene encoding kallikrein B1 (KLKB1), with the goal of lifelong control of angioedema attacks after a single dose.MethodsIn this phase 1 dose-escalation portion of a combined phase 1–2 trial of NTLA-2002 in adults with hereditary angioedema, we administered NTLA-2002 at a single dose of 25 mg, 50 mg, or 75 mg. The primary end points were the safety and side-effect profile of NTLA-2002 therapy. Secondary and exploratory end points included pharmacokinetics, pharmacodynamics, and clinical efficacy determined on the basis of investigator-confirmed angioedema attacks.ResultsThree patients received 25 mg of NTLA-2002, four received 50 mg, and three received 75 mg. At all dose levels, the most common adverse events were infusion-related reactions and fatigue. No dose-limiting toxic effects, serious adverse events, grade 3 or higher adverse events, or clinically important laboratory findings were observed after the administration of NTLA-2002. Dose-dependent reductions in the total plasma kallikrein protein level were observed between baseline and the latest assessment, with a mean percentage change of −67% in the 25-mg group, −84% in the 50-mg group, and −95% in the 75-mg group. The mean percentage change in the number of angioedema attacks per month between baseline and weeks 1 through 16 (primary observation period) was −91% in the 25-mg group, −97% in the 50-mg group, and −80% in the 75-mg group. Among all the patients, the mean percentage change in the number of angioedema attacks per month from baseline through the latest assessment was −95%.ConclusionsIn this small study, a single dose of NTLA-2002 led to robust, dose-dependent, and durable reductions in total plasma kallikrein levels, and no severe adverse events were observed. In exploratory analyses, reductions in the number of angioedema attacks per month were observed at all dose levels. (Funded by Intellia Therapeutics; ClinicalTrials.gov number, NCT05120830.)